Iron minerals inhibit the growth of bacteria via a free-radical mechanism : : Implications for soil carbon storage

Abstract. Natural minerals in soil can inhibit the growth of bacteria that protect organic carbon from decay. However, the mechanism inhibiting the bacterial growth remains poorly understood. Here, using a series of cultivation experiments and biological, chemical and synchrotron-based spectral analyses, we showed that kaolinite, hematite, goethite and ferrihydrite had a significant inhibitory effect on the growth of Pseudomonas J12, which was more prominent with a concentration of 25 mg mL−1 than it was with either 10 mg mL−1 or 5 mg mL−1. In contrast, montmorillonite promoted the growth of Pseudomonas J12. Compared to Al-containing minerals, Fe(III)-containing minerals produced more hydroxyl radical (HO•) that have high efficiency for the inhibition of bacteria. Moreover, a significant positive correlation between HO• radical and Fe(II) was found, suggesting that Fe(II) contributes to the generation of HO•. Furthermore, both micro X-ray fluorescence and X-ray photoelectron spectroscopies indicated that surface Fe(III) was reduced to Fe(II) which can produce HO• through the well-known Fenton reaction series. Together, these findings indicate that the reduced surface Fe(II) derived from Fe(III)-containing minerals inhibit bacteria via a free-radical mechanism, which may further contribute to soil carbon storage.



Introduction
A variety of minerals exhibit bacterial inhibition properties by releasing Al(III) or 40 Fe(II) (Morrison et al., 2014;McMahon et al., 2016;Williams, 2017).Hence, natural minerals have long been used as bactericidal agents for human pathogens (Williams and Haydel, 2010;Williams et al., 2011).The bacterial inhibition property of a mineral is associated with the particular chemistry and with the mineral properties, resulting in the various bacterial inhibition mechanisms of minerals (Williams et al., 45 2008).Iron oxides are abundant in terrestrial and aquatic environments and exist predominantly as ferric minerals such as goethite, ferrihydrite, and hematite (Cornell and Schwertmann, 2003;Meunier, 2005;Chesworth, 2008).Due to the ubiquity of soil iron minerals and their distinct inhibition properties, which may affect soil carbon storage and nutrient turnover, investigations of the inhibitory potential of iron 50 minerals on microorganisms are of great importance.
To better understand the inhibition of bacteria by minerals, the mineral type and size should be examined.Previous studies have demonstrated that Al(III)-and Fe(II)-containing minerals can inhibit the growth of bacteria (McMahon et al., 2016).
For Al(III)-containing minerals, their toxicity mainly depends on the release of Al(III), 55 an extensively toxic element to bacteria (McMahon et al., 2016).However, Fe(II)-containing minerals usually cause oxidative damage to bacteria, i.e., the oxidative role of reactive oxygen species (ROS), particularly by involving hydroxyl radicals (HO • ) that are generated by an Fe(II) catalyzed Fenton reaction where Fe(II) reacts with hydrogen peroxide (H 2 O 2 ) to form HO • radicals (Stohs and Bagchi, 1995; 60 Williams et al., 2011;Wang et al., 2017a;Usman et al., 2018).However, it is unclear whether the common Fe(III)-containing minerals in soil have a similar inhibition Taxonomically and ecologically diverse bacteria from terrestrial environments are a vast source of superoxide (O 2 •-) and H 2 O 2 (Diaz et al., 2013;Tang et al., 2013).
where Fe(III) OH   represents the iron mineral surface.
These Fenton-like reactions are well known as a type of heterogeneous catalysis (involving Fe minerals), which is distinct from homogeneous Fenton reactions (based on soluble Fe(II) in acidic media) (Garrido-Ramírez et al., 2010).The major 75 advantage of heterogeneous catalysis is that it operates well over a wide range of pH values, while homogeneous catalysis displays optimal performance only at a pH of ~3 (Garrido-Ramírez et al., 2010).Furthermore, some researchers had demonstrated that surface Fe(II) was generated in the systems of H 2 O 2 and ferric minerals (Kwan and Voelker, 2003;Polerecky et al., 2012).To date, the impact of Fe(III)-containing 80 minerals on bacteria remains largely unexplored.
Here, we hypothesize that Fe(III)-containing minerals can inhibit the growth of bacteria through a free-radical mechanism (i.e., Fenton-like reactions).(Cornell and Schwertmann, 2003;Meunier, 2005;Chesworth, 2008).Meanwhile, Pseudomonas brassicacearum J12 was selected as the model bacterium because it represents a 90 major group of rhizobacteria that aggressively colonize plant roots in soils (Zhou et al., 2012).In this study, the objectives were to 1) examine and compare the bacterial inhibition properties of Al and Fe minerals; 2) build the correlation between solution chemistry and HO • and the growth of bacteria; and 3) identify the mechanism by which Fe(III)-containing minerals inhibit bacteria.Throughout our experiments, the 95 HO • was trapped by terephthalic acid (TPA) (non-fluorescent), and the reaction's fluorescent product, i.e., 2-hydroxylterephthalic acid (HTPA) (Li et al., 2004), was quantitated in a high-performance liquid chromatography (HPLC) system.Correlative micro X-ray fluorescence (µ-XRF) and synchrotron-based Fourier transform infrared (SR-FTIR) spectroscopies were used to probe the in situ distribution and species of 100 the Fe and extracellular polymeric substances (EPS), respectively (Luo et al., 2014;Sun et al., 2017a).X-ray photoelectron spectroscopy (XPS) was also used for analyzing the oxidation state(s) and speciation of Fe (Wilke et al., 2001;Yamashita and Hayes, 2008).The bacterium used in this experiment is Pseudomonas brassicacearum J12, which is a major group of rhizobacteria that aggressively colonize plant roots, has been considered an important group for sustainable agriculture, and was provided by Dr. Zhou (Zhou et al., 2012).The stock strain of J12 was inoculated in Nutrient Broth (NB) medium to an optical density (OD 600 ) of ~0.6.The NB medium includes beef 125 extract, 3 g L -1 ; Tryptone, 5 g L -1 ; yeast extract, 0.5 g L -1 ; Glucose, 10 g L -1 .The cultivation system contained 9.5 mL of NB medium and 0.5 mL of J12, with a concentration of minerals of 5, 10 or 25 mg mL -1 .The final pH of the cultivation system was adjusted to 7.2.Next, the cultivation media were incubated for 12 h on a shaking incubator (180 rpm) at 28 °C.Then, 50 µL of the cultures were transferred to  S1.
After SR-FTIR analysis, Fe image was collected at beamline 15U1 of Shanghai 160 Synchrotron Radiation Facility (SSRF) for the same region of the thin section.
Fluorescence maps (µ-XRF) of Fe were obtained by scanning the samples under a monochromatic beam at E = 10 keV with a step size of 2.3 × 3.3 µm 2 and a dwell time of 1 s.Then, two positions were selected for Fe K-edge µ-X-ray absorption near-edge structure (µ-XANES) analysis, and µ-XANES spectra were recorded using a 0.1 eV

XPS analysis
The species of iron oxides were analyzed by XPS (PHI5000 Versa Probe, 175 ULVAC-PHI, Japan).All the samples were freeze-dried and ground to fine powders prior to the XPS measurement.The XPS spectra were obtained with a monochromatized Al Kα X-ray source (1486.6 eV) and the pressure in the analytical chamber was below 6 × 10 −8 Pa (Yangzhou University).For wide scan spectra, an energy range of 0-1100 eV was used with the pass energy of 80 eV and the step size 180 of 1 eV.The high-resolution scans were conducted according to the peak being examined with the pass energy of 40 eV and the step size of 0.06 eV.The precision of XPS was 0.06 eV.In order to obtain the oxidation status of surface sites, narrow scan spectra for Fe 2p 3/2 were acquired.The carbon 1s electron binding energy corresponding to graphitic carbon at 284.8 eV was used as a reference for calibration 185 purposes.Narrow scan spectra for Fe 2p 3/2 were collected in binding energy forms and fitted using a least-squares curve-fitting program (XPSPEAK41 software).The XPS spectra were analyzed after subtracting the Shirley background that was applied for transition metals.The full width at half-maximum of those spectra was fixed constant between 1 and 3 and the percentage of Lorentzian-Gaussian was set at 20% 190 for all the spectra.

Electron paramagnetic resonance (EPR) spectroscopy
The EPR spectra were recorded with a Bruker A300 X-band spectrometer (Guangxi University), which used a Gunn diode as microwave source and incorporated a given in the references (Goodman et al., 2016).The g values were calculated by reference to the Bruker ER4119HS-2100 marker accessory which has a g value of 1.9800.Spectral data were processed using the Bruker WinEPR software; with 200 samples recorded with the same values for the microwave power, modulation amplitude, time constant and conversion time, intensities were determined both from double integration of complete spectra after background correction, and the heights of individual peaks, and corrected for any differences in the receiver gain or number of scans.Simulations of spectra to test the validity of various models for the 205 C-centre spectrum were performed using the Bruker SimFonia software.

Chemical analysis
At cultivation time of 2 h and 12 h of the original cultures, portions of the samples were centrifuged at 16,000 g for 5 min, then filtered through a 0.45 µm membrane filter and analyzed with Inductively Coupled Plasma-Atomic Emission Spectroscopy 210 (710/715 ICP-AES, Agilent, Australia) to detect the concentration of soluble Fe and Al.Total Fe and Fe(II) were determined with a modified 1,10-phenanthroline method (Amonette, 1998).Turbidity at 600 nm (a standard proxy for bacterial cell density) was measured using a Microplate Reader (Hach DR/2010) in mid-exponential phase.
All experiments were performed in triplicate.

Statistical analysis
Significance was determined using one-way ANOVA followed by Tukey's HSD post hoc test, where the conditions of normality and homogeneity of variance were met; means ± SE (n = 3) that are followed by different letters indicate significant differences between treatments at p < 0.05.Microsoft Excel (2010), Origin Pro8 and 220 SPSS (18.0) were used for drawing the graphs and data analysis.

Bacterial inhibition by minerals
The effects of the nature and content of tested minerals on the OD 600 of Pseudomonas brassicacearum J12 subcultures taken after 12 h growth are shown in  significantly increased OD 600 .On the other hand, presence of all other investigated minerals decreased OD 600 in the following order: ferrihydrite > goethite > hematite > kaolinite at 5 and 25 mg mL -1 , and ferrihydrite > goethite > kaolinite > hematite at 10 mg mL -1 , suggesting that montmorillonite promoted the growth of Pseudomonas 230 brassicacearum J12, but the rest of the tested minerals inhibited its growth.
Meanwhile, an increase in mineral concentration resulted in a significant decrease in OD 600 , except for montmorillonite, as the OD 600 seemed to be independent of its concentration (Fig. 1).
To further explore the factors influencing the bacterial growth by (Fig. 2).Iron oxides, which are commonly associated with these minerals produce a broad signal centred on ~ 3500 gauss (g ~ 2.0).However, the relatively weak 240 resonance indicated that neither sample had appreciable amounts of iron oxides associated with it.The montmorillonite also showed a signal from Mn(II) and a free radical, whereas the free radical signal in the kaolinite was very weak, and there was no evidence of any Mn(II) signal in this sample.The Mn(II) in the montmorillonite was reported to act as a scavenger for any hydroxyl radical production 245 (Garrido-Ramírez et al., 2010), which may explain the promotion of microbial growth.

Generation of HO •
A 12 h cultivation of Pseudomonas brassicacearum J12 in the presence of different minerals revealed that generation of HO • radicals in the cases of montmorillonite, 250 kaolinite and hematite was almost similar to the control (Fig. 3).However, presence of goethite and ferrihydrite significantly increased the production of HO • radicals, which increased with an increase in their concentration.Specifically, in ferrihydrite treatments, the concentration of HO • was approximately 260 nM at 5 and 10 mg mL -1 but increased rapidly to 450 nM at 25 mg mL -1 .In addition, the generation of 255 HO • at early growth (i.e., 2 h) was only detected with ferrihydrite at both 10 and 25 mg mL -1 and with goethite at 25 mg mL -1 (Fig. S5).

Iron chemistry and its correlation with HO • and OD 600
To explore the factors affecting the generation of HO with HO • and OD 600 (Fig. 4).Much more soluble Fe was released from Fe(III)-containing minerals than from montmorillonite, kaolinite, and control (Fig. 4a).Additionally, a significant increase of soluble Fe was observed with the increase of ferrihydrite concentration.The solubility of Fe is closely related to pH value.
Therefore, the solution pH was determined after 12 h growth of Pseudomonas 265 brassicacearum J12 with different minerals and with no minerals (control) (Fig. S6).
The range of solution pH varied from 4 to 6 for all of the treatments, expect for ferrihydrite treatment with a pH near 7.The pH decline suggests the production of organic acids by Pseudomonas brassicacearum J12.Thus, a high pH value in ferrihydrite treatment also support the inhibition of Pseudomonas brassicacearum 270 J12.For all of the examined minerals, the trends of total Fe and Fe(II) were similar in the following order: ferrihydrite >> goethite > hematite > montmorillonite ≈ kaolinite ≈ control (Fig. 4b-4c).

290
S8a) and found a high concentration of Al in the montmorillonite and kaolinite solutions.However, a weak correlation was found between soluble Al and HO • (R = -0.35,t = -3.36,p = 0.004) and OD 600 (R = 0.30, t = 2.24, p = 0.041) (Fig. S8b-8c), suggesting that the generation of HO • and the inhibition of Pseudomonas brassicacearum J12 is not contributed by Al.S3).However, considerable percentages of hematite (~13%) and goethite (~19%) were present on the edge of these mineral particles (Spot A in Fig. 5b and Table S3).Furthermore, XPS analysis was conducted to investigate the oxidation state 310 spectra for Fe 2p peak of samples (Fig. 6).The shift of the Fe 2p 3/2 peak of 0.5 eV was observed between raw ferrihydrite and ferrihydrite after 12 h of cultivation with bacteria (Fig. 6a), suggesting the presence of reducing iron.Four Fe 2p 3/2 peaks at 709.5 eV, 710.3 eV, 711.5 eV, 713.1 eV appeared in the F + bacteria treatment (Fig. 6b-6c).The peaks at 710.3 eV, 711.5 eV and 713.1 eV are regarded as multiplet peaks 315 of Fe(III), but the peak at 709.5 eV is interpreted as Fe(II) (Grosvenor et al., 2004).
Interestingly, the area of the peak at 709.5 eV was bigger in the F + bacteria treatment than that in F -bacteria treatment (Fig. 6b-6c), suggesting that Fe(II) was generated on the surface of ferrihydrite during the cultivation with bacteria.Based on the reaction 1, HO 2 • should be the oxidant products.

Effect of Al(III)-containing minerals on the inhibition of bacterial growth
Our results showed that kaolinite (1 : 1 layer-type) resulted in significant inhibition of bacterial growth, but montmorillonite (2 : 1 layer-type) remarkably accelerated the bacterial growth (Fig. 1).Similarly, recent studies have shown the toxic effects of aluminosilicate on microorganisms (Liu et al., 2016;Wilson and Butterfield, 2014), but the bacterial activity was not inhibited by the interfacial interactions between 335 montmorillonite and bacteria (Wilson and Butterfield, 2014).It should be noted that the presence of minerals may potentially interfere with the measurement of cell numbers in Fig. 1.In this study, we subsampled the experimental cultures and diluted them in fresh medium so that both clay particles and bacteria were 200× less concentrated (Fig. S3), following the protocol of McMahon et al. (2016).As a result, 340 the effect of mineral concentration may be minimal.In addition, plating the bacteria by evaluating populations by counting colonies may act as a complementary method for OD 600 and needs to be investigated in the future.
It is generally accepted that diverse bacteria are susceptible to Al(III).A previous study showed that a 58 µM (~1.6 mg L -1 ) concentration of Al has toxicological effects 345 on Pseudomonas sp.(Illmer and Schinner, 1999).In the present study, the amount of aqueous Al(III) exceeded 2 mg L -1 for all kaolinite experiments while its concentration was negligible in the presence of montmorillonite during the early growth of bacteria (Fig. S8).Thus, the inhibition of bacterial activity by kaolinite may possibly be attributed to the toxicity of aqueous Al(III).Specifically, Al(III) reacts 350 with membrane phospholipids and then increases membrane permeability that leads to the inactivation of bacteria (Londono et al., 2017).
In addition, some essential elements (e.g., Mg and P) can be affected by Al(III) for bacterial absorption, which could also limit bacterial growth (Piña and Cervantes, 1996;Londono et al., 2017).Furthermore, the formation of some Al intermediates by bacterial growth (Amonette et al., 2003;Liu et al., 2016).It is worth noting that >2 mg L -1 of aqueous Al(III) was detected for montmorillonite experiments with the passage of time (Fig. S8); however, the bacterial growth was not inhibited (Fig. 1).
This may be attributed to the adsorption of aqueous Al(III) by bacterial EPS, which 360 further protected bacteria from damage (Wu et al., 2014).However, direct evidence is lacked in this study and thus further investigation is needed to address this issue.

Inhibition of bacteria by Fe(III)-containing minerals via a free-radical mechanism
Our results showed that Fe(III)-containing minerals resulted in higher generation of 365 HO • and had higher bacterial inhibition efficiency than Al(III)-containing minerals (Figs. 1 and 3).Fe is widely known as a transition metal that might cause microbial inactivation through ROS-mediated cellular damage, i.e., genotoxicity, protein dysfunction and impaired membrane function (Lemire et al., 2013).Inhibition of bacteria by Fe minerals is generally attributed to the generation of HO • through a 370 Fenton reaction (Morrison et al., 2016) or Fenton-like reaction (Garrido-Ramírez et al., 2010).Due to its amorphous structure, high reactive surface area and solubility, ferrihydrite (~200-300 m 2 g -1 ) is more likely to physically interact with bacterial surfaces than hematite (~30 m 2 g -1 ) and goethite (~20 m 2 g -1 ) (Schwertmann and Cornell, 2007;Lemire et al., 2013).A recent study demonstrated that metal oxide 375 nanoparticles produced more ROS than bulk metal oxides (Wang et al., 2017a).In this study, we observed higher HO • formation and stronger inhibition of bacteria in ferrihydrite treatments (Figs."carriers" where HO • -inducing materials are adsorbed (Schoonen et al., 2006).In our experiment, there was a lesser amount of HO • produced with the different concentrations of aqueous Fe(NO 3 ) 3 (Fig. S7) than with the iron minerals (Fig. 3).In line with other studies (Kwan and Voelker, 2003;Wang et al., 2017b), we deduced that HO • may mainly generate on the mineral surface, partly due to the positive charge 385 of mineral surface (Tombácz and Szekeres, 2006) but the negative charge of microbes (Jucket et al., 1996).
A recent study demonstrated that surface rather than aqueous Fe(II) plays a dominant role in producing extracellular HO • that damage cell membrane lipid revealed by in-situ imaging (Wang et al., 2017b).The following reactions (equations 390 3-4) reveal that the generation of HO • is catalyzed by surface Fe(II) (Kwan and Voelker, 2003;Polerecky et al., 2012): In this study, a substantial amount of Fe(II) was generated by ferrihydrite, 395 approximately 4-fold higher than soluble Fe (Fig. 4).This amount of Fe(II) included two portions: one existed in solution, another was derived from the mineral surface.
To further confirm the generation of surface Fe(II), Fe K-edge µ-XANES analysis were used, and they showed that ferrihydrite presented various Fe species, and Fe(II) increased from 17.3% among the mineral particles (A position) to 25.9% in the edge 400 of mineral particles (B position) (Fig. 5 and Table S3).High percentage of the less stable ferrihydrite (Table S3) may be attributable to the stabilization role of produced EPS (Fig. 5c) by bacteria to minerals, which had been shown during the cultivation of fungi with minerals (Li et al., 2016).Note that the LCF results are dependent on the range of compounds used to generate the reference spectrum library, which is one 405 drawback of LCF.To further support the LCF results, XPS being a near-surface sensitive technique is also used to detect the production of ferrous iron at the surface of the iron oxides, owing to a greater certainty than LCF and XANES to demonstrate the presence of ferrous iron by fitting multiplet-splitting models (Grosvenor et al., 2004).According to the XPS analysis, the Fe 2p 3/2 peak shifted from high energy (F -410 bacteria) to low energy (F + bacteria) (Fig. 6), revealing that Fe(II) was produced on the surface of ferrihydrite during cultivation.
In addition to Fenton-like reactions (Garrido-Ramírez et al., 2010), Fe(II) can also be generated by catalyzing a series of cellular intracellular (e.g., glutathione and NAD(P)H) and free (e.g., L-cysteine and FADH 2 ) reductants (Imlay, 2003).Other 415 metabolically formed oxidants released by bacteria may also contribute to Fe(II) oxidation (Melton et al., 2014).Subsequently, the oxidation of Fe(II) to Fe(III) is followed by a reduction of the Fe(III) to Fe(II) (Melton et al., 2014).In addition, many microorganisms are thought to transfer electrons between their cytoplasmic membranes and extracellular minerals through a network of redox and structural 420 c-type cytochromes (c-Cyts) and flavins (Shi et al., 2016).The redox cycling of Fe during interfacial interactions between Fe(III)-containing minerals and bacteria accelerates the generation of HO • (Page et al., 2013).
The responses of bacterial inhibition activity followed the order Fe(III)-containing minerals > Fe(NO 3 ) 3 > Control (Figs. 1 and S7).Intracellular oxidative toxicity 425 caused by soluble Fe(III) played an important role in bacterial inhibition activity simultaneously (Schoonen et al., 2006).We deduced that inhibition of bacteria with  S4).Substrate availability is improved in the presence of radicals, owing to the following two facts: 1) the depolymerization role of radicals on the 440 complex substrates; 2) the inhibition role of radicals on bacteria indirectly increasing the amounts of available substrates.
Microbes affect the cycling of soil organic carbon (SOC), and their products are important components of SOC (Kögel-Knabner, 2002;Kleber and Johnson, 2010;Schmidt et al., 2011;Liang et al., 2017).In this study, we suggest that soil carbon 445 storage is regulated by Fe minerals, not only because of the formation of organo-mineral complexes (Kögel-Knabner, 2002;Kleber and Johnson, 2010;Schmidt et al., 2011) but also due to the bacterial inhibition activity of Fe minerals.
However, it should be noted that NB medium containing casein and meat hydrolysates is only a medium that enables the growth of Pseudomonas brassicacearum J12 in this soil systems.Further investigation should be conducted to explore the effect of microbe-driven Fenton-like reaction on the storage of SOC in soil system in the future.

Conclusions
455 Kaolinite, hematite, goethite and ferrihydrite had a significant inhibitory effect on the growth of Pseudomonas J12, which was more prominent with a higher concentration, following the order 25 mg mL -1 > 10 mg mL -1 > 5 mg mL -1 .In contrast, montmorillonite promoted the growth of Pseudomonas J12, which was independent on its concentration.Compared to Al(III)-containing minerals, Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.activity with Al(III)-and Fe(II)-containing minerals.

≡
Fe(III)-OH + H 2 O 2  ≡ Fe(II) + H 2 II) + H 2 O 2  ≡ Fe(III)-OH + HO • Five minerals were selected in this study, including kaolinite (98%, Aladdin Reagent Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.Company, Shanghai, China), montmorillonite (98%, Aladdin Reagent Company, Shanghai, China) and synthetic hematite, goethite and ferrihydrite.All of the three iron minerals were synthesized by a previously described method (Schwertmann and 110 Cornell, 2007).In brief, ferrihydrite was prepared by dissolving 40 g Fe(NO 3 ) 3 •9H 2 O in 500 mL deionized water, and then 330 mL of 1 M KOH was added.Goethite was prepared by mixing 180 mL of 5 M KOH with 100 mL of 1 M Fe(NO 3 ) 3 •9H 2 O, and then the resulting mixture was aged for 60 h at 70 °C.Hematite was synthesized by mixing 2 L of 0.002 M HNO 3 (98 °C) with 16.16 g of 115 Fe(NO 3 ) 3 •9H 2 O and then aging for 7 d at 98 °C.Once prepared, all three suspensions were dialyzed with deionized water for 3 d to remove impurity ions, and then the pellets were air-dried.Powder X-ray diffraction (XRD) and FTIR analysis results for the used minerals are shown in Figs.S1-S2.All minerals were crushed and sieved through a 0.149 mm screen.120

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Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.fresh medium (10 mL) so that the effects of minerals were negligible.After 8 h growth, bacterial growth was monitored by measuring OD 600 of the new culture and the photographs are shown as Fig. S3.The control experiment was performed without any mineral.All experiments were performed in triplicate.The particle size distribution of the applied raw minerals and the minerals after 12 h of incubation is 135 listed in Table

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step size with a Si drift detector.Standard samples of hematite, goethite, ferrihydrite, iron(II) oxalate, and iron(III) oxalate were recorded in transmission mode.Iron(II) oxalate and iron(III) oxalate represent organic complexing ferrous and ferric, respectively, whereas hematite, goethite and ferrihydrite were used as the main iron mineral species.Linear combination fitting of standards was also performed for the 170 µ-XANES spectra of samples, using ATHENA software (version 2.1.1).A standard was considered to have a substantial contribution if it accounted for more than 10% of a linear combination fit.Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.
Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.high-sensitivity cavity.Individual spectra were recorded over scan ranges of 500 and 195 30 mT to observe the signals originating from transition metal ions and free radicals, respectively.Details of additional spectra and all other acquisition parameters are

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may dominates the generation of HO • and ultimately the inhibition of Pseudomonas brassicacearum J12.To test whether the release of Fe(III) to solution inhibit the growth of Pseudomonas brassicacearum J12 via a free-radical mechanism, we replaced Fe(III)-containing minerals by adding a series of concentrations of Fe(NO 3 ) 3 , i.e., 0, 50 and 100 mg L -1 , in the cultivation experiments with the final pH of 7.2.The Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.results showed that addition of Fe(III) can inhibit the growth of Pseudomonas brassicacearum J12 (25-50%) by producing an additional HO • concentration of 15 nM (Fig. S7), supporting the role of Fe(III) ion from solution in the initialization of a free-radical reaction.
situ observation of Fe species and the distribution of organic functional groupsTo explore the critical role of Fe chemistry in the inhibition of the growth of Pseudomonas brassicacearum J12, we used correlative µ-XRF and SR-FTIR analyses for in situ measurement of the distribution of Fe species and EPS on the 300 surface of ferrihydrite.The µ-XRF spectromicroscopy showed a distinct density of Fe distributed on iron particles (Fig.5a).Two positions were selected for identifying the coordination state and species of Fe by µ-XANES spectra.Using hematite, goethite, ferrihydrite, iron(II) oxalate, and iron(III) oxalate as reference compounds, the linear combination fitting (LCF) results from Fe K-edge µ-XANES spectra 305 indicated that ferrihydrite was dominant (~82%), with a lesser percentage (~17%) of FeC 2 O 4 among the mineral particles (Spot B in Fig.5b and Table Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.
1 and 3), suggesting that reactive surface area and solubility has a significant effect on enhancing formation of HO • and bacterial inhibition activity.Reactive mineral surfaces can catalyze HO • generation or act as Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.
Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.Fe(III)-containing minerals mainly depends on the coupled effect of soluble Fe, surface Fe(II), and extracellular HO • .4.3.Inhibition of bacterial growth by a free-radical mechanism and its430 implications for soil carbon storageIn this study, we proposed a schematic of Fe(III)-containing minerals inhibiting bacterial growth through a free-radical mechanism (Fig.7).Surface Fe(II) is produced from the reduction of Fe(III) on the surface of Fe(III)-containing minerals, promoting the production of HO • through the Fenton or Fenton-like reactions (Garrido-Ramírez 435 et al., 2010).Oxidative damage of extracellular HO • may lead to bacterial inactivation, and protection of carbon from microbial degradation.In addition, the generation of free radicals may also have indirect effects on bacterial growth via substrate availability (Table 450study, but it is far away from organic matter decomposition or substrates available in Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.

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Fe(III)-containing minerals promoted more HO • generation and thus increased suppression to Pseudomonas J12 via a free-radical mechanism.Furthermore, our results revealed that surface Fe(II) was produced on the mineral surface that may act as a catalyst, promoting the generation of HO • rather than soluble Fe.The generation of HO • by Fe(III)-containing minerals follows the order ferrihydrite > goethite > 465 hematite.In addition, the generation of free radicals may also have indirect (i.e., substrate availability) effects on bacterial growth and the presence of minerals may potentially interfere with the measurement of cell numbers.In summary, our findings indicate that the inhibition of bacteria with Fe(III)-containing minerals mainly depends on the coupled effect of soluble Fe and extracellular HO • , which may 470 further contribute to soil carbon storage.Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.compound oxidation in mineral-catalyzed Fenton-like systems, Environ.Sci.Technol., 37, 1150-1158, 2003.Zhou, T., Chen, D., Li, C., Sun, Q., Li, L., Liu, F., Shen, Q., and Shen, B.: Isolation and characterization of Pseudomonas brassicacearum J12 as an antagonist 635 against ralstonia solanacearum and identification of its antimicrobial components, Microbiol.Res., 167, 388-394, 2012.Biogeosciences Discuss., https://doi.org/10.5194/bg-2018-479Manuscript under review for journal Biogeosciences Discussion started: 10 December 2018 c Author(s) 2018.CC BY 4.0 License.

Figure 2 .
Figure 2. Wide scan EPR spectra of both the kaolinite and montmorillonite.

Figure 2 .
Figure 2. Wide scan EPR spectra of both the kaolinite and montmorillonite.675

Figure 5 .
Figure 5. Correlative micro X-ray fluorescence (µ-XRF) and synchrotron-based Fourier transform infrared (SR-FTIR) analysis of the thin section from the cultures of690

Figure 7 .
Figure 7. Schematic of the bacterial inhibition by Fe(III)-containing minerals through a free-radical mechanism.