3.1 Pseudomonas syringae bacteria characterization

The AIDA cloud chamber was preconditioned overnight to reach the desired experimental temperature. Before the expansions, a suspension of bacteria (Pseudomonas syringae for Expansion 1 and PF CGina for Expansions 2 and 3) was aerosolized and dried, and the resulting particles were introduced into the chamber. Figure 1 shows the calculated volume equivalent diameter (d_ve) size distributions of particles present in AIDA before Expansion 1, which was obtained by combining measurements by the SMPS (black line) and the APS (red line). The size distribution is bimodal, with a smaller size mode that peaks at ∼70 nm and a broad peak at ∼800 nm. The miniSPLAT-measured vacuum aerodynamic size (d_va) distribution, shown in Fig. 1b, is also bimodal, with modes at 180 and ∼850 nm. The difference between the small particle peak positions of the measured d_va and d_ve size distributions is due to particle density (ρ>1 g cm⁻³) and miniSPLAT's small particle detection limit (50 % cutoff at 85 nm, marked with a green line in Fig. 1a) (Vaden et al., 2011b). This type of bimodal size distribution has been observed previously with other aerosolized suspensions of cultivated bacteria (Wex et al., 2015; Wolf et al., 2015; Möhler et al., 2008). The large size mode corresponds to intact/whole bacteria cells and the smaller mode is composed of bacterial fragments mixed with agar.

Figure 1Size distributions of an aerosolized suspension of Pseudomonas syringae in the AIDA cloud chamber before Expansion 1, calculated based on the SMPS and the APS measurements (a) and miniSPLAT (b). The miniSPLAT small particle detection limit is denoted by the green line in panel (a).

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Figure 2Positive (a) and negative (b) average mass spectra of the particles in the AIDA chamber, measured before Expansion 1. Reference mass spectra of the small size mode, made up of bacterial fragments mixed with agar, which includes particles smaller than 300 nm, are marked in black, and the mass spectra of the large size mode particles (d_va > 70 nm), composed of intact Pseudomonas syringae bacterial cells, are marked in red.

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Figure 3Positive (a) and negative (b) average mass spectra (MS) of intact Pseudomonas syringae bacteria measured before Expansion 1; positive (c) and negative (d) average MS of the small particle mode measured before Expansion 1; positive (e) and negative (f) average MS of pure agar particles measured in a separate experiment.

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Representative positive and negative ion mass spectra (MS) of the two particle modes are shown in Fig. 2. The reference MS of the small particle mode includes MS of particles smaller than 300 nm and that of the large particle mode/intact bacteria includes the MS of particles larger than 700 nm. Comparison between the reference MS of the two particle modes shows that nearly all the peaks are present in the MS of both modes. However, the positive ion MS of the two modes exhibit significantly different relative peak intensities, while the negative ion MS are very similar. The positive ion MS indicate the presence of many organics and metal ions, including carbon containing fragments (¹²C⁺, ${}^{\mathrm{15}}{\mathrm{CH}}_{\mathrm{3}}^{+}$, ²⁸CO⁺), ⁴³CH₃CO⁺, ${}^{\mathrm{44}}{\mathrm{CO}}_{\mathrm{2}}/{\mathrm{CH}}_{\mathrm{3}}{\mathrm{COH}}^{+}$), ammonium (${}^{\mathrm{18}}{\mathrm{NH}}_{\mathrm{4}}^{+}$), organic nitrogen (²⁸HCNH⁺, ${}^{\mathrm{30}}{\mathrm{H}}_{\mathrm{2}}{\mathrm{NCH}}_{\mathrm{2}}/{\mathrm{NO}}^{+}$, ${}^{\mathrm{70}}\mathrm{HNCH}({\mathrm{CH}}_{\mathrm{2}}{)}_{\mathrm{3}}^{+}$, ${}^{\mathrm{85}}{\mathrm{C}}_{\mathrm{5}}{\mathrm{NH}}_{\mathrm{10}}^{+}$), sodium (²³Na⁺), potassium (${}^{\mathrm{39}/\mathrm{41}}{\mathrm{K}}^{+}$), calcium (⁴⁰Ca⁺), and calcium oxides (⁵⁶CaO⁺, ¹¹³(CaO)₂H⁺). The negative ion MS show the presence of organic nitrogen (²⁶CN⁻, ⁴²CNO⁻), chloride (${}^{\mathrm{35}/\mathrm{37}}{\mathrm{Cl}}^{-}$), organics (${}^{\mathrm{45}}{\mathrm{HCO}}_{\mathrm{2}}^{-}$, ⁵⁰C₃N⁻, ${}^{\mathrm{71}}{\mathrm{C}}_{\mathrm{3}}{\mathrm{H}}_{\mathrm{3}}{\mathrm{O}}_{\mathrm{2}}^{-}$), and phosphates (${}^{\mathrm{63}}{\mathrm{PO}}_{\mathrm{2}}^{-}$, ${}^{\mathrm{79}}{\mathrm{PO}}_{\mathrm{3}}^{-}$, ${}^{\mathrm{97}}{\mathrm{H}}_{\mathrm{2}}{\mathrm{PO}}_{\mathrm{4}}^{-}$). The ions were identified via comparison to previous studies on bacteria (Pratt et al., 2009; Zawadowicz et al., 2017; Wolf et al., 2015; Fergenson et al., 2004; Srivastava et al., 2005; Czerwieniec et al., 2005). The positive ion MS for intact cells show significantly higher intensities for the organic peaks (¹²C⁺, ${}^{\mathrm{15}}{\mathrm{CH}}_{\mathrm{3}}^{+}$, ${}^{\mathrm{28}}\mathrm{CO}/{\mathrm{HCNH}}^{+}$, ${}^{\mathrm{30}}{\mathrm{H}}_{\mathrm{2}}{\mathrm{NCH}}_{\mathrm{2}}/{\mathrm{NO}}^{+}$, ${}^{\mathrm{44}}{\mathrm{CO}}_{\mathrm{2}}/{\mathrm{CH}}_{\mathrm{3}}{\mathrm{COH}}^{+}$, ${}^{\mathrm{70}}\mathrm{HNCH}({\mathrm{CH}}_{\mathrm{2}}{)}_{\mathrm{3}}^{+}$, ${}^{\mathrm{85}}{\mathrm{C}}_{\mathrm{5}}{\mathrm{NH}}_{\mathrm{10}}^{+}$) as compared to those of the small particle mode, which are dominated by the potassium peak. The same MS are presented in Fig. 3 in expanded scale together with the MS of pure agar particles. Comparison between MS of agar and the small particle mode shows their positive ion MS have the same peaks just with varying ion intensities and the absence of a peak at m∕z 131 (possibly C₇H₁₇NO⁺) in the agar. The agar negative ion MS have sulfate peaks (${}^{\mathrm{64}}{\mathrm{SO}}_{\mathrm{2}}^{-}$, ${}^{\mathrm{81}}{\mathrm{HSO}}_{\mathrm{3}}^{-}$, ${}^{\mathrm{97}}{\mathrm{HSO}}_{\mathrm{4}}^{-}$) and lack some organic ions (${}^{\mathrm{45}}{\mathrm{HCO}}_{\mathrm{2}}^{-}$, ${}^{\mathrm{71}}{\mathrm{C}}_{\mathrm{3}}{\mathrm{H}}_{\mathrm{3}}{\mathrm{O}}_{\mathrm{2}}^{-}$) as compared to the bacteria MS. The size distributions and MS provide direct evidence that the smaller particles are composed of bacterial fragments mixed with agar, while the larger size particle mode corresponds to intact bacteria cells. The MS of intact bacteria shown in Figs. 2 and 3 are similar to previously published single particle mass spectra of laboratory-generated Pseudomonas syringae acquired by two other single particle mass spectrometers (Pratt et al., 2009; Zawadowicz et al., 2017). The negative ion MS presented here and in previous studies are virtually the same, while the positive ion MS presented in this study exhibit significantly higher intensity in organic peaks, most likely due to the differences in the ablation laser power or wavelength (266 nm in Pratt et al., 2009, and 193 nm in this study and Zawadowicz et al., 2017). Moreover, these studies do not mention the presence of two particle size modes nor distinguish between their mass spectra.

Figure 4Data from Expansion 1 with Pseudomonas syringae bacteria. (a) The pressure (black) and the temperature of the gas (red) inside the AIDA chamber. The measured RH with respect to ice (RH_i) and water (RH_w) are indicated in solid and dashed blue lines, respectively; (b) the size distributions of the particles measured with welas2 during the expansion. Particles smaller than ∼15 µm are liquid droplets, while particles with larger optical diameters are ice. The PCVI cut size is shown by the blue trace. (c) The welas2-measured total particle number concentrations (black) and the number concentrations of particles larger than 30 µm (pink). The number concentrations of particles transmitted by the PCVI and measured by CPC and miniSPLAT is marked in yellow and green, respectively.

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The size distributions shown in Fig. 1a indicate a total number concentration of particles in the chamber of ∼2500 and ∼100 particles cm⁻³, or 4 %, are intact bacteria. In comparison, the d_va size distribution (Fig. 1b) yields 7 % for the fraction of intact bacteria, since more than half of the particles in the smaller size mode are too small to be detected by miniSPLAT. It is important to point out that the size, number concentration, and the relative fraction of the small particles containing bacterial fragments can vary from experiment to experiment and are determined by the solution/suspension concentrations of agar, intact bacteria, and cell fragments, as well as how the bacteria culture is grown and prepared (Wolf et al., 2015), and may change with the age of the solution/suspension.

3.2 Cloud formation and cloud droplet characterization

Cloud formation was induced by lowering the pressure in the AIDA chamber, using active pumping, which lowered the temperature and increased the RH, shown in Fig. 4a. As the chamber became supersaturated with respect to both ice and water, cloud droplets and ice crystals formed, which is illustrated in Fig. 3b with a false color plot of the welas2-measured size distribution as a function of expansion time. The number concentration of cloud droplets and ice crystals (particles larger than 5 µm, plotted as a black line) quickly reached ∼900 particle cm⁻³ and then decreased slowly to 400 particles cm⁻³. An estimate of the fraction of particles that activated as cloud droplets (CCN∕CN) yields 0.45, indicating that less than half of the particles were incorporated into droplets. After ∼500 s, as pumping stopped, the temperature increased, RH dropped, and the droplet number concentration decreased mainly due to water evaporation. During the expansion, the vast majority of the droplets remained smaller than 14 µm. The few ice crystals that formed are apparent in the figure as a second mode of much larger particles (marked in Fig. 4b) mainly visible at ∼100 to 200 s, after which point their numbers rapidly decreased mainly because of settling losses.

Figure 5Normalized miniSPLAT-measured d_va size distributions of Pseudomonas syringae particles in the AIDA chamber before Expansion 1 (green) and of cloud residuals transmitted through the PCVI during the expansion (blue). The inset presents in expanded scale the size distributions of the intact bacteria, showing that cloud residuals do not exhibit the distinct peak for intact bacteria.

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Figure 6Positive (a) and negative (b) ion average mass spectra of cloud residuals sampled during Expansion 1 (blue) superimposed on the average MS of intact bacteria (red). Panels (c) and (d) show the same cloud residuals average MS superimposed on the reference average MS of the small particle mode, composed of Pseudomonas syringae bacterial fragments mixed with agar (black).

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Ice crystal concentrations derived from welas2-measured number concentrations of particles larger than 30 µm (pink line in Fig. 4c) reach a maximum of 3 particles cm⁻³, or more than 200 times lower than droplets. An estimate of the fraction of particles that were incorporated into ice crystals shows that INP/CN ≈0.0014. Figure 4b and c show that during Expansion 1 the PCVI transmitted predominantly droplets, the residuals of which were characterized by the CPC and miniSPLAT.

The d_va size distribution of cloud residuals (Fig. 5) is virtually the same as the size distribution of particles in the chamber before the expansion except that the intact bacteria, i.e., the large particle mode, is “missing” from the size distribution of the cloud residuals.

In addition to the d_va size distribution, miniSPLAT-measured positive and negative ion MS of individual cloud residuals, which are presented in Fig. 6 superimposed on the reference MS of intact bacteria (large mode particles) (Fig. 6a and b) and on the reference MS of the small particle mode composed of agar and bacterial fragments (Fig. 6c and d) measured before the expansion. Comparison between the three particle types shows that the intact bacteria have higher relative intensities of many organic and nitrogen-containing (${}^{\mathrm{15}}{\mathrm{CH}}_{\mathrm{3}}^{+}$, ${}^{\mathrm{18}}{\mathrm{NH}}_{\mathrm{4}}^{+}$, ²⁸HCNH⁺, ${}^{\mathrm{30}}{\mathrm{H}}_{\mathrm{2}}{\mathrm{NCH}}_{\mathrm{2}}/{\mathrm{NO}}^{+}$, ${}^{\mathrm{44}}{\mathrm{CO}}_{\mathrm{2}}^{+}$, ${}^{\mathrm{70}}\mathrm{HNCH}({\mathrm{CH}}_{\mathrm{2}}{)}_{\mathrm{3}}^{+}$, ${}^{\mathrm{85}}{\mathrm{C}}_{\mathrm{5}}{\mathrm{NH}}_{\mathrm{10}}^{+}$) and calcium ions (⁴⁰Ca⁺, ⁵⁶CaO⁺) than the cloud residuals, but the small particle mode has nearly the same MS as the cloud residuals. The small differences between the MS of residuals and the small particle mode suggest a slightly higher content of organics in the residuals, indicated by higher relative intensities of ²⁸HCNH⁺, ${}^{\mathrm{30}}{\mathrm{H}}_{\mathrm{2}}{\mathrm{NCH}}_{\mathrm{2}}/{\mathrm{NO}}^{+}$, and ${}^{\mathrm{44}}{\mathrm{CO}}_{\mathrm{2}}^{+}$, which is due to the fact that the MS of residuals include all detected particles, while the reference MS of the small particle mode include only particles smaller than 300 nm. These MS differences will be further examined below in a detailed analysis of data obtained during Expansion 2, where the observed differences are larger and therefore easier to analyze. Taken together with the size distributions, these data provide direct evidence that under these experimental conditions, from the two particle types present in the chamber, particles composed of bacterial fragments mixed with agar preferentially activated to form cloud droplets.

3.3 PF CGina bacteria and cloud droplet characterization

Expansion 2 was performed in the same manner as Expansion 1, except that different bacteria, PF CGina, was used. Figure 7a presents the size distributions of particles in the chamber prior to Expansion 2, showing two particle modes, the small size mode, composed of agar and bacterial fragments, which peaks at d_ve≈60 nm, and the intact bacteria, which peaks at d_ve≈700 nm. There were 1800 particles cm⁻³, ∼4 % of which were intact bacteria. Similarly, the d_va size distribution, shown in Fig. 7b, shows the small particle mode peaking at d_va≈160 nm, composed of agar and bacterial fragments, and the intact bacteria mode, which peaks at d_va≈850 nm and represents ∼9 % of particles detected and sized prior to Expansion 2. Comparison between the reference MS of the two particle modes present before Expansion 2 is presented in Fig. 8, where the small mode includes particles smaller than 300 nm, and the large mode represents particles larger than 700 nm. As in the case of Expansion 1, the MS show that the small particles are composed of agar and bacterial fragments. The difference between the two positive ion reference MS for PF CGina is very similar to the pattern observed for Pseudomonas syringae bacteria (Fig. 2) and contains all of the same ions.

Figure 7(a) Size distributions of an aerosolized suspension of PF CGina particles in the AIDA cloud chamber before and after Expansion 2 calculated from the SMPS and the APS measurements; (b) miniSPLAT-measured size distribution of PF CGina particles in the chamber before Expansion 2. The miniSPLAT small particle detection limit is denoted by the green line in panel (a).

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Figure 8Positive (a) and negative (b) average mass spectra of the particles in the AIDA chamber, measured before Expansion 2. Reference mass spectra of the small size mode, made up of particles smaller than 300 nm (black), and the mass spectra of the large size mode (> 700 nm) composed of intact PF CGina bacterial cells are marked in red.

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Figure 9Data from Expansion 2 with PF CGina bacteria. (a) The pressure (black) and the temperature of the gas (red) inside the AIDA chamber. The measured RH with respect to ice (RH_i) and water (RH_w) is indicated in solid and dashed blue lines, respectively; (b) the size distributions of the particles measured with welas2 during the expansion. Particles smaller than ∼15 µm are liquid droplets, while larger particles are ice. The PCVI cut size is shown by the blue trace. (c) The welas2-measured total particle number concentrations (black) and the number concentrations of particles larger than 30 µm (pink). The number concentrations of particles transmitted by the PCVI and measured by CPC and miniSPLAT are marked in yellow and green, respectively.

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Figure 10Normalized miniSPLAT-measured d_va size distributions of PF CGina particles in the AIDA chamber before Expansion 2 (green) and of cloud residuals transmitted through the PCVI during the expansion (blue). The inset presents in expanded scale the size distributions of the intact bacteria, showing nearly no distinct peak for intact bacteria.

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Figure 11(a) Average reference MS of the small particle mode, composed of agar and PF CGina bacterial fragments (black) and the average MS of cloud residuals acquired during Expansion 2 (blue); (b) average MS of intact bacteria (red) and the average MS of cloud residuals acquired during Expansion 2 (blue).

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The temporal evolution of pressure, temperature, and RH during Expansion 2 is shown in Fig. 9a. The welas2-measured size distributions of cloud particles (Fig. 9b) reveal that most of the activated particles were liquid droplets smaller than 10 µm, and only a small number of cloud particles were ice crystals. The average droplet and ice crystal concentrations (Fig. 9c) were ∼730 and ∼1 particle cm⁻³, respectively, with ice crystal peak concentrations reaching ∼3 particles cm⁻³ at 150 to 250 s from the start of the expansion. As a result, particles transmitted through the PCVI to the CPC and miniSPLAT during Expansion 2 were predominantly liquid droplets. The CPC- and miniSPLAT-measured number concentrations of particles transmitted by the PCVI (Fig. 9c) show rapid increases early in the expansion and slower decreases thereafter, displaying the same behavior as in Expansion 1. A lower percentage of particles was activated into droplets (30 %) than in the first expansion, but roughly the same percentage of particles was activated into ice crystals (0.16 %).

The miniSPLAT-measured d_va distributions of cloud residuals (blue) and particles in the chamber prior to the expansion (green) are shown in Fig. 10. As with Expansion 1, the d_va distribution of cloud residuals is nearly identical to that of the small particle mode present in the chamber before the expansion. An expanded scale of the intact bacteria size region, shown in the figure inset, indicates no detectable intact bacteria in cloud residuals.

The average positive ion MS of cloud residuals is shown in Fig. 11. For comparison, it is superimposed on the reference MS of the small particle mode (Fig. 11a) and intact bacteria (Fig. 11b). The MS of residuals show that they contain slightly more organics (¹²C⁺, ${}^{\mathrm{15}}{\mathrm{CH}}_{\mathrm{3}}^{+}$, ²⁸HCNH⁺, ${}^{\mathrm{30}}{\mathrm{H}}_{\mathrm{2}}{\mathrm{NCH}}_{\mathrm{2}}/{\mathrm{NO}}^{+}$, ${}^{\mathrm{44}}{\mathrm{CO}}_{\mathrm{2}}^{+}$, ${}^{\mathrm{70}}\mathrm{HNCH}({\mathrm{CH}}_{\mathrm{2}}{)}_{\mathrm{3}}^{+}$, ${}^{\mathrm{85}}{\mathrm{C}}_{\mathrm{5}}{\mathrm{NH}}_{\mathrm{10}}^{+}$) than the particles that were included in the reference MS of the small particle mode. It is important, however, to note that the residual MS include all particles, while the reference MS contain only particles smaller than 300 nm. A possible explanation for the observed difference between the two MS is that the organic content of the small particle mode increases with particle size; i.e., larger particles contain more or larger bacterial fragments and relatively less agar. Indeed, Fig. 12 shows that the fraction of characteristic organic peaks (m∕z 28 and 44) relative to K⁺, which serves as a simple qualitative measure of the relative fraction of bacterial fragments and agar in these particles, is increasing with particle size. The figure includes the d_va distribution of cloud residuals (via a dashed line) for reference. For comparison, the same analysis shows that the K⁺ peak contributes 86 % of the mass spectral intensity for pure agar and 13 % for intact bacteria. To examine this further, we compare in Fig. S2a the MS of cloud residuals smaller than 300 nm to the reference MS of the small particle mode measured before the expansion and find them to be nearly identical. This indicates that they are bacterial fragments mixed with agar. The MS of cloud residuals that are larger than 300 nm have greater ion intensities for the organic peaks (${}^{\mathrm{28}}\mathrm{CO}/{\mathrm{HNCH}}^{+}$, ${}^{\mathrm{44}}{\mathrm{CO}}_{\mathrm{2}}/{\mathrm{CH}}_{\mathrm{3}}{\mathrm{COH}}^{+}$, ${}^{\mathrm{70}}\mathrm{HNCH}({\mathrm{CH}}_{\mathrm{2}}{)}_{\mathrm{3}}^{+}$, ${}^{\mathrm{85}}{\mathrm{C}}_{\mathrm{5}}{\mathrm{NH}}_{\mathrm{10}}^{+}$) than the bacterial fragments mixed with agar but slightly smaller ion intensities than those of the reference MS of intact bacteria, as shown in Fig. S2b. These differences in ion intensities are not sufficient to eliminate the possibility that some of the larger cloud residuals could be smaller intact bacteria.

Overall, the d_va size distributions and MS of cloud residuals are like those of the small particle mode, although the larger residuals, which contain more organics, could be smaller intact bacteria. Nevertheless, the data clearly show that the intact bacteria contribute a significantly lower fraction, if at all, to the cloud residuals than they contribute to the total particle population present before the expansion. In all respects, the miniSPLAT data for PF CGina are consistent with those for Pseudomonas syringae (Expansion 1) and confirm the preferential droplet activation of particles composed of bacterial fragments mixed with agar compared to intact bacteria.

3.4 PF CGina ice crystal and droplet characterization

Expansion 3 followed the same pumping strategy and expansion temperatures as the previous expansions but was conducted on the PF CGina particles which remained in the AIDA chamber after Expansion 2. The d_ve size distributions of these particles, shown in Fig. 7a as dashed lines, are nearly identical to those measured before Expansion 2, despite a decrease in the total particle number concentration. During Expansion 3, the cloud residual particles, sampled by the CPC and miniSPLAT, were transmitted through a different inlet, the IS-PCVI, in order to reduce the number of droplets transmitted to allow for characterization of ice crystal residuals.

Figure 12The relative mass spectral intensity of peaks assigned to organics and potassium as a function of particle d_va, showing that larger particles contain more organics for PF CGina bacteria. The dashed line represents the measured d_va distribution of cloud residuals.

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Figure 13Data from Expansion 3 with PF CGina bacteria. (a) The pressure (black) and the temperature of the gas (red) inside the AIDA chamber. The measured RH with respect to ice (RH_i) and water (RH_w) is indicated in solid and dashed blue lines, respectively; (b) the size distributions of the particles measured with welas2 during the expansion. Particles smaller than ∼15 µm are liquid droplets, while larger particles are ice. The IS-PCVI cut size is shown in blue. (c) The welas2-measured total particle number concentrations (black), the welas2 number concentrations of particles larger than 15 µm but smaller than 20 µm (light blue), and the welas2 number concentrations of particles larger than 20 µm (pink). The CPC- and miniSPLAT-measured number concentrations of particles transmitted by the IS-PCVI are marked in yellow and green, respectively.

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Changes in chamber pressure, temperature, and RH for Expansion 3 are shown in Fig. 13a. A false color plot of the activated particles size distribution measured by welas2, as a function of time, is presented in Fig. 13b. Figure 13c displays the welas2-measured total number of activated particles as a function of time (black), which reaches a maximum of ∼700 particles cm⁻³. Before the expansion, 1456 cm⁻³ particles were present in the chamber, indicating that 47 % of the particles activated as CCN. In addition, it shows the number concentrations of cloud particles with optical diameters larger than 15 µm but smaller than 20 µm (light blue) and the number of particles larger than 20 µm (pink). The former may correspond to the number of droplets transmitted by the IS-PCVI, while the latter may be relevant to transmitted ice crystals. Comparison between the two shows that the IS-PCVI transmitted nearly equal numbers of ice crystals and droplets to miniSPLAT and the CPC. A small percentage of particles (0.04 %) formed ice crystals. The miniSPLAT- and CPC-measured particle number concentrations are also shown in Fig. 13c. Both instruments show a rapid rise in the concentration of cloud residuals, followed by a significant drop in concentration with time. Early in the expansion, most of the transmitted particles are droplets, but later, between 302 and 557 s after the expansion started, the number of droplets and ice crystal residuals becomes comparable, as shown in Fig. 13c.

Two particle modes are present in the d_va size distribution of particles in the chamber before Expansion 3 (Fig. 14, green trace). Figure 14 also shows the d_va size distributions of all cloud residuals detected throughout the expansion (blue) and cloud residuals detected late in the expansion (302–557 s, light blue), when the number concentration of droplets and ice crystals was approximately equal. Both cloud residual size distributions are nearly the same as that of the small particle mode and show no distinct peak in the size region of intact bacteria.

The average MS of particles sampled through the IS-PCVI throughout the expansion and later in the expansion are shown in Fig. 15. The fact that the two MS are virtually identical suggests that the compositions of particles that remain as droplets and those that activate into ice crystals are not different, which is consistent with the finding that the size distributions are the same. The residual size distributions and compositions combined again confirm that the majority of particles that served as CCN and INPs were bacterial fragments mixed with agar. While there is no clear indication that intact bacterial cells were activated, it cannot be ruled out that larger cloud residuals were small intact bacteria mixed with agar.

These results are consistent with previous studies that show that whole cells are not necessary for ice formation because IN active proteins and protein complexes can serve as INPs (Lindow et al., 1989; Maki and Willoughby, 1978; Hartmann et al., 2013; Govindarajan and Lindow, 1988). However, it has generally been assumed that whole cells will also nucleate ice (Möhler et al., 2008; Wex et al., 2015; Lindow et al., 1989) and are more active than a single IN active protein (Govindarajan and Lindow, 1988) or bacterial fragment (Yankofsky et al., 1981; Hartmann et al., 2013). The explanation for enhanced IN activity of intact bacteria is that IN active proteins collect in the cell membrane (Lindow et al., 1989; Govindarajan and Lindow, 1988) and become concentrated on the surface of intact cells, thus enhancing their IN activity. However, the data presented here show that bacterial fragments mixed with agar preferentially activate as droplets and are the only particles observed in ice residuals under these experimental conditions.

Figure 14d_va size distribution, measured by miniSPLAT before Expansion 3 (green) with PF CGina bacteria, of cloud residuals (blue) and of cloud residuals measured during the later part of the expansion (302–557 s) when the number concentration of sampled droplets and ice crystals was nearly the same (light blue). The inset shows an expanded scale of the region of intact bacteria.

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Figure 15Average mass spectrum of cloud residuals measured during Expansion 3 (blue) with PF CGina bacteria super imposed on the average mass spectrum of particles characterized later in the expansion (302–557 s) when the number of sampled ice crystals and cloud droplets was comparable (light blue).

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