Kai Muffler

Future aspects of bioprocess monitoring.

Nature has the impressive ability to efficiently and precisely control biological processes by applying highly evolved principles and using minimal space and relatively simple building blocks. The challenge is to transfer these principles into technically applicable and precise analytical systems that can be used for many applications. This article summarizes some of the new approaches in sensor technology and control strategies for different bioprocesses such as fermentations, biotransformations, and downstream processes. It focuses on bio- and chemosensors, optical sensors, DNA and protein chip technology, software sensors, and modern aspects of data evaluation for improved process monitoring and control.

Importance of correlation effects in hcp iron revealed by a pressure-induced electronic topological transition

We discover that hcp phases of Fe and Fe(0.9)Ni(0.1) undergo an electronic topological transition at pressures of about 40 GPa. This topological change of the Fermi surface manifests itself through anomalous behavior of the Debye sound velocity, c/a lattice parameter ratio, and Mössbauer center shift observed in our experiments. First-principles simulations within the dynamic mean field approach demonstrate that the transition is induced by many-electron effects. It is absent in one-electron calculations and represents a clear signature of correlation effects in hcp Fe.

Ethanol production from grass silage by simultaneous pretreatment, saccharification and fermentation: first steps in the process development.

Grass silage provides a great potential as renewable feedstock. Two fractions of the grass silage, a press juice and the fiber fraction, were evaluated for their possible use for bioethanol production. Direct production of ethanol from press juice is not possible due to high concentrations of organic acids. For the fiber fraction, alkaline peroxide or enzymatic pretreatment was used, which removes the phenolic acids in the cell wall. In this study, we demonstrate the possibility to integrate the enzymatic pretreatment with a simultaneous saccharification and fermentation to achieve ethanol production from grass silage in a one‐process step. Achieved yields were about 53 g ethanol per kg silage with the alkaline peroxide pretreatment and 91 g/kg with the enzymatic pretreatment at concentrations of 8.5 and 14.6 g/L, respectively. Furthermore, it was shown that additional supplementation of the fermentation medium with vitamins, trace elements and nutrient salts is not necessary when the press juice is directly used in the fermentation step.

Application of Biofilm Bioreactors in White Biotechnology

The production of valuable compounds in industrial biotechnology is commonly done by cultivation of suspended cells or use of (immobilized) enzymes rather than using microorganisms in an immobilized state. Within the field of wastewater as well as odor treatment the application of immobilized cells is a proven technique. The cells are entrapped in a matrix of extracellular polymeric compounds produced by themselves. The surface-associated agglomerate of encapsulated cells is termed biofilm. In comparison to common immobilization techniques, toxic effects of compounds used for cell entrapment may be neglected. Although the economic impact of biofilm processes used for the production of valuable compounds is negligible, many prospective approaches were examined in the laboratory and on a pilot scale. This review gives an overview of biofilm reactors applied to the production of valuable compounds. Moreover, the characteristics of the utilized materials are discussed with respect to support of surface-attached microbial growth.

Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye.

Dye affinity chromatography for fast and simple purification of fucoidan from marine brown algae

Fucoidan is a sulfated polysaccharide with promising pharmacological applications. Due to its medicinal properties, there is a demand for a separation technique that yields a high purification grade. Here, we present a novel purification tool for recovering fucoidan from the marine brown macroalgae Fucus vesiculosus. The developed method is based on amino‐derivatized Sepabeads® EC‐EA. The beads were modified with toluidine blue (TB), a thiazine derivative, to exploit the strong donor acceptor interactions between the cationic dye and the anionic polysaccharide. The adsorption kinetics and the binding capacity of the resin were analyzed. A Sips model was used to approximate the adsorption isotherm, resulting in a maximum capacity of 127.7 mg fucoidan per g adsorbent. Investigation of the effect of adsorption steps pH on purity and chemical structure was performed by TB and Fourier transform infra‐red spectroscopy assays. Results showed that adsorption at pH 1 and 6 had negligible effects on fucoidans chemical structure. However, purity was actually improved by 1.55‐ and 1.69‐fold at pH 1 and 6, respectively, with an average yield of 5 g/100 g dried algae powder. In contrast, only a 1.46‐fold increment was observed in fucoidan purified by the traditional method at pH 2, with a yield of 7.5 g/100 g dried algae powder. Furthermore, fucoidan purified by this method at pH 6 complies with, or even exceeds the quality of the commercially available (≥95% pure) fucoidan (Sigma‐Aldrich®) with respect to molecular weight and sulfur content. Therefore, dye affinity chromatography provides more advantages than the classically used techniques for fucoidan purification.

Characterization of terrestrial cyanobacteria to increase process efficiency in low energy consuming production processes

Terrestrial cyanobacteria have seldom been used for biotechnological processes, even though they offer great potential for new pharmaceutical products or other value-added substances. Particularly cyanobacteria of xeric habitats are of biotechnological interest, because they tolerate high temperatures, are desiccation-tolerant and feature low water consumption. In addition, the cyanobacteria collected in deserts are able to produce more photoprotective agents than their counterparts from other habitats, because of their genetical preadaptation. In this study, carotenoid and chlorophyll content of two representative terrestrial cyanobacteria strains, i.e. Nostoc muscorum and Leptolyngbya spec. sampled in Columbia (USA) and Soebatsfontein (RSA), were studied after exposure of the strains to different light conditions and cultivation temperatures. A temperature raise from 17°C to 30°C led to an increase of 46% in chlorophyll a content as well as an increase of 39% in carotenoid content of Nostoc muscorum. An irradiation raise from 19 μmol m-2s-1 to 125 μmol m-2s-1 resulted in an increase of a 10 to 20 times higher chlorophyll content. Additional results from light-curves support the potential future use of terrestrial cyanobacteria within low energy biotechnological processes using a novel type of photobioreactor to reduce the downstream process costs and nutrients needed during the cultivation. Results indicate that especially light intensity optimization currently holds unused potential.

A new photobioreactor concept enabling the production of desiccation induced biotechnological products using terrestrial cyanobacteria

Cyanobacteria offer great potential for the production of biotechnological products for pharmaceutical applications. However, these organisms can only be cultivated efficiently using photobioreactors (PBR). Under submerged conditions though, terrestrial cyanobacteria mostly grow in a suboptimal way, which makes this cultivation-technique uneconomic and thus terrestrial cyanobacteria unattractive. Therefore, a novel emersed photobioreactor (ePBR) has been developed, which can provide the natural conditions for these organisms. Proof of concept as well as first efficiency tests are conducted using the terrestrial cyanobacteria Trichocoleus sociatus as a model organism. The initial maximum growth rate of T. sociatus (0.014±0.001h(-1)) in submerged systems could be increased by 35%. Furthermore, it is now possible to control desiccation-correlated product formation and related metabolic processes. This is shown for the production of extracellular polymeric substances (EPS). In this case the yield of 0.068±0.006g of EPS/g DW could be increased by more than seven times.

A semi-continuous process based on an ePBR for the production of EPS using Trichocoleus sociatus

Biodiversity forms the basis for a large pool of potential products and productive organisms offered by terrestrial cyanobacteria. They are stuck together by EPS (extracellular polymeric substances) that can obtain antiviral, antibacterial or anti-inflammatory substances. Most stress conditions, e.g. drought, induce the production of protective EPS or biotechnological-products for pharmaceutical application. However, the growth of a phototrophic biofilm is limited under submerged conditions. Therefore, a semi-continuous process to produce EPS by cyanobacteria was developed in an aerosol-based ePBR (emerse photobioreactor) that imitates the natural habitat of terrestrial cyanobacteria. The process consists of a growth-phase (biomass production), followed by a dry-phase (EPS-production) and a consecutive extraction. The EPS-productivities of Trichocoleus sociatus (ranging from 0.03 to 0.04gL-1d-1) were 32 times higher than described in topic-related literature. In comparison to submerge cultivations in shaking flasks, the EPS-productivities were sevenfold higher. To ensure that the extraction solvent has no influence on cell viability, a cell-vitality-test was performed. However, no statistically significant difference between the amount of living and dead cells before and after the extraction was detected. A bioactivity assay was then performed to identify antimicrobial activity within EPS extracts from emerse and submerge cultivations. The EPS revealed an antibacterial effect against gram-negative bacteria (E. coli) which was two times higher than EPS from a submerged cultivation.

Cationic Dye for the Specific Determination of Sulfated Polysaccharides

ABSTRACT A new, highly specific toluidine blue assay for the determination of sulfated polysaccharides is reported. Sulfated polysaccharides may be specifically determined at pH 1 in the presence of carboxylated polysaccharides and phosphorylated molecules such as nucleic acids. In addition, performing the assay at pH 1 extended the linear dynamic range for fucoidan up to 3 g/L but lowered the sensitivity in comparison with pH 7. For a color change at neutral pH, at least a sulfated disaccharide is required, whereas at pH 1, the detection was negative even for a sulfated maltose. A comparison of the fucoidan concentration in crude algae determined by the toluidine blue assay at pH 1 and gel permeation chromatography confirmed the accuracy and specificity of the colorimetric method. The limit of quantitation for the toluidine blue assay at pH 1 was 0.62 g/L for fucoidan. For a deeper understanding of the interaction between the sulfated polysaccharides and the toluidine blue, spectroscopic constants were evaluated by Ketelaar plots for sulfate esters. The maximum value for the molar absorptivity was 4.07 × 104 L/(mol cm), indicating strong affinity. Similar behavior was obtained for fucoidan at pH 7 with a value of 4.46 × 103 L/mol.