Monitoring the apical growth characteristics of hairy roots using non-invasive laser speckle contrast imaging.

Hairy roots are used to produce plant agents and additives. Due to their heterogeneous structure and growth characteristics, it is difficult to determine growth-related parameters continuously and in real time. Laser speckle contrast analysis is widely used as a non-destructive measurement technique in material testing or in medical technology. This type of analysis is based on the principle that moving objects or particles cause fluctuations in stochastic interference patterns known as speckle patterns. They are formed by the random backscattering of coherent laser light on an optically rough surface. A Laser Speckle Imager, which is well established for speckle studies of hemodynamics, was used for the first time for non-invasive speckle measurements on hairy roots to study dynamic behavior in plant tissue. Based on speckle contrast, a specific flux value was defined to map the dynamic changes in the investigated tissue. Using this method, we were able to predict the formation of lateral strands and to identify the growth zone in the apical root region, as well as dividing it into functional regions. This makes it possible to monitor physiological processes in the apical growth zone in vivo and in real time without labeling the target structures.

Biospeckle-characterization of hairy root cultures using laser speckle photometry.

Monitoring is indispensable for the optimization and simulation of biotechnological processes. Hairy roots (hr, plant tissue cultures) are producers of valuable relevant secondary metabolites. The genetically stable cultures are characterized by a rapid filamentous growth, making monitoring difficult with standard methods. This article focuses on the application of laser speckle photometry (LSP) as an innovative, non-invasive method to characterize Beta vulgaris (hr). LSP is based on the analysis of time-resolved interference patterns. Speckle interference patterns of a biological object, known as biospeckles, are characterized by a dynamic behavior that is induced by physical and biological phenomena related to the object. Speckle contrast, a means of measuring the dynamic behavior of biospeckles, was used to assess the biospeckle activity. The biospeckle activity corresponds to processes modifying the object and correlates with the biomass growth. Furthermore, the stage of the cultures' physiological development was assessed by speckle contrast due to the differentiation between active and low active behavior. This method is a new means of monitoring and evaluating the biomass growth of filamentous cultures in real time. As a potential tool to characterize hairy roots, LSP is non-invasive, time-saving, can be used online and stands out for its simple, low-cost setup.

Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy.

Monoclonal antibodies have become an increasingly important part of fundamental research and medical applications. To meet the high market demand for monoclonal antibodies in the biopharmaceutical sector, industrial manufacturing needs to be achieved by large scale, highly productive and consistent production processes. These are subject to international guidelines and have to be monitored intensely due to high safety standards for medical applications. Surface plasmon resonance spectroscopy - a fast, real-time, and label-free bio-sensing method - represents an interesting alternative to the quantification of monoclonal antibody concentrations by enzyme-linked immunosorbent assay during monoclonal antibody production. For the application of monitoring bioactive and total monoclonal antibody concentrations in cell culture samples, a surface plasmon resonance assay using a target-monoclonal antibody model system was developed. In order to ensure the subsequent detection of bioactive monoclonal antibody concentrations, suitable immobilization strategies of the target were identified. A significant decrease of the limit of detection was achieved by using an adapted affinity method compared to the commonly used amine coupling. Furthermore, the system showed limit of detection in the low ng/mL range similar to control quantifications by enzyme-linked immunosorbent assay. Moreover, the comparison of total to bioactive monoclonal antibody concentrations allows analysis of antibody production efficiency. The development of an alternative quantification system to monitor monoclonal antibody production was accomplished using surface plasmon resonance with the advantage of low analyte volume, shorter assay time, and biosensor reusability by target-layer regeneration. The established method provides the basis for the technical development of a surface plasmon resonance-based system for continuous process monitoring.

Microalgae wastewater treatment: Biological and technological approaches.

Current global environmental issues raise unavoidable challenges for our use of natural resources. Supplying the human population with clean water is becoming a global problem. Numerous organic and inorganic impurities in municipal, industrial, and agricultural waters, ranging from microplastics to high nutrient loads and heavy metals, endanger our nutrition and health. The development of efficient wastewater treatment technologies and circular economic approaches is thus becoming increasingly important. The biomass production of microalgae using industrial wastewater offers the possibility of recycling industrial residues to create new sources of raw materials for energy and material use. This review discusses algae-based wastewater treatment technologies with a special focus on industrial wastewater sources, the potential of non-conventional extremophilic (thermophilic, acidophilic, and psychrophilic) microalgae, and industrial algae-wastewater treatment concepts that have already been put into practice.

Modeling population dynamics in a microbial consortium under control of a synthetic pheromone-mediated communication system.

Microbial consortia can be used to catalyze complex biotransformations. Tools to control the behavior of these consortia in a technical environment are currently lacking. In the present study, a synthetic biology approach was used to build a model consortium of two Saccharomyces cerevisiae strains where growth and expression of the fluorescent marker protein EGFP by the receiver strain is controlled by the concentration of α-factor pheromone, which is produced by the emitter strain. We have developed a quantitative experimental and theoretical framework to describe population dynamics in the model consortium. We measured biomass growth and metabolite production in controlled bioreactor experiments, and used flow cytometry to monitor changes of the subpopulations and protein expression under different cultivation conditions. This dataset was used to parameterize a segregated mathematical model, which took into account fundamental growth processes, pheromone-induced growth arrest and EGFP production, as well as pheromone desensitization after extended exposure. The model was able to predict the growth dynamics of single-strain cultures and the consortium quantitatively and provides a basis for using this approach in actual biotransformations.

Uptake of iron by Kluyveromyces marxianus DSM 5422 cultivated in a whey-based medium.

The ability of Kluyveromyces marxianus for converting lactose into ethyl acetate offers a chance for the economical reuse of whey. Iron plays a significant role in this process as ester synthesis requires a low intracellular iron content, xFe . The iron content in turn is decreased by growth due to cell expansion and increased by iron uptake. Thus, the iron-uptake rate, ψ, is important for the considered process. Iron uptake by K. marxianus DSM 5422 was studied in aerobic cultivation on a whey-borne medium with varied initial iron content, in part combined with a feed of iron under intensive growth conditions. A possible precipitation of iron that would pretend iron uptake was verified not to have occurred. Regularly measured dissolved iron concentrations, CFe,L , allowed the xFe and ψ parameters to be obtained by model-based iron balancing. The achieved data were used for establishing a ψ(CFe,L , xFe ) model. Mathematical simulations based on this iron-uptake model reproduced the performed cultivation processes. The created iron-uptake model allows for a future predictive system to be developed for the optimization of biotechnological ester production.

A new method for non-invasive biomass determination based on stereo photogrammetry.

A novel, non-destructive method for the biomass estimation of biological samples on culture dishes was developed. To achieve this, a photogrammetric system, which consists of a digital single-lens reflex camera (DSLR), an illuminated platform where the culture dishes are positioned and an Arduino board which controls the capturing process, was constructed. The camera was mounted on a holder which set the camera at different title angles and the platform rotated, to capture images from different directions. A software, based on stereo photogrammetry, was developed for the three-dimensional (3D) reconstruction of the samples. The proof-of-concept was demonstrated in a series of experiments with plant tissue cultures and specifically with calli cultures of Salvia fruticosa and Ocimum basilicum. For a period of 14 days images of these cultures were acquired and 3D-reconstructions and volumetric data were obtained. The volumetric data correlated well with the experimental measurements and made the calculation of the specific growth rate, µ max, possible. The µ max value for S. fruticosa samples was 0.14 day-1 and for O. basilicum 0.16 day-1. The developed method demonstrated the high potential of this photogrammetric approach in the biological sciences.

Additive Biotech-Chances, challenges, and recent applications of additive manufacturing technologies in biotechnology.

The diversity and complexity of biotechnological applications are constantly increasing, with ever expanding ranges of production hosts, cultivation conditions and measurement tasks. Consequently, many analytical and cultivation systems for biotechnology and bioprocess engineering, such as microfluidic devices or bioreactors, are tailor-made to precisely satisfy the requirements of specific measurements or cultivation tasks. Additive manufacturing (AM) technologies offer the possibility of fabricating tailor-made 3D laboratory equipment directly from CAD designs with previously inaccessible levels of freedom in terms of structural complexity. This review discusses the historical background of these technologies, their most promising current implementations and the associated workflows, fabrication processes and material specifications, together with some of the major challenges associated with using AM in biotechnology/bioprocess engineering. To illustrate the great potential of AM, selected examples in microfluidic devices, 3D-bioprinting/biofabrication and bioprocess engineering are highlighted.

Green bioprinting: extrusion-based fabrication of plant cell-laden biopolymer hydrogel scaffolds.

Plant cell cultures produce active agents for pharmaceuticals, food and cosmetics. However, up to now process control for plant cell suspension cultures is challenging. A positive impact of cell immobilization, such as encapsulation in hydrogel beads, on secondary metabolites production has been reported for several plant species. The aim of this work was to develop a method for bioprinting of plant cells in order to allow fabrication of free-formed three-dimensional matrices with defined internal pore architecture for in depth characterization of immobilization conditions, cell agglomeration and interactions. By using extrusion-based 3D plotting of a basil cell-laden hydrogel blend consisting of alginate, agarose and methylcellulose (alg/aga/mc), we could demonstrate that bioprinting is applicable to plant cells. The majority of the cells survived plotting and crosslinking and the embedded cells showed high viability and metabolic activity during the investigated cultivation period of 20 d. Beside its compatibility with the plant cells, the novel alg/aga/mc blend allowed fabrication of defined 3D constructs with open macropores both in vertical and horizontal direction which were stable under culture conditions for several weeks. Thus, Green Bioprinting, an additive manufacturing technology processing live cells from the plant kingdom, is a promising new immobilization tool for plant cells that enables the development of new bioprocesses for secondary metabolites production as well as monitoring methods.

A novel protocol to prepare cell probes for the quantification of microbial adhesion and biofilm initiation on structured bioinspired surfaces using AFM for single-cell force spectroscopy: Dedicated to Prof. em. Dr. Dr. H.C. Karl Schügerl on the occasion of his 90th birthday.

We present a novel protocol that uses single-cell force spectroscopy to characterize the bacteria-to-surface interactions involved in early steps of biofilm formation. Bacteria are immobilized as a monolayer by electrostatic interactions on a polyethylenimine-coated silica bead, and the Escherichia coli-bead complex is then glued on a tipless cantilever. We validated our new protocol by comparing to earlier published methods using single bacteria, but in contrast to these, which carry out bacterial attachment to the bead after fixation to the cantilever, our protocol results in more reliable production of usable cell probes. Measurements of interactions of E. coli with bio-inspired surfaces by single-cell force spectroscopy yielded comparable detachment forces to those found with the previous methods.

"Fungal elicitors combined with a sucrose feed significantly enhance triterpene production of a Salvia fruticosa cell suspension".

Oleanolic (OA) and ursolic acid (UA) are plant secondary metabolites with diverse pharmacological properties. To reach reasonable productivities with plant cell suspension cultures, elicitation is a widely used strategy. Within the presented work, the effects of different elicitors on growth and production of OA and UA in a Salvia fruticosa cell suspension culture were examined. Beside commonly used elicitors like jasmonic acid (JA) and yeast extract, the influence of medium filtrates of the endophytic fungi Aspergillus niger and Trichoderma virens was investigated. The best eliciting effects were achieved with JA and fungal medium filtrates. Both increased the triterpene content by approximately 70 %. Since JA showed significant growth inhibition, the volumetric triterpene yield did not increase. But, adding fungal filtrates increased the volumetric triterpene yield by approximately 70 % to 32.6 mgOA l(-1) and 65.9 mgUA l(-1) for T. virens compared to the control with 19.4 mgOA l(-1) and 33.3 mgUA l(-1). An elicitation strategy combining fungal medium filtrate of T. virens with sucrose feeding significantly enhanced cell dry weight concentration to 22.2 g l(-1) as well as triterpene content by approximately 140 %. In total, this led to an approximately 500 % increase of volumetric triterpene yield referring to the control with final values of 112.9 mgOA l(-1) and 210.4 mgUA l(-1). Despite the doubled cultivation duration, productivities of 6.7 mgOA l(-1) day(-1) and 12.4 mgUA l(-1) day(-1) were reached. These results demonstrate methods by which increased productivities of triterpenes can be achieved to attain yields competing with intact plants.

Two parametric cell cycle analyses of plant cell suspension cultures with fragile, isolated nuclei to investigate heterogeneity in growth of batch cultivations.

Plant cell suspensions are frequently considered to be heterogeneous with respect to growth in terms of progression of the cells through the cell cycle and biomass accumulation. Thus, segregated data of fractions in different cycle phases during cultivation is needed to develop robust production processes. Bromodeoxyuridine (BrdU) incorporation and BrdU-antibodies or 5-ethynyl-2'-deoxyuridine (EdU) click-it chemistry are frequently used to acquire such information. However, their use requires centrifugation steps that cannot be readily applied to sensitive cells, particularly if nuclei have to be extracted from the protective cellular milieu and envelopes for DNA analysis. Therefore, we have established a BrdU-Hoechst stain quenching protocol for analyzing nuclei directly isolated from delicate plant cell suspension cultures. After adding BrdU to test Harpagophytum procumbens cell suspension cultures the cell cycle distribution could be adequately resolved using its incorporation for the following 72 h (after which BrdU slowed biomass accumulation). Despite this limitation, the protocol allows resolution of the cell cycle distribution of cultures that cannot be analyzed using commonly applied methods due to the cells' fragility. The presented protocol enabled analysis of cycling heterogeneities in H. procumbens batch cultivations, and thus should facilitate process control of secondary metabolite production from fragile plant in vitro cultures. Biotechnol. Bioeng. 2016;113: 1244-1250. © 2015 Wiley Periodicals, Inc.

Light-field-characterization in a continuous hydrogen-producing photobioreactor by optical simulation and computational fluid dynamics.

Externally illuminated photobioreactors (PBRs) are widely used in studies on the use of phototrophic microorganisms as sources of bioenergy and other photobiotechnology research. In this work, straightforward simulation techniques were used to describe effects of varying fluid flow conditions in a continuous hydrogen-producing PBR on the rate of photofermentative hydrogen production (rH2 ) by Rhodobacter sphaeroides DSM 158. A ZEMAX optical ray tracing simulation was performed to quantify the illumination intensity reaching the interior of the cylindrical PBR vessel. 24.2% of the emitted energy was lost through optical effects, or did not reach the PBR surface. In a dense culture of continuously producing bacteria during chemostatic cultivation, the illumination intensity became completely attenuated within the first centimeter of the PBR radius as described by an empirical three-parametric model implemented in Mathcad. The bacterial movement in chemostatic steady-state conditions was influenced by varying the fluid Reynolds number. The "Computational Fluid Dynamics" and "Particle Tracing" tools of COMSOL Multiphysics were used to visualize the fluid flow pattern and cellular trajectories through well-illuminated zones near the PBR periphery and dark zones in the center of the PBR. A moderate turbulence (Reynolds number = 12,600) and fluctuating illumination of 1.5 Hz were found to yield the highest continuous rH2 by R. sphaeroides DSM 158 (170.5 mL L(-1) h(-1) ) in this study.

Better One-Eyed than Blind--Challenges and Opportunities of Biomass Measurement During Solid-State Fermentation of Basidiomycetes.

Filamentous fungi, especially basidiomycetes, produce a wide range of metabolites, many of which have potential biotechnological and industrial applications. Solid-state fermentation (SSF) is very suitable for the cultivation of basidiomycetes since it mimics the natural habitat of these fungi. Some of the major advantages of SSF are the robustness of the process, the use of low-cost residual materials as substrates, and the reduced usage of water. However, monitoring key variables is difficult, which makes process control a challenge. Specifically, it is very difficult to determine the biomass during SSF process involving basidiomycetes. This is problematic, as the biomass is normally a key variable in mass and energy balance equations. Further, the success of fungal SSF processes is often evaluated, in part, based on the growth of the fungus. Direct determination of the dry weight of biomass is impossible and indirect quantification techniques must be used. Over the years, various determination techniques have been developed for the quantification of fungal biomass in SSF processes. The current review gives an overview of various direct and indirect biomass determination methods, discussing their advantages and disadvantages.

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.

Biomass estimation during macro-scale solid-state fermentation of basidiomycetes using established and novel approaches.

Solid-state fermentation (SSF) has been utilised in food production for millennia and is well suited for the cultivation of basidiomycetes, due to the robustness of the process and the possibility of using lignocellulose as the substrate. Basidiomycetes produce diverse enzymes and various primary and secondary metabolites, many of which have biotechnological potential. The quantification of the fungal biomass present is essential for the characterisation of growth kinetics in processes such as SSF. In SSF, fungi grow into the substrate and use it as a nutrient source. Therefore, direct biomass determination is not possible and indirect methods have to be employed. In the presented study, we compared 11 methods for quantifying fungal biomass during SSF of the basidiomycete Trametes hirsuta in a newly developed laboratory reactor (working volume 10 L). The methods were based on measuring the levels of six cell-specific components (ergosterol, glucosamine, nucleic acids, number of fungal nuclei, protein and genomic DNA) and estimations of biological activity (respiration, activities of lignolytic and cellulolytic enzymes, and the glucose and protein contents of the liquid). The methods were evaluated with regards to reproducibility and plausibility of the results, time and resource requirements, possible influential factors, and matrix effects. The most reliable biomass estimates were obtained from measurements of ergosterol content, number of nuclei, and respiration. Thus, these three methods were deemed most suitable for process control and modelling.