Explore the vast range of titles available.

Microbial induced calcite precipitation (MICP) based on ureolysis has a high potential for many applications, e.g. restoration of construction materials. The gram-positive bacterium Sporosarcina pasteurii is the most commonly used microorganism for MICP due to its high ureolytic activity. However, Sporosarcina pasteurii is so far cultivated almost exclusively in complex media, which only results in moderate biomass concentrations at the best. Cultivation of Sporosarcina pasteurii must be strongly improved in order to make technological application of MICP economically feasible. The growth of Sporosarcina pasteurii DSM 33 was boosted by detecting auxotrophic deficiencies (L-methionine, L-cysteine, thiamine, nicotinic acid), nutritional requirements (phosphate, trace elements) and useful carbon sources (glucose, maltose, lactose, fructose, sucrose, acetate, L-proline, L-alanine). These were determined by microplate cultivations with online monitoring of biomass in a chemically defined medium and systematically omitting or substituting medium components. Persisting growth limitations were also detected, allowing further improvement of the chemically defined medium by the addition of glutamate group amino acids. Common complex media based on peptone and yeast extract were supplemented based on these findings. Optical density at the end of each cultivation of the improved peptone and yeast extract media roughly increased fivefold respectively. A maximum OD600 of 26.6 ± 0.7 (CDW: 17.1 ± 0.5 g/L) was reached with the improved yeast extract medium. Finally, culture performance and media improvement was analysed by measuring the oxygen transfer rate as well as the backscatter during shake flask cultivation.

Background Escherichia coli ( E. coli ) is the most abundant expression host for recombinant proteins. The production efficiency is dependent on a multitude of parameters. Therefore, high-throughput applications have become an increasingly frequent technique to investigate the main factors. Within this study, the effects of temperature, induction time and inducer concentration on the metabolic state and the product formation were extensively examined. Induction profiling of E. coli Tuner(DE3) pRhotHi-2-EcFbFP was performed in 48-well Flowerplates and standard 96-well plates using a robotic platform. In parallel shake flask cultivations, the respiration activity of the microorganisms was analyzed. Therefore, two online-monitoring systems were applied: the BioLector for microtiter plates and the RAMOS-device for shake flasks. The impact of different induction conditions on biomass and product formation as well as on the oxygen transfer rate was surveyed. Results Different optimal induction conditions were obtained for temperatures of 28, 30, 34, and 37 °C. The best inducer concentrations were determined to be between 0.05 and 0.1 mM IPTG for all investigated temperatures. This is 10–20 times lower than conventional guidelines suggest. The induction time was less relevant when the correct inducer concentration was chosen. Furthermore, there was a stronger impact on growth and respiration activity at higher temperatures. This indicated a higher metabolic burden. Therefore, lower IPTG concentrations were advantageous at elevated temperatures. Very similar results were obtained in standard 96-well plates. Conclusion Two online-monitoring systems were successfully used to investigate the optimal induction conditions for the E. coli Tuner(DE3) pRhotHi-2-EcFbFP strain ( lacY deletion mutant) at four different temperatures. The experimental effort was reduced to a minimum by integrating a liquid handling robot. To reach the maximum product formation, a detailed induction analysis was necessary. Whenever the cultivation temperature was changed, the induction conditions have to be adapted. Due to the experimental options provided by the BioLector technology, it was found that the higher the cultivation temperature, the lower the inducer concentration that has to be applied.

Background An advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given. Results Bacterial and yeast biomass concentrations of up to 50 g/L cell dry weight could be linearly correlated to scattered light intensities. In media screening, the BioLector could clearly demonstrate its potential for detecting different biomass and product yields and deducing specific growth rates for quantitatively evaluating media and nutrients. Growth inhibition due to inappropriate buffer conditions could be detected by reduced growth rates and a temporary increase in NADH fluorescence. GFP served very well as reporter protein for investigating the promoter regulation under different carbon sources in yeast strains. A clone screening of 90 different GFP -expressing Hansenula polymorpha clones depicted the broad distribution of growth behavior and an even stronger distribution in GFP expression. The importance of mass transfer conditions could be demonstrated by varying filling volumes of an E. coli culture in 96 well MTP. The different filling volumes cause a deviation in the culture growth and acidification both monitored via scattered light intensities and the fluorescence of a pH indicator, respectively. Conclusion The BioLector technology is a very useful tool to perform quantitative microfermentations under engineered reaction conditions. With this technique, specific yields and rates can be directly deduced from online biomass and product concentrations, which is superior to existing technologies such as microplate readers or optode-based cultivation systems. In particular, applications with strong demand on high-throughput such as clone and media screening and systems biology can benefit from its simple handling, the high quantitative information content and its capacity of automation.

Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein. While anecdotal reports about IBs themselves showing catalytic functionality/activity (CatIB) are found throughout literature, only recently, the use of protein engineering methods has facilitated the on-demand production of CatIBs. CatIB formation is induced usually by fusing short peptide tags or aggregation-inducing protein domains to a target protein. The resulting proteinaceous particles formed by heterologous expression of the respective genes can be regarded as a biologically produced bionanomaterial or, if enzymes are used as target protein, carrier-free enzyme immobilizates. In the present contribution, we review general concepts important for CatIB production, processing, and application.Key points• Catalytically active inclusion bodies (CatIBs) are promising bionanomaterials.• Potential applications in biocatalysis, synthetic chemistry, and biotechnology.• CatIB formation represents a generic approach for enzyme immobilization.• CatIB formation efficiency depends on construct design and expression conditions.

Viscosity is an inherent characteristic of fluids and is therefore an important parameter in many different processes. Current methods to measure viscosity involve direct contact with the liquid sample, which is often undesirable. Here we present a simple, precise and robust contact-free method to determine viscosity, using a single drive motor, inexpensive components and disposable sample vessels. The measurement principle involves the detection of viscosity-dependent angular positions in a rotating liquid relative to the direction of centrifugal acceleration in an orbitally shaken vessel. The signal can be detected using different optical methods, as shown here using fluorescence and transmitted light. The sensitivity of the system can be adjusted over a wide range by varying the sample volume or the shaking diameter, and multiple samples can be analysed in parallel. This novel viscometer is also applicable to characterize non-Newtonian shear rate-dependent fluids.

Background The promising yet barely investigated anaerobic species Phocaeicola vulgatus (formerly Bacteroides vulgatus ) plays a vital role for human gut health and effectively produces organic acids. Among them is succinate, a building block for high-value-added chemicals. Cultivating anaerobic bacteria is challenging, and a detailed understanding of P. vulgatus growth and metabolism is required to improve succinate production. One significant aspect is the influence of different gas concentrations. CO 2 is required for the growth of P. vulgatus. However, it is a greenhouse gas that should not be wasted. Another highly interesting aspect is the sensitivity of P. vulgatus towards O 2 . In this work, the effects of varying concentrations of both gases were studied in the in-house developed Respiratory Activity MOnitoring System (RAMOS), which provides online monitoring of CO 2 , O 2, and pressure under gassed conditions. The RAMOS was combined with a gas mixing system to test CO 2 and O 2 concentrations in a range of 0.25-15.0 vol% and 0.0-2.5 vol%, respectively. Results Changing the CO 2 concentration in the gas supply revealed a CO 2 optimum of 3.0 vol% for total organic acid production and 15.0 vol% for succinate production. It was demonstrated that the organic acid composition changed depending on the CO 2 concentration. Furthermore, unrestricted growth of P. vulgatus up to an O 2 concentration of 0.7 vol% in the gas supply was proven. The viability decreased rapidly at concentrations larger than or equal to 1.3 vol% O 2 . Conclusions The study showed that P. vulgatus requires little CO 2 , has a distinct O 2 tolerance and is therefore well suited for industrial applications.

Computational fluid dynamics (CFD) has recently become a pivotal tool in the design and scale-up of bioprocesses. While CFD has been extensively utilized for stirred tank reactors (STRs), there exists a relatively limited body of literature focusing on CFD applications for shake flasks, almost exclusively concentrated on fluids at waterlike viscosity. The importance of CFD model validation cannot be overstated. While techniques to elucidate the internal flow field are necessary for model validation in STRs, the liquid distribution, caused by the orbital shaking motion of shake flasks, can be exploited for model validation. An OpenFOAM CFD model for shake flasks has been established. Calculated liquid distributions were compared to suitable, previously published experimental data. Across a broad range of shaking conditions, at waterlike and moderate viscosity (16.7 mPa∙s), the CFD model's liquid distributions align excellently with the experimental data, in terms of overall shape and position of the liquid relative to the direction of the centrifugal force. Additionally, the CFD model was used to calculate the volumetric power input, based on the energy dissipation. Depending on the shaking conditions, the computed volumetric power inputs range from 0.1 to 7 kW/m 3 and differed on average by 0.01 kW/m 3 from measured literature data.

Background In the fermentation industry, the demand to replace expensive complex media components is increasing for alternative nutrient sources derived from waste or side streams, such as corn steep liquor (CSL). However, the use of CSL is associated with common problems of side products, such as batch-to-batch variations and compositional inconsistencies. In this study, to detect batch-to-batch variations in CSL for Ogataea polymorpha cultivations, a “fingerprinting” system was developed by employing the Respiration Activity Monitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (µTOM). Results At 2.5 g d.s./L CSL and 5 g/L glucose, a limitation by a secondary substrate, other than the carbon source, was observed. For this specific CSL medium, this limitation was caused by ammonium nitrogen and could be removed through targeted supplementation of ammonium sulphate. Under ammonium nitrogen limitation, O. polymorpha showed a change in morphology and developed a different cell size distribution. Increasing CSL storage times impaired O. polymorpha cultivation results. It was speculated that this observation is caused by micronutrient precipitation as sulfide salts. Through targeted nutrient supplementation, these limiting microelements were identified to be copper, iron and zinc. Conclusions This study shows the versatility of CSL as an alternative nutrient source for O. polymorpha cultivations. “Fingerprinting” of CSL batches allows for early screening. Fermentation inconsistencies can be eliminated by selecting the better performing CSL batches or by supplementing and improving an inferior CSL prior to large-scale productions.

Background Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation. Results The efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation. Conclusion This study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Background In the past decade, an enormous number of new bioprocesses have evolved in the biotechnology industry. These bioprocesses have to be developed fast and at a maximum productivity. Up to now, only few microbioreactors were developed to fulfill these demands and to facilitate sample processing. One predominant reaction platform is the shaken microtiter plate (MTP), which provides high-throughput at minimal expenses in time, money and work effort. By taking advantage of this simple and efficient microbioreactor array, a new online monitoring technique for biomass and fluorescence, called BioLector, has been recently developed. The combination of high-throughput and high information content makes the BioLector a very powerful tool in bioprocess development. Nevertheless, the scalabilty of results from the micro-scale to laboratory or even larger scales is very important for short development times. Therefore, engineering parameters regarding the reactor design and its operation conditions play an important role even on a micro-scale. In order to evaluate the scale-up from a microtiter plate scale (200 μL) to a stirred tank fermenter scale (1.4 L), two standard microbial expression systems, Escherichia coli and Hansenula polymorpha , were fermented in parallel at both scales and compared with regard to the biomass and protein formation. Results Volumetric mass transfer coefficients (k L a) ranging from 100 to 350 1/h were obtained in 96-well microtiter plates. Even with a suboptimal mass transfer condition in the microtiter plate compared to the stirred tank fermenter (k L a = 370-600 1/h), identical growth and protein expression kinetics were attained in bacteria and yeast fermentations. The bioprocess kinetics were evaluated by optical online measurements of biomass and protein concentrations exhibiting the same fermentation times and maximum signal deviations below 10% between the scales. In the experiments, the widely applied green fluorescent protein ( GFP ) served as an online reporter of protein expression for both strains. Conclusions The successful 7000-fold scale-up from a shaken microtiter plate to a stirred tank fermenter was demonstrated in parallel fermentations for standard microbial expression systems. This confirms that the very economical and time efficient platform of microtiter plates can be very easily scaled up to larger stirred tank fermenters under defined engineering conditions. New online monitoring techniques for microtiter plates, such as the BioLector, provide even more real-time kinetic data from fermentations than ever before and at an affordable price. This paves the way for a better understanding of the bioprocess and a more rational process design.