Felix Lenk

Bioprocessing of differentiated plant in vitro systems

Plant cells contain a wide range of interesting secondary metabolites, which are used as natural pigments and flavoring agents in foods and cosmetics as well as phyto‐pharmaceutical products. However, conventional industrial extraction from whole plants or parts of them is limited due to environmental and geographical issues. The production of secondary metabolites from in vitro cultures can be considered as alternative to classical technologies and allows a year‐round cultivation in the bioreactor under optimal conditions with constant high‐level quality and quantity. Compared to plant cell suspensions, differentiated plant in vitro systems offer the advantage that they are genetically stable. Moreover, the separation of the biomass from culture medium after fermentation is much easier. Nevertheless, several investigations in the literature described that differentiated plant in vitro systems are instable concerning the yield of the target metabolites, especially in submerged cultivations. Other major problems are associated with the challenges of cultivation conditions and bioreactor design as well as upscaling of the process. This article reviews bioreactor designs for cultivation of differentiated plant in vitro systems, secondary metabolite production in different bioreactor systems as well as aspects of process control, management, and modeling and gives perspectives for future cultivation methods.

Automatic image recognition to determine morphological development and secondary metabolite accumulation in hairy root networks

This study focuses on the morphological development and secondary metabolite production of the red pigments from the group of betacyanins in hairy roots of Beta vulgaris. We demonstrate a working, medium throughput, customized, automatic image recognition solution for hairy roots on agar plates including the evaluation of 12 experimental samples. Image acquisition is conducted under comparable para‐meters using a tripod with light emitting diode background lighting and a digital single lens reflex camera. The server‐based image recognition system developed together with Wimasis GmbH, Munich, Germany helps to obtain not only quantitative values for morphological parameters, such as segment lengths and widths or metabolite concentrations, but also global parameters of root growth, such as total root length or the number of branching points. Using timed diagrams the development of the total root length, the total number of branching points, and the mean pigment concentration during the cultivation period were determined. The generated data present the basis for detailed mathematical modeling in order to achieve a structured growth model for hairy roots. A mathematical model for growth of hairy roots can be used to decrease experimental efforts as well as to optimize cultivation conditions and the bioreactor design.

Modeling hairy root tissue growth in in vitro environments using an agent-based, structured growth model

An agent-based model for simulating the in vitro growth of Beta vulgaris hairy root cultures is described. The model fitting is based on experimental results and can be used as a virtual experimentator for root networks. It is implemented in the JAVA language and is designed to be easily modified to describe the growth of diverse biological root networks. The basic principles of the model are outlined, with descriptions of all of the relevant algorithms using the ODD protocol, and a case study is presented in which it is used to simulate the development of hairy root cultures of beetroot (Beta vulgaris) in a Petri dish. The model can predict various properties of the developing network, including the total root length, branching point distribution, segment distribution and secondary metabolite accumulation. It thus provides valuable information that can be used when optimizing cultivation parameters (e.g., medium composition) and the cultivation environment (e.g., the cultivation temperature) as well as how constructional parameters change the morphology of the root network. An image recognition solution was used to acquire experimental data that were used when fitting the model and to evaluate the agreement between the simulated results and practical experiments. Overall, the case study simulation closely reproduced experimental results for the cultures grown under equivalent conditions to those assumed in the simulation. A 3D-visualization solution was created to display the simulated results relating to the state of the root network and its environment (e.g., oxygen and nutrient levels).

PetriJet Platform Technology: An Automated Platform for Culture Dish Handling and Monitoring of the Contents

Due to the size of the required equipment, automated laboratory systems are often unavailable or impractical for use in small- and mid-sized laboratories. However, recent developments in automation engineering provide endless possibilities for incorporating benchtop devices. Here, the authors describe the development of a platform technology to handle sealed culture dishes. The programming is based on the Petri net method and implemented via Codesys V3.5 pbF. The authors developed a system of three independent electrical driven axes capable of handling sealed culture dishes. The device performs two difference processes. First, it automatically obtains an image of every processed culture dish. Second, a server-based image analysis algorithm provides the user with several parameters of the cultivated sample on the culture dish. For demonstration purposes, the authors developed a continuous, systematic, nondestructive, and quantitative method for monitoring the growth of a hairy root culture. New results can be displayed with respect to the previous images. This system is highly accurate, and the results can be used to simulate the growth of biological cultures. The authors believe that the innovative features of this platform can be implemented, for example, in the food industry, clinical environments, and research laboratories.

Phototrophic growth of Arthrospira platensis in a respiration activity monitoring system for shake flasks (RAMOS

Optimizing illumination is essential for optimizing the growth of phototrophic cells and their production of desired metabolites and/or biomass. This requires appropriate modulation of light and other key inputs and continuous online monitoring of their metabolic activities. Powerful noninvasive systems for cultivating heterotrophic organisms include shake flasks in online monitoring units, but they are rarely used for phototrophs because they lack the appropriate illumination design and necessary illuminatory power. This study presents the design and characterization of a photosynthetic shake flask unit, illuminated from below by warm white light‐emitting diodes with variable light intensities up to 2300 μmol m−2 s−1. The photosynthetic unit was successfully used, in combination with online monitoring of oxygen production, to cultivate Arthrospira platensis. In phototrophic growth under continuous light and a 16 h light/8 h dark cycle (light intensity: 180 μmol m−2 s−1), the oxygen transfer rate and biomass‐related oxygen production were −1.5 mmol L−1 h−1 and 0.18 mmol O2 gx−1 h−1, respectively. The maximum specific growth rate was 0.058 h−1, during the exponential growth phase, after which the biomass concentration reached 0.75 g L−1.

A Modular Flow Cell System for Studying Biomimetic and BioinspiredAnti-Adhesive and Antimicrobial Surfaces

ABSTRACT Biofouling causes serious problems in many kinds of technical equipment. Consequently, there is great interest in developing surfaces with anti-adhesive and antimicrobial properties. Many such surfaces exist in nature, and artificial systems that mimic or are inspired by these natural systems could potentially be valuable technical surfaces. Recent studies on the interactions of bacteria with cicada wings revealed that rather than the wings being effective at repelling bacteria, after attachment the wing surface disrupts bacterial cell walls. Inspired by these structures, SmartMembranes GmbH used electrochemical precision etching to produce porous anodized aluminum oxide (AAO) membranes with structurally well-defined surface nanopatterns having pore diameters of 200–300 nm. We investigated the surfaces of these AAO membranes to assess their potential to prevent biofouling, using Escherichia coli as a model microbe. Bacterial adhesion tests were conducted using a modular flow cell system that is designed to enable on-line observation of biofilm formation in continuous flow on opaque surfaces using a fluorescence microscope. The system was adapted to permit testing of multiple samples with various dimensions and material properties simultaneously. Bacterial adhesion tests showed that AAO surfaces with pore diameters of 300 nm exhibit 99% less biofilm growth than widely used electropolished stainless steel.

A new method for mixing of suspended superparamagnetic beads using variable electromagnetic fields

The detection, identification, and analysis of bacterial pathogens are highly important for identifying potential health risks. Often, the first step of the process is an enrichment of the pathogens to such densities that exceed the detection limits of analytical instruments. In this context, biomagnetic separation is a promising approach to achieve this enrichment due to its selectivity and efficiency. Here, we present the electromagnetic sample mixer (ESM), an innovative device that may optimize this process. The aim of the ESM is to improve the binding between the superparamagnetic particles (beads) and the intended biological targets through the movement of the beads using variable magnetic fields to reduce these costly beads and save process time. The magnetic fields are generated using three electromagnets equipped with magnetic cores with special pole shoe geometry. This geometry was improved by numerical simulation of the magnetic field using FEMLAB 3. Also, proof‐of‐principle studies with Escherichia coli were performed by comparing the separation efficiencies received using the ESM and the conventional Dynabeads MX4 Mixer (Life Technologies AG, Carlsbad, CA, USA). The results show a similar performance of these two devices.

Ramified Challenges: Monitoring and Modeling of Hairy Root Growth in Bioprocesses--A Review.

The review presents a comprehensive overview on available solutions for the monitoring and modeling of various aspects of hairy root growth processes. Several online and offline measurement principles get explained exemplary and are being compared. It was found that no direct online measurement principle for hairy root biomass in submerged and solid-state culturing environment is available. However, certain indirect methods involving one or more measurement values have been developed for biomonitoring of hairy roots especially in bioreactors. In the field of modeling of hairy root growth processes, four independent architectures (continuous models, metabolic flux analysis, agent-based models, and artificial neural networks) are described and compared including literature references. The discussion is structured into microscopic model approaches, addressing only certain aspects of growth, and macroscopic model approaches, describing the hairy root network as a whole. An agent-based macroscopic model based on phenomenological data acquired with systematic imaging of hairy roots on culture dishes together with a 3D visualization of simulation results is presented in detail.

Automatische Inspektion von Kulturschalen mit der PetriJet-Plattform

Benchtop lab automation systems address specific, recurring tasks in small or mid-sized laboratories. With the versatile, standardized PetriJet platform technology the handling of culture dishes has been automated completely. Through a range of different processing stations different tasks such as multi-perspective imaging can be carried out without user interference and with a higher throughput. The integrated sorting function and the LIMS-connectivity allow to speed up subsequent processes.