Development of monitoring and control systems for biotechnological processes

The field of biotechnology represents an important research area that has gained increasing success in recent times. Characterized by the involvement of biological organisms in manufacturing processes, its areas of application are broad and include the pharmaceuticals, agri-food, energy, and even waste treatment. The implication of living microorganisms represents the common element in all bioprocesses. Cell cultivations is undoubtedly the key step that requires maintaining environmental conditions in precise and defined ranges, having a significant impact on the process yield and thus on the desired product quality. The apparatus in which this process occurs is the bioreactor. Unfortunately, monitoring and controlling these processes can be a challenging task because of the complexity of the cell growth phenomenon and the limited number of variables can be monitored in real-time. The thesis presented here focuses on the monitoring and control of biotechnological processes, more specifically in the production of bioethanol by fermentation of sugars using yeasts. The study conducted addresses several issues related to the monitoring and control of the bioreactor, in which the fermentation takes place. First, the topic concerning the lack of proper sensors capable of providing online measurements of key variables (biomass, substrate, product) is investigated. For this purpose, nonlinear estimation techniques are analyzed to reconstruct unmeasurable states. In particular, the geometric observer approach is applied to select the best estimation structure and then a comparison with the extended Kalman filter is reported. Both estimators proposed demonstrate good estimation capabilities as input model parameters vary. Guaranteeing the achievement of the desired ethanol composition is the main goal of bioreactor control. To this end, different control strategies, evaluated for three different scenarios, are analzyed. The results show that the MIMO system, together with an estimator for ethanol composition, ensure the compliance with product quality. After analyzing these difficulties through numeric simulations, this research work shifts to testing a specific biotechnological process such as manufacturing bioethanol from brewery’s spent grain (BSG) as renewable waste biomass. Both acid pre-treatment, which is necessary to release sugars, and fermentation are optimized. Results show that a glucose yield of 18.12 per 100 g of dried biomass is obtained when the pre-treatment step is performed under optimized conditions (0.37 M H2SO4, 10% S-L ratio). Regarding the fermentation, T=25°C, pH=4.5, and inoculum volume equal to 12.25% v/v are selected as the best condition, at which an ethanol yield of 82.67% evaluated with respect to theoretical one is obtained. As a final step, the use of Raman spectroscopy combined with chemometric techniques such as Partial Least Square (PLS) analysis is evaluated to develop an online sensor for fermentation process monitoring. The results show that the biomass type involved significantly affects the acquired spectra, making them noisy and difficult to interpret. This represents a nontrivial limitation of the applied methodology, for which more experimental data and more robust statistical techniques could be helpful

K/Na-silicate, ethyl-silicate and silane nano-molecular treatments in the restoration of high porous limestone

Carbonate sedimentary rocks (i.e., limestones) have been frequently used in historical times due to easy availability and workability. These latter depend primarily by petrophysical characteristics (porosity, bulk density) that influence the mechanical strength. However, the limestones with high porosity (>30%) and a poorly cemented carbonate-matrix show chemical alteration (i.e., dissolution) and physical decay (e.g., decohesion). In this work it was taken as case study a biomicritic limestone belonging to the carbonatic miocenic series (lower Tortonian) of Cagliari (southern-Sardinia, Italy). This limestone has a low-medium cementing matrix containing hygroscopic clay and sea-salt phases, which make the rock degradable. To limit the decay it can intervene with consolidating products (K-Na-silicate, ethyl-silicate) and protective-chemicals (silane nano-molecular gel-coat) to reduce the porosity and permeability to the liquid aqueous phase. Results highlight an increase of strength after consolidation and a decrease of gas-permeability after protection-treatment, maintaining in both cases a good permeability to the vapor-phase

A geometric observer-assisted approach to tailor state estimation in a bioreactor for ethanol production

In this work, a systematic approach based on the geometric observer is proposed to design a model-based soft sensor, which allows the estimation of quality indexes in a bioreactor. The study is focused on the structure design problem where the set of innovated states has to be chosen. On the basis of robust exponential estimability arguments, it is found that it is possible to distinguish all the unmeasured states if temperature and dissolved oxygen concentration measurements are combined with substrate concentrations. The proposed estimator structure is then validated through numerical simulation considering two different measurement processor algorithms: the geometric observer and the extended Kalman filter

Building pathology and environment: Weathering and decay of stone construction materials subjected to a Csa mediterranean climate laboratory simulation

Building stone materials have to satisfy long-term durability requirements in different environments in terms of mechanical strength and resistance to aggressive conditions. Several studies and field observations show that weathering on geo-materials is related to average annual precipitation and temperature. The decay also depends on salts air/soil concentrations and biological agents, but the more harmful impact is given by greenhouse gas (e.g. CO2, SOX, NOX, O3). These last induce the acidification of the rain and runoff waters. Nowadays, decay prediction is required in order to estimate the behaviour of stone materials over time. This research represents a second part of a previous work where the response to weathering of some construction materials used in ancient and contemporary architecture and cultural heritage has been evaluated by a labo- ratory simulation of hot-summer Csa Mediterranean climate. Simulation consists of accelerate ageing test on climate chamber by reproducing macro (e.g. daily and seasonal cycles of temperature, relative humidity, CO2 air concentration) and micro (e.g. rain, soil capillary rising) environments. Some non-destructive testing were executed to evaluate some physical–mechanical “decay markers” before and after the ageing. Test caused both decreasing and increasing of Leeb D hardness, decreases of permeability and a general decrease of ultrasonic speed, mainly due to the formations of patinas, crusts and efflorescences on the surfaces

Different control strategies for a yeast fermentation bioreactor

Biological systems are usually highly sensitive to process conditions variations, such as temperature, pH, substrate concentration. For this reason, it is important to adequately control and monitor the process in order to guaranteeing product quality while maintaining adequate performance and productivity. The production of ethanol by fermentation is certainly one of the most important industrial bioprocesses, being ethanol an alternative source of energy. For this reason, valuable models of this process based on different kinetic considerations are available in literature, and they can be considered a valid benchmark to investigate control system and estimation techniques for biological reactors. Three different control strategies have been analysed: direct reactor temperature control, cascade control where the primary loop uses delayed ethanol measurements, and 2x2 control system with inferential control for the product concentration. The proposed configurations have been compared at different operating conditions and results show that the use of the inferential control is the most effective in case of severe disturbances

Differential Requirement for the Cell Wall Integrity Sensor Wsc1p in Diploids Versus Haploids.

The primary role of the Cell Wall Integrity Pathway (CWI) in Saccharomyces cerevisiae is monitoring the state of the cell wall in response to general life cycle stresses (growth and mating) and imposed stresses (temperature changes and chemicals). Of the five mechanosensor proteins monitoring cell wall stress, Wsc1p and Mid2p are the most important. We find that WSC1 has a stringent requirement in zygotes and diploids, unlike haploids, and differing from MID2's role in shmoos. Diploids lacking WSC1 die frequently, independent of mating type. Death is due to loss of cell wall and plasma membrane integrity, which is suppressed by osmotic support. Overexpression of several CWI pathway components suppress wsc1∆ zygotic death, including WSC2, WSC3, and BEM2, as well as the Rho-GAPS, BEM3 and RGD2. Microscopic observations and suppression by BEM2 and BEM3 suggest that wsc1∆ zygotes die during bud emergence. Downstream in the CWI pathway, overexpression of a hyperactive protein kinase C (Pkc1p-R398P) causes growth arrest, and blocks the pheromone response. With moderate levels of Pkc1p-R398P, cells form zygotes and the wsc1∆ defect is suppressed. This work highlights functional differences in the requirement for Wsc1p in diploids Versus haploids and between Mid2p and Wsc1p during mating