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Evidence for photochemical production of reactive oxygen species in desert soils


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Received 14 Jul 2014 | Accepted 1 Apr 2015 | Published 11 May 2015
DOI: 10.1038/ncomms8100

Evidence for photochemical production of reactive oxygen species in desert soils
Christos D. Georgiou1, Henry J. Sun2, Christopher P. McKay3, Konstantinos Grintzalis1, Ioannis Papapostolou1, Dimitrios Zisimopoulos1, Konstantinos Panagiotidis1, Gaosen Zhang4, Eleni Koutsopoulou5, George E. Christidis6 & Irene Margiolaki1

The combination of intense solar radiation and soil desiccation creates a short circuit in the biogeochemical carbon cycle, where soils release signi?cant amounts of CO2 and reactive nitrogen oxides by abiotic oxidation. Here we show that desert soils accumulate metal superoxides and peroxides at higher levels than non-desert soils. We also show the photogeneration of equimolar superoxide and hydroxyl radical in desiccated and aqueous soils, respectively, by a photo-induced electron transfer mechanism supported by their mineralogical composition. Reactivity of desert soils is further supported by the generation of hydroxyl radical via aqueous extracts in the dark. Our ?ndings extend to desert soils the photogeneration of reactive oxygen species by certain mineral oxides and also explain previous studies on desert soil organic oxidant chemistry and microbiology. Similar processes driven by ultraviolet radiation may be operating in the surface soils on Mars.

1 Department of Biology, University of Patras, Patras 26504, Greece. 2 Desert Research Institute, Las Vegas, Nevada 89119, USA. 3 NASA Ames Research Center, Moffett Field, California 94035, USA. 4 Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, Lanzhou 73000, China. 5 Laboratory of Electron Microscopy and Microanalysis, University of Patras, Patras 26500, Greece. 6 Department of Mineral Resources Engineering, Technical University of Crete, Chania 73100, Greece. Correspondence and requests for materials should be addressed to C.D.G. (email: c.georgiou@upatras.gr).

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n most ecosystems, microorganisms carry out mineralization of soil organic matter. In desert soils, however, non-biological and biological organic decomposition can be comparable in magnitude. For example, soils from the Atacama Desert, Chile, can oxidize D-alanine and L-glucose at signi?cant rates, although terrestrial organisms do not utilize these enantiomers1,2. In the Mojave Desert, California, nitrogen oxide (NOy) gases are emitted from desiccated soils3 at rates comparable to wet soils only if the soils are irradiated by sunlight. In other arid and semiarid locations, photochemical degradation reportedly accounts for 60% of plant litter decomposition4. Studies on the photochemical oxidation of xenobiotic organic carbon (herbicides, insecticides and pesticides) in agricultural soils suggested that 1O2 generated by sunlight via the photosensitization of soil organics (and possibly of Ti- and Zn-oxides) to be the main oxidant5. The involvement of other reactive oxygen species (ROSs) such as ? ? ) and hydroxy ( ? OH) radical has been superoxide (O2 hypothesized as well6–11. OH is postulated to result indirectly from the reaction of water with humic substances in the excited triplet state or from degradation of H2O2 by transition metal (Metr) cations such as Fe3 ? via a photo-Fenton reaction10. However, the photochemical oxidative process and its components are not well understood because of a lack of methods for the detection of the involved ROS in soils and their very short life (1 ns to 1 ms (ref. 12)). Photogenerated ROS have been identi?ed in laboratory studies on mineral oxides (mainly TiO2) exposed to ultraviolet radiation: ? ?, atomic oxygen radical anion (O ? ? ), ozone radical anion (O3 ? ? ), O ? ? and ? OH13–15. The postulated process and possibly O4 2 in these studies involves ultraviolet-induction of electron mobilization from the oxide surface and its subsequent capture by O2 to form oxygen radical surface adsorbates16. This photoinduced mechanism is also supported by the previously shown ? ? in ultraviolet-exposed plagioclase feldspars17. generation of O2 1 Singlet oxygen ( O2) also has been identi?ed18,19 and may oxidize soil organics via intermediate oxidant products but is not adsorbed on mineral surfaces because it is uncharged. Degradation of organic compounds, mainly via ? OH, can be catalysed by the photochemistry (via reduction) of iron (III) species such as Fe(OH)3, FeOOH and Fe2O3 (ref. 20), besides TiO2-mineral oxides. Here we show that metal superoxides and peroxides accumulate in topsoils of the Atacama and Mojave deserts (at midsummer sunlight irradiance 800–1,000 W m ? 2) at a 10-fold higher level than in a non-arid control site. Moreover, we show that desiccated and aqueous desert soils photogenerate equimolar ? ? adsorbates and ? OH, respectively, and also that the O2 reactivity of desert soils is associated with the generation of ? OH and the concomitant decrease of soil H O in their aqueous 2 2 extracts in the dark, possibly via the Fenton reaction due to the presence of Metr in these soils. For this study, new methods ? ? , H O and ? OH. These enable the identi?cation of soil O2 2 2 methods are not yet suitable, however, for in situ studies on the

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seasonal and soil depth-dependent formation rates of detected soil oxidants. Moreover, their speci?city restricts the evaluation of only a fraction of the photogenerated ROS involved in the oxidative desert soil chemistry. Although the elucidation of the entire model of desert soil ROS photogeneration is not possible with the available methods, they unveil some key steps as they take place by a photo-induced electron transfer mechanism. In addition, our selected soil desert sites are among the most studied of all the world’s arid locations1,21,22, and thus provide a good basis for considering the environmental effects of aridity on the photochemical production of ROS in desert soils. Results .? Detection and stability soil O2 /peroxides and detection of ? ? from desicMetr. We extracted soil metal superoxides as O2 cated Atacama and Mojave Desert topsoils by acetonitrile and quanti?ed ?uorometrically by a modi?ed hydroethidine (HE)? ? and validity of the HEbased assay23,24, and the identity of O2 based assay were veri?ed by a second assay25,26, a modi?cation of the superoxide dismutase (SOD)-inhibited equimolar reduction ? ? (Methods, Supplementary Methods). The of cyt. c by O2 ? ? detection was determined in methodological baseline for O2 ? ? by H2O-prewashed and dried soil, a treatment that destroys O2 dismutation12: 2O:2? ? 2H2 O ! 2OH ? ? H2 O2 ? O2 ? 1? Soil metal peroxides (peroxides/hydroperoxides) were quanti?ed upon aqueous extraction as H2O2 (their hydrolysis product shown in reactions 2–4; refs 27,28):
? 2? O2 ? 2H2 O ! 2Me ? OH ? ? H2 O2 Me2 ? 2? ?OH ? ?2 ? H2 O2 Me2 ? O2 2 ? 2H2 O ! Me

? 2? ? 3? ? 4?

MeO2 H ? H2 O ! MeOH ? H2 O2

Soil metal peroxides can be present, however, in mixture with metal superoxides that on aqueous extraction also release H2O2 (reaction 1). Thus, the extracted soil H2O2 is the sum of two fractions that can be quantitatively differentiated. The ?rst H2O2 fraction results from equimolar hydrolysis of metal peroxides (equations 2–4). The second is differentiated from the ?rst because it results from dismutation of ? 2 molar metal superoxides (equation 1). Total soil H2O2 extracted from metal superoxides plus peroxides was quanti?ed by a modi?cation of the Fe2 ? -xylenol orange assay (Methods, Supplementary Methods), using catalase as control29. ? ? g?1 Atacama and Mojave Desert soils contained B2.0 nmol O2 (that is, metal superoxides). In contrast, soils from a non-arid control location contained an order of magnitude less concentration of B0.2 nmol g ? 1 (Table 1). However, the dry desert soil is ? ? , while constantly humid soils conducive to preservation of O2 are not (because of reaction 1). Even in the deserts, given the ? ? , only a small fraction would be extreme labile nature of O2

Table 1 | Soil metal superoxides, total superoxides/peroxides (H2O2) and Fe3 ? .
Soil oxidants*
? ?w O2 H2O2 Fe3 ? w

Atacama Desert sites* 1 1.8±0.6 (0.03) 14±2 (0.05) 20±2 (1.2) Yungay 1.9±0.3 (0.03) 19±2 (0.6) 10±1 (0.6)

Mojave Desert sites* Basalt 2.3±0.4 (0.04) 110±9 (4) 10±3 (0.6) ? CIMA sand 2.0±0.3 (0.03) 5±0.7 (0.4) 12±2 (0.6)

Control site* 0.15±0.06 (0.002) 0.2±0.01 (0.01) 0.2±0.03 (0.01)

? ? (nmol g ? 1) and total superoxides/peroxides as their hydrolysis product H O (in nmol g ? 1 soil). Soil Fe3 ? concentration is expressed in nmol g ? 1. Data *Metal superoxides are expressed as soil O2 2 2 are mean values±standard error from at least ?ve replicates. Numbers in parentheses are in ppm. ? ? ? ? assays. For statistical comparison of both assays, the wValues for soil O2 are averages of the mean values obtained by the HE-based and the SOD-inhibited reduction of cytochrome c-based soil O2 within-day % coef?cient variation is o3.7% and the variance of intermediate precision (or between day repeatability) is o4.8%.

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remove any potentially interfering soil inorganics (for example, perchlorates and nitrates known to exist in these deserts34,35) and organics (for example, photosensitizers, ROS scavengers; ? ? and H O (peroxides) concentrations almost Methods). Soil O2 2 2 doubled or tripled (above baseline; 1.5 and 0.7 nmoles g ? 1, respectively) during the ?rst hour of soil irradiation (Fig. 2). ? ? generated on ultraviolet-irradiated A similar trend for O2 plagioclase feldspar minerals has been observed previously17. Longer irradiation (up to 6 h) resulted in a concentration plateau ? ? (similarly for H O ), irrespective of the radiation source for O2 2 2 (although somewhat higher for the more effective ultraviolet C), which could be due to the saturation of the positively charged ? ? anion. The concentration of sites on the soil surface by the O2 ? ? the soil photogenerated O2 (Fig. 2a) was approximately twice ? ? /H O that of H2O2 (Fig. 2b). This O2 2 2 ratio ?ts the ? ? (reaction 1), which stoichiometry of the dismutation of O2 ? ? is the main soil oxidant formed during indicates that O2 sunlight/ultraviolet C exposure. We analysed the mineral substrates potentially involved in the photogeneration of ROS by the Atacama and Mojave Desert and control soils both in the bulk and the clay fractions of the samples (Methods, Supplementary Methods). All the samples contained plagioclase feldspar with composition that varied from albite to labradorite. Clay minerals were most abundant in the control soil, absent from the Mojave Desert basalt, and found in traces in the remaining samples, in accordance with the Loss On Ignition data in Table 2. All the soils contain traces of Fe-Ti- and/or Fe-Mnand/or Fe-oxides and/or Ti-oxides. ?? It has been established that aqueous TiO2 photogenerate O2 ? 15,36 and OH , the latter being photogenerated by aqueous TiO2
Table 2 | Bulk sample chemical analysis.
Major elements (wt %) SiO2 TiO2 Al2O3 Fe2O3t MnO MgO CaO Na2O K 2O P2O5 LOI* Total Atacama desert Site 1 63.10 1.11 15.98 5.26 0.07 1.20 4.55 3.99 2.41 0.16 2.09 99.92 Yungay 61.80 0.81 13.45 5.43 0.07 1.52 5.31 3.82 2.37 0.16 5.73 100.47 Mojave desert Basalt 46.31 2.48 15.80 10.29 0.13 7.31 9.28 4.09 1.96 0.68 1.39 99.72 ? CIMA 67.07 0.66 14.05 4.47 0.05 1.43 3.22 3.50 3.10 0.17 2.30 100.02 Control site

stabilized in soils. The Mojave and Atacama Desert soils also contain metal peroxides (Table 1) because the concentration levels of the extracted total soil H2O2 are 5–95 times higher than the H2O2 expected from the (dismutation of) the detected soil metal superoxides. The levels of H2O2 we detected in both Atacama Desert sites are similar to those previously reported1. The detected soil metal superoxides and peroxides were stable even when the desiccated desert soils were heated at 200 °C for 1 h. This agreed with the ?nding that the desert soils retained B50% of their initial concentrations in metal superoxides and peroxides after storage for 1year (desiccated in the dark at B16 °C; data not shown). The extracted H2O2 was not stable through time, however, as almost 50% was destroyed within 1 day in the dark (Fig. 1a). This could be because of its decomposition to ? OH by co-extracted Metr via the Fenton reaction12,30, as supported by the identi?cation of Fe and other Metr (for example, Cu, Cr, Co, Ni) in these soils (Tables 1 and 2) and the participation of these in the Fenton reaction31. Indeed, desert soil H2O2 extracts generated ? OH through time (Fig. 1b, Methods, Supplementary Methods), the rate of which in the presence of soil decreased by B6-fold probably because of ? OH scavenging by soil organics32 and/or metal (Me) ions (via the general reaction: ? OH ? Men ? H O-Men ? OH-Me(n ? 1) ? ? OH ? ; ref. 33). 2
.? and . OH, and mineral analysis. Photogeneration of soil O2 ROS were photogenerated (by natural sunlight and ultraviolet C) by desiccated soils. Before exposure soils also were washed to
2

0.9

Control H2O2 Yungay H2O2 exract +CIMA H2O2 extract

1.5 0.6 H2O2 (?M) ˙OH (?M)

˙HO (Yungay H2O2 extract) ˙HO (+CIMA H2O2 extract) ˙HO (Yungay H2O2 extract +soil) ˙HO (+CIMA H2O2 extract +soil)

1

0.3 III 0.5 II

I 0 0 10 20 30 40 Incubation time (h) 50 0 10 20 Retention time (min)
.

Figure 1 | Instability of acid-extracted soil H2O2 and generation of OH. Soil H2O2 results from the dismutation/hydrolysis of metal superoxides/ peroxides, respectively. (a) Decreasing exponential solid line curves depict the change of 10 mM H3PO4-extracted soil H2O2 concentration (initially adjusted to 2 mM, and in the absence of the extracted soil) from the Mojave and Atacama ? CIMA and Yungay sites (?lled rhombs and squares), respectively, versus time, against a 2.0 mM H2O2 (in 10 mM H3PO4) control solution (?lled circles). Increasing exponential line curves depict the generation (versus time) of ? OH in the H2O2 extracts from the same Mojave and Atacama sites (open rhombs and squares, respectively) in the absence and presence of soils (solid and dotted lines, respectively). (b) ? OH was detected from its reaction with TPA(added to the H2O2 extracts at 2 mM) and the equimolar production of the speci?c ?uorescent product HTPA70. HTPA was quanti?ed and identi?ed ?uorometrically by HPLC (I, using as controls the ? OH scavenger DMSO, II, and pure HTPA, III). Similar data (not shown) were obtained for H2O2 extracted from total peroxides from the other tested Atacama and Mojave Desert soil sites. Error bars designate s.d.

Fluorescence units

HTPA

30

55.39 0.53 9.21 4.02 0.08 2.03 12.13 1.32 1.62 0.10 12.92 99.35

Trace elements (p.p.m.) V 172 Cr 38 Co 8 Ni 14 Cu 50 Zn 50 Rb 62 Sr 390 Y 17 Zr 201 Ba 681 La 23 Ce 33 Nd 18 Pb 18 Th 7
*LOI, loss on ignition.

164 31 11 15 81 79 88 604 20 185 744 30 44 17 39 9

215 146 164 120 89 89 41 807 35 320 599 41 73 29 16 5

76 27 7 14 21 47 107 448 27 340 711 36 71 39 33 12

78 202 17 100 46 105 73 195 22 150 230 21 42 24 48 8

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40 Yungay site Yungay site (+DMSO) +CIMA site 30 +CIMA site (+DMSO) 1 ?M H2O2 ˙OH (nM)

nmol O2.– per gram of soil

4

3

2 UV-C 1 Untreated Washed Solar

20

0

100

10
50 0 8 10

nmol H2O2 per gram of soil

0

2

4

h

6

2

0

0

2

4 6 8 Illumination time (h)
.

10

1 UV-C Untreated Washed 0 0 1 2 3 4 Illumination time (h) 5 6 Solar

Figure 3 | Photogeneration of OH by desert soils under aqueous conditions. Soils (from the Atacama Yungay site and the Mojave ? CIMA site) were extensively washed and exposed to light (400–700 nm), against ±100 mM DMSO ( ? OH scavenger) and an H2O2 control, using commercial TiO2 (inset) as additional control, given its presence in the tested desert soils. The other tested desert/control sites gave similar data (not shown). Error bars designate s.d.

? ? was Figure 2 | Photochemical formation of ROS in desert soils. (a) O2 generated in the Mojave Desert ? CIMA site (untreated or washed and then dried), which was exposed to solar (natural sunlight) and ultraviolet C irradiation versus exposure time. (b) Generation of total metal superoxides/peroxides (expressed as H2O2) in the same ? CIMA site under irradiation conditions as in a. Similar data (not shown) were obtained in the other tested desert/control sites. Error bars designate s.d.

Sunlight
O2
Desert soil Oxides +e– CB e.g. TiO2 +h+ VB Fe2O3 H+ H2O – –e OH– –e–

CO2 O2
.–

˙OH (?M)

TiO2

150

NOy

Metal superoxides Dismutation Hydrolysis

Soil organics oxidation

H2O2

Metr (Fenton reaction)

.

OH

Sunlight

Metal peroxides

Dark

?lms as well37,38. The presence in the tested desert soils of Ti-based transition metal oxides prompted us to investigate whether these soils photogenerate ? OH under aqueous conditions. Here we show that both Atacama and Mojave soils (0.2 g, prewashed from any soluble inorganic and organic constituents) photogenerate an average of 3.2 nm ? OH h ? 1 (Fig. 3) upon exposure to 180 W m ? 2 400–700 nm (or 18% that of sunlight) . This rate is proportional to light intensity as well (data not shown), obeying zero-order reaction rate kinetics as in aqueous TiO2 ?lm photocatalysis38. We identi?ed ? OH and quanti?ed its rate of photogeneration by a modi?cation of the terephthalic acid (TPA)-based assay37,38 and by the B15-fold rate decrease caused by the ? OH scavenger dimethyl sulfoxide (DMSO; Methods). ? OH is not photogenerated by exposing a control H2O2 solution, given that H2O2 is released from metal superoxides/peroxides under aqueous conditions. Generation of 3.2 nM ? OH in 1 h by 0.2 g soil exposed to 180 W m ? 2 or 18% that of sunlight corresponds to B1 nmol g ? 1 for 1 h exposure to sunlight (see Supplementary Methods). This is ? ? g ? 1 that is similar to the plateau rate of 1.45 nmol O2 generated by desiccated (and prewashed) desert soils after exposure to sunlight for 1 h (Fig. 2a). This near 1:1 molar ? ? /OH ratio is in agreement with the mechanism of TiO O2 2 ?? photocatalysis15,36, which assumes equimolar ? OH and O2 aqueously generated; the latter also being photogenerated in desiccated soils by the same mechanism (Fig. 4). The ratio 1:1.5 deviation from the equimolar ratio could be due to various factors
4

Figure 4 | Photochemical mechanism of desert soil oxidizing action. Sunlight radiation of soils generates unpaired electrons that convert O2 to ? ? . This radical can be initially adsorbed on the surface of soils and O2 stabilized in the long term as metal superoxides and peroxides. These can be converted under aqueous conditions (via dismutation and hydrolysis, respectively) to H2O2, which can be converted subsequently to hydroxyl radical ( ? OH) in the dark by reaction with reduced soil transition metals (Metr). A second route of ? OH generation may involve a single electron abstraction from H2O or OH ? by an h ? VB hole (see text). ? OH (and also ? ? , although to a lesser degree as weak oxidants) can then H2O2 and O2 oxidize soil organics possibly to CO2 and NOy.

such as the non-aqueous/aqueous generation of additional ROS ? ? , O ? ? ; refs 13–15), the not species (for example, O ? ? , O3 4 accounted ultraviolet component of natural sunlight, and the possible incomplete trapping of ? OH by the assay reagent TPA. Generation of ? OH by photodissociated H2O2, under the employed experimental conditions, may be negligible as shown by the corresponding assay control (Fig. 3). The aqueous photogeneration of ? OH by metal oxides in desert soils also was supported by our control experiment with TiO2. Tested at a quantity equivalent to its w% concentration in the soils (Table 2), TiO2 produced ? OH at B13 mM h ? 1 (Fig. 3 inset). This B4,000fold higher rate compared with the desert soils is because of the very small particle size of TiO2 (photocatalysis grade, o30 nm in size; ref. 39), that results in a highly disordered structure and high

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superoxides such as KO2 and NaO2, (ii) metal peroxides such as Na2O2, CaO2, K2O2 and TiO32H2O and TiO22H2O (both less water soluble) that are usually formed by the reaction of the metal with H2O2 and (iii) metal hydroperoxides such as NaOOH, (KOOH)23H2O and Ti(OH)3OOH)27,28. ? ? as soil adsorbate and The photochemical production of O2 stabilized as metal superoxides/peroxides could explain the reactivity of desert soils with organics (Fig. 4) under several considerations. Soil H2O2 (for example, aqueously generated via reactions 1–4) can decompose carbohydrates to formic acid and ultimately to CO2 (ref. 47). The release of NOy from the Mojave Desert soil3 and N2O from the Atacama Desert soil48) could have resulted from ? OH by the following mechanism: ? OH can oxidize amino acids with ammonia as a byproduct. Ammonia can be further oxidized by ? OH to amidogen radical49, that can subsequently react with O2 to form nitric oxide radical ( ? NO), dioxide radical ( ? NO2) and N2O (refs 49–51). Hydroxyl radical can be generated from H2O2 (the product of dismutation/hydrolysis of metal superoxides/peroxides, respectively) either by the Fenton reaction (5) catalysed by Metr or by ? ? via the Me -catalysed Haber–Weiss reaction (6)30: O2 tr
n?1 H2 O2 ? Men ? OH? ? ? OH tr ! Metr

speci?c surface area compared with the natural TiO2 polymorphs and ilmenite (that is, Fe–Ti oxides) found in the tested soils. Discussion Superoxide radical is photogenerated in desert soils (Fig. 2a) and stabilized initially as surface-saturating desiccated anionic adsorbate, given that it is the least reactive hence the most stable species formed by ultraviolet exposure17. Long-term diffusion of ? ? may be the subsequent stabilization process, as metal O2 superoxides and peroxides were detected in the Atacama and Mojave Desert soils. This is in agreement with the ?nding that the average concentration of metal superoxides in a basalt rock (pulverized) from the Mojave Desert ? CIMA ?eld is similar to their concentration in the surrounding sand (Table 1), suggesting ? ? diffusion inside basalt rocks. Such diffusion a long-term O2 through soils does not occur within the short irradiation period used in our and similar studies (with labradorite), since this is considered geologically very short17. ? ? is In general, the main conditions under which stable O2 formed on the surface of metal oxides and zeolites are (i) photoinduced electron transfer, (ii) direct surface-oxygen electron transfer, (iii) surface intermolecular electron transfer and (iv) the decomposition of H2O2 (ref. 40). The photo-induced electron transfer mechanism (Fig. 4) could involve ejection from transition or non-transition metal oxides (for example, TiO2, Fe2O3 (ref. 20), or ZnO (ref. 41), respectively) of an electron (e ? CB) into its CB (conduction band) from its valence band ? ?, (VB), and its transfer to O2 with concomitant generation of O2 leaving a positively charged hole (h ? VB) at the band edge of the ? ? , an adsorbed ? OH may be formed on the VB. In addition to O2 oxide surface, from an e ? abstracted by the h ? VB hole from H2O or OH ? (refs 15,36,40,42). Nonetheless, O2 may not be the only acceptor of the photogenerated single electrons, as 1O2 also possesses suf?cient one-electron reduction potential to form ? ? (ref. 43). Then, the photogenerated O ? ? could be adsorbed O2 2 on the surface of minerals where it could saturate their positively ? ? may migrate via diffusive charged sites. In the long term, O2 transport to greater depths in the soils, where it may stabilize as metal superoxides and peroxides (Table 1). Metal superoxides/peroxides are better preserved (protected from dismutation/hydrolysis, respectively) in desiccated soils than in soils containing water42. This, in turn, depends on the clay mineral composition. Swelling clay minerals (smectite and vermiculite), that were more abundant in the control site soil, readily adsorb H2O molecules, thus contributing to greater water ? ? and metal adsorption44 and possibly to lower stability of O2 peroxides. This assumption could explain the observed 10-fold lower concentration of metal superoxides and the much lower concentration of metal peroxides in the control soil compared with the desert soils (Table 1). Moreover, the presence of albite and labradorite plagioclase in the Atacama and Mojave Desert soils, respectively, is in agreement with the previously proposed ultraviolet-induced ? ? generation by these minerals17,42. The mechanism of O2 same mechanism might also apply for the albite-bearing control site soil. Also, metal superoxides and peroxides may be continuously photogenerated in all soils because TiO2, Feoxides and Fe-Ti-oxides—present in the tested soils and typical of desert soils45—are known to be involved in the ? ? (refs 13–15,17,36). In addition photogeneration process of O2 to Ti (in Ti- and/or Fe-Ti-oxides) as well as Fe and Mn (in Feand/or Mn-oxides either free or impurities in silicate minerals), ? ? as the long-term stabilization process for adsorbed soil O2 metal superoxides and peroxides also may involve (besides Ti46) the exchangeable metals Ca, Na and K that are present in ? ? may be stabilized as (i) metal smectite. Speci?cally, O2

? 5? ? 6?

O?2? ? H2 O2 !? OH ? OH ? ? O2

The Fenton mechanism is considered probable because Metr (Fe3 ? and trace elements Cu, Cr, Co and Ni) were identi?ed in Mojave and Atacama Desert soils (Tables 1 and 2). Metr might exist in the desert soils in their reduced and/or oxidized form and can participate in the Fenton reaction (5) with their reduced form directly. For example, Fe2 ? might occur as interlayer species such as the Fe2 ? -aquo complex in the 2:1 clay minerals52. Also Fe3 ? may participate in the Fenton reaction indirectly after its ? ? (O ? ? ? Men ? 1 reduction by H2O2 (ref. 30) or O2 2 tr ? O ; the sum of this reaction with the Fenton reaction 5 Men tr 2 is the Metr-catalysed Haber–Weiss reaction 6; ref. 12). Therefore, ? ? to the highly Metr can convert the weak oxidants H2O2 and O2 reactive ? OH. We showed that the instability of the Atacama and Mojave Desert soil metal superoxides/peroxides (as H2PO4-extracted H2O2) in the dark is associated with the production of ? OH (Fig. 1). The possible involvement of the Fenton reaction mechanism can be implied by the concurrence of the higher (threefold) production of ? OH in Atacama (Yungay site) over Mojave ( ? CIMA site) with the higher concentration of Metr (four and twofold higher in Cu and Co), respectively (Table 2). H2O2 could be destabilized by other factors such as alkaline pH (via the hydrolysis reaction: 2H2O2-2H2O ? O2; ref. 53). However, this is not a likely scenario given the stabilizing effect of phosphates and acidic pH on H2O2 (ref. 54), with the latter con?rmed by the relative stability of a control H2O2 solution in 10 mM H3PO4 (Fig. 1a). Therefore, the identi?cation of ? OH in the Atacama aqueous soil extracts (Fig. 1a) could explain the previous observation of the oxidation (to CO2) of sodium formate and aqueous mixtures of chiral sodium alanine and glucose after incubation for several days with Atacama Desert soil even in the dark, albeit at a slower rate1. The reactivity of the Atacama Desert soils with organics in the dark may not proceed by direct involvement of 1O2, as its lack of charge would hinder its adsorption on the surface of soils. Moreover, non-light-induced abiotic oxidation of organics also may be involved in desert soils because the process has shown that Mn-oxides and Fe-oxides/ hydroxides common in desert soils (and in the tested ones, Table 2) could cause oxidation of organics (for example, 17betaestradiol55) and aromatic amines56. Similarly, perchlorates
5

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(present in both Atacama and Mojave Deserts34,35) could be potential oxidants of soil organics57–59. The oxidation of soil organics by photogenerated ROS may be a continuous process given that Metr are ubiquitous in all soils. ? ? may represent a fraction of the ROS produced in photoO2 irradiated desert soils, however. Another ROS could be 1O2 because it is generated—and oxidizes organics—by photoirradiated minerals such as MgO, Al2O3 and SiO2 (refs 18,19,60), which are common in desert soils and also found in the tested soils (Table 2). Moreover, 1O2 could be formed directly ? ? with H O (refs 61,62), and possibly by the by reaction of O2 2 2 ? ? on irradiated surface decomposition of light-generated O2 ? ? because it possesses the soils18. In addition, 1O2 can form O2 required one-electron reduction potential ( ? 0.65 V at pH 7.0), while its two-electron reduction could generate H2O2 (ref. 43). ? ? from 1O could have been Therefore, photogeneration of O2 2 possible under non-aqueous conditions as long as it involves a highly reactive single electron photo-ejected from soil minerals or donated by soil organic photosensitizers. ? ? is photogenerated by The present study also showed that O2 ? ? is generated at similar levels even by desert soils (Fig. 2a). O2 soils prewashed before exposure from polar inorganic and polar/ non-polar organic soil constituents, suggesting a mechanism not involving photosensitization and the involvement of perchlorates. These prewashed soils also photogenerated ? OH under aqueous conditions (Fig. 3) and at a near 1:1 molar rate ratio with the ? ? that is generated (in the 1st hour) by sunlight-exposed O2 desiccated soils (Fig. 2a), in agreement with the mechanism for TiO2 photocatalysis15,36. The same mechanism also could apply for the desert soils (Fig. 4) since they contain transition metal oxides such as TiO2 and Fe-Ti-oxides (Table 2). Moreover, the photo-catalytic generation of ? OH by aqueous desert soils could provide a light-based mechanism for their reactivity with organics. This would involve the photo-catalytic ejection of an ? ? by dissolved O ) electron from metal oxides (to generate O2 2 and the subsequent abstraction of an electron by the resulting h ? VB hole from H2O or OH ? (resulting also from H2O via reaction with a bridging-oxygen atom15). Then, ? OH (and H2O2 to a lesser degree) could oxidize soil organics to CO2 and NOy. The photogenerated ROS detected in desert soils also may explain the ionizing radiation resistance of certain desert bacteria—notably Deinococcus radiodurans and Chroccocidiopsis —that can withstand extraordinarily high levels of ionizing radiation63. Radiation resistance is regarded as a proxy for desiccation resistance63. Ionizing radiation also produces reactive oxidants similar to photochemical oxidants64. Thus, the ability to withstand photochemical oxidants might explain the resistance of desert bacteria to radiation. Finally, an extreme example of this soil oxidant chemistry appears to take place on Mars. The oxidative reactivity of the Martian soil as measured by the Viking Biology Experiments (and exhibited on the organics of a certain nutrient by releasing O2 and CO2 (refs 65,66)), requires a mix of oxidants in the soil including ?? oxidants capable of destroying organics57,67. Production of O2 by the photo-induced transition metal oxide surface-O2 electron transfer mechanism has been suggested17. Other alternative potential pathways may involve (i) photogeneration of ROS on mineral substrates (for example, plagioclase) that can generate ? ? from adsorbed O on TiO and (ii) photoactive on Mars O2 2 2 Fe-oxides such as hematite being analogous to TiO2 in surface catalysis42. The source of O2 needed for the photogeneration of ? ? in Martian topsoil under an O -depleted atmosphere, O2 2 however, has not been elucidated. It could be that O2 might have been released by perchlorate (given its abundance on Mars68,69) on exposure to cosmic radiation. This is supported by the generation of O2 by perchlorate exposed to gamma rays in a CO2
6

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8100

atmosphere57. Nonetheless, the buildup of soil ROS oxidants on Mars would be less impeded by liquid water than even the driest desert on Earth. Accumulated metal superoxides and peroxides were detected in soils from the Atacama (hyper-arid) and Mojave (arid) deserts. ? ? at saturaMoreover, desiccated desert soils photogenerate O2 tion levels, followed by long-term stabilization by diffusion as soil metal superoxides and peroxides. Desert soils photogenerate ? OH ? ? by under aqueous conditions. The photogeneration of O2 desiccated or aqueous soils may proceed via capture by O2 of a single electron that is photo-ejected from metal oxides (for example, TiO2/Fe2O3 detected in the tested soils). The resulting h ? VB hole in H2O-wetted soil metal oxides can, then, abstract a single electron from H2O or OH ? and generate ? OH. Moreover, aqueous desert soil metal superoxides/peroxides are converted (by dismutation/hydrolysis, respectively) to H2O2, the observed instability of which in the dark concurs with the generation of ? OH, possibly via the Fenton reaction due to the presence in these soils of Metr. The generation of ? OH, in the dark (via the Fenton reaction) and by photocatalysis could explain the abiotic ? ? acting oxidation of organics by desert soils, with H2O2 and O2 as weak oxidants. Similar ultraviolet-induced processes may take place in the topsoils of Mars. Methods
Samples and reagents. Full details of sample collection and treatment are available in the Supplementary Methods section, with details of the reagents used. HE-based soil O2 ? assay. Assay principle and other supporting control experiments are described in full in the Supplementary Methods. Reagent solutions. 100% alkaline acetonitrile. It is made by mixing 100% acetonitrile (ACN) with 0.1 M NaOH at a 100:1 ratio. At this NaOH proportion (it mixes almost completely as it leaves a small aqueous droplet in the bottom of the tube, resulting in B99% ACN) and higher, the resulting ACNalk solvent is saturated with hydroxyl ions, reaching a stable pH 8–8.5. For the pH measurements a Sentron Argus-type pH Meter was used, equipped with a non-glass Hot-Line electrode (Sentron, The Netherlands). 100 mM CE. It is prepared by dissolving 186 mg dicyclohexano-18-crown-6 ether (CE) in 5 ml ACNalk. Prepare fresh, keep at 4 °C. Caution: accurate pipetting of the reagent is required. ? ? ACN -CE extraction solutions. Solution ACN -CE (0.2 mM) is Soil O2 alk alk prepared by mixing 29.94 ml ACNalk with 60 ml 100 mM CE. This standard solution is designated also as ACNalk-CE. ACNalk solutions containing other CE concentrations can be prepared from the 100% ACNalk and 100 mM CE stocks. For example, ACNalk-CE (1 mM) solution is made by mixing 29.7 ml ACNalk with 0.3 ml 100 mM CE. The ACNalk-CE extraction solutions should be prepared fresh, and kept in an airtight and light-protected glass container at 4 °C. 0.5 mM HE. Prepare fresh (and kept at 4 °C, protected from light) by dissolving 1.2 mg HE in 7.5 ml ACNalk-CE (0.2 mM) solution. Caution: accurate pipetting of the reagent is required. 50 mM HCl. Prepare by diluting ? 240 (with ddH2O) the concentrated 37% HCl (or 12 M). Caution: accurate pipetting of thr reagent is required. 1 M Tris-base, pH 8.0. Caution: accurate pipetting of the reagent is required. Protocol. Soil samples were treated as follows: 1 g soil is placed in a 15-ml plastic centrifuge tube (with conical bottom) with 2.5 ml ACNalk or ACNalk-CE (for example, 0.2 mM) and mixed by shaking for 2–3 min at room temperature (RT; ? ? ), and the soil is removed by precipitation (or ?ltration). The to extract O2 supernatants are diluted appropriately with ACNalk-CE to set the CE concentration in the ACNalk extract to ?nal 0.2 mM and are kept capped in an ice-water bucket. NOTES: I. Soil metal superoxides can be extracted effectively by an ACNalk-CE solution. Testing various concentrations of CE (for example, 0.2, 1 mM) in the ACNalk extraction solution allows the determination of the optimum CE ? ?. concentration for solubilizing the maximum quantity of soil metal salts of O2 ? ? with ACN This can be tested by extracting soil O2 alk mixed with various CE concentrations, to determine a minimum CE concentration above which the ? ? g ? 1 soil is constant. II. For extracting soil-adsorbed O ? ? the extracted O2 2 ACNalk extraction solution does not need the presence of CE and the ACNalk solution can be used alone. However, ACNalk may also extract soil metal superoxides although not as effectively as being in mixture with CE. The ACNalk-CE supernatants from the preceeding step are split in two halves (in two 1.5-ml microcentrifuge tubes), one designated ‘sample’ (S) and the other
.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8100

ARTICLE
two steps in Procedure B) and only those dilutions resulting in proportional FU2-OH-E ? values will be used for determining the ?nal average FU2-OH-E ? value. II. The FURCE-0.2 mM and FURCE-1 mM values can be kept as low as possible by setting the HE ?nal concentration in the assay (see second step in the Assay Protocol) at low levels (for example, 20–40 mM HE). The FURHCl-1 mM ? FURHCl0 mM value should be in the order of 100 FU. Much higher values suggest an excessive autoxidation of HE, and a fresh HE stock solution (see Assay Reagent Solutions) should be used. To convert the net FU2-OH-E ? value to the concentration of 2-OH-E ? ? ? ) in the assay mixture volume (1 ml, in this case), (and subsequently of O2 the spectro?uorometer in use must be calibrated with known concentrations of 2-OH-E ? by determining the ?uorescence extinction coef?cient of 2-OH-E ? ? (FEC2-OH-E , expressed in FU per 1 mM ? 1 2-OH-E ? ) as follows. ? The FEC2-OH-E is determined in ?nal 97% ACN (same as the ACN ?nal concentration in the assay reaction mixture) for the spectro?uorometer (set at high sensitivity) and quartz cuvette in use in this study, as follows: from a synthetic 2-OH-E ? stock solution (see Supplementary Methods) of known concentration, 1 mM 2-OH-E in 1 ml ACNalk (or 1 ml ACNalk-CE extraction solution) is prepared in a 1.5-ml microcentrifuge tube, to that 2 ml ddH2O and 30 ml 0.33 M Tris solution are added, and vortexed, and centrifuged at 12,000g for 1 min. Similarly is treated a reagent blank, which is prepared with 1 ml ACNalk (or 1 ml ACNalk-CE extraction ? solution), containing 2 ml ddH2O and 30 ml 0.33 M Tris solution. FEC2-OH-E is determined by measuring the FU value of the clear 1 mM 2-OH-E ? supernatant (at ex/em 505/580 nm) and subtracting from this value the FU of the clear supernatant of the blank. Having determined FEC2-OH-E ? in the preceeding step, and given the molar ? ? /2-OH-E ? ? 2/1, the netFU stoichiometric ratio O2 2-OH-E ? (determined three steps before) is converted to a mM concentration of 2-OH-E ? (in the assay mixture ? ? by the formula (see derivation in Supplementary volume) and then to mM O2 Methods):   O:2? ?mM? ?2??2-OH-E ? ?in mM??? 2? FU2-OH-E ? =FECex=em 505=580 nm ?2?FU=1300
? ? (extracted from a known soil quantity) are converted to Finally, the mM O2 ? ? g ? 1 soil. nmoles O2

‘blank’ (B), which are centrifuged at 12,000g for 3 min at RT to remove any soil remnants. Then, 880 ml clear supernatant from each half tube are transferred in new tubes to each of which 80 ml 0.5 mM HE are added (40 mM ?nal in the assay). NOTES: I. For this step, sample/blank tubes are kept in an ice-water bath. II. If using quartz micro-cuvette holding a sample volume 0.3–0.5 ml, (cuvette dimensions 45 ? 4 ? 4, mm with the Shimadzu RF-1501 spectro?uorometer), the total sample volume required can be minimized to B1 ml (0.45 ml per S and B), which results in halving the volumes of the reagents added in this and in the subsequent two steps. Thereafter, the following alternative Procedures A and B can be followed: Procedure A: to the B and S tubes, 20 ml 50 mM CE and 20 ml ACNalk-CE (0.2 mM) are added, respectively, followed by the addition in both tubes of 20 ml 50 mM HCl (1 mM H ? concentration in the ?nal assay mixture) and mixing by inversion up to 1 min. For preparing the assay reagent blanks, in each of the two microcentrifuge tubes 880 ml ACNalk-CE (0.2 mM) and 80 ml 0.5 mM HE are added, followed by the addition in one tube of 20 ml 50 mM CE and 20 ml 50 mM HCl (designated RCE-1 mM) and in the other tube of 20 ml ACNalk-CE (0.2 mM) and 20 ml 50 mM HCl (designated RCE-0.2 mM). ? ? (the reaction requires at least NOTES: I. In the S tube, HE reacts with O2 0.5 mM H ? ) and forms the ?uorescent product 2-hydroxy ethidium (2-OH-E ? ; at ? ? /2-OH-E ? ? 2/1) at 0.2 mM CE, while in the B molar stoichiometric ratio O2 tube, this reaction is inhibited by the inclusion of 1 mM CE. II. For the reaction of ? ? in 1 mM HCl to take place, the pH of the assay mixture must be HE with O2 acidic (pH 1.5–2). Possible alkalinity of soil may change this pH, therefore, a test of the assay mixture pH must be performed and if needed the ?nal concentration of HCl can be adjusted appropriately to bring the assay pH to 1.5–2. III. This step and the subsequent two steps are performed as soon as possible to minimize the possible oxidation of HE by any extracted soil constituents. After the 1-min incubation period, the assay mixtures in the S, B, RCE-1 mM and RCE-0.2 mM tubes are mixed with 10 ml 1 M Tris by brief vortexing (to adjust the assay mixture pH to B8), centrifuged at 12,000g for 1 min and the resulting supernatants are collected for ?uorescence measurement. The ?uorescence units (FUs) of the S, B, RCE-0.2 mM and RCE-1 mM supernatants are measured at ex/em 505/580 nm (designated FUS, FUB, FURCE-0.2 mM and FURCE-1 mM, respectively). From these FU, the net FU of 2-OH-E ? is calculated from the following formula: net FU2-OH-E ? ? FUS ? FUB ? ?FURCE ? 0:2mM ? FURCE ? 1mM ?. NOTES: I. To verify that the net FU2-OH-E ? corresponds to the concentration of ? ? , various dilutions of the ACN -CE supernatants (derived in the extracted O2 alk the ?rst two steps of the Assay Protocol) should be tested (as in this and the previous two steps in Procedure A) and only those dilutions resulting in proportional FU2-OH-E ? values will be used for determining the ?nal average FU2-OH-E ? value. II. The FURCE-0.2 mM and FURCE-1 mM values can be kept as low as possible by setting the HE ?nal concentration in the assay (see second step in the ? ? is quite Assay Protocol) at low levels (for example, 20–40 mM HE), given that O2 stable in the ACNalk extraction solvent. The FURCE-0.2 mM ? FURCE-1 mM value is in the order of 50 FU. Much higher values suggest an excessive autoxidation of HE, and a fresh HE stock solution (see Assay Reagent Solutions) should be used. III. In case FUSoFUB, the soil extract may contain a soil constituent, which in the presence of 1 mM CE (in tube B) oxidizes HE to E ? . In this case, the alternative Procedure B is followed. Procedure B: to the B and S tubes, 40 and 20 ml ACNalk-CE (0.2 mM) are added, respectively, followed by the addition in the S tube of 20 ml 50 mM HCl (1 mM H ? concentration in the ?nal assay mixture) and mixing by inversion up to 1 min. For preparing the assay reagent blanks, in each of the two microcentrifuge tubes 880 ml ACNalk-CE (0.2 mM) and 80 ml 0.5 mM HE are added, followed by the addition in one tube of 20 ml ACNalk-CE (0.2 mM) and 20 ml 50 mM HCl (designated RHCl-1 mM) and in the other tube of 40 ml ACNalk-CE (0.2 mM) (designated RHCl-0 mM). ? ? (the reaction requires at least NOTES: I. In the S tube, HE reacts with O2 0.5 mM H ? ) and forms the ?uorescent product 2-OH-E ? (at 0.2 mM CE), while in the B tube, this reaction does not take place (also in the presence of 0.2 mM CE) ? ? in 1 mM HCl to take because HCl is absent. II. For the reaction of HE with O2 place, the pH of the assay mixture must be acidic (pH 1.5–2). Possible alkalinity of soil may change this pH, therefore, a test of the assay mixture pH must be performed, and if needed the ?nal concentration of HCl can be adjusted appropriately to bring the assay pH to 1.5–2. III. This step and the subsequent two steps are performed as soon as possible to minimize the possible oxidation of HE by any co-extracted soil constituents. After the 1-min incubation period, the assay mixtures in the S, B, RHCl-1 mM and RHCl-0 mM tubes are mixed with 10 ml 1 M Tris by brief vortexing (to adjust the assay mixture pH to 7–8), centrifuged at 12,000g for 1 min and the resulting supernatants are collected for ?uorescence measurement. The FU of the S, B, RHCl-0 mM and RHCl-1 mM supernatants are measured at ex/em 505/580 nm (designated FUS, FUB, FURHCl-0 mM and FURHCl-1 mM, respectively). From these FU, the net FU of 2-OH-E ? is calculated from the following formula: net FU2-OH-E ? ? FUS -FUB -?FUR HCl-1mM-FUR HCl-0mM?. NOTES: I. To verify that net FU2-OH-E ? corresponds to the concentration of the ? ? , various dilutions of the ACN -CE supernatants (derived as in the extracted O2 alk ?rst two steps of the Assay Protocol) should be tested (as in this and the previous

Assay sensitivity. Applying the protocol on 1 g soil, extracted in 1 ml ACNalk-CE
solution by ?ltration (to recover a B1.0 ml ACNalk-CE extract), with an assay reaction volume 0.5 ml (containing 0.44 ml ACNalk-CE extract) and a minimum net FU2-OH-E ? ? 200, the theoretical assay sensitivity (using the above formula) ? ? (in the assay reaction mixture, or 0.34 nmoles is ? 200 ? 2/1300 ? B0.3 mM O2 in 1.0 ml ACNalk-CE extract, or 0.34 nmoles g ? 1 soil).
.

SOD-inhibited reduction of cyt. c-based soil O2 ? assay. Protocol. Soil samples were mixed with a minimum 0.3 ml O2-degassed ACNalk-CE (0.2 mM) per gram of soil by shaking for 2–3 min at RT. This proportion achieves the maximum ? ? extracted in a minimum solvent volume. Then, B0.5 g concentration of O2 batches of this soil/ ACNalk-CE slurry were placed in centrifuge tube ?lters ?tted to a 1.5-ml microcentrifuge collection tube, centrifuged at 15,000g for 5 min and ? ? /ACN -CE eluents of all the batches were combined and used in the the O2 alk following step. NOTE: In this step and in subsequent steps, all the reagents used should be O2degassed as in two steps before. ? ? to cyt. c Having established the molar stoichiometric ratio 1:1 of O2 red up to a ?nal 40% ACNalk-CE (see Supplementary Methods), the maximum ACNalk-CE ? ? solvent volume for the recovery of O2 from any soil sample is 0.4 ml. This volume is suf?cient for the control if B0.5-volume capacity cuvettes are used. Given that the minimum proportion of ACNalk-CE extraction volume/soil weight is B0.3 ml per 1 g, the maximum amount of soil that this assay can extract for ? ? in a 0.4 ml extract is 1.3 g. measuring accurately the concentration of O2 Therefore, 1.3-g soil samples were used extracted in 0.4 ml ACNalk-CE, and were diluted with 100% ACNalk-CE to 0.8 ml (unless a smaller than 1-ml ?? spectrophotometer cuvette is used). A 0.4 ml portion of the clear ACNalk-CE-O2 extract (after centrifugation at at 15,000g for 5 min) was mixed with 0.6 ml 50 mM phosphate buffer, pH 7.8, containing 26.7 mM cyt. cox (?nal 16 mM). Its absorbance was measured at 550 nm against a reagent blank (0.4 ml ACNalk-CE plus 0.6 ml 50 mM phosphate buffer, pH 7.8) and also against the following SOD blank: It consists of the remaining 0.4-ml portion of the ACNalk-CE extract, mixed with 0.6 ml 50 mM phosphate buffer, pH 7.8, containing 26.7 mM cyt. cox and 3.3 units ? ? is present in the 0.4 ml SOD (or SOD added before the addition of cyt. cox). If O2 ACNalk-CE extract the assay will identify it from the SOD-inhibited reduction of cyt. cox. Competing with cyt. cox, SOD will reduce or eliminate the concentration of ? ? by dismutation. This blank also quanti?es possible soil contaminants that O2 may cause chemical reduction of cyt. cox and/or absorb at 550 nm. Then, the DA550 nm (absorbance of the sample minus absorbance of the SOD blank) is ? ? derived from converted nmoles of cyt. cred, which correspond to equimolar O2 ? ? concentration (see Supplementary the standard curve of cyt. cred versus O2 ? ? (or 0.5 mM in the Methods). The sensitivity of the assay is B0.5 nmoles O2 ACNalk-CE/phosphate buffer assay mixture).
7

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ARTICLE
? ? soil extract should be rapidly mixed NOTE: The 0.4 ml of the ACNalk-CE-O2 with the 0.6-ml phosphate-buffered cyt. cox in the spectrophotometer cuvette and its peak absorbance immediately measured as it rapidly decreases due to the reoxidation of cyt. cred.

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8100

Sample treatments (STs) and reagent blanks (RBs): Reagents in ml Soil extract (or appropriate dilutions in 10 mM phosphate buffer, pH 6.5) 10 mM phosphate buffer, pH 6.5 3 KU ml ? 1 CAT (in 10 mM phosphate buffer, pH 6.5) 5-min incubation 0.1 M EDTA 1-min incubation FOX ? Fe2 ? reagent FOX-Fe2 ? reagent

ST1 ST2 ST3 ST4 RB1 RB2 935 935 935 935 15 — — 15 15 — — — — —

Assay for soil total metal superoxides/peroxides and Fe3 ? . Reagent solutions. FOX (Ferrous Oxidation in Xylenol orange) assay reagents. In a plastic vial (glass vials may leak Fe3 ? ), 3.644 g sorbitol (?nal 2 M, and in assay reaction 0.1 M) and 15.2 mg xylenol orange are dissolved (for about 30 min) in 7.6 ml 0.66 M HClO4 (?nal 0.5 M, and in assay reaction 25 mM). Then, any non-dissolved matter is removed by centrifugation (or by ?ltering), the resulting supernatant is split in two halves (5 ml each), and one half is the FOX-Fe2 ? reagent. To the other half, 7.9 mg ferrous ammonium sulfate are dissolved, resulting in the FOX ? Fe2 ? reagent. Both the reagents are prepared fresh. 0.1 M EDTA stock. It is prepared in ddH2O. EDTA is a Fe3 ? chelator, and is used here as blank for the estimation of soil Fe3 ? . Standard curves. H2O2 (0–2 mM) standard curve. It is made with a 4 mM H2O2 solution (in 10 mM phosphate buffer, pH 6.5) prepared from a 5 mM H2O2 working stock, which is made from the concentrated 30% H2O2 using the H2O2 molar extinction coef?cient at 240 nm (e240 ? 43.6 M ? 1 cm ? 1). The reagents are mixed as presented in the following box:

950 935 — 15

No Yes No No No Yes — — — 15 — — No No No Yes No No 50 50 — — 50 50 — — 50 50 — —

Tube numbers (mM H2O2, or nmol H2O2 in 1 ml assay reaction). Reagents in ml

0

0.2 0.4 0.6 0.8

1

1.2 1.4 1.6

2

After mixing, the ST1, ST2, ST3, ST4, RB1 and RB2 tubes are incubated for 30 min at RT. NOTE: For higher accuracy, ST1, ST2, ST3 and ST4 mixtures are preferably made each in triplicates from three different soil extract dilutions, while RB1 and RB2 are made in triplicates. The corresponding AST1, AST2, AST3, AST4, ARB1 and ARB2 absorbance values are recorded at 560 nm, and their triplicate averages are then entered in the following equations to calculate the corresponding netA560 nm for soil H2O2 and Fe3 ? , which are then converted to H2O2 and Fe3 ? concentrations (from the corresponding standard curves), expressed per g soil: Soil H2 O2 netA560nm ? AST1 -AST2 ? ?ARB2 -ARB1 ? Soil Fe3 ? netA560nm ? AST3 -AST4 Instability of aqueous desert soil superoxides/peroxides. To test whether the instability of soil H2O2 extracts (from total metal peroxides) during various incubation intervals concurs with the generation of ? OH radicals, these were trapped in the H2O2 extracts by TPA and the resulting speci?c ?uorescent product 2-hydroxyl terephthalate (HTPA) was isolated and quanti?ed by the following procedured developed for this study. Procedure: Soil H2O2 extracts were analysed for stability and ? OH generation versus extract incubation time (0–48 h) in the absence (A) and presence (B) of the extracted soil. Speci?cally, H2O2 was extracted from two 1-g soil samples (per incubation time interval) after each was mixed with 4 ml 10 mM H3PO4 for 5 min at RT, followed by centrifugation at 4,200g for 3 min. The H2O2 extract in the isolated supernatant (in the absence of the soil precipitate) was kept for treatment A and that in the upper aqueous phase (in the presence of the soil precipitate) was kept for treatment B. Stability of H2O2 in extract samples from treatment A was measured as the remaining H2O2 concentration after certain incubation time intervals (up to 48 h) at RT, using as control a solution of 2 mM H2O2 in 10 mM H3PO4 (made from the 30% H2O2, the preparation of which is described in the H2O2 standard curve presented in the Methods section ‘Assay for soil total metal superoxides/peroxides and Fe3 ? ’). The concentration of H2O2 was measured as described in the Methods section ‘Assay for soil total superoxides/peroxides and Fe3 ? ’. More experimental details are also presented in the legend of Fig. 1. To measure the generation of ? OH in the extracts of treatments A and B, they were both brought to 2 mM TPA (from a 100 mM TPA stock made by dissolving 115 mg TPA in 1.4 ml 1 M NaOH and then mixing with 1.4 ml 50 mM phosphate buffer, pH 7.4, readjusted with 1 M HCl and ddH2O to ?nal 7 ml) and incubated at RT in the dark for the selected time intervals. As control, corresponding soil extracts were also incubated with 100 mM DMSO), an ? OH scavenger. After each incubation period, 0.9 ml of the soil extracts for treatments A and B were mixed with 9 ml 10 M NaOH (to ensure deprotonation of HTPA) and washed with an equal volume ethyl acetate–ethanol (2:1) by vortexing, followed by centrifugation at 4,200g for 5 min. The resulting bottom aqueous phase (B1.2 ml) was acidi?ed by 25 ml 10 M HCl (to protonate HTPA) and HTPA (and TPA) was extracted (by vortexing) with two 0.9-ml ethyl acetate washes, followed by centrifugation for 5 min at 4,200g. The combined 1.8 ml ethyl acetate phase was washed with 1.8 ml 0.1 M HCl and evaporated in a rotary vacuum concentrator. The resulting solid precipitate was dissolved in 15 mM KOH (at a minimum volume for example, B0.1 ml) and adjusted to pH 7 (with 0.2 M KH2PO4, pH 7) for high-performance liquid chromatography (HPLC) analysis. Samples (10 ml) were injected into an Shimadzu HPLC Prominence UFLC system, consisting of a solvent delivery system (model LC-20AD), a sample manual injector (Rheodyne 7725i), an on-line degasser (DGU-20A5), a reverse-phase C18 column (Waters Nova-Pak C18, 60 ?, 4 mm, 3.9 ? 150 mm) and a ?uorescent detector (RF-20Axs) set at ex/em 311/425 nm. The mobile phase, consisting of 50 mM KH2PO4 in 30% methanol, pH 3.2 (an alternative mobile phase is 0.2 M KH2PO4, pH 4.37, containing 2% of KCl70), was passed through the column at a ?ow rate 0.8 ml min ? 1 and the sample was eluted for a total of 20 min. HTPA in samples was identi?ed and quanti?ed by HPLC?uorescence (eluted as a peak at B14 min) against pure HTPA (article Fig. 1b), which was synthesized as reported in the Supplementary Methods. HTPA was also identi?ed by mass spectroscopy using an Agilent 1260 In?nity HPLC-MS system equiped with a binary pump, an auto-sampler and a micro-column Zorbax Extend-

0 50 100 150 200 250 300 350 400 500 4 mM H2O2 10 mM phosphate buffer, 950 900 850 800 750 700 650 600 550 450 pH 6.5 FOX ? Fe2 ? reagent 50 ml in each tube

Each tube is then incubated for 30 min at RT after which the absorbance at 560 nm (A560 nm) of each of the assay reaction mixtures in tubes 0–2 is measured. The net A560 nm value of tubes 0.2–2 is calculated by subtracting from their A560 nm value the A560 nm value of tube 0 (the blank). Then, the standard curve is drawn by plotting the 0–2 mM (or nmoles) H2O2 against their corresponding net A560 nm values. NOTE: With 10 mM phosphate, pH 6.5, as H2O2/sample solvent, the ?nal 25 mM perchloric acid in the assay achieves assay reaction pH B1.8. At these conditions, the assay achieves its highest, for H2O2, molar extinction coef?cient B250,000 M ? 1 cm ? 1). With unbuffered samples (that is, in H2O), the ?nal concentration of perchloric acid in the assay should be 15.5 mM (in H ? ), which corresponds to the optimal assay pH 1.8. Fe3 ? (0–12 lM) standard curve. It is made from a 20 mM Fe3 ? stock solution (in 10 mM phosphate buffer, pH 6.5). The reagents are mixed as presented in the following box:

Tube numbers (mM Fe3 ? , or nmol Fe3 ? in 1 ml assay reaction). Reagents in ml

0

1

2

4

6

8

10

12

20 mM Fe3 ? (in 10 mM phosphate 0 50 100 200 300 400 500 600 buffer, pH 6.5) 10 mM phosphate buffer, pH 6.5 950 900 850 750 650 550 450 350 50 in each tube FOX-Fe2 ? reagent
Each tube is then incubated for 30 min at RT after which the absorbance at 560 nm (A560 nm) of each of the assay reaction mixtures in tubes 0–12 is measured. The net A560 nm value of tubes 1–12 is calculated by subtracting from their A560 nm value the A560 nm value of tube 0 (the blank). Then, the standard curve is plotted as 0–12 mM (or nmoles) Fe3 against their corresponding net A560 nm values. Protocol. Soil samples were treated as follows: 1 g soil (placed in a capped 15-ml plastic centrifuge tube) was extracted in (for example, 1–4 ml) 10 mM H3PO4 for 2 min by gentle inversion and the soil extract was collected by centrifugation at 5,000g for 5 min. NOTE: Extraction of the Mojave and Atacama soil samples with 10 mM H3PO4 resulted in pH B6.5 of the soil H2O2 extract. The soil extract (its pH is adjusted to B6.5 with 0.1 M NaOH/HCl if needed) was treated with the assay reagents (in 1.5-ml microcentrifuge tubes) shown as presented in the following box:
8

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stable at a pH range 5.5–9.5 (ref. 70). Similarly, the control (c) emission spectrum was subtracted from the emission spectra of controls (d) and (e) to identify the formation of HTPA due to H2O2 and TiO2, respectively. The net FU emission peak (at 425 nm with excitation at 311 nm) in the difference spectra was, then, converted to HTPA concentration by a spectro?uorometer (Shimadzu RF-1501) calibrated with 1 mM pure HTPA; it emits B300 FU at the spectro?uorometer’s low sensitivity setting (or 15,000 FU at the high-sensitivity setting). Additional data analysis is presented in the corresponding section of the Supplementary Methods. Mineralogical and geochemical analysis of soils. Procedure: The bulk mineralogy of the samples was determined by X-ray diffraction on the D8 Advance diffractometer (Bruker AXS) equipped with a LynxEye strip silicon detector, using Ni-?ltered CuKa radiation, an applied voltage 35 kV and a 35 mA current, and 0.298° divergence and antiscatter slits. The random powder mounts of samples were scanned from 2 to 70° 2y with a scanning step of 0.015° 2y and with 18.7-s count time per step. Powder diffraction data were collected at RT. Results were con?rmed with synchrotron X-ray high-resolution powder diffraction measurements. The samples were loaded into 1.0 mm-diameter glass capillaries. Powder diffraction data (for Atacama Yungay and site 1, and Mojave ? CIMA basalt rock samples) were collected at RT (295 K) on the undulator beamline ID31 at the ESRF (European Synchrotron Radiation Facility) in Grenoble, France. The parallel beam geometry instrument is equipped with nine Si(111) analyser crystals and provides data with very high angular resolution, with a minimum instrumental contribution to the full-width half-maximum around 0.003°. High speed spinning of the sample was applied to ensure suf?cient powder averaging. The nine detector modules (separated by B2°) were scanned over a 40° 2y range, at a wavelength of 0.400054(15) ?, using an incident beam size of 2.0 mm (2.0 mm (horizontally) ? 1.0 mm (vertically)). The clay fraction (o2 mm) was separated by settling and dried on glass slides at RT. Clay minerals were identi?ed from air dried slides and after ethylene glycol solvation at 60 °C overnight to ensure maximum saturation. X-ray diffraction analysis was performed from 2 to 35°2y with the Bruker D8 Advance diffractometer, using the same divergence and antiscatter slits, and a step size of 0.019° 2y, with 47.7-s count time per step. Chemical composition of minerals was examined with a JEOL 6300 Scanning Electron Microscope equipped with an Oxford Link Energy Dispersive Spectrometer (EDS; Oxford Instruments). Semi-quantitative analyses of minerals were performed on sample powders mounted directly on glass slides and coated with carbon. The chemical composition of the minerals was determined using natural and synthetic standards and 20 kV accelerating voltage with 10 nA beam current. Back-scattered electron images were used to identify any heavy minerals present. The concentrations of major and trace elements in bulk samples (from Atacama sites, Mojave sites and control site) were determined with Energy Dispersive X-ray Florescence Spectroscopy (EDS-XRF, S2 Ranger, Bruker). The samples were fused using a ?ux:sample powder ratio of 5:1 and were casted in dies forming glass beads. Li-tetraborate was used as ?ux. Additional data analyses can be found in the corresponding section of the Supplementary Methods.

C18 600Bar 2.1 ? 50 mm, 1.8 mM, connected on line with an Agilent 6538 UHD Accurate-Mass Q-TOF LC/MS detector, operated in negative (ESI ? ) electrospray ionization mode. Samples (10 ml) were eluted under linear gradient conditions (at a ?ow rate 0.3 ml min ? 1 for 10 min) at RT, by an increasing 100% ACN (solvent A) and a decreasing 50 mM KH2PO4, pH 3.2, 30% MetOH (solvent B) mobile solvent phase. The microcolumn was initially washed with the A and subsequently equilibrated with the solvent B. HTPA (eluted as a peak at B1.5 min) was identi?ed as giving, for the parent ion, m/z with highest peak at M-1 ? 181.01437 (versus the theoretical 181.01425), and a fragmentation product at M-1 ? 137.02442 (expected from the fragmentation, at CE 20 eV, of one carboxyl group from HTPA). Soil O2 ? generation by sunlight and ultraviolet-C. Procedure: Soils (B1 g), untreated or washed (to remove any soluble inorganics/organics and polar/ hydrophobic organics by vortexing (and intermittent centrifugation at 4,200g for 5 min) with 2 ? 10 ml 0.1 M HCl, 1 ? 10 ml ddH2O, 2 ? 10 ml 0.1 M NaOH, 3 ? 10 ml ddH2O, 2 ? 5 ml ddH2O plus 5 ml ethyl acetate:ethanol (2:1), 1 ? 10 ml 25% ethanol, and ?nally dried at 150 °C for 1 h) were spread as a thin layer on aluminum foil and exposed to natural sunlight (at 800–1,000 W m ? 2, in a July day, from 10:00 to 16:00 hours and at an average 45 °C); unexposed were used as control. The relative humidity at 10:00 hours was B15% and reached a minimum B5% during the exposure period. Soils were also exposed to iltraviolet C radiation (using a germicidal lamp with ?ux density 30 mW cm ? 2 set at a close distance of 10 cm) at relative humidity 10–25% and at an average 40 °C. After irradiation, soils ? ? (as in the Methods section ‘HE-based soil O ? ? assay’) and were extracted for O2 2 measured by the aforementioned HE-based and SOD-inhibited reduction of cyt. ? ? c-based soil O2 assays. Irradiated soils were also extracted for total superoxides/ peroxides and the resulting H2O2 extract was measured as in the Methods section ‘Assay for soil total metal superoxides/peroxides and Fe3 ? ’. Photo-generation of OH by aqueous desert soils. Procedure: Soil samples were ? ? generation by sunlight and extensively washed as in the Methods section ‘Soil O2 ultraviolet C’ to remove any soluble inorganic/organic and polar/hydrophobic organic soil constituents, which may affect the rate of photogeneration of ? OH. Washed soil samples (0.2 g) were placed in capped small plastic Petri plates (inner diameter 5 cm), mixed with 10 ml ddH2O and spread as an even thin layer on the plate bottom with a small glass rod. Then, 50 ml from the 100 mM TPA stock solution (made in 400 mM NaOH) was added. This resulted in ?nal 0.5 mM TPA and 1 mM NaOH (due to its partial neutralization by the 1 mM carboxyl groups of 0.5 mM TPA), which were set as an optimal for the ? OH-TPAassay in a previous study using illuminated TiO2 ?lms37,38. Samples were exposed to light irradiation 180 W m ? 2 (at 400–800 nm, emitted by two halogen lamps, type R7s J118 tube linear 500W/230V B9000 Lumens per m2 each, both covered with glass and ultraviolet and infrared cutoff ?lters and set at 50 cm above the soil plates) or higher (adding more lamps) and for up to 10 h (at RT). The following lightexposed controls were used: (a) soil samples mixed with 10 ml 1 mM NaOH (for soil interference), (b) 10 ml 1 mM NaOH (for aqueous phase treatment interference), (c) 10 ml 0.5 mM TPA and 2 mM NaOH (for TPA interference) ±100 mM DMSO ( ? OH scavenger), (d) 10 ml 1 mM H2O2 in 0.5 mM TPA and 2 mM NaOH (to test possible photolysis of H2O2 to ? OH; both can be products of, for example, TiO2 photocatalysis38, given that H2O2 can be generated directly or ? ? ) and (e) 10 ml suspension indirectly, for example, via dismutation, from O2 of 1.6 mg TiO2 (or 20 mM TiO2) in 0.5 mM TPA and 2 mM NaOH (for testing the employed assay to detect ? OH photo-generation and by simulating a photocatalysis mechanism with TiO2). The amount of TiO2 used is equivalent to the average concentration (w%) in the 0.2 g soils from Atacama Yungay and Mojave ? CIMA sites (Table 2). Exposure of the soil samples to ultraviolet C irradiation was not used because the control (c) produced ? OH, possibly because the ultraviolet C lamp may also emit near the absorbance of water (186 nm) and, thus, photo-dissociate it to ? OH. Aqueous samples (1 ml) were drawn from the plates at certain time intervals, centrifuged at 16,000g for 5 min at RT, acidi?ed with ?nal 0.1 M HCl, extracted ? 2 with 1 ml ethyl acetate (by vortexing and subsequent centrifugation at 4,000g) and dried by a centrifugal vacuum concentrator at 50 °C. The isolated HTPA solid precipitate in soil and control samples was dissolved in 0.1 ml 20 mM NaOH (its moles are ? 2 those of the total carboxy groups of added TPA) and neutralized by 0.2 ml 150 mM K-phosphate buffer, pH 7.4. The solubilized HTPA was quanti?ed by HPLC-?uorescence (as in the Methods section ‘Instability of aqueous desert soil superoxides/peroxides’), and, in routine HTPA quanti?cations by measuring the ?uorescence difference emission spectra (recorded at the excitation wavelength 311 nm) of samples minus those of appropriate controls as follows: Starting with the determination of the emission spectra of controls (a) and (b), they were found identical (indicating that washed soil did not interfere) and lower in FUs than of the control (c) spectrum, which shows that both the controls were part of the control (c). Therefore, the emission spectrum of latter control was subtracted from the soil sample emission spectra taken at different exposure time periods, to estimate the corresponding net emission spectra due to HTPA present in the samples. These HTPA spectra were identi?ed by comparing their shape and peak at 425 nm with the spectra of pure HTPA (synthesized as in the Methods section ‘Instability of aqueous desert soil superoxides/peroxides’), and with the known emission spectra of HTPA, which are
. .

References
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them with K.G., I.P., D.Z and K.P. H.J.S. and G.Z. also performed soil peroxide detection experiments. K.G., I.P., D.Z and K.P. had equal contribution in this study. E.K., G.E.C. and I.M. designed and performed the mineralogical experiments, and analysed the relevant data.

Acknowledgements
C.D.G. was ?nancially supported by the Greek Ministry of Education. H.J.S. and G.Z. were supported by a grant from the NASA Astrobiology Program NNX07AT65. H.J.S was also in part supported by the National Science Foundation under grant number IIA-1301726. He thanks R. Kreidberg for editorial assistance. C.P.M. acknowledges the support from the NASA Planetary Protection Program. We thank the ESRF for the provision of synchrotron X-ray beamtime at the high-resolution powder diffraction beamline (ID31). We are grateful to Professor P.V. Ioannou (Department of Chemistry) and Associate Professor M. Kornaros (Department of Chemical Engineering) at the University of Patras, Greece, for HTPA synthesis and HPLC-MS identi?cation in samples, respectively.

Additional information
Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications Competing ?nancial interests: The authors declare no competing ?nancial interests. Reprints and permission information is available at http://npg.nature.com/ reprintsandpermissions/ How to cite this article: Georgiou, C. D. et al. Evidence for photochemical production of reactive oxygen species in desert soils. Nat. Commun. 6:7100 doi: 10.1038/ncomms8100 (2015).

Author contributions
C.D.G., H.J.S. and C.P.M. conceived and designed the experiments, analysed the data, contributed materials/analysis tools and co-wrote the paper. C.D.G. conceived the soil ? ? /peroxide and ? OH detection assays and experimentally developed and performed O2

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