Felix Krujatz

Green bioprinting: Fabrication of photosynthetic algae-laden hydrogel scaffolds for biotechnological and medical applications

Embedding of mammalian cells into hydrogel scaffolds of predesigned architecture by rapid prototyping technologies has been intensively investigated with focus on tissue engineering and organ printing. The study demonstrates that such methods can be extended to cells originating from the plant kingdom. By using 3D plotting, microalgae of the species Chlamydomonas reinhardtii were embedded in 3D alginate‐based scaffolds. The algae survived the plotting process and were able to grow within the hydrogel matrix. Under illumination, the cell number increased as indicated by microscopic analyses and determination of the chlorophyll content which increased 16‐fold within 12 days of cultivation. Photosynthetic activity was evidenced by measurement of oxygen release: within the first 24 h, an oxygen production rate of 0.05 mg L−1 h−1 was detected which rapidly increased during further cultivation (0.25 mg L−1 h−1 between 24 and 48 h). Furthermore, multichannel plotting was applied to combine human cells and microalgae within one scaffold in a spatially organized manner and hence, to establish a patterned coculture system in which the algae are cultivated in close vicinity to human cells. This might encourage the development of new therapeutic concepts based on the delivery of oxygen or secondary metabolites as therapeutic agents by microalgae.

Hydrogen production by Rhodobacter sphaeroides DSM 158 under intense irradiation.

To identify optimal hydrogen production conditions using growing cultures of Rhodobacter sphaeroides DSM 158 the effects of varying the reactors volumetric power input (0.01-1.4kWm(-3)) and irradiation intensity (5-2500Wm(-2)) were investigated in batch and continuous production modes. Irradiation intensity had a greater effect on hydrogen production than volumetric power input. Hydrogen production and photofermentative biomass formation were maximized by irradiation at 2250Wm(-2) with a volumetric power input of 0.55kWm(-3). The bacterial dry weight (2.64gL(-1)) and rate of hydrogen production (195mLL(-1)h(-1)) achieved under these conditions were greater than any that have previously been reported for batch-mode hydrogen production by R. sphaeroides. Continuous mode experiments (D=0.1h(-1)) yielded a bacterial dry weight, hydrogen production rate, productivity and hydrogen yield of 2.35±0.18gL(-1), 165±6.2mLL(-1)h(-1), 3.96LL(-1)d(-1) and 36.6%, respectively.

Biotechnological hydrogen production by photosynthesis

Microbiological photosynthesis is a promising tool for producing hydrogen in an ecologically friendly and economically efficient way. Certain microorganisms (e.g. algae and bacteria) can produce hydrogen using hydrogenase and/or nitrogenase enzymes. However, their natural capacity to produce hydrogen is relatively low. Thus, there is a need to optimize their core photosynthetic processes as well as their cultivation, for more efficient hydrogen production. This review aims to provide a holistic overview of the recent technological and research developments relating to photobiological hydrogen production and downstream processing. First we cover photobiological hydrogen synthesis within cells and the enzymes that catalyze the hydrogen production. This is followed by strategies for enhancing bacterial hydrogen production by genetic engineering, technological development, and innovation in bioreactor design. The remaining sections focus on hydrogen as a product, that is, quantification via (in‐process) gas analysis, recent developments in gas separation technology. Finally, a discussion of the sociological (market) barriers to future hydrogen usage is provided as well as an overview of methods for life cycle assessment that can be used to calculate the environmental consequences of hydrogen production.

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. Biotechnol. Bioeng. 2015;112: 2439–2449.

Green bioprinting: Viability and growth analysis of microalgae immobilized in 3D‐plotted hydrogels versus suspension cultures

In this study, microalgae were cultivated in the form of suspension cultures and a new structurally organized immobilization technique called “Green Bioprinting.” This technique allows the cocultivation of microorganisms in close vicinity to, but without direct contact with microalgae, to improve the oxygen supply of different cell types by photosynthetic oxygen evolution. However, more research on the optimum culture conditions for immobilized microalgae is necessary. Therefore, Chlamydomonas reinhardtii 11.32b and Chlorella sorokiniana UTEX1230 were suspended in culture medium or embedded in hydrogels by the 3D‐bioprinting process followed by cultivation under different temperatures (26°C, 30°C, or 37°C) and modes of illumination (continuous illumination or a 14/10 h light/dark cycle). The viability was monitored by either flow cytometry (suspension cultures) analysis of DiBAC4(3)‐stained cells or fluorescence image analysis (hydrogel‐embedded cultures). Suspended microalgae subjected to continuous illumination exhibited an increased number of membrane‐depolarized cells compared to those cultivated at a 14/10 h light/dark cycle. Hydrogel immobilization resulted in a facilitated viability and stable growth rates between 0.4 and 0.7 d−1 for both microalgae strains. Concluding, the 3D‐bioprinting immobilization represents a technique to cultivate microalgae at a high viability and growth rate even under nonoptimal temperature conditions.

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.

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.