Scale-up from microtiter plate to laboratory fermenter: evaluation by online monitoring techniques of growth and protein expression in Escherichia coli and Hansenula polymorpha fermentations

<p>Abstract</p> <p>Background</p> <p>In the past decade, an enormous number of new bioprocesses have evolved in the biotechnology industry. These bioprocesses have to be developed fast and at a maximum productivity. Up to now, only few microbioreactors were developed to fulfill these demands and to facilitate sample processing. One predominant reaction platform is the shaken microtiter plate (MTP), which provides high-throughput at minimal expenses in time, money and work effort. By taking advantage of this simple and efficient microbioreactor array, a new online monitoring technique for biomass and fluorescence, called BioLector, has been recently developed. The combination of high-throughput and high information content makes the BioLector a very powerful tool in bioprocess development. Nevertheless, the scalabilty of results from the micro-scale to laboratory or even larger scales is very important for short development times. Therefore, engineering parameters regarding the reactor design and its operation conditions play an important role even on a micro-scale. In order to evaluate the scale-up from a microtiter plate scale (200 μL) to a stirred tank fermenter scale (1.4 L), two standard microbial expression systems, <it>Escherichia coli </it>and <it>Hansenula polymorpha</it>, were fermented in parallel at both scales and compared with regard to the biomass and protein formation.</p> <p>Results</p> <p>Volumetric mass transfer coefficients (k_La) ranging from 100 to 350 1/h were obtained in 96-well microtiter plates. Even with a suboptimal mass transfer condition in the microtiter plate compared to the stirred tank fermenter (k_La = 370-600 1/h), identical growth and protein expression kinetics were attained in bacteria and yeast fermentations. The bioprocess kinetics were evaluated by optical online measurements of biomass and protein concentrations exhibiting the same fermentation times and maximum signal deviations below 10% between the scales. In the experiments, the widely applied green fluorescent protein (<it>GFP</it>) served as an online reporter of protein expression for both strains.</p> <p>Conclusions</p> <p>The successful 7000-fold scale-up from a shaken microtiter plate to a stirred tank fermenter was demonstrated in parallel fermentations for standard microbial expression systems. This confirms that the very economical and time efficient platform of microtiter plates can be very easily scaled up to larger stirred tank fermenters under defined engineering conditions. New online monitoring techniques for microtiter plates, such as the BioLector, provide even more real-time kinetic data from fermentations than ever before and at an affordable price. This paves the way for a better understanding of the bioprocess and a more rational process design.</p

Gramophone noise detection and reconstruction using time delay artificial neural networks

Gramophone records were the main recording medium for more than seven decades and regained widespread popularity over the past several years. Being an analog storage medium, gramophone records are subject to distortions caused by scratches, dust particles, degradation, and other means of improper handling. The observed noise often leads to an unpleasant listening experience and requires a filtering process to remove the unwanted disruptions and improve the audio quality. This paper proposes a novel approach that employs various feed forward time delay artificial neural networks to detect and reconstruct noise in musical sound waves. A set of 800 songs from eight different genres were used to validate the performance of the neural networks. The performance was analyzed according to the outlier detection and interpolation accuracy, the computational time and the tradeoff between the accuracy and the time. The empirical results of both detection and reconstruction neural networks were compared to a number of other algorithms, including various statistical measurements, duplication approaches, trigonometric processes, polynomials, and time series models. It was found that the neural networks' outlier detection accuracy was slightly lower than some of the other noise identification algorithms, but achieved a more efficient tradeoff by detecting most of the noise in real time. The reconstruction process favored neural networks with an increase in the interpolation accuracy compared to other widely used time series models. It was also found that certain genres such as classical, country, and jazz music were interpolated more accurately. Volatile signals, such as electronic, metal, and pop music were more challenging to reconstruct and were substantially better interpolated using neural networks than the other examined algorithms.http://ieeexplore.ieee.org/xpl/RecentIssue.jsp?punumber=6221021hj2017Computer Scienc

The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

Endo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the β-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-β-N-acetylmuramidases, which catalyze an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www. cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria

Spectral Function of 2D Fermi Liquids

We show that the spectral function for single-particle excitations in a two-dimensional Fermi liquid has Lorentzian shape in the low energy limit. Landau quasi-particles have a uniquely defined spectral weight and a decay rate which is much smaller than the quasi-particle energy. By contrast, perturbation theory and the T-matrix approximation yield spurious deviations from Fermi liquid behavior, which are particularly pronounced for a linearized dispersion relation.Comment: 6 pages, LaTeX2e, 5 EPS figure

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Biodesalination: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance