2.3. Isoprenoid emission
Leaf-level gas fluxes were measured on site from one leaf per tree and
repeated in the same tree across seasons. Measurements were made using a
commercial, portable gas exchange system with infrared gas analyzer
(IRGA), the LI6400XT (LiCor, U.S.A.). A hydrocarbon filter (Restek Pure
Chromatography, Restek Corporations, U.S.A.) was installed at the air
inlet of the IRGA to remove hydrocarbons from incoming ambient air. All
tubing in contact with the sampling air was PTFE (a material that is
non-reactive to hydrocarbons). Before each measurement, a blank was
sampled from the empty leaf chamber (see more details on blank samples
in Table S1, Supporting Information). One leaf was separately enclosed
in the leaf chamber with standard conditions — photosynthetic photon
flux density (PPFD) at 1000 μmol m-2s-1 and leaf temperature at 30ºC — until net
assimilation (An), stomatal conductance
(gs) and internal CO2 concentration
(Ci) were stable. The stability criterion for
measurements was assigned as up to one standard deviation of the mean
An. The flow rate of air going into the leaf chamber was
400 μmol s-1, CO2 and
H2O concentrations were 400 μmol mol-1and 21 mmol mol-1 (relative humidity of
~ 60%), respectively. Air exiting the IRGA leaf chamber
was routed to flow through adsorbent cartridges (stainless silico steel
tubes filled with Tenax TA and Carbograph 5 TD adsorbents) at a rate of
200 sccm for 10 min, which resulted in 2 L air samples for isoprenoid
chemical analysis. The isoprenoids accumulated in the adsorbent
cartridges were determined subsequently by laboratory analysis. Samples
from December 2018 and May 2019 were analysed in the State University of
Amazonas (UEA, Brazil), and samples from September 2019 were analysed in
the Max Planck Institute for Chemistry (MPIC, Germany).
In UEA, cartridges were analyzed with a thermal desorption system (TD;
Markes International, U.K.) interfaced with gas chromatograph-mass
spectrometer and flame ionization detectors (GC-MS-FID; 7890B-GC and
5977A-MSD series, Agilent Technologies, U.S.A.). The cartridges were
loaded in the TD automatic sampler (TD-100, Markes International, U.K.),
which connects to the thermal desorption system. Then, samples were
dried by purging for 5 min with 50 sccm of ultrahigh-purity helium (all
flow vented out of the split vent) and transferred (300ºC for 10 min
with 50 sccm of ultrapure nitrogen) to the thermal desorption cold trap
held at -10ºC (Unity Series 1, Markes International, U.K.). During GC
injection, the trap was heated to 300ºC for 3 min while backflushing
with carrier gas (helium) at a flow rate of 6.0 sccm directed into the
column (Agilent HP-5, 5% phenyl methyl siloxane capillary, 30.0 m x 320
μm x 0.25 μm). The oven ramp temperature was programmed with an initial
hold of 6 min at 27ºC, followed by an increase to 85ºC at 6ºC
min-1, followed by a hold at 200 ºC for 6 min. We
confirmed the identification of emitted isoprenoids from the samples by
comparison of retention time with a solution of authentic liquid
standards in methanol (Sigma-Aldrich, U.S.A.) and comparison to the
library of the National Institute of Standards and Technology (NIST).
The GC-MS-FID was calibrated at least three times before the analysis of
the sample cartridges; with calibration curves that were generated by
injecting different amounts of gas standard (27 biogenic VOCs gas
mixture, by Apel & Riemer Environmental Inc., U.S.A.) into separate
cartridges, a mean correlation coefficient >= 0.98 was
obtained, and the LOD quantified as 27 pptv.
In MPIC, the cartridges were analyzed through thermal-desorption gas
chromatography time of flight mass spectrometry (TD-GC-TOF-MS, Bench ToF
Tandem Ionisation from Markes International, U.K.). The analysis
consisted of three main steps: desorption of the analytes from the
cartridges (TD), separation of the analytes through gas chromatography
(GC), and quantification and identification of the analytes through
time-of-flight mass spectrometry (ToF-MS). Samples were first dried by
purging them for 5 min with a flow of ultrapure N2 at 50
ml min-1, then transferred to the thermal-desorption
unit. Thermal-desorption was carried out in two stages - tube desorption
and trap desorption, both performed at 250°C for 10 minutes through a TD
100xr (Markes International, U.K.). The desorbed components were carried
in a flow of Helium into the GC column (dimethyl T.B.S. β-cyclodextrin
0.15µm, 0.15mm ID, 25m L, from MEGA, Italy). The temperature ramp
consisted of an initial 5 min at 40°C, after which the temperature was
increased at a rate of 1.5 °C/min from 40°C to 150°C, and further
increased at a rate of 30°C/min from 150°C to 200°C. After the GC
column, the analytes were fragmented through electron impact ionization
at -70 eV in the ToF. Identification was obtained by comparing the MS
spectra with the MS NIST library for the same ionization energy and by
injection of gas mixtures (162 VOCs gas mixture and 25 biogenic VOCs gas
mixture, by Apel & Riemer Environmental Inc., U.S.A.) and liquid
standards. The obtained chromatograms were integrated with TOF-DS
(Markes International, U.K.). Gas standard cartridges were used to
calibrate the instrument, determine the precision and LOD of the
analysis, which was quantified as 23% and ~1 pptv,
respectively. More information on the material and method used can be
found in Zannoni et al. (2020).
For the final flux calculation, isoprenoid concentrations were
determined using the sample volume that was passed through each
cartridge. This volume is the integration of the mass flow rate measured
and controlled by the pump used to suck the air coming out from the IRGA
leaf chamber. Once the volume mixing ratios of isoprenoids (ppbv) were
obtained, leaf emission fluxes were determined using the equation
(F = R ppbv × Q/A), where F (nmol m-2s-1) is leaf flux of isoprenoid emission; Rppbv(nmol mol-1) is isoprenoid concentration of the
sample; Q is flow rate of air into the leaf chamber (400 x
10-6 mol s-1); and A is the
area of leaf within the chamber (0.06 m²). In order to calculate
isoprenoid emission on a mass basis, we measured Leaf Mass per Area
(LMA). LMA was calculated as the ratio of leaf dry weight to leaf area.
We did not include petioles in the LMA calculation since they can be
quite large for rainforest species and are usually more related to leaf
positioning rather than biomass efficiency (Poorter, Castilho, Schietti,
Oliveira & Costa 2018). With LMA, isoprenoid emissions were then
calculated to μgC g-1 h-1.