Experimental setup
Figure 1 provides a schematic of the experimental setup. The platinum catalyst, CO2 detector and PTR-MS sampled approximately 200 mL/min of the diluted reactor off-gas stream. The flow path was alternated every 3 h using an automated three-way valve to flow either directly to the CO2 detector to measure respiratory CO2 only or through the VOC oxidizing platinum catalyst and then to the CO2 detector, measuring CO2 from both VOCs and fungal respiration. The PTR-MS sampled both the gas steams that entered and bypassed the platinum catalyst once per day. Gas flow that passed through the catalyst was measured to verify complete removal of VOCs from the gas steam and as a background with which to compare the bypass flow stream PTR-MS measurements. Three biological replicate experiments were performed to assess the reproducibility of the system.
Solid state fungal reactor system
Solid state reactors constructed from a 2-L, air tight, borosilicate glass container with three stainless steel mesh shelves were inoculated with 250 mg of biomass as determined by a correlation with protein concentration using a modified Bradford method (Bradford 1976). As a substrate, 50 g of beet pulp were autoclaved for 20 min with 250 mL of water and allowed to cool. The reactor, hydration flask and all tubing were autoclaved at 121 °C for 20 min before use. Fungal biomass was added to the beet pulp, mixed by inversion and evenly distributed to the three shelves of the reactor. Biological triplicate experiments were run for approximately 5 days each until total VOC production approached zero.
The solid state reactor (see Fig. 1) was continuously sparged with 100 mL/min of hydrated medical grade compressed air. The air was delivered via a stainless steel tube (0.5 cm I.D.) running to the bottom center of the reactor. Inlet air pressure forced reactor gas out of the top of the reactor. Reactor off-gas was then diluted with 900 mL/min of dry medical grade air to keep water from condensing in the system and to keep concentrations within the linear range of the CO2 detector and PTR-MS. A portion of the diluted flow stream, ~200 mL/min, was then pulled through the CO2 detector and PTR-MS with the PTR-MS diaphragm pump.
Microorganism
Nodulisporium isolate TI-13 was discovered as an endophyte of Cassia fistula in the highlands of Thailand using established collection and isolation methods as described previously (Ezra et al. 2004a). The TI-13 ITS1-5.8 S-ITS2 ribosomal gene sequence is available in GenBank as KJ558391, and the filamentous fungus is stored as sample NRRL 50502 in the Agriculture Research Service Culture Collection at the US Department of Agriculture (Nigg et al. 2014). The fungus was characterized as having the perfect stage of Hypoxylon sp. and as Nodulisporium sp. based on ITS1-5.8 S-ITS2 ribosomal gene sequence information (Nigg et al. 2014).
Growth conditions
Inoculum cultures were generated by growing the fungus on potato dextrose agar until hyphae covered ≥60 % of the plate surface. A 20 % glycerol solution (10 mL) was added to each plate, scraped with a sterile glass rod and the suspended fungal biomass solution was collected. The solution was mixed thoroughly, added to Microbank microbead vials, and stored at −80 °C until use.
The composition of the inoculum medium was 60 g/L glucose and 0.5 g/L yeast extract. Two hundred and fifty millilitre of sterile filtered (0.22 µm) medium was added to 500 mL sterile baffled flasks. Three microbeads were added to each inoculum culture and grown for 9 days at 160 rpm and 30 °C. All inoculum flasks were covered with sterile Kimguard (Kimberly-Clark; Roswell, GA, USA) to allow gas exchange, but prevent microbial contamination.
CO2 detector calibration and verification
An LI-840 CO2/H2O non-dispersive infrared gas analyzer (Li-cor Biosciences; Lincoln, NE, USA), quantified CO2 in the reactor off-gas continuously without impacting it. This optical technique is non-destructive and allowed the PTR-MS to be placed in series after the CO2 detector. The CO2 detector was calibrated using a certified CO2 free gas standard to set the zero point (1 % methane, balance nitrogen Scotty Analyzed Gases, Plumsteadville, PA, USA) and another gas standard for the span (a gas standard of 1010 ppm CO2, balance nitrogen, Scotty Analyzed Gases, Plumsteadville, PA, USA). Linearity of the CO2 detector was assessed by diluting the gas standard (1010 ppm CO2, balance nitrogen) with medical grade compressed air using mass flow controllers to produce final CO2 concentrations of 50.5, 101, 505 and 1010 ppm. The CO2 detector response agreed with the prepared gas standard to within 3.3 % for the four values measured, with values creating a line with a slope of 1.0006 and an R2 value of 0.9998 when plotting measured concentration versus expected concentration (data not shown).
Platinum catalyst VOC conversion verification
Complete oxidation of VOCs to CO2 by a heated platinum catalyst (Shimadzu, Kyoto, Japan, High Sensitivity Catalyst 630-00996 maintained at 400 °C) was confirmed by diluting a gas standard (0.1 % propane, balance air, Cal Gas Direct, Huntington Beach, CA, USA) and medical grade compressed air with mass flow controllers to produce final propane concentrations in the range of 10–100 ppm, which spanned the total VOC concentration presented to the catalyst in the diluted sample flow. Based on the tenfold dilution of the bioreactor effluent and accounting for the three carbons in propane, this experiment produced a test matrix that was equivalent to total bioreactor VOC concentration of 300–3000 ppm C. Overall, the platinum catalyst and CO2 detector measurement agreed with the gas standard for conversion of propane to CO2 by the platinum catalyst to within 5 % for the four measured values, and created a line with a slope of 0.95 and an R2 value of 0.9991 when measured concentration was plotted against expected concentration. The catalyst exhibited near complete conversion efficiency for propane concentrations up to 50 ppm of propane, which decreased to 95 % at the highest, 100 ppm test point. This experiment established the effective maximum working concentration of the employed catalyst system at 1500 ppm C after adjusting for dilution. Catalyst systems employing higher temperatures and greater amounts of catalyst could be used to achieve higher maximum working concentrations. Similar catalyst systems have been used previously to confirm concentrations of calibration gases with high accuracy (Baasandorj et al. 2015; Veres et al. 2010).
Bioreactor measurements
The reactor off-gas flow path was alternated with a valve on a 3 h timer to run either directly to the CO2 detector or through the platinum catalyst to oxidize VOCs to CO2 before the CO2 detector. By alternating the air stream either around or through the platinum catalyst every 3 h, a near-continuous record of respiratory CO2 and total gas phase carbon (CO2 and VOC) production was created for each experiment. The CO2 detector measurement was recorded once per second during the experiments. Background CO2 and VOCs measurements for each tank of medical grade compressed air were subtracted from these values.
Proton transfer reaction-mass spectrometry (PTR-MS)
PTR-MS was used to provide compositional information about the VOCs in the reactor off-gas. PTR-MS uses H3O+ ions to protonate molecules (e.g. VOCs) with proton affinities greater than water (Lindinger et al. 1998). The singly charged ions are then typically detected as protonated molecules (ions with a mass-to-charge ratio, m/z, equal to the molecular weight plus 1 for the proton) by a quadrupole mass spectrometer (Lindinger et al. 1998). Identity is assigned based on the mass of the ion and prior knowledge of the likely products of a specific reactor system (Luchner et al. 2012). Constituents of air like O2, N2 and CO2 have proton affinities lower than water so are not protonated and do not interfere with measurements. Alkanes also have proton affinities lower than water, so PTR-MS does not efficiently detect alkanes (Lindinger et al. 1998).
Initial experiments with this solid state system showed the major VOC products were ethanol, methanol, acetaldehyde and monoterpenes, so care was taken to calibrate the PTR-MS for these compounds. Quantification was performed using sensitivity factors derived from calibration experiments by dynamically diluting a multicomponent standard containing methanol, acetaldehyde and terpenes (Apel-Riemer Environmental Inc., Broomfield, CO, USA). Ethanol calibration was performed separately using a permeation tube at a variety of humidities as described below. Sensitivity factors of 4.1, 17.4, 18.2 and 2.2 ncps (normalized counts per second)/ppbv for ethanol (m47, m65, m93), methanol (m33, m51), acetaldehyde (m45) and terpenes and terpenoids (m137) were used, respectively. The off-gas of the abiotic control and fungal cultures were both analyzed by the PTR-MS. The total VOCs measured in the abiotic control were subtracted from the fungal VOC production measurements.
Ethanol PTR-MS calibration
Ethanol calibrations were performed using a calibrated permeation tube (KIN-TEK; La Marque, TX, USA) at a variety of humidity values approaching dry air as the experiments were performed with 90 % dry air. Compressed air (19.5 mL/min) was sparged through a temperature-controlled oven (50 °C) holding a calibrated ethanol permeation tube (emission rate 209 ng/min at 50 °C). Mass flow controllers were used to control medical grade compressed air flowing through the permeation tube and a second dilution stream, introduced after the permeation tube, to produce concentrations of 101, 199, 484 and 924 ppb of ethanol based on the calibrated permeation tube emission rate. The concentration of ethanol produced with the permeation tube was checked using the platinum catalyst system as described previously (Baasandorj et al. 2015) resulting in a line with a slope of 1.1 and an R2 value of 0.997 (data not shown). The temperature of the permeation tube was then increased to 80 °C to produce ethanol concentrations comparable to those observed in the fungal experiment (1.2–6.2 ppm). The humidity of the air was varied by utilizing a hydration flask before the dilution. Humidity was monitored via the ratio of the H3O+(H2O) to H3O+ ion intensities with calibration experiments performed under conditions that closely matched the humidity in the fungal experiments. At low humidity, the sensitivity factor had a relatively constant value of approximately 4.0. Calibrations were repeated after the experiments and yielded a sensitivity factor of approximately 3.9.