Thomas Bley

Hairy root type plant in vitro systems as sources of bioactive substances.

Abstract“Hairy root” systems, obtained by transforming plant tissues with the “natural genetic engineer” Agrobacterium rhizogenes, have been known for more than three decades. To date, hairy root cultures have been obtained from more than 100 plant species, including several endangered medicinal plants, affording opportunities to produce important phytochemicals and proteins in eco-friendly conditions. Diverse strategies can be applied to improve the yields of desired metabolites and to produce recombinant proteins. Furthermore, recent advances in bioreactor design and construction allow hairy root-based technologies to be scaled up while maintaining their biosynthetic potential. This review highlights recent progress in the field and outlines future prospects for exploiting the potential utility of hairy root cultures as “chemical factories” for producing bioactive substances.

Origin and analysis of microbial population heterogeneity in bioprocesses.

Heterogeneity of industrial production cultures is accepted to a certain degree; however, the underlying mechanisms are seldom perceived or included in the development of new bioprocess control strategies. Population heterogeneity and its basics, perceptible in the diverse proficiency of cells, begins with asymmetric birth and is found to recess during the life cycle. Since inefficient subpopulations have significant impact on the productivity of industrial cultures, cellular heterogeneity needs to be detected and quantified by using high speed detection tools like flow cytometry. Possible origins of population heterogeneity, sophisticated fluorescent techniques for detection of individual cell states, and cutting-edge Omics-technologies for extended information beyond the resolution of fluorescent labelling are highlighted.

The growth and differentiation of mesenchymal stem and progenitor cells cultured on aligned collagen matrices.

Cell-matrix interactions are paramount for the successful repair and regeneration of damaged and diseased tissue. Since many tissues have an anisotropic architecture, it has been proposed that aligned extracellular matrix (ECM) structures in particular could guide and support the differentiation of resident mesenchymal stem and progenitor cells (MSCs). We therefore created aligned collagen type I structures using a microfluidic set-up with the aim to assess their impact on MSC growth and differentiation. In addition, we refined our aligned collagen matrices by incorporating the glycosaminoglycan (GAG) heparin to demonstrate the versatility of the applied methodology to study multiple ECM components in a single system. Our reconstituted, aligned ECM structures maintained and allowed multilineage (osteogenic/adipogenic/chondrogenic) differentiation of MSCs. Most noticeable was the observation that during osteogenesis, aligned collagen substrates choreographed ordered matrix mineralization. Likewise, myotube assembly of C2C12 cells was profoundly influenced by aligned topographic features resulting in enhanced myotube organization and length. Our results shed light on the regulation of MSCs through directional ECM structures and demonstrate the versatility of these cell culture platforms for guiding the morphogenesis of tissue types with anisotropic structures.

Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4′,6′-Diamidino-2-Phenylindole) and Tetracycline Labeling

ABSTRACT Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a “Candidatus Accumulibacter”-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

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.

Betalain production in plant in vitro systems

Betalains have been widely used as natural colorants for many centuries, but their attractiveness for use as colorants of foods (or drugs and cosmetics) has increased recently due to their reportedly high anti-oxidative, free radical scavenging activities and concerns about the use of various synthetic alternatives. The main commercial sources of betalains are powders and concentrates of red beet (Beta vulgaris) or cactus pear (Opuntia ficus-indica) extracts. However, in recent years the technical and commercial feasibility of various in vitro systems to produce them biotechnologically has been explored. These research activities have included assessments of novel approaches for cultivating plant cell or tissue cultures, and diverse bioreactor systems for increasing production levels of secondary metabolites. This paper reviews recent progress in plant in vitro systems for producing betalain pigments. In addition, the factors that could be manipulated, the bioreactor systems that could be used, and the strategies that could be applied to improve betalain production are discussed.

Methylobacterium rhodesianum cells tend to double the DNA content under growth limitations and accumulate PHB

The investigation of microbial population dynamics gains more and more importance for biotechnological processes insofar as people may assume that the individual cells of a population contribute differently to the overall productivity. Flow cytometry is known to be suitable to get information on specific features of single cells of a population. In the paper presented, the distributions of the DNA and PHB over the whole population of Methylobacterium rhodesianum MB126 were determined. Three different kinds of limitation, namely that of nitrogen, phosphate and carbon, were investigated and compared with an unlimited growth process. Some differences in the population dynamics were observed, obviously caused by the remaining chances of continuing metabolism under restricted growth conditions. Most impressive was the appearance of two subpopulations due to phosphate limitation, characterized, in addition to their DNA content, by their cellular PHB content. On the other hand, nitrogen and carbon limitations produced homogeneous populations with a high or without a PHB content, respectively. It was found that under growth-limiting conditions the individuals first unwind the program to ensure the genetic information by doubling the chromosome content, thus the organisms maintain the chance to restart the multiplication as the forward strategy of survival if ‘better’ conditions arise. Then they lay in an energy reserve in the form of PHB. An hypothesis about the transitions between different physiological states characterized by the cellular DNA content and the cell size depending on process conditions is formulated and demonstrated by a formal scheme.

Editorial: Engineering is driving innovations in industrial biotechnology

The realization of innovations in industrial, environmental, plant and food biotechnology, especially those related to white biotechnology and bioenergy is the mission of Engineering in Life Sciences. In light of several events and highlights on industrial biotechnology in 2012, the content of the journal will cover many of these topics to respond to the demands of a changing world. Currently, more than 50% of new therapeutics admitted by regulatory authorities are biotechnological products. For cheap and reliable production of these molecules, innovative and efficient production systems are needed. The economics of these biopharmaceutical production processes depend not only on progress in research in all -omicsrelated fields for strain development and analytics, but also significantly on advances in bioprocess engineering. A successful example of such an innovative production process is an article by Thomas Scheper and coworkers in this issue, which presents production and purification of the cytokine FGF-2 [1]. This year’s traditional annual meeting of the German bioprocess engineers in the week before Ascension Day (Himmelfahrt) is organized under the topic Biopharmaceutical Production (http://events.dechema. de/biopro). This meeting will drive innovation in upstream and downstream processing, as well as concepts for Process Analytical Technology (PAT), Quality by Design (QbD), single-use technologies and continuous bioprocesses. Engineering in Life Sciences will not only be present at this meeting, but also cover the highlights in our future issues. One further key topic in 2012 will be the development of new bioproduction systems. This is documented by the strategy process Biotechnology 20201 of the German Federal Ministry of Education and Research and the newly started research funding program Next Generation of Biotechnological Processes. To drive this progress, Co-Editor of our Journal, Prof. An-Ping Zeng, founded a new temporary DECHEMA working party New Bioproduction Systems. ‘‘Cell free and hybrid systems’’, ‘‘artificial microbial communities’’, and ‘‘synthetic biotechnology’’ are the keywords outlining this exciting future. Articles on new bioproduction systems will be the focus of a special issue for the IBS2012 meeting in Daegu, Korea, which will take place in September (www.ibs2012.org).

Immuno and flow cytometric analytical methods for biotechnological research and process monitoring

In this article, the applications of immunoanalysis and flow cytometry for research and process monitoring in biotechnology are discussed. Brief reviews of the two analytical methods are followed by descriptions of actual applications in various areas of biotechnology. In the case of immunoanalysis, emphasis is placed on systems for on-line bioprocess monitoring, and examples are given for a thermostable pullulanase, a mouse IgG, and antithrombin III. Although flow cytometry is not currently an on-line analytical technique, its value as an off-line method is illustrated by examples of the measurement of shear stress effects, lipid content, and sterol content.

Temporary immersion systems in plant biotechnology

Plant tissue and organ cultures in vitro usually face technological challenges. When submerged cultivation of plant cells in a controlled environment is desired, the characteristic growth morphology and physiology of differentiated organ cultures present a problem in process scale‐up. Temporary immersion systems (TIS) were developed several decades ago. These systems are providing the most natural environment for in vitro culture of plant shoots and seedlings. Over the past few years, TIS have been recognized as a perspective technology for plant micropropagation, production of plant‐derived secondary metabolites, expression of foreign proteins, and potential solutions in phytoremediation. Nowadays, several TIS, operating on similar or divergent technological principles, have been developed and successfully applied in the cultivation of various plant in vitro systems, including somatic embryos and transformed root cultures. In this article, the operational principle and technological design of the most popular TIS are reviewed. In addition, recent examples of the application of temporary immersion technology for in vitro cultivation of plant tissue and organ cultures at laboratory and pilot scales are discussed. Finally, future prospects and challenges to the industrial realization of that fast‐developing technique are outlined.