Rapid microbiological detection method based on ultrasonic instrumentation

Los métodos de detección rápida de microorganismos se están convirtiendo en una herramienta esencial para el control de calidad en el área de la biotecnología, como es el caso de las industrias de alimentos y productos farmacéuticos y bioquímicos. En este escenario, el objetivo de esta tesis doctoral es desarrollar una técnica de inspección rápida de microoganismos basada en ultrasonidos. La hipótesis propuesta es que la combinación de un dispositivo ultrasónico de medida y un medio líquido diseñado específicamente para producir y atrapar burbujas, pueden constituir la base de un método sensible y rápido de detección de contaminaciones microbianas. La técnica presentada es efectiva para bacterias catalasa-positivas y se basa en la hidrólisis del peróxido de hidrógeno inducida por la catalasa. El resultado de esta reacción es un medio con una creciente concentración de burbujas. Tal medio ha sido estudiado y modelado desde el punto de vista de la propagación ultrasónica. Las propiedades deducidas a partir del análisis cinemático de la enzima se han utilizado para evaluar el método como técnica de inspección microbiana. En esta tesis, se han investigado aspectos teóricos y experimentales de la hidrólisis del peróxido de hidrógeno. Ello ha permitido describir cuantitativamente y comprender el fenómeno de la detección de microorganismos catalasa-positivos mediante la medida de parámetros ultrasónicos. Más concretamente, los experimentos realizados muestran cómo el oxígeno que aparece en forma de burbujas queda atrapado mediante el uso de un gel sobre base de agar. Este gel fue diseñado y preparado especialmente para esta aplicación. A lo largo del proceso de hidrólisis del peróxido de hidrógeno, se midió la atenuación de la onda y el “backscattering” producidos por las burbujas, utilizando una técnica de pulso-eco. Ha sido posible detectar una actividad de la catalasa de hasta 0.001 unidades/ml. Por otra parte, este estudio muestra que por medio del método propuesto, se puede lograr una detección microbiana para concentraciones de 105 células/ml en un periodo de tiempo corto, del orden de unos pocos minutos. Estos resultados suponen una mejora significativa de tres órdenes de magnitud en comparación con otros métodos de detección por ultrasonidos. Además, la sensibilidad es competitiva con modernos y rápidos métodos microbiológicos como la detección de ATP por bioluminiscencia. Pero sobre todo, este trabajo muestra una metodología para el desarrollo de nuevas técnicas de detección rápida de bacterias basadas en ultrasonidos. ABSTRACT In an industrial scenario where rapid microbiological methods are becoming essential tools for quality control in the biotechnological area such as food, pharmaceutical and biochemical; the objective of the work presented in this doctoral thesis is to develop a rapid microorganism inspection technique based on ultrasounds. It is proposed that the combination of an ultrasonic measuring device with a specially designed liquid medium, able to produce and trap bubbles could constitute the basis of a sensitive and rapid detection method for microbial contaminations. The proposed technique is effective on catalase positive microorganisms. Well-known catalase induced hydrogen peroxide hydrolysis is the fundamental of the developed method. The physical consequence of the catalase induced hydrogen peroxide hydrolysis is an increasingly bubbly liquid medium. Such medium has been studied and modeled from the point of view of ultrasonic propagation. Properties deduced from enzyme kinematics analysis have been extrapolated to investigate the method as a microbial inspection technique. In this thesis, theoretical and experimental aspects of the hydrogen peroxide hydrolysis were analyzed in order to quantitatively describe and understand the catalase positive microorganism detection by means of ultrasonic measurements. More concretely, experiments performed show how the produced oxygen in form of bubbles is trapped using the new gel medium based on agar, which was specially designed for this application. Ultrasonic attenuation and backscattering is measured in this medium using a pulse-echo technique along the hydrogen peroxide hydrolysis process. Catalase enzymatic activity was detected down to 0.001 units/ml. Moreover, this study shows that by means of the proposed method, microbial detection can be achieved down to 105 cells/ml in a short time period of the order of few minutes. These results suppose a significant improvement of three orders of magnitude compared to other ultrasonic detection methods for microorganisms. In addition, the sensitivity reached is competitive with modern rapid microbiological methods such as ATP detection by bioluminescence. But above all, this work points out a way to proceed for developing new rapid microbial detection techniques based on ultrasound.

Propagation of nuclear data uncertainties in fuel cycle calculations using MONTE-CARLO Technique

The uncertainty propagation in fuel cycle calculations due to Nuclear Data (ND) is a important important issue for : issue for : • Present fuel cycles (e.g. high burnup fuel programme) • New fuel cycles designs (e.g. fast breeder reactors and ADS) Different error propagation techniques can be used: • Sensitivity analysis • Response Response Surface Method Surface Method • Monte Carlo technique Then, p p , , in this paper, it is assessed the imp y pact of ND uncertainties on the decay heat and radiotoxicity in two applications: • Fission Pulse Decay ( y Heat calculation (FPDH) • Conceptual design of European Facility for Industrial Transmutation (EFIT)

Self-validating technique for the measurement of the linewidth enhancement factor in semiconductor lasers

A new method for measuring the linewidth enhancement factor (α-parameter) of semiconductor lasers is proposed and discussed. The method itself provides an estimation of the measurement error, thus self-validating the entire procedure. The α-parameter is obtained from the temporal profile and the instantaneous frequency (chirp) of the pulses generated by gain switching. The time resolved chirp is measured with a polarization based optical differentiator. The accuracy of the obtained values of the α-parameter is estimated from the comparison between the directly measured pulse spectrum and the spectrum reconstructed from the chirp and the temporal profile of the pulse. The method is applied to a VCSEL and to a DFB laser emitting around 1550 nm at different temperatures, obtaining a measurement error lower than ± 8%.

Laser Shock Processing: An Emerging Technique for the Enhancement of Surface Properties and Fatigue Life of High Strength Metal Alloys

Profiting by the increasing availability of laser sources delivering intensities above 10 9 W/cm 2 with pulse energies in the range of several Joules and pulse widths in the range of nanoseconds, laser shock processing (LSP) is being consolidating as an effective technology for the improvement of surface mechanical and corrosion resistance properties of metals and is being developed as a practical process amenable to production engineering. The main acknowledged advantage of the laser shock processing technique consists on its capability of inducing a relatively deep compression residual stresses field into metallic alloy pieces allowing an improved mechanical behaviour, explicitly, the life improvement of the treated specimens against wear, crack growth and stress corrosion cracking. Following a short description of the theoretical/computational and experimental methods developed by the authors for the predictive assessment and experimental implementation of LSP treatments, experimental results on the residual stress profiles and associated surface properties modification successfully reached in typical materials (specifically steels and Al and Ti alloys) under different LSP irradiation conditions are presented

Pulse shortening of gain switched single mode semiconductor lasers using a variable delay interferometer

We propose a pulse shaping and shortening technique for pulses generated from gain switched single mode semiconductor lasers, based on a Mach Zehnder interferometer with variable delay. The spectral and temporal characteristics of the pulses obtained with the proposed technique are investigated with numerical simulations. Experiments are performed with a Distributed Feedback laser and a Vertical Cavity Surface Emitting Laser, emitting at 1.5 µm, obtaining pulse duration reduction of 25-30%. The main asset of the proposed technique is that it can be applied to different devices and pulses, taking advantage of the flexibility of the gain switching technique.

Laser Shock Micro-Forming as an Emerging Technique for Microsystems Shaping and Adjustment

Continuous and long-pulse lasers have been extensively used for the forming of metal sheets for macroscopic mechanical applications. However, for the manufacturing of Micro-Mechanical Systems (MMS), the applicability of such type of lasers is limited by the long relaxation time of the thermal fields responsible for the forming phenomena. As a consequence, the final sheet deformation state is attained only after a certain time, what makes the generated internal residual stress fields more dependent on ambient conditions and might difficult the subsequent assembly process. The use of short pulse (ns) lasers provides a suitable parameter matching for the laser forming of an important range of sheet components used in MEMS. The short interaction time scale required for the predominantly mechanic (shock) induction of deformation residual stresses allows the successful processing of components in a medium range of miniaturization (particularly important according to its frequent use in such systems). In the present paper, Laser Shock Micro-Forming (LSμF) is presented as an emerging technique for Microsystems parts shaping and adjustment along with a discussion on its physical foundations and practical implementation possibilities developed by the authors.

Integrable high order UWB pulse photonic generator based on cross phase modulation in a SOA-MZI

We propose and experimentally demonstrate a potentially integrable optical scheme to generate high order UWB pulses. The technique is based on exploiting the cross phase modulation generated in an InGaAsP Mach-Zehnder interferometer containing integrated semiconductor optical amplifiers, and is also adaptable to different pulse modulation formats through an optical processing unit which allows to control of the amplitude, polarity and time delay of the generated taps.

Temporally flat top pulse generation from gain switched semiconductor lasers based on a polarization interferometer with variable transfer function

We propose the use of a polarization based interferometer with variable transfer function for the generation of temporally flat top pulses from gain switched single mode semiconductor lasers. The main advantage of the presented technique is its flexibility in terms of input pulse characteristics, as pulse duration, spectral bandwidth and operating wavelength. Theoretical predictions and experimental demonstrations are presented and the proposed technique is applied to two different semiconductor laser sources emitting in the 1550 nm region. Flat top pulses are successfully obtained with input seed pulses with duration ranging from 40 ps to 100 ps.

A clustering technique for partial discharge and noise sources identification in power cables by means of waveform parameters

On-line partial discharge (PD) measurements have become a common technique for assessing the insulation condition of installed high voltage (HV) insulated cables. When on-line tests are performed in noisy environments, or when more than one source of pulse-shaped signals are present in a cable system, it is difficult to perform accurate diagnoses. In these cases, an adequate selection of the non-conventional measuring technique and the implementation of effective signal processing tools are essential for a correct evaluation of the insulation degradation. Once a specific noise rejection filter is applied, many signals can be identified as potential PD pulses, therefore, a classification tool to discriminate the PD sources involved is required. This paper proposes an efficient method for the classification of PD signals and pulse-type noise interferences measured in power cables with HFCT sensors. By using a signal feature generation algorithm, representative parameters associated to the waveform of each pulse acquired are calculated so that they can be separated in different clusters. The efficiency of the clustering technique proposed is demonstrated through an example with three different PD sources and several pulse-shaped interferences measured simultaneously in a cable system with a high frequency current transformer (HFCT).