A new approach for calculating microalgae culture growth based on an inhibitory effect of the surrounding biomass.

Ever since the potential of algae in biotechnology was recognized, models describing the growth of algae inside photobioreactors have been proposed. These models are the basis for the optimization of process conditions and reactor designs. Over the last few decades, models became more and more elaborate with the increase of computational capacity. Thus far, these models have been based on light attenuation due to the absorption and scattering effects of the biomass. This manuscript presents a new way of predicting the apparent growth inside photobioreactors using simple models for enzymatic kinetics to describe the reaction between photons and the photosynthetic unit. The proposed model utilizes an inhibition kinetic formula based on the surrounding biomass to describe the average growth rate of a culture, which is determined by the local light intensities inside the reactor. The result is a mixed-inhibition scheme with multiple inhibition sites. The parameters of the new kinetic equation are replaced by empirical regression functions to correlate their dependency on incident light intensity and reactor size. The calibrations of the parameters and the regression functions are based on the numerical solutions of the growth rate computed with a classical Type II model. As a final verification, we apply the new equation in predicting the growth behavior of three phototrophic organisms in reactors of three different sizes.

Extracorporeal perfusion - reduced to a one-way infusion.

BACKGROUND: Extracorporeal perfusion (EP) is moving into focus of research in reconstructive and transplantation medicine for the preservation of amputates and free tissue transplants. The idea behind EP is the reduction of ischemia-related cell damage between separation from blood circulation and reanastomosis of the transplant. Most experimental approaches are based on a complex system that moves the perfusate in a circular course. OBJECTIVE AND METHODS: In this study, we aimed to evaluate if a simple perfusion by an infusion bag filled with an electrolyte solution can provide acceptable results in terms of flow stability, oxygen supply and viability conservation for EP of a muscle transplant. The results are compared to muscles perfused with a pump system as well as muscles stored under ischemic conditions after a one-time intravasal flushing with Jonosteril. RESULTS: With this simple method a sufficient oxygen supply could be achieved and functionality could be maintained between 3.35 times and 4.60 times longer compared to the control group. Annexin V positive nuclei, indicating apoptosis, increased by 9.7% in the perfused group compared to 24.4% in the control group. CONCLUSIONS: Overall, by decreasing the complexity of the system, EP by one-way infusion can become more feasible in clinical situations.

Terbium Excitation Spectroscopy as a Detection Method for Chromatographic Separation of Lanthanide-Binding Biomolecules.

Studies of biosorption and bioaccumulation of heavy metals deal mostly with challenging, inhomogeneous, and complex materials. Therefore, most reports describe only application studies, while fundamental research is limited to indirect methods and speculations on the binding mechanisms. In this study, we describe a method for detecting and isolating heavy metal-binding biomolecules directly from crude extracts. The underlying principle is terbium sensitization and fluorescence excitation spectroscopy used offline after a chromatographic run. Compounds interacting with metal ions inevitably change the coordination sphere of terbium, which is reflected in the excitation spectrum leading to metal-specific luminescence. Main advantages of our approach include simple, fast, and inexpensive experiment design, nondestructive measurements, and detection limits far below 1 mg. Here, we have applied our method for three promising biosorbents (green algae, moss, and cyanobacterium) and obtained first information on the character of active compounds isolated from each species.

The response of newly established cell lines of Spodoptera littoralis to group I and group II baculoviruses.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV) belong to group I and group II nucleopolyhedroviruses, respectively and can replicate in a wide range of insect species. In this study, the ability of newly established S. littoralis cell lines to support replication of AcMNPV and SpliMNPV was examined. The microscopic observations showed that the S. littoralis cells infected with AcMNPV exhibited morphological changes such as cells breaking into small bodies and forming apoptosis-like bodies post-infection. Nuclear DNA fragmentation was observed in all AcMNPV-infected cell lines through DNA gel electrophoresis analysis. Therefore, the virus replication was unsuccessful in most of cells, which were able to abort the virus replication. On the other hand, cells that were infected with SpliMNPV did not show similar morphological changes and no small bodies were formed. In addition, SpliMNPV succeeded to infect the cells, replicate, and form viral occlusion bodies inside the infected cells. In suspension culture, S. littoralis cells, which were infected with AcMNPV, accumulated as composed balls in shaker flasks after infection overnight, with cell density decreasing dramatically. In contrast, there was no cell clumping seen in the infected cells with SpliMNPV and the uninfected cells. In conclusion, the newly established embryonic S. littoralis cells were highly susceptible to SpliMNPV, whereas the cells were non-permissive to AcMNPV, yet they still underwent programmed cell death.

Validated Nuclear-Based Transgene Expression Regulated by the Fea1 Iron-Responsive Promoter in the Green Alga Chlamydomonas reinhardtii.

Microalgae are in the focus for the production of recombinant proteins in research and potential commercial application. Inducible promoters represent important tools that potentially allow the expression of recombinant proteins at higher rates. In general, they are used to separate the culture growth phase from the production phase by initiating product formation after high cell densities have been achieved. This potentially offers a higher space-time yield, consequently improving the economics of a process. In the case of the green micro alga Chlamydomonas reinhardtii, a controlled switch between activation and deactivation of gene expression is possible by changes in cultivation parameters. In this work, parameters of induction and deactivation of the iron-responsive Fea1 promoter were analyzed over time in C. reinhardtii. The results presented for the strain CC4351 validate our previous findings presented for strain CC 400. The Fea1 promoter was successfully deactivated upon transferring the cells to medium containing 10 and 20 µM Fe3+. Within 120 h, cells showed only 1.7-6% of the initial fluorescence. Activation of the Fea1 promoter occurred promptly and prominently when cells were transferred to iron-deplete medium. In general, both strains showed a pronounced difference between the active and the inactive states of the Fea1 promoter.

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method.

A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic engineering of microalgae (Steinbrenner and Sandmann in Appl Environ Microbiol 72:7477-7484, 2006; Prasad et al. in Appl Microbiol Biotechnol 98(20):8629-8639, 2014). However, this mutated genomic clone harbors several introns and the entire expression cassette including its native promoter and terminator has a length > 5.6 kb, making it unsuitable as a standard selection marker. Therefore, we designed a synthetic, short pds gene (syn-pds-int) by removing introns and unwanted internal restriction sites, adding suitable restriction sites for cloning purposes, and introduced the first intron from the Chlamydomonas reinhardtii RbcS2 gene close to the 5'end without changing the amino acid sequence. The syn-pds-int gene (1872 bp) was cloned into pCAMBIA 1380 under the control of a short sequence (615 bp) of the promoter of pds (pCAMBIA 1380-syn-pds-int). This vector and the plasmid pCAMBIA1380-pds-L504R hosting the mutated genomic pds were used for transformation studies. To broaden the existing transformation portfolio, the rhodophyte Porphyridium purpureum was targeted. Agrobacterium-mediated transformation of P. purpureum with both the forms of pds gene, pds-L504R or syn-pds-int, yielded norflurazon-resistant (NR) cells. This is the first report of a successful nuclear transformation of P. purpureum. Transformation efficiency and lethal norflurazon dosage were determined to evaluate the usefulness of syn-pds-int gene and functionality of the short promoter of pds. PCR and Southern blot analysis confirmed transgene integration into the microalga. Both forms of pds gene expressed efficiently as evidenced by the stability, tolerance and the qRT-PCR analysis. The molecular toolkits and transformation method presented here could be used to genetically engineer P. purpureum for fundamental studies as well as for the production of high-value-added compounds.

Analysis of the Sugar Content in Food Products by Using Gas Chromatography Mass Spectrometry and Enzymatic Methods.

The aim of this study is to develop and optimise a method of sugar content determination in food products. Date juice (syrup) was used as a sample natural food resource for the analysis because of its potential usage as an alternative substrate for a variety of fermentation processes. Hence, qualifying and quantifying its sugar content is a crucial step. Therefore, gas chromatography mass spectrometry (GCMS) was used as a pre-qualitative method to identify the types of sugar in the date sample. The results demonstrate that the analysed date juice contains glucose, fructose and sucrose. This analysis was obtained by measuring the retention time of individual standard sugar samples such as glucose, fructose, mannose and sucrose. In addition, the mass spectra of the standard and date juice samples contained characteristic fragments of glucose, fructose and sucrose. Thus, GCMS results determined the appropriate enzymatic assays for quantifying the sugars in date juice. These results were similar to those of the two enzymatic methods (standard enzymatic assay and measuring the change in pH by CL10 analyser). Therefore, they confirmed the identified sugars and provided the sugar contents of the sample. Consequently, sugar quantification results indicate that 1 g of date juice sample contains a total of 0.5275⁻0.5507 g of six-carbon sugars (glucose + fructose) and 0.064⁻0.068 g of sucrose. As a consequence, the total sugar content in 1 g of date juice is 0.600⁻0.615 g. These results are comparable to the sample analysis that is provided by the date juice production company.

The components of shear stress affecting insect cells used with the baculovirus expression vector system.

Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

Quantification of oxygen production and respiration rates in mixotrophic cultivation of microalgae in nonstirred photobioreactors.

Online monitoring and controlling of different cellular parameters are key issues in aerobic bioprocesses. Since mixotrophic cultivation, in which we observe a mixture of cellular respiration and oxygen production has gained more popularity, there is a need for an on-process quantification of these parameters. The presented and adapted double gassing-out method applied to a mixotrophic cultivation of Galdieria sulphuraria, will be a tool for monitoring and further optimization of algal fermentation in nonstirred photobioreactors (PBR). We measured the highest net specific oxygen production rate (opr net) as 5.73 · 10-3 molO2 g-1 h-1 at the lowest oxygen uptake rate (OUR) of 1.00 · 10-4 molO2 L-1 h-1. Due to higher cell densities, we also demonstrated the increasing shading effect by a decrease of opr net, reaching the lowest value of 1.25 10-5 molO2 g-1 h-1. Nevertheless, with this on process measurement, we can predict the relation between the zone in which oxygen is net produced to the area where cell respiration dominates in a PBR, which has a major impact to optimize cell growth along with the formation of different products of interest such as pigments.

Prolonged Ex vivo expansion and differentiation of naïve murine CD43(-) B splenocytes.

Ex vivo expansion of naive primary B cells is still a challenge, yet would open new possibilities for in vitro studies of the immune response or the production of monoclonal antibodies. In our hands, unstimulated murine B cells did not expand in significant numbers, while culture viability decreased rapidly within a few days. Activation mimicking in vivo stimulation through either T cell-independent or T-cell dependent signaling, led to several division cycles, albeit accompanied by irreversible differentiation. By co-culturing B cells under moderate hypothermia (30°C) on live feeder fibroblasts expressing recombinant CD40 ligand (CD154) and by repeatedly transferring cultured B cells to new feeder cell cultures, we could extend the growth of primary mouse B cells compared to cultures maintained at 37°C. B cells under these conditions showed an activated phenotype as shown by the presence of AID and IRF4, two factors required for IgH class switch recombination in antigen-activated B cells. In contrast to cells cultured at 37°C, B cells under hyperthermia did surprisingly not differentiate into Blimp-1 expressing plasmablasts. Thus, the repeated batch process under hyperthermic conditions represents a first step towards the development of a continuous cultivation system for the expansion of primary B cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:978-989, 2016.

Hydroxyethyl starch solution for extracorporeal tissue perfusion.

In the field of free flap transfer in reconstructive surgery, the trans- or replanted tissue always undergoes cell damage during ischemia to a more or less strong extent. In previous studies we already showed that conserving muscle transplants by means of extracorporeal perfusion over a period of 6 hours by using a crystalloid solution for perfusion. However, we observed significant edema formation. In this study we aimed at reducing the edema formation by using an iso-oncotic colloid as perfusion solution. This way we wanted to evaluate a possible new application of hydroxyl-ethyl starch in an extracorporeal setup to exploit potential benefits of the colloid.Examined parameters include the muscles' functionality with external field stimulation, histological examination and edema formation. Perfused muscles showed a statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, as perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. Substituting the electrolyte perfusion solution with a colloidal one shows the tendency to reduce the edema formation however without statistical significance.

Assessing viability of extracorporeal preserved muscle transplants using external field stimulation: a novel tool to improve methods prolonging bridge-to-transplantation time.

Preventing ischemia-related cell damage is a priority when preserving tissue for transplantation. Perfusion protocols have been established for a variety of applications and proven to be superior to procedures used in clinical routine. Extracorporeal perfusion of muscle tissue though cumbersome is highly desirable since it is highly susceptible to ischemia-related damage. To show the efficacy of different perfusion protocols external field stimulation can be used to immediately visualize improvement or deterioration of the tissue during active and running perfusion protocols. This method has been used to show the superiority of extracorporeal perfusion using porcine rectus abdominis muscles perfused with heparinized saline solution. Perfused muscles showed statistically significant higher ability to exert force compared to nonperfused ones. These findings can be confirmed using Annexin V as marker for cell damage, perfusion of muscle tissue limits damage significantly compared to nonperfused tissue. The combination of extracorporeal perfusion and external field stimulation may improve organ conservation research.

Photobioreactors with internal illumination - A survey and comparison.

The idea of internally illuminated photobioreactors has existed since the 1990s and various systems were developed since. Recently, the interest in these systems has been on the rise again, due to the increased production of and research on high-value products and recombinant proteins from microalgae and plant cell cultures. While promising results in lab-scale have been achieved, the potential of photoautotrophic or mixotrophic production of these compounds is limited due to the lack of scalable photobioreactors, which could be overcome by internally illuminated systems. In this article, we review different internally illuminated photobioreactors from the last two decades and classify them into two major groups. The photobioreactors are compared based on the ratio of illuminated surface-to-culture volume and the occupied volume by internal light-emitting elements, and possible obstacles, challenges and future trends are discussed. Looking forward, new technologies and smaller light sources have improved the potential of internally illuminated photobioreactors with internal light sources and will enable these technologies to compete with systems with internal light guides, but only a few of these systems have currently achieved a relevant scale.

Unsupervised unstained cell detection by SIFT keypoint clustering and self-labeling algorithm.

We propose a novel unstained cell detection algorithm based on unsupervised learning. The algorithm utilizes the scale invariant feature transform (SIFT), a self-labeling algorithm, and two clustering steps in order to achieve high performance in terms of time and detection accuracy. Unstained cell imaging is dominated by phase contrast and bright field microscopy. Therefore, the algorithm was assessed on images acquired using these two modalities. Five cell lines having in total 37 images and 7250 cells were considered for the evaluation: CHO, L929, Sf21, HeLa, and Bovine cells. The obtained F-measures were between 85.1 and 89.5. Compared to the state-of-the-art, the algorithm achieves very close F-measure to the supervised approaches in much less time.

Using the low-pass monogenic signal framework for cell/background classification on multiple cell lines in bright-field microscope images.

PURPOSE: Several cell detection approaches which deal with bright-field microscope images utilize defocusing to increase image contrast. The latter is related to the physical light phase through the transport of intensity equation (TIE). Recently, it was shown that it is possible to approximate the solution of the TIE using a low-pass monogenic signal framework. The purpose of this paper is to show that using the local phase of the aforementioned monogenic signal instead of the defocused image improves the cell/background classification accuracy. MATERIALS AND METHODS: The paper statement was tested on an image database composed of three cell lines: adherent CHO, adherent L929, and Sf21 in suspension. Local phase and local energy images were generated using the low-pass monogenic signal framework with axial derivative images as input. Machine learning was then employed to investigate the discriminative power of the local phase. Three classifier models were utilized: random forest (RF), support vector machine (SVM) with a linear kernel, and SVM with a radial basis function (RBF) kernel. RESULTS: The improvement, averaged over cell lines, of classifying 5×5 sized patches extracted from the local phase image instead of the defocused image was 7.3% using the RF, 11.6% using the linear SVM, and 10.2% when a RBF kernel was employed instead of the linear one. Furthermore, the feature images can be sorted by increasing discriminative power as follows: at-focus signal, local energy, defocused signal, local phase. The only exception to this order was the superiority of local energy over defocused signal for suspended cells. CONCLUSIONS: Local phase computed using the low-pass monogenic signal framework considerably outperforms the defocused image for the purpose of pixel-patch cell/background classification in bright-field microscopy.

Combined shifted-excitation Raman difference spectroscopy and support vector regression for monitoring the algal production of complex polysaccharides.

The applicability of shifted-excitation Raman difference spectroscopy (SERDS) in combination with signal regression analysis as an alternative and non-invasive approach for monitoring the cultivation of phototrophic microorganisms producing complex molecules of pharmaceutical relevance in a bioreactor is demonstrated. As a model system, the cultivation of the red unicellular algae Porphyridium purpureum is used for focusing on the segregation of sulfated exopolysaccharides (EPS) which exhibit antiviral activity. The spectroscopic results obtained by partial linear least squares regression (PLSR) and by nonlinear support vector regression (SVR) are discussed against the corresponding results from the reference analytics based on the phenol-sulfuric acid assay. The SERDS-approach turns out to have strong potential as a non-invasive tool for online-monitoring of biotechnological processes.

Online oxygen measurements in ex vivo perfused muscle tissue in a porcine model using dynamic quenching methods.

INTRODUCTION: Transplantation of autologous free tissue flaps is the best applicable technique for treating large and complex tissue defects and still has one major failure criterion. Tissue--and in particular muscle tissue--is strongly sensitive to ischemia, thus after a critical period of oxygen depletion the risk of a partial or total flap loss is high. MATERIALS AND METHODS: For that reason a miniaturized ex vivo perfusion system has been developed, that supplies the tissue during operational delays. The purpose of this study was to determine the oxygenation levels during such a perfusion using different perfusates and therefore to objectify if a complementary oxygenation unit is required to improve perfusion quality. The oxygen levels of the tissue, as well of the perfusate, were measured by using minimal invasive optical oxygen sensors that are based on dynamic quenching. The ex vivo perfused tissue was the porcine rectus abdominis muscle. RESULTS: Results show, that during perfusion with heparinized crystalloid fluid (Jonosteril) and heparinized autologous whole blood, additional oxygenation of the perfusion reactor led to different ex vivo oxygen tissue saturations, which can be detected by dynamic quenching. CONCLUSION: Dynamic quenching methods are a promising and valuable technique to perform online oxygen measurements in ex vivo perfused muscle tissue in a porcine model.

Identification of sulfoquinovosyldiacyglycerides from Phaeodactylum tricornutum by matrix-assisted laser desorption/ionization QTrap time-of-flight hybrid mass spectrometry.

The pharmaceutical industry is interested in identifying novel target compounds. Due to their versatile pharmacological activities (e.g. antiviral, anti-carcinogen and immunosuppressive) sulfoquinovosyldiacylglycerides (SQDGs) are potential drug candidates. The present publication deals with the purification and structural characterization of SQDGs from three different strains of Phaeodactylum tricornutum. Besides detection of SQDGs (sn-1: C16:1/sn-2: C16:0 and sn-1: C20:5/sn-2: C16:0), two novel 2'-O-acylsulfoquinovosyldiacylglyerides (Ac-SQDGs, sn-1: C16:0/ sn-2: C16:0/2' C20:5 and sn-1: C20:5/sn-2: C16:0/2' C20:5) were identified by using matrix-assisted laser desorption/ionization (MALDI) QTrap time-of-flight (ToF) hybrid mass spectrometry (MS) with multistage MS(n). The analytical method enables the sn-position verification of fatty acids (MS(2)) as well as the confirmation of the regioposition of eicospentanoic acid at the sulfoquinovose (MS(3)).

Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides.

BACKGROUND: Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. RESULTS: The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipµ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. CONCLUSIONS: The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.