Ricerc@Sapienza

A detailed description of each component of the Integrated Smart PHYTOTRON.

1) Walk-in chambers

Concerning the Walk-in chambers one chamber will be used as a control, the second for treatment according to the experimental protocol defined from time to time. The two Walk-in chambers will be placed on the Botanical Garden of the Department of Environmental Biology, Sapienza University of Rome. The two chambers will be equipped with an automatic control system for temperature ranging between 15 ÷ 40 °C, and relative humidity ranging between 40÷80 %. In the chambers, artificial lighting will be installed for a realistic simulation of irradiation. The lamps can reproduce realistic solar spectrum, reaching an intensity of 800 microEm²s^-1, and can be programmed for simulating specific photoperiod. The light modules will be placed on mobile carriage allowing to set the right height of lamps relative to the species used for the experiments from time to time. Each chamber is equipped with a pre-welded counter composed of side rails, headboards and crosspieces in custom-made aluminum profiles, height-adjustable hot-dip galvanized steel racks, Danish original polystyrene shelves. The counter will be equipped with a flow and backflow irrigation system composed of a water storage tank, a push pump, a four-valve station, controlled flow valves, electrical wiring, hydraulic connections and a dedicated electronic unit for irrigation control. Each chamber will be equipped with the main electrical panel for managing the illumination, the general mechanical operation and the heating and cooling system, and three secondary control panels, for the connection with the real-time analysis integrated system (see the following detailed description) and a connection line for the fumigation system. The air ventilation and recirculation system consist of an extractor connected in suction to each chamber. Each chamber will have a single conditioning system consisting of an outdoor and an indoor unit, and a humidification and dehumidification system. The central engine will be placed close to the walk-in chambers in a dedicated space able to contain all the equipment and components for the production and distribution of heat/cold air. The walk-in chambers will be planned and realized following all the safety regulations established by the National law.

2) The real-time analysis integrated system is composed by: CIRAS-3 IRGAs (Infrared Gas Analyzers), CIRAS-3 DC CO2/H2O Gas Analyzer and High-Resolution Accurate Mass GC-QTOF Mass Spectrometry coupled to a TDU System (Thermal Desorber Unit) with a selective adsorption system. CIRAS-3's and CIRAS-3 DC CO₂/H₂O gas analyzer consist in a portable system based on IRGAs (Infrared Gas Analyzers) technology that measures CO₂ and H₂O concentration inside and outside the phytotron, with a measurements range of 0-1000 micromol mol-1 for CO2 and 0-75 mb for H₂O. The gas analyzers include an infrared source, highly polished and gold plated sample cells and detectors that are optimized for CO₂ (4.26 microns) and H2O (2.60 microns). The analyzers act as absorptiometers measuring infrared absorption only. The optical bench is temperature controlled and pressure compensated ensuring the most accurate CO₂ and H₂O measurements under changing ambient conditions. The technology of the CIRAS-3 and CIRAS-3 DC CO₂/H₂O gas analyzer ensures an inherent calibration stability thanks to the innovative Auto-Zero function that allows for fast warm-up adaptation to changing ambient conditions and excellent stability. CIRAS-3 console can be connected to leaf cuvette (Universal, Narrow and Conifer) for carrying out the measures at leaf level. Using this arrangement, it is possible to control air humidity, light intensity, and leaf temperature. The leaf cuvette is also equipped with a Chlorophyll fluorescence module able to measure, with a MultiPulse technology, the efficiency of photosystems. The integrated system is composed of a power system, accessories, suitcase transport, software. The CO₂/H₂O gas analyzer will be used to take measures in continuous from both control and treatment phytotron, connecting the console directly to the walk-in chambers. Moreover, CIRAS-3 DC C₂O/H₂O gas analyzer console can be connected to whole plant cuvette for carrying out the measures at the individual level inside the phytotron.

3) High-Resolution Accurate Mass GC-QTOF Mass Spectrometry coupled to a TDU System with a selective adsorption system. The GC-QTOF System selected as the best technical solution in the market is the GC-QTOF 7250 Low Energy EI source coupled with a GC 7890 series system combined to UNITY-xr automation system with Air server-xr by Markes International, a partner company of Agilent Technologies. Biogenic Organic Volatiles (BVOCs) produced in both walk-in chambers (from here, the "control" chamber and the "treatment" chamber) will be intercepted by an extractor fan within the Smart PHYTOTRON driven by an external pump. A system of stainless steel pipes, deriving from the extractor fan inside the chambers, will drive, forcedly through the external power counter pressure, BVOCs into the Air-server-xr through which an on-line sampling for the GC-MS analysis will be done. The Air server-xr unit will collect and absorb the BVOCs present even in traces simultaneously from the two walk-in chambers, will absorb them and pump into the GC-MS analyzer. Specifically, the Air server-xr is provided with disposable and re-usable sorbent-traps sampling. Samples are introduced directly onto the electrically-cooled sorbent-packed focusing trap of the UNIT-xr thermal desorber, typically held between ambient and -30°C. Then, focusing trap rapidly heated (up to 100°C/s) in a reverse flow of carrier gas (backflush operation) will transfer the analyses to the GC-column. Analytes in a range of C2-C44, plus 5/6 ring PAHs, phthalates and PCBs, thiols can be adsorbed and released by the sorbent-tubes. To avoid water to be present in traces ("humid" BVOCs), since it is necessary to remove the moisture before the gas flow reaches the GC column and detector, the Kor-xr option for the Air server selectively removes through the NafionTM dryer water prior to analytic focusing. This device allows a high-sensitive on-line analysis of polar species, oxygenates and pinenes (as well as all other typical VOCs) in a humid environment. The TC-20 multi-tube conditioning unit and dry purge will allow reusing the sorbent tubes. TC-20 is an independent device for simultaneous conditioning or dry purge of up to 20 adsorbing tubes for thermal desorption. Thus, BVOCs produced in both control and treatment walk-in chambers will be driven after being adsorbed into the UNITY-xr directly into the GC column. Here, BVOCs are separated chromatographically and then, according to their own retention times, analysed into the detector, the core of this unit, i.e. the QTOF mass spectrometer. There are several types of columns, interchangeable, that provide different affinity for different molecules; this allows the very versatile type of analysis, i.e. the system can detect thousands of molecules differing for any chemical-physical features. The Agilent 7250 Quadrupole Time-of-Flight GC/MS system delivers full-spectrum, high-resolution, accurate-mass data with a wide dynamic range for identifying, quantifying and investigating GC-amenable compounds. The electron ionization source offers low energy EI capability. The control of both systems (adsorption and detection) is PC-driven as well as the mass spectra analysis. The software package, the Agilent Mass Profiler, allows the exact mass identification and comparison in specific databases for thousands of metabolites. BVOCs produced in the Integrated Smart PHYTOTRON will be identified at a molecular level even if produced in traces (<0.1 ppt) or if with a low molecular weight (mass resolution from m/z 20).