Establishing a Fed‐Batch Process for Protease Expression with
            Bacillus licheniformis
            in Polymer‐Based Controlled‐Release Microtiter Plates

Abstract Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

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Production of alkaline protease with Bacillus licheniformis in a controlled fed-batch process

Applied Microbiology and Biotechnology ◽

10.1007/bf00169884 ◽

1991 ◽

Vol 35 (6) ◽

Cited By ~ 18

Author(s):

UlrichEberhard Giesecke ◽

Gabriele Bierbaum ◽

Heinz Rudde ◽

Uwe Spohn ◽

Christian Wandrey

Keyword(s):

Bacillus Licheniformis ◽

Alkaline Protease ◽

Batch Process ◽

Fed Batch

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New Exopolysaccharides Produced by Bacillus licheniformis 24 Display Substrate-Dependent Content and Antioxidant Activity

Microorganisms ◽

10.3390/microorganisms9102127 ◽

2021 ◽

Vol 9 (10) ◽

pp. 2127

Author(s):

Penka Petrova ◽

Alexander Arsov ◽

Ivan Ivanov ◽

Lidia Tsigoriyna ◽

Kaloyan Petrov

Keyword(s):

Antioxidant Activity ◽

Bacillus Licheniformis ◽

Biological Activities ◽

Batch Process ◽

Industrial Applications ◽

Batch Cultivation ◽

Batch Processes ◽

Exponential Growth Phase ◽

Fed Batch ◽

Platform Chemicals

Bacillus licheniformis is a soil bacterium with many industrial applications. In addition to enzymes, platform chemicals, antibiotics and phytohormones, the species produces exopolysaccharides (EPSs) of various biological activities. This study revealed that Bulgarian isolate B. licheniformis 24 produced EPSs consisting of galactose, glucose and mannose with substrate-dependent ratio. From glucose, B. licheniformis 24 secreted EPS1, consisting of 54% galactose, 39% glucose and 7% mannose. From fructose, the strain formed EPS2, containing 51% glucose, 30% mannose and 19% galactose. Batch cultivation in flasks yielded 2.2–2.6 g/L EPS1 and 1.90–2.11 g/L EPS2. Four to five times higher yields of EPS were obtained from both substrates during batch and fed-batch processes in a fermenter at 37.8 °C, pH 6.2 and aeration 3.68 vvm. The batch process with 200 g/L of starting substrates received 9.64 g/L EPS1 and 6.29 g/L EPS2, reaching maximum values at the 33rd and 24th h, respectively. Fed-batch fermentation resulted in the highest yields, 12.61 g/L EPS1 and 7.03 g/L EPS2. In all processes, EPSs were produced only in the exponential growth phase. Both EPSs exhibited antioxidant activity, but EPS2 was much more potent in this regard, reaching 811 μM Vitamin C Equivalent Antioxidant Capacity (versus 135 μM for EPS1). EPS1 displayed antibacterial activity against a non-O1 strain of Vibrio cholerae.

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Production of Rhamnolipid Biosurfactant from Fed Batch Culture by Pseudomonas aeruginosa using Multiple Substrates

Current Nutrition & Food Science ◽

10.2174/1573401314666181107100127 ◽

2020 ◽

Vol 16 (6) ◽

pp. 928-933

Author(s):

Jujjavarapu S. Eswari

Keyword(s):

Pseudomonas Aeruginosa ◽

Batch Process ◽

Time Interval ◽

Fed Batch ◽

Multiple Substrates ◽

Surface Active Agents ◽

Limiting Nutrients ◽

Limiting Nutrient ◽

Surface Active ◽

Rhamnolipid Biosurfactant

Objective: Biosurfactants are the surface active agents which are used for the reduction of surface and interfacial tensions of liquids. Rhamnolipids are the surfactants produced by Pseudomonas aeruginosa. It requires minimum nutrition for its growth as it can also grow in distilled water. The rhamnolipids produced by Pseudomonas aeruginosa are extra-cellular glycolipids consisting of L-rhamnose and 3-hydroxyalkanoic acid. Methods: The fed-batch method for the rhamnolipid production is considered in this study to know the influence of the carbon, nitrogen, phosphorous substrates as growth-limiting nutrients. Pulse feeding is employed for limiting nutrient addition at particular time interval to obtain maximum rhamnolipid formation from Pseudomonas aeruginosa compared with the batch process. Results: Out of 3 fed batch strategies constant glucose fed batch strategy shows best and gave maximum rhamnolipid concentration of 0.134 g/l.

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