Ricerc@Sapienza

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

The real-time analysis integrated system
The real-time analysis integrated system is composed by:

1) CIRAS-3 IRGAs (Infrared Gas Analyzers), CIRAS-3 DC CO₂/H₂O Gas Analyzer;

2) High-Resolution Accurate Mass GC-QTOF Mass Spectrometry coupled to a TDU System (Thermal Desorber Unit) with a
selective adsorption system.

1) 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 CO₂/H₂O gas analyzer console can be connected to whole plant cuvette for carrying out
the measures at the individual level inside the phytotron.

2) High-Resolution Accurate Mass GC-QTOF Mass Spectrometry coupled to a TDU System with a selective adsorption system.

The GCQTOF 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).