Continuous measurements of volatile gases as

detection of algae crop health

Jon S. Sauera,1, Ryan Simkovskyb,1, Alexia N. Moorea, Luis Camardaa, Summer L. Shermana,
Kimberly A. Prathera,c, and Robert S. Pomeroya,2
a
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093; bDepartment of Biological Sciences, University of
California San Diego, La Jolla, CA 92093; and cScripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093

Edited by Alexis T. Bell, University of California, Berkeley, CA, and approved August 25, 2021 (received for review April 20, 2021)

Algae cultivation in open raceway ponds is considered the most Recent estimates of commercial monocultures in ORPs have
economical method for photosynthetically producing biomass for found grazer-initiated crop failures reduce 10 to 30% of produced
biofuels, chemical feedstocks, and other high-value products. One biomass, resulting in losses in the hundreds of millions of dollars
of the primary challenges for open ponds is diminished biomass (9). Part of the challenge posed by grazer contamination involves
yields due to attack by grazers, competitors, and infectious organ- their ability to rapidly eradicate a microalgae culture, sometimes
isms. Higher-frequency observations are needed for detection of within 48 h of contamination (10). A component of proper inte-
grazer infections, which can rapidly reduce biomass levels. In this grated pest management is the utilization of technologies that can
study, real-time measurements were performed using chemical rapidly detect grazers at the earliest time at the lowest pest con-
ionization mass spectrometry (CIMS) to monitor the impact of centrations possible (11). Once the grower is informed of an in-
grazer infections on cyanobacterial cultures. Numerous volatile fection, they can choose to treat the culture to kill or slow the
gases were produced during healthy growth periods from fresh- growth of the contaminant, if complementary grazer identity
water Synechococcus elongatus Pasteur Culture Collection (PCC) information is available, or immediately harvest to salvage the
7942, with 6-methyl-5-hepten-2-one serving as a unique metabolic crop (3).

CHEMISTRY
indicator of exponential growth. Following the introduction of a The detection of grazers has been approached using many
Tetrahymena ciliate grazer, the concentrations of multiple volatile analytical methodologies (3, 12, 13). Microscopy and automated
species were observed to change after a latent period as short as optical techniques, such as flow cytometry, have made progress
18 h. Nitrogenous gases, including ammonia and pyrroline, were in detecting grazers at low concentrations (<10 units/milliliter)
found to be reliable indicators of grazing. Detection of grazing by (14, 15). These techniques, while simple and cost-effective, are
CIMS showed indicators of infections much sooner than traditional off-line, can be slow to operate, and require specific protocols or
methods, microscopy, and continuous fluorescence, which did not knowledge of the grazer’s appearance. Alternatively, technolo-

AGRICULTURAL
detect changes until 37 to 76 h after CIMS detection. CIMS analysis
gies from molecular biology, such as qPCR, have shown promise
of gases produced by PCC 7942 further shows a complex temporal

SCIENCES
in definitively identifying grazers, with theoretical detection limits
array of biomass-dependent volatile gas production, which dem-
of a single molecule; however, challenges associated with cost and
onstrates the potential for using volatile gas analysis as a diagnos-
producing a library of primers specific to harmful grazers are
tic for grazer infections. Overall, these results show promise for
significant limitations (12, 16). Notably, both methods are highly
the use of continuous volatile metabolite monitoring for the de-
vulnerable to sampling biases, in which the preference of some
tection of grazing in algal monocultures, potentially reducing cur-
grazers to localize on surfaces or on biofilms precludes identifi-
rent grazing-induced biomass losses, which could save hundreds
of millions of dollars.
cation, as most samples are taken from the liquid bulk.

chemical ionization | crop protection | volatile organic compounds | Significance

algal grazers
Wide adoption of algae cultivation to produce environmentally
sustainable biofuels and fine chemicals is currently hampered
M icroalgae are prokaryotic or eukaryotic photosynthetic
organisms that can grow rapidly in a variety of conditions
at large scales. Their efficiency in producing large amounts of
by large losses (10 to 30%) incurred by grazer infections. We
show the usage of real-time chemical ionization mass spec-
trometry to rapidly identify gaseous indicators of grazer
biomass in small spaces with few added resources, as compared
infections in cyanobacteria cultures. Grazing was detected sig-
to terrestrial crops such as corn or soybeans, makes microalgae a
nificantly faster (up to 3 d) using real-time mass spectrometry
promising, sustainable platform for bioproduction of fuels or
than the current methods of microscopy and qPCR. By employing
industrial products. In addition to the most sought-after product,
this technology, cultivators may be empowered to treat grazer
biodiesel, microalgae can also be utilized for production of other
infestations sooner, thereby protecting the crop and enhancing
valuable fine chemicals such as dyes, cosmetics, pharmaceuticals,
profitability.
and food additives (1–3). Alternatively, microalgae can also be
used in tandem with other industrial processes such as removal Author contributions: J.S.S., R.S., K.A.P., and R.S.P. designed research; J.S.S., R.S., A.N.M.,
of dissolved nutrients from wastewater and CO2 removal from L.C., and S.L.S. performed research; J.S.S., R.S., K.A.P., and R.S.P. analyzed data; and J.S.S.
flue gases or the atmosphere (4, 5). Algal research to date has wrote the paper.
focused extensively on improvements in crop productivity, in- The authors declare no competing interest.
cluding strain selection, nutrient control, and management of This article is a PNAS Direct Submission.
physiochemical parameters (pH, temperature, etc.). Toward this This open access article is distributed under Creative Commons Attribution-NonCommercial-
goal, many cultivators use high-volume open raceway ponds (ORPs) NoDerivatives License 4.0 (CC BY-NC-ND).
as opposed to more expensive laboratory-style photobioreactors 1
J.S.S. and R.S. contributed equally to this work.
(PBRs) (6). While the cost of algal biomass from ORPs is ap- 2
To whom correspondence may be addressed. Email: rpomeroy@[Link].
proximately an order of magnitude lower than PBRs, a key dis-
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This article contains supporting information online at [Link]

advantage is the elevated risk from contamination by unwanted doi:10.1073/pnas.2106882118/-/DCSupplemental.
grazers, such as ciliates, rotifers, viruses, bacteria, and fungi (7, 8). Published October 1, 2021.

PNAS 2021 Vol. 118 No. 40 e2106882118 [Link] | 1 of 9

 Recently, attention has been directed to the suite of volatile in soft ionization of the analytes, which then are detected by
gases emitted from microalgae as they progress through their various types of mass spectrometry. Notably, this method can
bloom life cycle (17–19). For algae, volatile gas emissions are bypass the need for a gas chromatography column, as the selection
mediated by their environmental conditions or biological state, of a particular reagent ion (e.g., H3O+, C6H6+, or I−) can be used
such as exponential growth, nutrient availability, photooxidative to select for specific classes of molecules while precluding inter-
stress, and senescence (17). Furthermore, the presence of bac- ferences from the major constituents of air (N2, O2, CO2, Ar, CH4,
teria has been shown to transform and modulate the production etc) that are normally ionized by electron impact in GC/MS (24).
of volatile gases and metabolites by microalgae (20, 21). Col- The removal of the separation step enables the measurement of
lectively, the microalgae “volatilome” represents a reflection of
ambient gases in a highly continuous (1 Hz or faster) manner,
the organism’s state of health (SoH) and, due to the significant
while the soft nature of chemical ionization limits ion fragmen-
effect of grazing on microalgae health, a reflection of the infection
state of the bloom. Development of techniques and instrumenta- tation, which can overcomplicate the mass spectra of unseparated
tion to measure the microalgae volatilome, therefore, represents gases. Studies of volatile gas emissions from natural marine and
an opportunity to identify grazer infections in a highly sensitive freshwater systems using the most common form of chemical
and descriptive manner (17). Furthermore, issues of sampling ionization, proton transfer reaction mass spectrometry (PTR-MS),
biases are less problematic, as volatile gases partition through have indicated the ability of CIMS to identify distinct states of
solutions far better than physical grazers that are fixed to con- biogenic activity from phytoplankton and bacteria (25, 26). These
tainer surfaces. Currently, the predominant technique for analysis findings suggest that CIMS may possess the ability to monitor
of the algae volatilome has been gas chromatography mass spec- highly concentrated algal monocultures and benefit from the far
trometry (GC/MS). While extremely sensitive and effective for higher throughput than modern GC/MS technology. Indeed, the
identification of volatile gases, common GC/MS systems are not usage of CIMS and PTR-MS has been applied to the continuous
well suited for continuous, in situ measurements without signifi- monitoring of fermentive bioprocesses and in detection of crop
cant modifications (22). In addition, the dominant sampling herbivory by unwanted parasites among dozens of other use cases
technique for microalgae volatile gases, solid phase micro- (27–30).
extraction (SPME), often requires long equilibration times of up In this study, we use CIMS to monitor freshwater cyano-
to 24 h to obtain sufficient signals (17). Unfortunately, such long bacterial monocultures of Synechococcus elongatus PCC 7942
sampling times not only delay detection but can average out before and after the addition of a field-isolated ciliate grazer,
transient, and potentially important, changes in volatile gas con- Tetrahymena. Over a 28-d experiment, three 20-L carboys were
centrations. Given the fast timescales (1 to 2 d) within which monitored continuously by CIMS with commensurate biological
grazer infections occur, there is a need for a headspace monitoring
measurements as they proceeded through healthy growth and
technique with high time resolution and the ability to expediently
were sequentially infected with Tetrahymena. Switching between
switch between and measure multiple vessels.
Chemical ionization mass spectrometry (CIMS) is a chemically sampling vessels on a 15-min interval allowed the probing of
selective method frequently utilized in the atmospheric, food, algal volatile gas production at time resolutions higher than pre-
medical, defense, and drug enforcement disciplines for the online viously used, enabling direct comparisons of the ability of gas
detection of various gas phase species that are spatiotemporally analysis to detect grazer infections compared to traditional tech-
heterogenous in concentration (23). In CIMS, an ionized reagent niques. Analysis of the CIMS data revealed unique insights into
gas is mixed with a continuously sampled stream of gas from the the timescales of gas production that highlight the potential for
sample headspace. Favorable chemical energetics between the high temporal resolution gas analysis of SoH and grazer infections
reagent ions and analyte molecules in the sample headspace result in microalgae cultures.

A CI-TOFMS B Sampling Setup C Sampling Schedule

Zero Air Input
3.0 SLPM
(Solenoid
Liquid
Sampling Valve State)
Analyte Introduction
1.8 LPM To CIMS
Turbo 1.8 SLPM Vent Transition
Solenoid Carboy 1
Valve Carboy 1
Turbo Array Transition

Vent Carboy 2
Reagent Ion
Production Transfer Transition
Po-210

1.8 SLPM Quadrupole Carboy 2

Carboy 3

Vent Transition
Ion-Molecule Time-of-Flight
Region Turbo Mass Spectrometer Zero Air
Ion Funnel Zero Air
Carboy 3 1:00 PM 1:30 PM 2:00 PM 2:30 PM
(Time)
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Fig. 1. Experimental arrangement of carboy infection experiments. (A) Diagram of the CIMS instrument, (B) carboy sampling setup with solenoid valve array,
(C) example sampling schedule of the solenoid valve array for switching between carboys for CIMS headspace sampling.

2 of 9 | PNAS Sauer et al.

[Link] Continuous measurements of volatile gases as detection of algae crop health
 Results is defined operationally as a >10% increase or decrease in nor-
Experimental Setup. To demonstrate the capabilities of CIMS malized intensity over the timescale of the experiment. Time series
analysis on SoH and grazer infections of cyanobacteria, we si- that showed exponential decay from the outset of the experiment
multaneously monitored three 20-L carboy cultures of S. elongatus or spikes caused by pressure instabilities were excluded from this
PCC 7942, hereafter referred to as PCC 7942, through axenic analysis, indicating that the 49 ions that changed intensity over the
exponential growth and subsequent planned infections with a course of the culture’s lifespan were most likely due to active
field-isolated Tetrahymena that rapidly grazes on PCC 7942. For biogenic processes and not off-gassing from the media or experi-
comparison, liquid samples were collected from each carboy at mental arrangement. These results together demonstrate the
least once daily for microscopy, absorbance spectroscopy, and ability of water cluster CIMS to detect a wide range of unique
fluorescence spectroscopy. Upon addition of the predator, the volatile gases that can be related to the health of cyanobacterial
infected carboy was also monitored using a continuous fluores- and other microalgae cultures.
cence spectroscopy system. Cultures were continuously bubbled Of the ions detected by the CIMS, direct assignments of sev-
with sterile zero air, which pushed headspace gases above the eral more intense species were made utilizing complementary
culture to a custom-built solenoid valve array that was pro- MS methods such as solid phase microextraction gas chroma-
grammed to switch every 15 min between each of the three car- tography mass spectrometry (SPME-GC/MS) and modified at-
mospheric pressure chemical ionization high-resolution mass
boys and a direct zero-air input (Fig. 1B). The headspace or zero-
spectrometry (APCI-Orbitrap) incorporating recent innovations
air sample source enabled by the solenoid valve array was pulled
for direct analysis of gas phase species (31) (Protocol S1). These
directly into the ion-molecule region (IMR) of a chemical ioni-
identifications are listed in SI Appendix, Table S1. Overall, mo-
zation time of flight mass spectrometer, in which the continuous
lecular formulae or putative identifications were made for a
flow of air from the carboys or zero air was ionized by (H2O)nH+,
small fraction, <15%, of the total number of ions observed by
which proceeds by proton transfer, and directly introduced into
CIMS during the experiment duration. Many of these species
the CIMS without any column chromatography (Fig. 1A). Mass were observed to be aliphatic ketones and aldehydes similar to
spectra were accumulated as a sum of 60,000 spectra collected those observed in investigations of algae by others (17, 18,
over 1 s (Fig. 1C). 32–34); however, nitrogen-containing gases, including ammonia

CHEMISTRY
and C4H7N, were an important constituent of the headspace,
Mass Spectral Characteristics of PCC 7942. The mass spectra col-
which has been generally overlooked by previous analyses of
lected from the headspace over the PCC 7942 cultures displayed
commercial algal volatile gases despite evidence of the produc-
numerous peaks indicative of volatile gases present in the sam-
tion of methylated amines from phytoplankton in natural sea-
ples. Fig. 2A shows a comparison of average mass spectra water (33, 35, 36).
obtained from both the headspace of Carboy 1 and clean zero air
during a period of axenic algal growth on the fourth day after Evaluation of Tubing and CIMS Inlet Equilibration Times. While
PCC 7942 inoculation. The mass spectrum for both sample types

AGRICULTURAL
CIMS used in this work possesses a relatively low-mass resolu-
is dominated by the water cluster reagent ions at mass to charge tion (∼1,200 full width at half maximum) and does not induce

SCIENCES
ratios (m/z) 19, 37, 55, 73, and 91, which are typical clusters significant ion fragmentation that can be used for molecular
formed in chemical ionization. In all carboys, the remainder of identification, valuable analyte information can be obtained
the mass spectrum is composed of numerous ions generated by through monitoring the rise or decay time of an ion as the CIMS
proton transfer (M+1), which fall between m/z 40 and 200. At switches between sampling vessels. This rise or decay time (SI
this time point, the fraction of ion intensity occupied by non- Appendix, Fig. S2) can be modeled using a double exponential fit
water cluster ions was 7% for zero air and 11% for the headspace and the resultant time for the ion to reach 1/e2(86.5%) of its
of Carboy 1, in which total ion count (TIC) was ∼2.5 × 106 counts equilibrated intensity can be calculated (37, 38). The 1/e2 decay
per second. The ratio of ion intensity for nonwater cluster ions times are reflected as the sum of multiple physical processes as
between Carboy 1 and zero air was 1.51. Of this ion intensity, gases travel down the length of the tubing from the sample
Carboy 1 possessed 75 unique m/z’s that were 25% greater in source to the instrument IMR inlet. Molecules with a high sat-
average intensity than zero air, suggesting their origin from ei- uration concentration (volatility) remain in the gas phase, which
ther cyanobacterial production, the BG-11 growth medium, or results in short equilibration times between samples. Conversely,
the carboy assembly. Of these 75 unique m/z’s, 49 ions were low volatility molecules tend to stick to tubing and result in
found to exhibit distinct time-variant behavior over the course of longer equilibration times (37). In addition to saturation con-
Carboy 1’s experimental lifetime, for which time-variant behavior centration, smaller molecules with high dipole moments, such as

A B
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Fig. 2. (A) Average CIMS mass spectrum for zero air and Carboy 1 headspace. (B) The 1/e2 histogram of ion equilibration times obtained from switching
between sampling zero air and Carboy 1.

Sauer et al. PNAS | 3 of 9

Continuous measurements of volatile gases as detection of algae crop health [Link]
 NH3 or HCl, may partition into water microlayers on tubing Optical density (OD) at a wavelength of 750 nm was also moni-
walls, thus increasing their equilibration times significantly (38). tored daily (SI Appendix, Fig. S5). While OD and fluorescence
Together, an analysis of gas rise or decay times, in combination show good agreement in these experiments, fluorescence was
with their corresponding m/z’s, provides valuable information as chosen as the primary measurement, as it better reflects the
to the general chemical characteristics of the observed ions. This presence of live cyanobacteria as opposed to OD, which can
analysis also enables calculation of the minimum time the CIMS capture senescent cells and detritus. Given the agreement be-
must monitor a sampling vessel before it can switch to the tween OD and fluorescence, we find that any self-shading during
next vessel. growth likely did not drastically alter chlorophyll production in
We applied this analysis to a set of 42 unique m/z’s using rise these experiments, which could distort assessment of culture
or decay times as the CIMS switched between sample carboys productivity.
and zero air. The results of this analysis performed on data ac- Numerous gases showed significant changes in intensity in
quired on the fourth day after inoculation of Carboy 1 with PCC response to grazer infection; however, only the time series of the
7942 are shown in Fig. 2B, with the single omission of the 1/e2 fastest-responding and most intense species will be primarily dis-
value of ammonia (NH3). Notably, the majority of 1/e2 decay cussed here. The most significant change in ion intensities were
times fall under 12 s, comparable with equilibration times neces- NH3 at m/z 18 (Fig. 3C) and its associated water cluster adducts at
sary for high-speed environmental measurements (39). In contrast, m/z’s 36 and 54. Initial NH3 ion intensities were observed to be
ammonia and other even mass gases at m/z’s 70 and 76, whose elevated at the outset of the experiment and are indicative of
even m/z values suggest an odd number of nitrogen atoms if volatized NH3 off-gassing from the BG-11 medium, which con-
ionized by proton transfer [the predominant ionization scheme in tains 6 mg/L ferric ammonium citrate, as observed by monitoring
(H2O)nH+], displayed longer equilibration times (14 and 19 s), sterile BG-11 (SI Appendix, Fig. S6). After the first 2 d of algal
with ammonia’s 1/e2 time being ∼335 s. The assignment of ni- growth, this NH3 ion signal decreased by nearly two orders of
trogen in the molecular formulas of these species is consistent with magnitude and remained below 2 × 104 counts · s−1 until after
their longer equilibration times, as their pronounced basicity Tetrahymena addition. This decrease was likely caused by uptake
promotes dissolution into water coated on tubing walls. Based on of ammonium by PCC 7942 as well as gradual ventilation of NH3
the longest equilibration time of ammonia of 5.6 min, this key by the bubbled zero air. After grazer addition to each carboy, an
operational parameter means that the CIMS, with an appropriate initial intensity spike of NH3 was observed for each carboy on days
sample switching device, is capable of analyzing 10 different ves- 11, 15, and 25. Notably, some NH3 signal carryover from Carboy 1
sels in under an hour. Furthermore, these equilibration times are to Carboys 2 and 3 occurred during Carboy 1’s ammonia spike as
not fixed and can be further improved through higher flow rates, well as carryover from Carboy 2 to Carboy 3 during Carboy 2’s
tubing diameters, heating, and chemical coatings of the tubing or ammonia spike. These carryover events were caused by excess
the CIMS inlet. Finally, this analysis demonstrates that the 15-min ammonia in the sampling lines and CIMS inlet that did not suf-
sampling schedule used in this experiment was more than suffi- ficiently evaporate before the CIMS switched to the next sampling
cient to accurately determine ion intensities for a diverse suite of vessel, consistent with the slow equilibration rate observed for
gases present in the culture headspace samples. ammonia. After the first intensity spike, the NH3 signal decreased
before spiking a second time for each infected carboy in concert
Time Series Analysis. Volatile gases from Carboys 1, 2, and 3 were with the culture crash and decrease in fluorescence signals. No-
monitored over a total period of 28 d. All carboys were inocu- tably, the magnitude of the first ammonia spike increased in in-
lated with PCC 7942 at the same time, and each carboy was tensity as the experiment duration continued in concert with the
sequentially infected on days 8, 13, and 23, respectively, with increase in algal biomass in later carboys (Fig. 3 A and C). We
Tetrahymena at a concentration of 0.1 cells/milliliter (Fig. 3, solid tentatively suggest that the magnitude of the first ammonia spike
vertical lines). This infection density was chosen because it is well appears to be biomass dependent and may be an algal stress-
below the typical limit of detection (LOD) of microscopy at 5 to related response. Although ammonia release has been described
20 cells/milliliter and the theoretical limit of blank for the for some strains of Anabaena (43), though not as a stress or pre-
technique used of 20 cells/milliliter (40–42). Twice daily after dation response, algal growers have used free ammonia addition
infection, liquid samples were taken manually from the infected as a method to control pests (44). We hypothesize that this initial
carboy and analyzed by microscopy for the presence of Tetra- ammonia spike represents a possible defense mechanism. In
hymena. Grazers were observed first from each carboy on days contrast to the first spike, the magnitude of the second ammonia
12, 16, and 27 (Fig. 3, dashed vertical lines), with further infor- spike is relatively consistent for all three carboys, suggesting that
mation on the specific duration until detection summarized in its production is a function of biomass degradation caused by the
Table 1. While qPCR was not used to detect grazers in these predation of the grazer on the cyanobacteria.
experiments, a separate analysis was performed to assess the While ammonia signals increased after predator addition, the
LOD of qPCR toward the detection of Tetrahymena and is detailed intensity of m/z 137, an unidentified monoterpene, showed a
in SI Appendix. The observed qPCR LOD of 597.6 cells · mL−1 clear decrease in Carboys 1 and 2 multiple days before detection
(SI Appendix, Fig. S4) for Tetrahymena greatly exceeded detec- of Tetrahymena by microscopy (Fig. 3D). For Carboy 3, the in-
tion thresholds by microscopy; thus, we chose microscopy for the tense NH3 signal from the first spike disrupted the ion chemis-
experiment time course as the benchmark grazer detection try of the CIMS, preventing reliable observations of m/z 137
method. To observe changes in algal biomass associated with (SI Appendix, Fig. S7). For this reason, in cases in which m/z 18
grazing at faster timescales, each grazer inoculated carboy was intensity exceeded 2,500 counts per second, m/z 137 intensity was
monitored by continuous fluorescence (Fig. 3B) until the complete discarded. This protocol was also followed for most other ions
loss of biomass, as indicated by the daily fluorescence measure- except nitrogen-containing (even-mass) ions, which showed sta-
ments, visible color change and culture collapse (Fig. 3A). Con- bility in intensity during periods of high NH3. This stability is
tinuous fluorescence, while providing a higher temporal resolution likely due to the higher gas phase proton affinity of these mol-
in showing biomass loss during the grazer-induced crash, did not ecules, which are able to receive protons from NH4+ (proton
confer any significant advantage to grazer detection over man- affinity [PA] = 853.6 kJ/mol) if NH3 had sufficiently titrated
ual daily fluorescence sampling. In addition, during the infection (H2O)nH+ as the active reagent ion; however, monoterpenes
of carboy 3, the continuous fluorescence signal began to gradu- (PAalpha pinene = 878 kJ/mol) would not. In Fig. 3E, the intensity
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ally diminish before changes in the manual daily sampling, likely of m/z 70, which was determined to have the molecular formula
due to observed biofouling of the sensor at high algal biomass. C4H7N and is likely 1- or 3-pyrroline (PA = 925.8 or 931.0 kJ/mol)

4 of 9 | PNAS Sauer et al.

[Link] Continuous measurements of volatile gases as detection of algae crop health
 A

CHEMISTRY
Fig. 3. Time-series data of the carboy experiment colored by carboy (Carboy 1: green, Carboy 2: red, Carboy 3: blue). (A) Daily fluorescence: 590 nm exci-
tation/670 nm emission. Arbitrary units, AU. (B) Continuous flow-through cuvette fluorescence: 420 nm excitation/670 nm emission. (C) CIMS m/z 18 (NH3)
intensity normalized counts per second, ncps. (D) CIMS m/z 137 (monoterpenes) intensity. (E) CIMS m/z 70 (C4H7N) intensity. Solid vertical lines denote the time
of Tetrahymena addition for each carboy; dashed vertical lines denote the first time of Tetrahymena detection via microscopy.

AGRICULTURAL
SCIENCES
showed strong positive changes in intensity for infected Carboys 2 25 to 76 h before microscopy detection, depending on the gas used
and 3 but only decreased in intensity in Carboy 1 (Fig. 3 E, Inset). for detection and the specific experiment (Table 1). C4H7N, in all
Similar to the double spike phenomenon observed for NH3, it cases, was the first gas to appear or diminish in response to grazing
appears that the amount of algal biomass present at infection time and was effective for use in all three carboys, while monoterpenes
is an important effector of m/z 70 production in response to are more effective at lower biomass, at which complications from
grazing. These results are further supported by measurements of high ammonia were less problematic. Notably, the duration at
m/z 32, methylamine (PA = 899.0 kJ/mol), which only appeared in which grazer detection was achieved by volatile gas analysis de-
response to grazing in Carboy 3, the vessel that reached the creased over the experimental duration, indicating that the in-
highest level of biomass before the grazer-induced crash (SI Ap- creased biomass in Carboys 2 and 3 seems to have shortened the
pendix, Fig. S8). Given the disruption to the (H2O)nH+ ion algal response to infection. For microscopy, detection of grazers
chemistry by NH3, in these experiments, the authors note that after inoculation was quite similar for Carboys 1 and 3; however, it
sample dilution is an option for future analyses to counteract ti-
occurred earlier for Carboy 2. High variance in detection time by
tration by NH3; however, this comes at a compromise of sensitivity
microscopy is reflective of challenges inherent in the technique at
to non-NH3 analytes in the sample stream, which are also diluted.
low cell densities, in which statistical fluctuation and heteroge-
Volatile Gases as Indicators of Grazing. Having observed volatile gas neous localization of the predator may be prominent factors. For
signatures that change consistently over the growth of all cultures example, a 1-mL sample screened for grazers at a detection limit
after the introduction of predators, we compared the timing of of 1 cell/milliliter may not contain a grazer, even if the bulk grazer
gas intensity changes as detected by CIMS to indicate the presence concentration is ∼1 cell/milliliter. While continuous fluorescence
of grazers relative to the timing of grazer detection by microscopy cannot detect grazers until after cyanobacteria biomass dimin-
and continuous fluorescence. By applying an empirically derived ishes, the remarkably similar times reflect a grazing consistency
detection threshold based on the ion signal changing by 10σ over a between the three carboy inoculations, which help validate
4-h window, we find that the CIMS detects the impact of grazers chemical comparisons between the three systems.

Table 1. Time after grazer addition before detection technique flags presence of Tetrahymena grazer
NH3 (hours after Monoterpenes (hours after C4H7N (hours after Microscopy (hours after Continuous fluorescence (hours
Carboy grazer addition) grazer addition) grazer addition) grazer addition) after grazer addition)

1 67 47 45 92 119
2 36 39 32 69 114
3 30 — 18 94 112
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For CIMS-measured species, grazer detection was based on a signal intensity change of 10σ over a duration of 4 h. Microscopy identification was at the first
date of visible grazers observed during liquid grab sampling. Continuous fluorescence detection was based on a 10σ over a duration of 4 h.

Sauer et al. PNAS | 5 of 9

Continuous measurements of volatile gases as detection of algae crop health [Link]
 Temporal Changes in Ion Intensity. To better understand if the Notably, m/z 127, 6-methyl-5-hepten-2-one (6-MHO), exhibits a
apparent correlation for changes in volatile gas intensity of some departure from continuous increase with algal growth, plateauing
chemical species and increased cyanobacterial biomass is a more ∼5.5 d into the experiment and remaining at approximately the
generalized phenomenon, we calculated the moving 1-h deriva- same signal intensity until grazing pressure on PCC 7942 by Tet-
tive of ion signals, which changed in response to grazing, and rahymena (added day 8) resulted in loss of productive algal biomass
evaluated these signal changes by separate carboy. From an able to produce 6-MHO. As continuous bubbling of the carboys
analysis of the maximum raw ion intensity derivative of gases in occurred during the full experiment duration, causing continuous
each carboy (Fig. 4A), it is apparent that denser algal cultures ventilation of dissolved gases, a plateau of 6-MHO suggests that
produce larger total gas change in response to grazing, suggest- algal production of 6-MHO reached a steady-state value that may
ing that the utilization of algal gases as a diagnostic will be more be biologically informative. Indeed, departure from growth of
effective for denser, mature ponds. In addition, the maximum 6-MHO signal appears to correspond with the changes in the
derivative of each ion was normalized to their respective raw productivity of the algal culture, in which exponential algal growth
intensities at the time of Tetrahymena addition, and then the similarly leveled off at approximately the same time in the ex-
time for the ion to double in intensity at this rate of change was periment time course (SI Appendix, Fig. S9B). To further compare
calculated (Fig. 4B). the production of volatile gases in Carboy 1 with healthy growth,
This calculation allows the investigation of how much, by the daily average ion intensity of the chemical species shown in SI
fraction, each volatile gas changed in time as a response to grazer Appendix, Fig. S9A were divided by the daily average algal fluo-
addition between the three carboys. The results in Fig. 4B show rescence. Shown in SI Appendix, Fig. S9 A, B, D–F, the ratio of
that the doubling time for gases significantly decreased from each ion to daily fluorescence decreased to a stable value until
Carboy 1 to Carboy 3, indicating that the quantity of initial algal algal biomass was degraded by grazing after day 8. A stabilization
biomass at time of infection accelerates the production or loss of of ion to fluorescence ratio indicates that the per-cell production
gases in response to grazing. Furthermore, this observation dem- rate of these gases likely remained constant. However, the ratio of
onstrates that as the doubling time decreases, the need for higher 6-MHO to fluorescence showed different behavior over the ex-
time resolution analytical methods, such as CIMS, become neces- perimental time course, similarly in concert with the departure
sary to capture these signal changes for effective grazer detection in from exponential growth to attenuated growth (SI Appendix, Fig.
a manner that current GC/MS-based gas analysis techniques S9B). While assessing the specific biological purpose or reason for
simply cannot. the release of each of these volatile gases is beyond the scope of
this work, ratios of ion intensity to algal fluorescence help distin-
Volatile Gases as Indicators of SoH. While the primary scope of this guish whether the per-cell production of volatile species varies
study focuses on changes to production of gases in grazer- with increasing biomass. We believe that better distinguishing the
infected algae, we also observed interesting changes in volatile roles of biomass-variant and -invariant volatile gases may open
gas intensities during healthy growth of PCC 7942. Shown in SI many new doors to better understanding algal biochemistry in the
Appendix, Fig. S9A, a collection of demonstrative gases in Car- future. While further investigation of the connections between
boy 1 are plotted across the time series of the experiment. Each PCC 7942 growth and 6-MHO production are warranted, these
of these gases generally increased in intensity with the growth of results suggest that some volatile gases may be used as metabolic
PCC 7942 in Carboy 1; however, the distinct shape of changes in indicators of the culture’s growth phase, better informing the
each ion’s intensity during growth demonstrates the biochemical cultivator of how to manage the algal crop to maintain or change
complexities that likely underlie the production of each compound. the growth phase to maximize biomass productivity or induce
generation of desired products.
Discussion
A B The utilization of chemical ionization mass spectrometry iden-
tified several promising molecular species as diagnostic tracers to
identify Tetrahymena grazing in PCC 7942. We found that
nitrogen-containing gases are highly reliable unique tracers in-
dicative of algal grazing. While the putative identification of all
ions was not possible, complementary analyses with GC/MS and
atmospheric pressure chemical ionization-high resolution mass
spectrometry (APCI-HRMS) were used to obtain further infor-
mation. However, ascertaining the identity of all volatile gases
measured by CIMS is not required for the goal of identifying
predation or infection in algal cultures. Provided the m/z’s
measured by the CIMS consistently respond to destructive con-
tamination, knowledge of their identity and biochemical purpose
only provides a better understanding of the biochemistry of the
system overall. This is analogous to medical measurements of
temperature or blood pressure, which are linked to pathogenesis
in often unknown and highly complicated systems that can be
used to indicate a change in the health of the patient.
Furthermore, this study evaluates and compares volatile gas
measurements with standard monitoring techniques, such as mi-
croscopy and spectroscopy, in terms of critical time-based metrics
Fig. 4. Derivative analysis of grazer-affected gas production from PCC 7942.
that include 1) time to detection following a below-LOD infection,
(A) Maximum derivative value of raw ion signal change. (B) Normalized
derivative doubling time for the same set of ions in A. The × markers des-
which reflects the sensitivity of the method to either the number or
ignate the average signal derivative and doubling time for each carboy. level of grazing activity of the contaminant, or 2) time of detection
prior to a culture crash, which is an important criteria for growers,
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Selected ions from Fig. 3 are indicated by colored boxes in each panel to
emphasize their time-dependent behavior in comparison to other ions as it determines how much time is available to prevent or recu-
observed in this study. perate potential biomass losses. The data provided in Fig. 3 and

6 of 9 | PNAS Sauer et al.

[Link] Continuous measurements of volatile gases as detection of algae crop health
 Table 1 demonstrate the improved performance of the real-time time to recover nearly all of the biomass lost, even using the most
measurements of the CIMS over microscopic and spectroscopic drastic intervention of harvesting the entire system.
monitoring methods, with volatile gas analysis of grazer-infected Future work on this method will require the study of multiple
PCC 7942 cultures providing detection signatures, at minimum, algae–grazer pairs to assess the specificity of volatile gas re-
24 h faster than microscopy and up to 3 d faster for the experi- sponses to grazing. Given that many microalgae are known to
mental conditions discussed herein. While the type of custom produce different volatile metabolites, it is likely that some chem-
CIMS used in this work with (H2O)nH+ chemistry was effective in icals may be unique to algae–grazer pairs (17). Other gases, likely
detecting grazer infections, other types of chemical ionization in- simpler molecules like NH3 that are derived from common pre-
struments, such at proton transfer reaction mass spectrometry cursors such as proteins, will probably be conserved among many
(PTR-MS) and selected ion flow tube mass spectrometry algae–grazer combinations. Development for future field applica-
(SIFT-MS), should be similarly useful. Both PTR-MS and SIFT- tions will need to address sampling of unwanted outdoor contam-
MS are commercially available, albeit expensive, systems but have inants that could confound analysis of volatiles from the crop.
enhanced capabilities for wider selectivity and reduced humidity Here, we envision the usage of sampling interfaces, which provide a
dependence. large surface area of pond water sampled by a stream of clean air,
We further identify fascinating features regarding the time- which will be measured by CIMS. This method will, at minimum,
scale and intensity with which volatile gases are produced in protect the system from drastic changes in gas intensity carried by
response to grazing that appear linked to algal biomass. First, we local airborne sources but is still vulnerable to gaseous compounds
observed transient early signal changes whose intensity increases that may partition into ponds over time. Diagnostic markers of
change disproportionately with increased biomass in each car- grazer infection will therefore need to be carefully selected based
boy. For example, the peaks of the first ammonia burst in Car- on the risks of external contamination by similar or isobaric com-
boys 1 and 2 differ by ∼25× in maximum intensity (Fig. 3C, Days pounds. While the analysis in this study focuses on a volatile gas
11.5 and 15.5), while the fluorescence or OD changes are both assessment for the health of microalgae, it is important to note that
1.1× . Based on the transient and disproportionate biomass- chemical ionization mass spectrometry can be used to monitor
dependent nature of this response, we have hypothesized that health-indicating gases in many other production systems, including
this feature is unlikely to be caused directly by degradation of beer, cheese, monoclonal antibodies, and laboratory-grown meats,
biomass or increased encounter rates but instead may represent all of which require stringent cleanliness.

CHEMISTRY
a defense response that is nonlinearly amplified by denser cell
populations. The possibility that algae respond to stressful stimuli Materials and Methods
in a population-dependent manner, as has been observed in the Culture Conditions. S. elongatus PCC 7942 was grown for maintaining Tet-
literature to abiotic stress (45), indicates that gas analysis as a rahymena or for inoculations for larger cultures in BG-11 medium (46) in
diagnostic method will likely require multiple data types to con- 250-mL or 2-L flasks with continuous shaking (125 rpm) at 30 °C under
textualize the overall SoH of the algal crop. Second, we observed continuous illumination of 200 μmol photons · m−2 · s−1 from fluorescent
cool white bulbs.

AGRICULTURAL
that overall signal changes occur earlier when infection occurs at a
higher initial biomass density (Fig. 4). Because this phenomenon is

SCIENCES
Tetrahymena Isolation and Culturing. A 30-L BG-11 culture of S. elongatus PCC
more generally observed with all volatile signals investigated, in- 7942 grown in a 100-L polybag at the University of California San Diego
cluding nontransient signals, its cause is more likely a direct con- Greenhouse Biology Field Station, as described by Schoepp, et al. (47), had
sequence of biomass degradation and is related to increased crashed due to an unknown contaminant. A sample of this crashed culture
encounter rates in higher-density cultures, which are subsequently was brought into the laboratory and analyzed by light microscopy, through
compounded by increased predator replication and grazing rates. which it was observed that the predominant organism was a free-swimming
Interestingly, though, microscopy and fluorescence detection ciliate. This culture sample was serially diluted or manually isolated under
times did not change in a similar manner. The ability of ponds to microscopic visualization into flask cultures of PCC 7942 or into wells of a
respond to stressful stimuli in a population-dependent manner flat-bottom multiwell cell-culture dish (Costar, Corning) containing dense S.
elongatus cultures as diluents to first isolate the grazer and subsequently to
informs us that gas analysis as a diagnostic method will likely re-
culture the grazer. Grazer cultures were incubated at 30 °C under low light
quire multiple data types to contextualize the overall SoH of the conditions (10 μmol photons · m−2 · s−1) or more routinely at room tem-
algal crop. We furthermore leveraged our high-temporal resolu- perature under ambient light conditions. In contrast to healthy PCC 7942
tion measurements to show that changes in gas composition due to cultures, grazed cultures were yellow, resulted in visible clumping of the few
grazing occur on a <24-h basis, with some intensities changing by a remaining S. elongatus cells, and were predominantly composed of ciliates
factor of 2 or more in less than an hour. The sampling frequency when viewed by light microscopy on a dissecting microscope. A 1-μL sample
of a volatile gas diagnostic measurement for algae likely needs to of a grazed culture was used as a template for Q5 (New England Biolabs)
occur more than once daily and ideally as frequently as once PCR amplification of the ITS2 region using the ITS1 (5′-AGGAGAAGTCGTAAC-
hourly or more, which is not easily done for GC/MS, the typical AAGGT-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers, producing an
method that is used in such studies. Given the constraints imposed ∼600-bp band. PCR was performed according to the Q5 standard protocol,
with an annealing temperature of 55 °C and an elongation time of 3 min and
by partitioning of volatile gases to sampling tubing, we find that
45 s. The PCR product was purified using the Zymo DNA Clean & Concentrator-
CIMS can monitor ∼10 vessels per hour, which will be improved 5 kit and sequenced by Sanger sequencing with the ITS1 primer. The resulting
through modifications of the sampling arrangement for field ap- sequence was analyzed by Basic Local Alignment Search Tool to determine the
plication, greatly offsetting the cost of this higher-complexity in- closest identified species as Tetrahymena tropicalis, Tetrahymena thermophila,
strumentation. Future applications of this approach to algal or Tetrahymena rostrata.
monitoring will significantly benefit from higher-complexity nu-
merical analyses that can draw connections between algal gas Carboy Cultures and Sampling Setup. Three 20-L transparent polycarbonate
production and various other indicators of health toward the goal carboys (Nalgene) fitted with a custom inlet/outlet polypropylene cap with
of giving cultivators the ability to “decide” a pond is infected in an stainless steel bulkhead compression fittings sealed with Viton O-rings for
automated manner. The grower may then initiate a predetermined gastight liquid and gas sampling designed to prevent the unwanted intro-
duction of organisms from the ambient environment were filled with 18 L
intervention in an automated fashion to minimize biomass losses
BG-11 and autoclaved (SI Appendix, Fig. S1). Once cooled, the carboys were
like those observed in these experiments and in the field. Given inoculated with 2 L S. elongatus PCC 7942 cultures previously grown in 2-L
reports of regular losses in the field of 30% or greater in biomass flasks to an OD at 750 nm of 0.08 to 0.1 so that the carboys were inoculated
production and the 1 to 3 d of prior notice to any significant to an OD at 750 nm of ∼0.01. Carboys were connected to sampling devices as
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biomass loss that is possible within the CIMS system, we believe shown in Fig. 1 and SI Appendix, Fig. S1. Cultures were grown continuously
that the CIMS monitoring system could effectively allow a grower under cool (4,100 K) fluorescent light, starting at 135 μE incident radiation at

Sauer et al. PNAS | 7 of 9

Continuous measurements of volatile gases as detection of algae crop health [Link]
 the front face of each carboy and increased to 190 μE after 24 h of growth, Torr) with chevron microchannel plate detector (Tofwerk AG) (Fig. 1A).
at which time self-shading lowered the risk of photobleaching. Tetrahymena Approximately 60,000 mass spectra were averaged per second on an
were added at a calculated concentration of 0.1 cells/milliliter separately to analog-to-digital converter and transferred to computer storage for later
Carboys 1, 2, and 3 after 8, 13, and 23 d following culture inoculation, analysis. For the analyses in this work, the mass spectral window was limited
respectively. between 10 and 400 m/z, as there was a dearth of ions observed above this
Clean zero air, obtained from a generator (Sabio 1001) and regulated by a mass range. Mass resolution was ∼1,200 full width at half maximum for the
mass flow controller (Alicat) to 3 standard liters per minute (SLPM) was di- spectra obtained in this experiment. MS data were baselined, calibrated, and
rected through a 47-mm 0.2-μm polytetrafluoroethylene filter into each analyzed in Tofware, a graphical user interface plugin for Igor Pro-7. Time
carboy, in which the air input line was terminated at a submerged circular series data of unit-mass m/z’s were averaged into ∼15-min bins to reduce
length of perforated 1/4”-outside diameter perfluoroalkyl (PFA) tubing so data density and were separated by the carboy sampled using a comple-
that all input air was released as bubbles directly into the culture. This mentary mask file generated by the solenoid valve array. Approximately
construction distributed bubbles throughout the carboy to adequately mix 5 min of MS data acquired at the beginning of each time bin was discarded
and supply air to enable culture growth. Liquid samples were collected from to remove signal that was equilibrating between samples.
the carboy using a manifold of three-way and one-way check valves con- Biological measurements. Tetrahymena cell counts were obtained by manual
nected by Tygon tubing to the compression fittings on the cap, all of which counting ciliates present in 1- to 10-μL droplets of culture on a microscope
were autoclaved attached to the carboy at the time of media preparation. slide under a dissecting microscope with 25× objective and 10× eyepiece.
The valve manifold allowed sanitation of liquid sample lines by 70% ethanol Alternatively, for low-density grazer cultures, 200 μL culture in a 96-well
with significantly reduced backflow risk of ethanol or microorganisms into plate could be rapidly scanned using the same dissecting microscope
the carboy. The output headspace gas from each carboy flowed through setup. For all cell counts, at least five individual sample counts were col-
3.175-mm inside diameter PFA tubing into a custom Labview controlled lected and averaged to calculate the cell density of the culture or
four-channel solenoid valve array, which switched between each carboy and inoculum.
clean zero air on a 15-min sampling cycle (Fig. 1C). Sample air from the so- Manually collected cyanobacterial culture samples were analyzed by OD at
lenoid array was drawn in by the CIMS at a fixed flow rate of 1.8 SLPM, with 750 nm in a clear, plastic cuvette or by absorbance and fluorescence spec-
excess sample gas overflowing to a downward-pointing exhaust port for troscopy of a 200-μL sample in a 96-well plate using a TECAN Infinite M200
condensed water droplets to drain (Fig. 1B). plate reader to collect OD750 values and autofluorescence values at an ex-
Ultra-high-purity N2 gas, produced by boil-off from a liquid nitrogen citation of 590 nm and emission of 670 nm.
dewar, was bubbled through a 250-mL Pyrex bottle filled with liquid chro- Culture samples were checked for bacterial and fungal contamination by
matography mass spectrometry purity H2O at 2.2 SLPM. Humidified N2 spotting 5-μL of sample on BG-11 Omni plates (BG-11 with 0.04% glucose,
exiting the bottle was passed into a Po-210 α-particle source (20 mCi) to pro- 5% Luria broth, and 1.5% agar) and incubating the plate in the dark at 30 °C
duce protonated water clusters, (H2O)nH+, where n = 1,2,3,. . ., that were mixed overnight (49). All S. elongatus flask and carboy cultures were demonstrated
with sample air in the IMR of the CIMS instrument (PIMR = 23 Torr). Input flow to be free of contaminants prior to Tetrahymena addition.
of sample and reagent gas into the IMR was controlled by inline critical orifices
(O’Keefe) at 1.8 SLPM for both flows. Chemical ionization of analytes in the Continuous Fluorescence. After each carboy was inoculated with Tetrahymena
IMR by protonated water clusters proceeds through several possible reaction grazer, a low-flow (∼1 mL/minute) peristaltic pump was connected and
mechanisms, which are controlled by the chemical energetics of the analytes withdrew carboy liquid through 1/8” outside diameter Tygon tubing to a
and reagent ion. These reactions are detailed in Eqs. 1–4 (39), quartz 1-cm2 flow-through cuvette before returning the liquid back to the
carboy in a closed loop. The cuvette was placed in a custom fabricated fluo-
M + X + → M+ + X Charge Transfer [1] rescence spectrometer, which irradiated the cell with 420 nm light produced
by a photodiode laser (Thorlabs). An orthogonally oriented photodiode de-
+ +
M + HX → MH + X Proton Transfer [2] tector (Thorlabs) with a 650-nm high-pass filter measured fluorescence, which
was logged at a rate of 1 Hz by a Labview interface. Continuous fluorescence
MH + X + → M+ + HX Hydride Abstraction [3] data were background subtracted, normalized, and averaged into 10-min in-
tervals after experiment completion.
X + + M + Z → MX + + Z Adduct Formation, [4]

where M is the analyte, X is the reagent, and Z is a third body required to Gas Chromatography Mass Spectrometry of Grazer-Infected PCC 7942. The
carry away excess energy. For (H2O)nH+, it is assumed that the predominant 10-mL crimp cap vials were filled with 4 mL BG-11 growth media, 4 mL
reaction channel is through proton transfer, which occurs when the proton previously grown culture of PCC 7942 at an OD of ∼0.25, or 4 mL PCC 7942
affinity of the analyte exceeds that of the water cluster. For (H2O)nH+, the culture with ∼370 Tetrahymena cells added. Vials were crimp sealed and held
range of proton affinities for n = 0,1,2,3 water clusters is 691, 694, 730, and under fluorescent light for the experiment duration. For each microalgae–
769 kJ · mol−1 respectively; however, it is difficult to predict the true distri- grazer experiment, 10 vials were prepared, of which 5 were controls of algae
bution of water clusters in the IMR, as the observed mass spectrum may not only and 5 were infected at the experiment start with Tetrahymena ciliate.
reflect this directly (48). Given the relatively high pressure of the IMR, water Each day of the experiment, an untested vial from each group was sampled by
clusters are expected to reach the equilibrium cluster distribution far faster SPME fibers possessing a 50-μm stationary phase of divinylbenzene/Carboxen/
than their residence time (∼100 ms) in the IMR. polydimethylsiloxane (solid gray, Supelco) equilibrated in the vial headspace
In chemical ionization, the humidity inside the reaction chamber can for 24 h. Samples were analyzed on an Agilent Technologies 7820A/5975
drastically affect the distribution of ions and the dynamics of chemical ion- GC/MS using an Agilent BP5 column. The following GC/MS parameters were
ization, which often requires humidity-dependent calibration or internal utilized for analysis of SPME fibers: inlet temperature: 250 °C; splitless injec-
standards to account for changes in sample humidity (24). While humidity of tion, carrier flow: 2 mL · min−1; GC temperature program: 35 °C for 3 min, then
the sampling lines was not monitored during these experiments, the tem- +7 °C · min−1 to 230 °C, which was held for 2 min; MS transfer line tempera-
peratures of the sampling lines were held constant at 30 °C, with the carboys ture: 260 °C; MS scan range: m/z 32.5 to m/z 300 over 1 s. MS data were
in a temperature-controlled laboratory at 23 °C. In previous experiments, baselined using the statistics-sensitive nonlinear iterative peak-clipping algo-
which did not use tubing heating, significant challenges with condensation rithm (50) and analyzed in OpenChrom (51) ([Link]
were encountered. From this, we assume that the tubing stayed quite close to openchrom). MS spectra were identified using the NIST14 Electron Ionization
the saturation humidity. Furthermore, an analysis of the water cluster ions mass spectral database. Results were subsequently exported to Igor Pro-7
over the duration of one of the carboy experiments (SI Appendix, Fig. S10) also (Wavemetrics) for plotting.
showed that the ratios of water cluster ions were quite stable against each
other. Given a large change in humidity, we might expect to see these ratios Data Availability. Comma Separated Value (CSV) and raw Hierarchical Data
drift toward larger clusters if more humid, and smaller clusters if less. Format (HDF) data have been deposited in the University of California San
After ionization, reagent and product ions were passed further into the Diego Library, [Link] (52).
instrument, starting with an electrodynamic ion funnel (PIF = 0.3 Torr), which
ACKNOWLEDGMENTS. We thank Joseph Manson for assistance in building
radially confined ions using a radio frequency (RF) pseudopotential applied
the hardware and software for the valve manifold and Joseph Mayer for
across 30+ concentric brass plates of decreasing diameter. Ions transmitted assistance in building the custom carboy caps. We thank Kerry Kizer, Laura
through the funnel were passed into an RF-only transfer quadrupole (PQuad = 5
Downloaded by guest on January 10, 2022

Lowe, and Paul Kasrazadeh for helpful assistance in preparing cultures for
× 10−5 Torr), which directs the ions into the final chamber: a commercial the experiments herein. This material is based on work supported by
orthogonal extraction time-of-flight mass spectrometer (PToF = 4.0 × 10−7 Department of Energy Grant DE-EE0007094.

8 of 9 | PNAS Sauer et al.

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