Endospore production of Bacillus spp. for industrial use

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected

Innovative Verfahren zur enzymatischen Abnahme von schädigenden Caseinüberzügen auf Wandmalereien

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Determination of aqueous two-phase system phase-forming components in the presence of bovine serum albumin

In the current work, the quantification of different poly(ethylene glycol) (PEG)-potassium phosphate/sodium citrate aqueous two-phase system (ATPS) phase-forming components was investigated by using conductivity and refractive index measurements. For this purpose, refractive index and conductivity calibration curves were obtained for ATPS at different pH values in the presence of different bovine serum albumin (BSA) concentrations. Whereas BSA had no effect on the conductivity, it had a considerable effect on the refractive index. Finally, a convenient dilution of the samples prior to the ATPS constituent determination is needed to ensure no significant influence from BSA

One-step-purification of penicillin G amidase from cell lysate using ion-exchange membrane adsorbers

This study describes the purification of penicillin G amidase (PGA) by ion exchange membrane adsorbers in a one-step-process. Preliminary experiments with high-throughput screening devices in microliter scale (8-strip modules) were performed to find suitable purification strategy and appropriate ion exchange ligands as well as basic process conditions for binding and elution. Best purification results were achieved by strong cation-exchange (S-) membrane adsorbers loaded with 2ml/min enzyme solution at pH 6.0 and eluted at pH 6.0 with 0.05M NaCl, which led to a high yield of bound PGA (98%) without any visible remains of host cell proteins and with a residual enzyme activity of 80-85%. The binding of PGA to the S-membrane was further investigated in an upscaling to milliliter scale with LP15 modules and breakthrough curves were determined by varying the flow rates: the PGA-binding to S-membrane adsorbers is independent of the flow rate. Dynamic binding capacities were estimated to be 0.69mg PGA/cm2 (25.5mg/ml) for 10% breakthrough respectively 0.95mg/cm2 (35.2mg/ml) for 100% breakthrough. Finally, real cell lysate samples from Escherichia coli culture containing PGA were processed under the found optimal conditions. Despite exceeded loading PGA was isolated from this complex mixture successfully fourfold concentrated and with a purification factor of 101.3 and a resulting specific activity of 4.97U/mg.BMBF/BIOCATALYSIS2021DFG/EXC/REBIRT

Improved production and in situ recovery of sesquiterpene (+)-zizaene from metabolically-engineered E. Coli

The sesquiterpene (+)-zizaene is the direct precursor of khusimol, the main fragrant compound of the vetiver essential oil from Chrysopogon zizanioides and used in nearly 20% of men’s fine perfumery. The biotechnological production of such fragrant sesquiterpenes is a promising alternative towards sustainability; nevertheless, product recovery from fermentation is one of the main constraints. In an effort to improve the (+)-zizaene recovery from a metabolically-engineered Escherichia coli, we developed an integrated bioprocess by coupling fermentation and (+)-zizaene recovery using adsorber extractants. Initially, (+)-zizaene volatilization was confirmed from cultivations with no extractants but application of liquid–liquid phase partitioning cultivation (LLPPC) improved (+)-zizaene recovery nearly 4-fold. Furthermore, solid–liquid phase partitioning cultivation (SLPPC) was evaluated by screening polymeric adsorbers, where Diaion HP20 reached the highest recovery. Bioprocess was scaled up to 2 L bioreactors and in situ recovery configurations integrated to fermentation were evaluated. External recovery configuration was performed with an expanded bed adsorption column and improved (+)-zizaene titers 2.5-fold higher than LLPPC. Moreover, internal recovery configuration (IRC) further enhanced the (+)-zizaene titers 2.2-fold, whereas adsorption velocity was determined as critical parameter for recovery efficiency. Consequently, IRC improved the (+)-zizaene titer 8.4-fold and productivity 3-fold from our last report, achieving a (+)-zizaene titer of 211.13 mg L−1 and productivity of 3.2 mg L−1 h−1. This study provides further knowledge for integration of terpene bioprocesses by in situ product recovery, which could be applied for many terpene studies towards the industrialization of fragrant molecules

Development and Testing of a 4-Columns Periodic Counter-Current Chromatography System Based on Membrane Adsorbers

Continuous chromatography can surmount the disadvantages of batch chromatography like low productivities and extensive usage of consumables. In this work, a 4-column continuous chromatographic system based on the principle of periodic counter-current chromatography (PCCC) was developed and tested with a model protein mixture of BSA and lysozyme. The PCCC system was specially designed for membrane adsorbers as an alternative to conventional columns to facilitate the use of disposable process units and to further increase the productivity due to higher convective mass transport in the membrane adsorber. Membrane adsorber Sartobind® Q was used to continuously purify BSA from the protein mixture. The usage of PCCC led to an increased capacity utilization (here 20%) and higher space–time-yields, and thus to a remarkable productivity increase and cost savings

Comparison of colorimetric methods for the quantification of model proteins in aqueous two-phase systems

In the current study, the quantification of different model proteins in the presence of typical aqueous two-phase system components was investigated by using the Bradford and bicinchoninic acid (BCA) assays. Each phase-forming component above 1 and 5 wt% had considerable effects on the protein quantification in both assays, respectively, resulting in diminished protein recoveries/absorption values by increasing poly(ethylene glycol) (PEG)/salt concentration and PEG molecular weight. Therefore, a convenient dilution of both components (up to 1 and 5 wt%) before protein quantification is recommended in both assays, respectively, where the BCA assay is favored in comparison with the Bradford assay

On-Line Monitoring of Biological Parameters in Microalgal Bioprocesses Using Optical Methods

Microalgae are promising sources of fuels and other chemicals. To operate microalgal cultivations efficiently, process control based on monitoring of process variables is needed. On-line sensing has important advantages over off-line and other analytical and sensing methods in minimizing the measurement delay. Consequently, on-line, in-situ sensors are preferred. In this respect, optical sensors occupy a central position since they are versatile and readily implemented in an on-line format. In biotechnological processes, measurements are performed in three phases (gaseous, liquid and solid (biomass)), and monitored process variables can be classified as physical, chemical and biological. On-line sensing technologies that rely on standard industrial sensors employed in chemical processes are already well-established for monitoring the physical and chemical environment of an algal cultivation. In contrast, on-line sensors for the process variables of the biological phase, whether biomass, intracellular or extracellular products, or the physiological state of living cells, are at an earlier developmental stage and are the focus of this review. On-line monitoring of biological process variables is much more difficult and sometimes impossible and must rely on indirect measurement and extensive data processing. In contrast to other recent reviews, this review concentrates on current methods and technologies for monitoring of biological parameters in microalgal cultivations that are suitable for the on-line and in-situ implementation. These parameters include cell concentration, chlorophyll content, irradiance, and lipid and pigment concentration and are measured using NMR, IR spectrophotometry, dielectric scattering, and multispectral methods. An important part of the review is the computer-aided monitoring of microalgal cultivations in the form of software sensors, the use of multi-parameter measurements in mathematical process models, fuzzy logic and artificial neural networks. In the future, software sensors will play an increasing role in the real-time estimation of biological variables because of their flexibility and extendibility

Study on the development and integration of 3D-printed optics in small-scale productions of single-use cultivation vessels

Integrating optical sensors and 3D-printed optics into single-use (SU) cultivation vessels for customized, tailor-made equipment could be a next big step in the bioreactor and screening platform development enabling online bioprocess monitoring. Many different parameters such as pH, oxygen, carbon dioxide and optical density (OD) can be monitored more easily using online measuring instruments compared to offline sampling. Space-saving integrated sensors in combination with adapted optics such as prisms open up vastly new possibilities to precisely guide light through SU vessels. This study examines how optical prisms can be 3D-printed with a 3D-inkjet printer, modified and then evaluated in a custom made optical bench. The prisms are coated or bonded with thin cover glasses. For the examination of reflectance performance and conformity prisms are compared on the basis of measured characteristics of a conventional glass prism. In addition, the most efficient and reproducible prism geometry and modification technique is applied to a customized 3D-printed cultivation vessel. The vessel is evaluated on a commercial sensor-platform, a shake flask reader (SFR) vario, to investigate its sensing-characteristics while monitoring scattered light with the turbidity standard formazine and a cell suspension of Saccharomyces cerevisiae as model organism. It is demonstrated that 3D-printed prisms can be used in combination with commercial scattered light sensor-platforms to determine OD of a microbial culture and that a 3D-printed unibody design with integrated optics in a cultivation vessel is feasible. In the range of OD600 0–1.16 rel.AU a linear correlation between sensor amplitude and offline determined OD can be achieved. Thus, enabling for the first time a measurement of low cell densities with the SFR vario platform. Moreover, sensitivity is also at least three times higher compared to the commonly used method

Whole-Cell Production of Patchouli Oil Sesquiterpenes in Escherichia coli: Metabolic Engineering and Fermentation Optimization in Solid–Liquid Phase Partitioning Cultivation

Patchouli oil is a major ingredient in perfumery, granting a dark-woody scent due to its main constituent (−)-patchoulol. The growing demand for patchouli oil has raised interest in the development of a biotechnological process to assure a reliable supply. Herein, we report the production of patchouli oil sesquiterpenes by metabolically engineered Escherichia coli strains, using solid–liquid phase partitioning cultivation. The (−)-patchoulol production was possible using the endogenous methylerythritol phosphate pathway and overexpressing a (−)-patchoulol synthase isoform from Pogostemon cablin but at low titers. To improve the (−)-patchoulol production, the exogenous mevalonate pathway was overexpressed in the multi-plasmid PTS + Mev strain, which increased the (−)-patchoulol titer 5-fold. Fermentation was improved further by evaluating several defined media, and optimizing the pH and temperature of culture broth, enhancing the (−)-patchoulol titer 3-fold. To augment the (−)-patchoulol recovery from fermentation, the solid–liquid phase partitioning cultivation was analyzed by screening polymeric adsorbers, where the Diaion HP20 adsorber demonstrated the highest (−)-patchoulol recovery from all tests. Fermentation was scaled-up to fed-batch bioreactors, reaching a (−)-patchoulol titer of 40.2 mg L–1 and productivity of 20.1 mg L–1 d–1. The terpene profile and aroma produced from the PTS + Mev strain were similar to the patchouli oil, comprising (−)-patchoulol as the main product, and α-bulnesene, trans-β-caryophyllene, β-patchoulene, and guaia-5,11-diene as side products. This investigation represents the first study of (−)-patchoulol production in E. coli by solid–liquid phase partitioning cultivation, which provides new insights for the development of sustainable bioprocesses for the microbial production of fragrant terpenes