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Mg isotope fractionation during uptake by a rock-inhabiting, model microcolonial fungus Knufia petricola at acidic and neutral pH. Rasesh Pokharel, Ruben Gerrits, Jan Schuessler, Geerke Floor, Anna Gorbushina, and Friedhelm von Blanckenburg Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01798 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Environmental Science & Technology

1

Mg isotope fractionation during uptake by a rock-inhabiting, model

2

microcolonial fungus Knufia petricola at acidic and neutral pH

3 4

Rasesh Pokharela,b,*, Ruben Gerritsc,d, Jan A. Schuesslera, Geerke H. Floora,#, Anna A.

5

Gorbushinab,c,d, Friedhelm von Blanckenburga,b

6 7

a

8

Potsdam, Germany

9

b

Institute of Geological Sciences, Freie Universität Berlin, Berlin, Germany

10

c

BAM Federal Institute for Materials Research & Testing, Department 4, Materials & Environment, Berlin,

11

Germany

12

d

Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany

13

#

present address: Institute of Life Science and Chemistry. Utrecht University of Applied Sciences, Utrecht, The

14

Netherlands

GFZ German Research Centre for Geosciences, Section 3.3, Earth Surface Geochemistry, Telegrafenberg, 14473

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 *

Corresponding author phone: 0049 331 288-28964; fax: 0049 331 288-2852; email: [email protected]

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Abstract

The model rock-inhabiting microcolonial fungus Knufia petricola fractionates stable Mg isotopes 26

Mg/24Mg in the fungal

37

in a time- and pH-dependent manner. During growth, the increase of

38

cells relative to the growth media amounted to 0.65 ± 0.14 ‰ at pH 6 and 1.11 ± 0.35 ‰ at pH 3.

39

We suggest a constant equilibrium fractionation factor during incorporation of Mg into ribosomes

40

and ATP as a cause of enrichment of 26Mg in the cells. We suggest too that the proton gradient

41

across the cell wall and cytoplasmic membrane controls Mg2+ transport into the fungal cell. As

42

the strength of this gradient is a function of extracellular solution pH, the pH-dependence on Mg

43

isotope fractionation is thus due to differences in fungal cell mass fluxes. Through a mass balance

44

model we show that Mg uptake into the fungal cell is not associated with a unique Mg isotope

45

fractionation factor. This Mg isotope fractionation dependence on pH might also be observed in

46

any organism with cells that follow similar Mg uptake and metabolic pathways, and serves to

47

reveal Mg cycling in ecosystems.

48 49

TOC/Abstract art

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53 54

Introduction

55

In the past 15 years metal and metalloid stable isotope ratios (e.g. Mg, Fe, Si, Li, Ca, Zn, etc.)

56

have been developed as novel source and process tracers in ecological and Earth surface systems.

57

Mg isotopes in particular are a promising tool to decipher both biological and Earth surface

58

processes as Mg is a mineral nutrient that is ubiquitous in rocks. Mg stable isotopes change their

59

relative abundances during biogeochemical transformations (e.g. precipitation and secondary

60

mineral formation from fluids, uptake and translocation by plants), hence producing distinct

61

isotope fingerprints

62

4,5,6,7,8,9

63

quantified. This knowledge gap is a significant obstacle to interpreting data from complex and

64

dynamic natural ecosystems. As mediators of metal and mineral transformations as well as

65

element cycling, fungi in particular play an important role in rock weathering and deliver large

66

amounts of Mg and other nutrients to higher plants and other phototrophs

67

the Mg processed by fungi has a unique isotopic fingerprint, this fingerprint serves to interpret

68

Mg elemental mass fluxes at the soil, ecosystem, and river catchment scale. To this end, we still

69

need to explore how cell physiology and isotope fractionation factors are related and whether

70

these in turn depend on environmental controls (pH, speciation, etc.). Addressing these questions

71

will reveal how metal isotope tracers can be used to explore the role of fungi in ecosystem

72

nutrient turnover studies.

1,2,3

. Biological processes vary in the way they fractionate Mg isotopes

. However, the metabolic processes controlling Mg isotope fractionation are not well-

10,11,14,12

. Therefore, if

73 74

The study of Mg isotopes in biology is at an early stage compared to the lighter traditional stable

75

isotope systems of H, C, O, N and S. All studies to date show that biological processes linked

76

with incorporation of Mg induces isotope fractionation. Black et al. (2006) demonstrated that 3 ACS Paragon Plus Environment


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chlorophyll molecules extracted from cyanobacteria are depleted in the heavier Mg isotopes

78

relative to the growth solution (i.e., chlorophyll is 0.71 ‰ lower in

79

solution) 4. In contrast, Ra and Kitagawa (2007) observed that the chlorophyll molecules of

80

marine red algae are enriched in the heavier isotopes relative to seawater by 0.58 ‰ in

81

26

82

that the plants are enriched in heavier Mg isotopes relative to the growth solution

83

studies discovered that heavier Mg isotopes are preferred during plant uptake, while the lighter

84

Mg isotopes were enriched from roots to shoots during translocation.

85

experimental study on Mg isotope fractionation in fungi has been conducted 9. This study

86

revealed that an ectomycorrhizal fungus showed only minor Mg isotope fractionation, whereas a

87

non-mycorrhizal fungus distinctly incorporated heavier Mg isotopes when grown on a growth

88

media. This pioneering study showed the direction of Mg isotope fractionation in different types

89

of fungus. However, an explanation for the biological processes that cause this fractionation is

90

still missing.

26

Mg/24Mg compared to the

Mg/24Mg 8. Two laboratory studies by Black et al. (2008) and Bolou-Bi et al. (2010) showed 5,6

. These

To date, only one

91 92

In this study we conducted laboratory experiments under controlled conditions that offered the

93

opportunity to isolate selected parts of the system and test the influence of important geochemical

94

parameters, in our case the effect of pH, on Mg isotope fractionation by a single fungal species.

95

We conducted growth experiments to show for the first time a pH dependence of the Mg isotope

96

fractionation during uptake by a model black fungus. Black microcolonial fungi are ubiquitous

97

world-wide and colonize sub-aerial rock surfaces and exposed natural or man-made structures in

98

diverse climates ranging from hot to cold deserts, acidic metal-enriched mine waters, neutral to

99

alkaline soils in the high Artic, limestone monuments, saltpans, and acidic exposed rocks 4 ACS Paragon Plus Environment

15,16

.

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We chose Knufia petricola A95 as a model black fungus because of its capacity to interact with

101

phototrophic algae and cyanobacteria to form stable subaerial microbial communities at acidic

102

and neutral pH in both natural and laboratory conditions

103

hyphae, they have a smaller surface area compared to mycorrhizae fungus

104

capacity of interacting with mineral surfaces

105

formation of ribosomes and ATP causes Mg isotope fractionation within K. petricola cells.

106

Furthermore, we use a mass balance model to explain that the magnitude of isotopic fractionation

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depends on differences in mass fluxes of Mg into and out of the fungal cell is controlled by pH-

108

dependent transport gradients.

17,18,19,20

. Due to their short torulose 20

, but possess the

21

. We suggest that chemical equilibrium during

109 110

Material and Methods

111

Knufia petricola A95

112

The fungus used in the study was Knufia petricola strain A95 (CBS 123872). Rock-inhabiting

113

microcolonial fungi were originally discovered in the early 1980s 22,23. They are the predominant

114

and permanent settlers of some of the most extreme environments on Earth, like Antarctica

115

glacial valleys, hot deserts and nuclear radiation-contaminated areas

116

relatively fast growing rock inhabiting ascomycete and can be easily handled in the lab. The

117

strain was isolated from a marble surface in Athens, Greece. It belongs to an early-diverging

118

lineage of the pathogen-rich ascomycetes order Chaetothyriales (class Eurotiomycetes) and is a

119

predecessor of lichens. The K. petricola strain and a melanin-minus mutant of K. petricola

120

(∆PKS) were cultivated in the lab following the procedure described in Noack-Schönmann et al.

121

(2014) and Nai et al. (2013)

122

test potential Mg isotope fractionation effects during adsorption on the cell wall of the fungus.

19,20

24

. K. petricola A95, is a

. A melanin-minus mutant of K. petricola was used in order to



123

Melanin is a cell wall-bound protective pigment that provides defense against environmental

124

stresses such as ultraviolet (UV) and ionizing radiation as well as desiccation and oxidizing

125

agents.

126

Growth media

127

Modified BG-11 media was used to obtain a favorable growth condition for the fungus.

128

Compared to normal BG-11 media, half the amount of MgSO4 was added. The aim of this

129

reduction was to allow detection of changes in Mg concentrations and isotope ratios in the

130

growth solution during uptake. Moreover, additional glucose, and NH4NO3 were added to

131

stimulate biomass growth. However, we show below that biomass growth was too low to resolve

132

changes in the solution’s Mg isotope composition. A buffered BG-11 media at pH 6 was also

133

prepared by adding about 55 mM of MES (2-(N-morpholino)ethanesulfonic acid) buffer. The

134

detailed composition of the modified BG-11 media is given in Table 1.

135 136

Growth Experiment

137

The experiments conducted here are biologically as well as geochemically reproducible. We

138

conducted independent growth experiments in triplicate to verify repeatability. Also all the

139

components of the experimental system are precisely quantifiable. All batch experiments with K.

140

petricola and abiotic controls were performed under aerobic and sterile conditions in triplicate.

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500 mL polycarbonate Erlenmeyer flasks (with sterile vented caps) were cleaned with HCl and

142

HNO3 acid, washed four times with deionized water (MilliQ), and autoclaved to avoid chemical

143

and microbial contamination. 400 mL of BG-11 media, either with or without MES buffer, was

144

transferred into each Erlenmeyer flask. The flasks with the MES buffer are hereafter called the

145

“buffered experiment” and the flasks without MES buffer are called the “unbuffered experiment”.

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The melanin-minus mutant was not included in the unbuffered experiment because, as will be 6 ACS Paragon Plus Environment

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shown below, results from the wild type and melanin-mutant strains did not differ in the buffered

148

experiment.

149 150

Before transfer of cultures of K. petricola from the stationary growth phase to the flasks for the

151

time-series growth experiments, they were separated from the MEB media by centrifugation (15

152

min, 7500 RPM) and then homogenized using an oscillating mill. The cultures were then washed

153

with 5 mM EDTA and BG-11 media to minimize risk of surficial contamination from the original

154

liquid malt-extract media (MEB). The cell number was measured just before inoculation

155

(hemocytometer and quantitative PCR) and a small aliquot of the culture was taken to determine

156

the initial elemental and Mg isotopic compositions (see methods below). Then, between 0.002 g

157

and 0.006 g dry biomass weight of K. petricola were incubated in the experiments. The

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Erlenmeyer flasks with modified BG-11 media and K. petricola were placed in a climate chamber

159

under constant temperature (25°C), constant light source (90 µmol photons/m2s) and were shaken

160

at 150 RPM. Experiments were ran for 10 days for the buffered experiment and 11 days for the

161

unbuffered experiment. Periodic sampling (5 mL) was done using a volumetric pipet whilst

162

manually shaking the flasks to homogenize the culture. The sampled supernatant mixed with

163

biomass was filtered using 0.2 µm sterile filters, acidified with concentrated HNO3 acid and

164

stored at 4oC until Mg concentration and isotope analysis. The biomass was separated from the

165

supernatant to determine growth at each sampling point. Finally, the experiment was terminated

166

by centrifuging to separate the remaining biomass in the Erlenmeyer flasks. Part of the biomass

167

was used for cell number measurements and the residual was stored at -80oC until used for Mg

168

concentration and isotope analysis.

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Analysis 7 ACS Paragon Plus Environment


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Elemental concentrations of the filtered supernatant and the digested biomass were analyzed by

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Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES, Varian 720-ES).

173

Quantitative PCR (qPCR) method was used to quantify the cell number of K. petricola by

174

measuring the amount of fungal DNA 25. For information about sample preparation and analysis

175

by ICP-OES and qPCR see the supplement.

176 177

Sample preparation and Mg purification for isotope measurements were done in the Helmholtz

178

laboratory for the geochemistry of the earth surface (HELGES) at GFZ Potsdam. The pre- and

179

post-experiment dried fungal biomass, and supernatant samples, were acid-digested along with

180

references materials (BHVO-2, BHVO-2 with glucose and Cambridge-1) and blanks in capped

181

screw-top PFA beakers. Mg was purified by cation exchange column chromatography (see

182

supplement for detailed information).

183 184

Mg isotope analysis was performed on a Multicollector-Inductively Coupled Plasma Mass

185

Spectrometer (MC-ICP MS, Thermo Scientific Neptune). Analytical results are reported relative

186

to the international measurement standard DSM3 in the delta notation according to Coplen et al.

187

(2011)

188

Multiplication with a factor 1000 gives the per mil (‰) deviation relative to DSM3. Based on

189

repeated measurements of reference materials, the uncertainty in δ26Mg is ± 0.1 ‰ (2SD). For all

190

cell number, pH, and concentrations (Mg, P, K, and Fe) results in Figure (1), the analytical

191

uncertainty or the repeatability of independent experiments was used for interpretation,

192

whichever was larger. See supplement for detailed information on sample preparation, instrument

193

setting and reference material used for MC-ICP MS analysis.

26

, δxMgsample = [ (xMg/24Mg)sample / (xMg/24Mg)DSM3 ̶ 1 , where x = 26 or 25.

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Results and Discussion

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Growth of K.petricola

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The acidic pH in the unbuffered experiments favored the growth of K. petricola. The dry weight

198

of K. petricola biomass after the unbuffered experiment was 0.27 ± 0.03 g, whereas after the

199

buffered experiment it was 0.03 ± 0.01 g, suggesting about 10 times more growth in the

200

unbuffered condition (Table 2). This favored growth of K. petricola under acidic conditions has

201

been previously observed

202

biomass growth (0.06 ± 0.03 g) than the corresponding wild type in the buffered experiment. The

203

number of cells determined by qPCR with respect to experimental runtime for both the

204

experiments is shown in Figure 1.

20

. The melanin-minus mutant experiment showed slightly greater

205 206

Evolution of growth solution and incorporation of nutritive metals

207

Figure 1 shows the evolution of pH, Mg, P, K, and Fe concentration and Mg isotope ratios in the

208

growth solution and biomass with respect to time. pH measurements of the unbuffered

209

experiment with K. petricola showed a rapid decrease from 6 to 3 and then remained stable after

210

6 days. This rapid acidification of the solution in the presence of fungi is well-known from past

211

studies, and is caused by the excretion of protons, release of organic acids, and formation of

212

carbonic acid by respiration of CO2 by the fungi 27. As expected, Mg concentrations of the abiotic

213

control experiment remained unchanged (within uncertainty) at both buffered and unbuffered

214

conditions. Mg concentrations in the supernatant solution in the K. petricola and melanin-minus

215

mutant experiments at both buffered and unbuffered condition also did not vary outside the

216

analytical uncertainty (Figure 1). This implies that the Mg uptake by the fungus was small

217

relative to the Mg in the initial growth media.



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Measurements of Mg in the fungal biomass yielded differences in the final amount of Mg in the

220

biomass between the buffered and unbuffered experiments (Table 2). In the unbuffered

221

experiment about 4.5 % of the total Mg of the growth solution was taken up by K. petricola,

222

compared to only 0.65 % in the buffered experiment. However, as mentioned above, the resulting

223

decrease in the growth solution’s Mg mass was not analytically resolvable. The concentration of

224

Mg per gram of dry cells was slightly higher in the buffered experiment (365.28 ± 65.25 µg/g)

225

compared to the unbuffered experiment (245.84 ± 22.46 µg/g) (Table 2). This minor difference

226

suggests that the concentration of Mg inside K. petricola does not strongly depend on growth

227

rate. This was already shown in previous studies, i.e. the concentration of intracellular Mg tends

228

to be homeostatic or tightly regulated inside the fungal cell

229

phosphorous and potassium were consumed in the buffered than in the unbuffered experiment.

230

The change in the nutrient concentrations with respect to the initial media amounted to 0.02 mM

231

for P and 0.01 mM for K in the buffered experiment, and 0.04 mM P and 0.13 mM K in the

232

unbuffered experiment, respectively. Due to the greater biomass growth in the melanin-minus

233

mutant experiment compared to the wild type under buffered conditions, a slightly higher uptake

234

of P (a change of 0.05 mM in the growth solution concentration) and K (a change of 0.03 mM)

235

was found. The BG-11 media contains iron as ammonium iron(III) citrate. This iron precipitated,

236

presumably as iron oxyhydroxides, in the abiotic and biotic buffered experiments and the abiotic

237

unbuffered experiment, due to a stable pH at around 6 in these experiments. In the unbuffered

238

biotic experiment where the pH changed from 6 to 3, iron precipitated in the beginning, but then

239

redissolved when acidic conditions were reached. The relevance of iron precipitation as iron

240

oxyhydroxides lies in their potential to sorb and thus fractionate Mg isotopes. However, we did

241

not observe a decline in Mg concentration in the growth solution during the unbuffered abiotic

242

control experiment, suggesting that Mg sorption onto precipitated Fe oxides was negligible. 10 ACS Paragon Plus Environment

28,29

. Less of the nutrients

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Information on measured elemental concentrations in the supernatant of individual experiments

244

can be found in Table S3 in the supplement.

245

Mg isotopic composition

246

All the measured isotopic shifts showed mass-dependent isotope fractionation (Figure S1). No

247

significant variations were observed in the Mg isotopic evolution of the supernatant solutions (all

248

results are identical within analytical uncertainties). Replicate measurements for Mg isotopes of

249

the supernatant were not performed since mass balance calculations verify that a change of less

250

than 5% in Mg amount over the course of the experiment will not significantly change the initial

251

Mg isotope composition of the growth media. Mass balance shows that the slight change are

252

within the analytical uncertainty of 0.1 ‰ (2SD) for δ26Mg (see supplement for mass balance

253

calculation). In contrast, at the end of both experiments, the biomass of K. petricola showed

254

significant enrichment in the heavier Mg isotopes relative to the growth media. The measured

255

isotopic difference between fungi (K. petricola or melanin-mutant) and growth solution (BG-11)

256

was calculated as:

257 258

Δ26Mgfungi

̶ solution

= (δ26Mgfungi ̶ δ26Mggrowth solution) (‰)

259 260

In the buffered experiment the calculated isotopic difference between fungus and growth solution

261

was 0.65 ± 0.06 ‰ (2SD, n = 3 full experimental replicates, whereas in the unbuffered

262

experiment the value was higher at 1.11 ± 0.35 ‰ (2SD, n = 3). Mg isotope fractionation for the

263

melanin-minus mutant buffered experiment was 0.78 ± 0.1 ‰ (2SD), which is similar to the

264

value obtained for the wild type within the given uncertainty (Table 2). This similarity suggests

265

the absence of Mg isotope fractionation due to adsorption of Mg into melanin in the cell wall and

266

verifies that adsorption is not a large term in the mass balance of Mg. The above uncertainties are 11 ACS Paragon Plus Environment


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derived from the external repeatability of the three independent growth experiments. However,

268

there is also an analytical uncertainty of 0.1 ‰ (2SD) for individual δ26Mg measurements.

269

Therefore, the error bars shown for ∆26Mgfungi

270

either the error propagation of analytical uncertainties for individual measurement or external

271

repeatability, whichever is higher. Information on individual Mg isotope analyses in supernatant

272

and biomass and their respective analytical and external repeatability uncertainties for all

273

experiments can be found in Table S3.

̶ solution

values in Figure 1 and Table 2 represent

274 275

Isotope fractionation during desolvation?

276

In the unbuffered experiment, PHREEQC speciation calculations predict that the most dominant

277

species was Mg2+(aq) (Figure S2). Before uptake, the cell membrane proteins that transport Mg

278

must be able to recognize the very large hydrated molecule of Mg2+(aq), strip the tightly bound

279

hydration shell from the cation, and then only transport the dehydrated form

280

dictates that the resulting quantitative transfer does not allow any isotope fractionations to be

281

manifested. Hence, desolvation in the unbuffered experiment is unlikely to be a dominant control

282

of the observed Mg isotope fractionation in the experiments.

30, 31

. Mass balance

283 284

However, in the buffered experiment, PHREEQC speciation calculations predict that the most

285

dominant aqueous species (>99%) was Mg-MES along with trace amounts of Mg2+(aq). A

286

potential mechanism to explain the enrichment of

287

speciation in solution prior to uptake. Schott et al. (2016) have shown that at equilibrium there is

288

an enrichment of 26Mg in MgSO40 or Mg(citrate)- compared to dissolved Mg2+ (Mg2+(aq))

26

Mg in the fungus is a change in the Mg


32

. In

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Mg in Mg(EDTA)2- or Mg(citrate)24- compared to Mg2+(aq).

26

289

contrast, there is a depletion of

290

The associated isotope fractionation depends on the bond strength of the dissolved chemical

291

species, with stronger bonds favoring incorporation of the heavier isotopes

292

experiment, we can envisage a scenario in which after complexing with MES, a small Mg2+(aq)

293

compartment remains that is lighter than the bulk growth solution. To explain the isotopic

294

difference ∆26Mgfungi

295

unbuffered experiment, the composition of the small Mg2+(aq) pool not complexed with MES

296

would be 0.46 ‰ lower than the isotopic composition of Mg complexed with MES. This scenario

297

can only be verified by comparing the bond strength of Mg-MES and Mg2+(aq) and calculating

298

the respective equilibrium fractionation factors, but no such fractionation factors have been

299

determined.

̶ solution

33

. For the buffered

of 0.65 ‰ observed in the buffered experiment and 1.11 ‰ in the

300 301

The scenario that we discuss instead is one in which the reaction between Mg-MES and Mg2+(aq)

302

is not associated with major isotope fractionation. We can further assume that after stripping from

303

Mg-MES, quantitative uptake of Mg2+(aq) species by the fungus is likely, similar to the

304

unbuffered experiment. If uptake of Mg from Mg2+(aq) is complete in both experiments then

305

isotopic fractionation during desolvation can explain neither the presence of fractionation itself,

306

nor the observed difference between the buffered and the unbuffered experiment.

307 308

Isotope fractionation during cell uptake and transport?

309

Previous studies have shown that the concentration of Mg is constant or tightly regulated within a

310

fungal cell, even though there is exchange of extracellular and intracellular Mg during growth 13 ACS Paragon Plus Environment


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28,29,34

312

Rayleigh-type isotope effects and is consistent with a steady-state (quasi-equilibrium) closed

313

system isotopic exchange 35.

314

Three different proteins ALR1, ALR2 and MRS2 have been identified as major Mg transporters

315

for eukaryotes 36. All the three proteins are homologs of the CorA protein. CorA protein is an ion

316

channel that mediates transport of divalent ions across cell membranes in prokaryotic organisms.

317

All three Mg transporters for eukaryotes independently mediate the translocation of free Mg2+

318

across the cytoplasmic membrane by an electrochemical gradient

319

gradient is established in the plasma membrane of the fungus. The proton pump (H+-ATPase)

320

releases protons and thus forms a negative membrane potential inside the cytoplasm. Protons

321

accumulate on the outside of the cell making the cell wall more acidic than the cytoplasm. This

322

electrochemical gradient across the cell wall and cytoplasm barrier leads to Mg2+ transport into

323

the cytoplasm.

. The continuous exchange of Mg between the cell and the external solution excludes

37,38,39,40

. The electrochemical

324 325

A unidirectional Mg2+ transport could, in principle, be associated with kinetic Mg isotope

326

fractionation. A kinetic isotope fractionation involves enrichment of lighter isotopes in the

327

product relative to the source compartment. However, K. petricola is isotopically heavier than the

328

growth solution in both experimental setups, suggesting that kinetic fractionation does not play a

329

dominant role in our experiments. Furthermore, we can safely assume that all Mg ions attracted

330

to the transporter are shuttled into the protein channel and finally into the cytoplasm. If this Mg

331

mass transfer from the cell wall into the cytoplasm is complete, any potential isotope

332

fractionation during transport within the protein channels is not preserved.


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4.3 Isotope fractionation within the fungus?

335

A substantial percentage (~95 %) of the intracellular Mg2+ transported by the protein transporter

336

is bound to ribosomes, ATP and other polyanionic cell constituents; the remainder consists of

337

tightly regulated free Mg2+ at a concentration of 1-5 mM Mg2+ 36,41,42. The bonding of Mg2+ in the

338

organic compounds occurs as highly coordinated or strong covalent bonds

339

has shown that in a cell, bonded and free Mg2+ are in equilibrium with each other

340

laboratory experiments conducted on free Mg2+ and RNA-bound Mg have suggested chemical

341

equilibrium between these species

342

equilibrium, isotope equilibrium between free Mg2+ and bonded magnesium is likely established.

343

As a general rule, at chemical and isotope equilibrium, the heavier isotopes (26Mg) are enriched in

344

the stronger bonds (in this case e.g. the ATP or ribosomes) 47,33. The process of Mg bonding into

345

organic molecules as a likely cause of isotope fractionation leading to higher δ26Mg signatures in

346

plants has also been suggested by Schmitt at al. (2012) and Bolou-Bi et al. (2010)

347

effluxed from the fungus is depleted in

348

Mg-ATP and ribosomes at equilibrium. Continuous removal of this isotopically light Mg from

349

the cell results in fungal biomass enriched in

350

growth solution and the bulk cell is governed, in addition to the individual isotope fractionation

351

factors, by the relative fluxes into, within, and from the cell.

45,46

43

. Nierhaus (2014) 44

. Various

. Thus in a closed, well-mixed system at chemical

26

1,6

. The Mg2+

Mg because of preferential partitioning of

26

Mg into

26

Mg. Thus the net isotope fractionation between

352 353

4.4 Isotope fractionation dependence on pH?

354

Previous work has demonstrated that net biological isotope fractionations can be modeled as a

355

function of cellular influx/efflux rate ratios of the element in question, so that the parameter that

356

controls these rates can control the isotope fractionation. For example, dissolved Si uptake and

357

efflux rates involving marine sponges are related to ambient dissolved Si concentrations, so 15 ACS Paragon Plus Environment


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silicon isotope fractionation by these organisms varies predictably as a function of Si

359

concentration 48,49. Similarly, the ratio of calcification to organic carbon fixation fluxes has been

360

related to carbon isotope fractionation in coccolithophores 50. In the following, we present a mass

361

balance model showing how observed Mg isotope fractionation can vary with Mg uptake rate

362

into the cell, and address how the pH of the solution might control this flux.

363 364

Under acidic conditions (pH 3), more H+ ions are present in the cell wall compared to neutral pH.

365

Acidic conditions thus increase the proton gradient between the cell wall and the cytoplasm,

366

resulting in a higher input rate of (isotopically unfractionated) Mg from the growth solution into

367

the cell. About 95% of the Mg within the cell is in the form of isotopically fractionated Mg-

368

bearing compounds (e.g. ATP, ribosome, referred to as “Mgribo+ATP”), which have a higher

369

δ26Mgribo+ATP relative to the growth solution. As dictated by mass balance, at equilibrium in a

370

closed system this remaining 5% free Mg2+ (Mg2+free) obtains a lower isotope ratio (defined as

371

δ26Mgfree). This isotopically light Mg2+free will eventually be exported from the cell. There, it is

372

mixed into the large pool of Mg in the growth solution (without significantly affecting its isotopic

373

composition), to be replaced with an influx of fresh unfractionated Mg from the growth solution.

374

Thus the δ26Mgfree depends on the rate of replenishment of Mg2+free versus the rate of Mg bonding

375

into Mgribo+ATP compounds. Because of mass conservation in the cell, influx and efflux are

376

coupled. At low pH, this coupling results in higher efflux of isotopically fractionated Mg2+free low

377

in δ26Mgfree. Thus the total fungal cell Mg (Mg2+free + Mgribo+ATP) will be isotopically enriched in

378

heavy Mg isotopes. In contrast, in the buffered experiment at higher pH, the gradient is smaller,

379

and thus the rate of Mg influx is small too. This results in lower efflux of isotopically fractionated


Page 17 of 29


380

Mg2+free causing the fungal cell to have lower positive total δ26Mgfungi values than in the

381

unbuffered experiment.

382

We model these scenarios with a simple mass balance following a similar approach taken for Si

383

isotope fractionation during uptake in marine organisms 48,49,51. To determine the pH dependency

384

on the fractionation factor, the cell was treated as a continuous batch reactor (Figure 2). A

385

continuous batch reactor is a setup where there is a continuous flow of reactant in and out of the

386

reactor.

387 388

In this model, Rin is the input rate of Mg into the cell [moles sec-1]; Rbonded is the bonding rate of

389

Mg into Mgribo+ATP [moles sec-1]; Rout is the rate of Mg efflux [moles sec-1]; Rfree is the rate of free

390

Mg formation [moles sec-1]; δ26Mgsolution is the Mg isotopic composition of the unfractionated

391

influx of BG-11 growth solution; δ26Mgribo+ATP is the isotopic composition of Mgribo+ATP [‰];

392

δ26Mgfree is the Mg isotope composition of Mg2+free in the cell [‰]; Mribo+ATP = amount of Mg in

393

Mgribo+ATP [moles]; Mfree = amount of Mg2+free in the cell [moles].

394

The mass balance equation for the total cell is

395

δ26Mgfungi • Mcell = δ26Mgfree • Mfree + δ26Mgribo+ATP • Mribo+ATP (eq. 1),

396

where, Mcell is the total amount of Mg in fungal cell [moles].

397

As mentioned above, the partitioning of Mg between Mg2+free and Mgribo+ATP (5:95) is constant

398

but the rate of change in the Mg mass in a growing cell is not constant, and given by

399

,-./00 ,1

= R 34 − R 671 = R 864,9, + R :;99 ≈ R 864,9,

(eq. 2)



Page 18 of 29

400

The simplification on the right hand side of equation 2 can be done because 95% of the Mg is

401

contained in Mgribo+ATP.

402

And thus, Rin = Rout + Rbonded

403

Introducing corresponding δ-values into equation 3,

404

Rin • δ26Mgsolution = Rout • δ26Mgfree + Rbonded • (δ26Mgfree

405

Where,

406

Mg2+free and Mgribo+ATP

407

Now, substituting Rout = Rin ̶ Rbonded in equation 4, we then solve for δ26Mgfree

408

δ26Mgfree= δ26Mgsolution ̶ (Rbonded /Rin) •

409

Substituting equation 5 in equation 1:

410

δ26Mgfungi ̶ δ26Mgsolution = εribo+ATP ̶ free [fbonded ̶ (Rbonded /Rin)

411

Δ26Mgfungi

412

Where fbonded is (Mribo+ATP/Mcell), which is ca. 0.95. In this model we assume that the fractionation

413

factor between the species Mg2+free and Mgribo+ATP i.e. (εribo+ATP ̶ free) is a constant that does not

414

depend on the growth solution pH. This assumption is based on the fact that the external pH does

415

not affect the intracellular pH due to the homeostatic nature of the cell. In other words, the

416

conditions for formation of Mgribo+ATP compounds are independent of external pH.

εribo+ATP ̶ free = δ26Mgribo+ATP

̶ solution =

(eq. 3)

+

εribo+ATP

̶ free)

(eq. 4)

̶ δ26Mgfree, is the equilibrium isotope fractionation between

εribo+ATP ̶ free [fbonded

εribo+ATP ̶ free

̶ (Rbonded /Rin)

(eq. 5)

(eq. 6)

(eq. 7)

417


Page 19 of 29


418

From equation 6, we can infer that the isotopic difference between fungi and solution (∆26Mgfungi

419

solution)

420

εribo+ATP ̶ free in Figure 3. Since data on equilibrium fractionation factors for Mgribo+ATP compounds

421

are unavailable, we chose two values of 2 and 4 ‰. These fractionation factors were chosen

422

because they are comparable to the equilibrium fractionation factors between Mg organic

423

complexes (Mg oxalate and Mg citrate) and Mg(H2O)62+ calculated by Schott et al. (2016) 32. To

424

conserve mass, the ratio of (Rbonded /Rin) during growth has to be between 0 and 1. In general,

425

Δ26Mgfungi

426

free

̶

is inversely proportional to (Rbonded /Rin). We show this dependence for two different

̶ solution ranges

between 0.95 • εribo+ATP ̶ free (when Rbonded/Rin = 0) and -0.05 • εribo+ATP ̶

(when Rbonded /Rin = 1).

427

εribo+ATP ̶

428

Using the fractionation factor

429

unbuffered experiment are 0.64 and 0.36, respectively. Assuming a

430

Rbonded/Rin ratios for the buffered and unbuffered experiments are 0.80 and 0.66, respectively.

431

Because we do not know

432

However, we conclude that the relationship between isotopic difference (between fungi and

433

solution) and pH observed in our experiments can be explained by variations in (Rbonded /Rin).

free

of 2 ‰, Rbonded/Rin ratios for the buffered and

εribo+ATP ̶ free of

4 ‰, the

εribo+ATP ̶ free, we cannot assign actual Rbonded /Rin to a given pH value.

434 435

Environmental Implications

436

The ubiquitous distribution of fungi in ecosystems makes them an important component of soil

437

microbial communities that are responsible for a large amount of nutrient transfer, in particular

438

mineral nutrients that can limit plant productivity. We have chosen a representative species of 19 ACS Paragon Plus Environment


Page 20 of 29

439

microcolonial rock-inhabiting fungi to investigate Mg isotope fractionation and elucidate the

440

responsible cellular processes under acidic and neutral conditions. Selecting a representative

441

primary coloniser on bare rocks which is proven to interact with mineral substrates at various

442

environmental conditions increases the relevance of our findings if placed into the context of how

443

Mg cycles in the critical zone at the interface between the atmosphere, biosphere and lithosphere.

444 445

Equally important is that 80 % of terrestrial plant species are associated with mycorrhizal fungi

446

52

447

3,7,53,54

448

to this suggestion as for the first time we show that the same species of black fungi fractionates

449

Mg isotopes differently at two different pH levels. This is important information for ecosystem

450

studies where Mg isotopes are used to trace Mg uptake pathways from the soils or soil pore water

451

to fungi and then to plants, for example. We now know that using a single fractionation factor for

452

fungal uptake in these types of studies is incorrect, as the net isotopic factor varies with the

453

environment in which the fungi are growing.

. This implies that the Mg isotope fractionations derived for plant uptake in previous studies might actually reflect the prior uptake of Mg into the symbiotic fungi. Our study pertains

454 455

Previous studies on Mg isotopes in plants in both laboratory and natural settings have shown a

456

wide variety of fractionation between the root of the plant and respective growth solution at both

457

acidic and neutral conditions 5, 6,7. The general trend observed in some studies is that the roots of

458

the plants grown in acidic conditions are slightly heavier in 26Mg than the roots of plants grown

459

in neutral conditions 6,7. However until now there was no explanation provided for this trend. We

460

suggest that Mg undergoes equilibrium isotopic fractionation during incorporation of Mg into

461

ATP and ribosomes inside the fungal cell, and observe that the degree of isotope fractionation is 20 ACS Paragon Plus Environment

Page 21 of 29


462

dependent on the initial rate of Mg uptake. For plants that are not associated with symbiotic

463

fungi, the same mechanism for varying isotope fractionation during uptake may be relevant as

464

some of the Mg transport proteins for plants have been identified to belong to the same CorA

465

superfamily as the fungi

466

pathways for Mg uptake, and the pH-dependence of Mg isotope fractionation we observed in K.

467

petricola might also be observed in other organisms.

55

. We therefore speculate that higher plants follow similar metabolic

468 469

Supporting Information

470

Methods: PCR, ICP-OES, Mg column separation, MC-ICP MS, closed system mass balance

471

derivation. Tables: Quality control analysis for ICP-OES, Mg isotopes reference materials and

472

concentration and isotope analysis of individual samples. Figures: Three isotope plot and

473

PHREEQC speciation graph.

474 475

Acknowledgements

476

This research is the contribution of the ISONOSE Marie Curie Research training network funded

477

from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework

478

Programme FP7/2007-2013/ under REA grant agreement no (608068). From the GFZ research

479

group at GFZ Potsdam, we gratefully acknowledge Dr. Patrick Frings, Josefine Buhk, Marcus

480

Oelze and Jutta Bartel. We also want to thank Franziska Maria Stamm, Dr. Vasileios

481

Mavromatis, Dr. Oleg Pokrovsky and Dr. Jacques Schott from Géosciences Environnement

482

Toulouse for their advice on PHREEQC calculations and experimental design.



Page 22 of 29

484 485 486

References

487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528

1. Schmitt, A.-D.; Vigier, N.; Lemarchand, D.; Millot, R.; Stille, P.; Chabaux, F., Processes controlling the stable isotope compositions of Li, B, Mg and Ca in plants, soils and waters: A review. C. R. Geosci. 2012, 344, (11–12), 704-722. 2. Shirokova, L. S.; Mavromatis, V.; Bundeleva, I.; Pokrovsky, O. S.; Bénézeth, P.; Pearce, C.; Gérard, E.; Balor, S.; Oelkers, E. H., Can Mg isotopes be used to trace cyanobacteria-mediated magnesium carbonate precipitation in alkaline lakes? Biogeosci. Disc. 2011, 8, (4), 6473-6517. 3. Uhlig, D.; Schuessler, J. A.; Bouchez, J.; Dixon, J. L.; von Blanckenburg, F., Quantifying nutrient uptake as driver of rock weathering in forest ecosystems by magnesium stable isotopes. Biogeosciences 2017, 14, (12), 3111-3128. 4. Black, J. R.; Yin, Q.-z.; Casey, W. H., An experimental study of magnesium-isotope fractionation in chlorophyll-a photosynthesis. Geochim. Cosmochim. Acta 2006, 70, (16), 4072-4079. 5. Black, J.; Emanuel Epstein; William D. Rains; Yin, Q.-z.; Casey, W. H., Magnesium-Isotope Fractionation During Plant Growth. Environ. Sci. Technol. 2008, 42, (21), 7831-7836. 6. Bolou-Bi, E. B.; Poszwa, A.; Leyval, C.; Vigier, N., Experimental determination of magnesium isotope fractionation during higher plant growth. Geochim. Cosmochim. Acta 2010, 74, (9), 2523-2537. 7. Bolou-Bi, E. B.; Vigier, N.; Poszwa, A.; Boudot, J.-P.; Dambrine, E., Effects of biogeochemical processes on magnesium isotope variations in a forested catchment in the Vosges Mountains (France). Geochim. Cosmochim. Acta 2012, 87, 341-355. 8. Ra, K.; Kitagawa, H., Magnesium isotope analysis of different chlorophyll forms in marine phytoplankton using multi-collector ICP-MS. J. Anal. At. Spectrom. 2007, 22, (7), 817-821. 9. Fahad, Z. A.; Bolou-Bi, E. B.; Köhler, S. J.; Finlay, R. D.; Mahmood, S., Fractionation and assimilation of Mg isotopes by fungi is species dependent. Environ. Microbiol. Rep. 2016, 8, (6), 956-965. 10. Hoffland, E.; Kuyper, T. W.; Wallander, H.; Plassard, C.; Gorbushina, A. A.; Haselwandter, K.; Holmström, S.; Landeweert, R.; Lundström, U. S.; Rosling, A.; Sen, R.; Smits, M. M.; van Hees, P. A. W.; van Breemen, N., The role of fungi in weathering. Front. Ecol. Environ. 2004, 2, (5), 258-264. 11. Gadd, G. M., Geomycology: biogeochemical transformations of rocks, minerals, metals and radionuclides by fungi, bioweathering and bioremediation. Mycol. Res. 2007, 111, (1), 3-49. 12. Gadd, G. M., Fungi, Rocks, and Minerals. Elements 2017, 13, (3), 171-176. 13. Gadd, G. M., Geomicrobiology of the built environment. 2017, 2, 16275. 14. Finlay, R.; Wallander, H.; Smits, M.; Holmstrom, S.; van Hees, P.; Lian, B.; Rosling, A., The role of fungi in biogenic weathering in boreal forest soils. Fungal Biol. Rev. 2009, 23, (4), 101-106. 15. Selbmann, L.; Zucconi, L.; Isola, D.; Onofri, S., Rock black fungi: excellence in the extremes, from the Antarctic to space. Curr. Genet. 2015, 61, (3), 335-345. 16. Zhang, T.; Wang, N.-F.; Liu, H.-Y.; Zhang, Y.-Q.; Yu, L.-Y., Soil pH is a Key Determinant of Soil Fungal Community Composition in the Ny-Ålesund Region, Svalbard (High Arctic). Front Microbiol 2016, 7, (227), DOI 10.3389/fmicb.2016.00227. 17. Gorbushina, A. A.; Kotlova, E. R.; Sherstneva, O. A., Cellular responses of microcolonial rock fungi to long-term desiccation and subsequent rehydration. Stud Mycol 2008, 61, (1), 91-97. 18. Gorbushina, A. A.; Broughton, W. J., Microbiology of the Atmosphere-Rock Interface: How Biological Interactions and Physical Stresses Modulate a Sophisticated Microbial Ecosystem. Annu. Rev. Microbiol. 2009, 63, (1), 431-450. 22 ACS Paragon Plus Environment

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19. Noack-Schönmann, S.; Bus, T.; Banasiak, R.; Knabe, N.; Broughton, W. J.; Den Dulk-Ras, H.; Hooykaas, P. J. J.; Gorbushina, A. A., Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions. AMB Express 2014, 4, (1), 80-85. 20. Nai, C.; Wong, H. Y.; Pannenbecker, A.; Broughton, W. J.; Benoit, I.; de Vries, R. P.; Gueidan, C.; Gorbushina, A. A., Nutritional physiology of a rock-inhabiting, model microcolonial fungus from an ancestral lineage of the Chaetothyriales (Ascomycetes). Fungal Genet. Biol. 2013, 56, (0), 54-66. 21. Seiffert, F.; Bandow, N.; Bouchez, J.; von Blanckenburg, F.; Gorbushina, A. A., Microbial Colonization of Bare Rocks: Laboratory Biofilm Enhances Mineral Weathering. Procedia Earth Planet. Sci. 2014, 10, (0), 123-129. 22. Krumbein, W. E.; Jens, K., Biogenic rock varnishes of the negev desert (Israel) an ecological study of iron and manganese transformation by cyanobacteria and fungi. Oecologia 1981, 50, (1), 25-38. 23. Staley, J. T.; Palmer, F.; Adams, J. B., Microcolonial Fungi: Common Inhabitants on Desert Rocks? Science 1982, 215, (4536), 1093-1095. 24. Yurlova, N. A.; de Hoog, G. S.; Fedorova, L. G., The influence of ortho- and para-diphenoloxidase substrates on pigment formation in black yeast-like fungi. Stud Mycol 2008, 61, 39-49. 25. Martin-Sanchez, P. M.; Gorbushina, A. A.; Kunte, H.-J.; Toepel, J., A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae. Biofouling 2016, 32, (6), 635-644. 26. Coplen, T. B., Guidelines and recommended terms for expression of stable-isotope-ratio and gasratio measurement results. Rapid Commun. Mass Spectrom. 2011, 25, (17), 2538-2560. 27. Gadd, G. M., Metals, minerals and microbes: geomicrobiology and bioremediation. Microbiology 2010, 156, 609-643. 28. Flatman, P., Magnesium transport across cell membranes. J. Membr. Biol. 1984, 80, (1), 1-14. 29. Okorokov, L. P.; Kholodenko, V. P.; Kadomtseva, V. M.; Petrikevich, S. B.; Zaichkin, E. I.; Karimova, A. M., Free and Bound Magnesium in Fungi and Yeasts. Folia Microbiol. 1975, 20, (6), 460-466. 30. Kehres, D. G.; Maguire, M. E., Structure, properties and regulation of magnesium transport proteins. BioMetals 2002, 15, (3), 261-270. 31. Moomaw, A. S.; Maguire, M. E., The Unique Nature of Mg(2+) Channels. Physiology (Bethesda) 2008, 23, (5), 275-285. 32. Schott, J.; Mavromatis, V.; Fujii, T.; Pearce, C. R.; Oelkers, E. H., The control of carbonate mineral Mg isotope composition by aqueous speciation: Theoretical and experimental modeling. Chem. Geol. 2016, 445, 120-134. 33. Schauble, E. A., Role of nuclear volume in driving equilibrium stable isotope fractionation of mercury, thallium, and other very heavy elements. Geochim. Cosmochim. Acta 2007, 71, (9), 2170-2189. 34. Nelson, D. L.; Kennedy, E. P., Magnesium Transport in Escherichia coli : INHIBITION BY COBALTOUS ION. J. Biol. Chem. 1971, 246, (9), 3042-3049. 35. Johnson, C. M.; Beard, B. L.; Albarède, F., Overview and General Concepts. Rev. Mineral. Geochem. 2004, 55, (1), 1-24. 36. Gardner, R. C., Genes for magnesium transport. Curr. Opin. Plant Biol. 2003, 6, (3), 263-267. 37. Maguire, M. E., Magnesium transporters: properties, regulation and structure. Front. Biosci. 2006, 11, 3149-3163. 38. Groisman, E. A.; Hollands, K.; Kriner, M. A.; Lee, E.-J.; Park, S.-Y.; Pontes, M. H., Bacterial Mg(2+) Homeostasis, Transport, and Virulence. Annu. Rev. Genet. 2013, 47, 625-646. 39. Shaul, O., Magnesium transport and function in plants: the tip of the iceberg. BioMetals 2002, 15, (3), 307-321. 40. Hawkesford, M.; Horst, W.; Kichey, T.; Lambers, H.; Schjoerring, J.; Møller, I. S.; White, P., Chapter 6 - Functions of Macronutrients A2 - Marschner, Petra. In Marschner's Mineral Nutrition of Higher Plants (Third Edition), Academic Press: San Diego, 2012; pp 135-189. 23 ACS Paragon Plus Environment


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41. Moncany, M. L. J.; Kellenberger, E., High magnesium content ofEscherichia coli B. Experientia 1981, 37, (8), 846-847. 42. Alatossava, T.; Jütte, H.; Kuhn, A.; Kellenberger, E., Manipulation of intracellular magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by ionophore A23187. J. Bacteriol. 1985, 162, (1), 413-419. 43. Kirkby, E., Chapter 1 - Introduction, Definition and Classification of Nutrients A2 - Marschner, Petra. In Marschner's Mineral Nutrition of Higher Plants (Third Edition), Academic Press: San Diego, 2012; pp 3-5. 44. Nierhaus, K. H., Mg(2+), K(+), and the Ribosome. J. Bacteriol. 2014, 196, (22), 3817-3819. 45. Zitomer, R. S.; Flaks, J. G., Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association. J. Mol. Biol. 1972, 71, (2), 263-279. 46. Stein, A.; Crothers, D. M., Equilibrium binding of magnesium(II) by Escherichia coli tRNAfMet. Biochemistry 1976, 15, (1), 157-160. 47. Bigeleisen, J.; Mayer, M. G., Calculation of Equilibrium Constants for Isotopic Exchange Reactions. J. Chem. Phys. 1947, 15, (5), 261-267. 48. Wille, M.; Sutton, J.; Ellwood, M. J.; Sambridge, M.; Maher, W.; Eggins, S.; Kelly, M., Silicon isotopic fractionation in marine sponges: A new model for understanding silicon isotopic variations in sponges. Earth Planet. Sci. Lett. 2010, 292, (3–4), 281-289. 49. Hendry, K. R.; Robinson, L. F., The relationship between silicon isotope fractionation in sponges and silicic acid concentration: Modern and core-top studies of biogenic opal. Geochim. Cosmochim. Acta 2012, 81, 1-12. 50. McClelland, H. L. O.; Bruggeman, J.; Hermoso, M.; Rickaby, R. E. M., The origin of carbon isotope vital effects in coccolith calcite. Nat. Commun. 2017, 8, 14511. 51. Milligan, A. J.; Varela, D. E.; Brzezinski, M. A.; Morel, F. M. M., Dynamics of silicon metabolism and silicon isotopic discrimination in a marine diatomas a function of pCO2. Limnol. Oceanogr. 2004, 49, (2), 322-329. 52. Landeweert, R.; Hoffland, E.; Finlay, R. D.; Kuyper, T. W.; van Breemen, N., Linking plants to rocks: ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol. Evol. 2001, 16, (5), 248-254. 53. Tipper, E. T.; Gaillardet, J.; Louvat, P.; Capmas, F.; White, A. F., Mg isotope constraints on soil pore-fluid chemistry: Evidence from Santa Cruz, California. Geochim. Cosmochim. Acta 2010, 74, (14), 3883-3896. 54. Tipper, E. T.; Lemarchand, E.; Hindshaw, R. S.; Reynolds, B. C.; Bourdon, B., Seasonal sensitivity of weathering processes: Hints from magnesium isotopes in a glacial stream. Chem. Geol. 2012, 312, 80-92. 55. Li, L.; Tutone, A. F.; Drummond, R. S. M.; Gardner, R. C.; Luan, S., A Novel Family of Magnesium Transport Genes in Arabidopsis. Plant Cell 2001, 13, (12), 2761-2775.

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620

Tables

621 622

Table 1: Compositiona of modified BG-11 media

623 624 625 626 627

Species

Concentration (mM)

NaEDTA

0.0027

NH4Fe citrate

0.0229

Citrate*H2O

0.0204

CaCl2*2H2O

0.2449

MgSO4*7H2O

0.15

K2HPO4*3H2O

0.1753

H3BO3

0.0463

MnCl2*4H2O

0.0058

ZnSO4*7H2O

0.0008

CuSO4*5H2O

0.0003

CoCl2*6H2O

0.0002

NaMoO4*H2O

0.0019

a The components of the BG 11 media were diluted to 1 L with MilliQ water. Then 0.75 g of NaNO3 (8.82 mM), 0.75 g of NH4NO3 (9.37 mM) and 0.02 g of Na2CO3 (0.189 mM) was added. The pH of the solution was adjusted to 6 and then autoclaved. Finally after autoclaving, 2000 mg filtered, sterilized glucose*H2O (11.22 mM) was added. For buffered BG-11 media, 10662 mg filter sterilized MES*H2O (54.62 mM) was further added.

628 629 630 631 632 633 634 635 636 25 ACS Paragon Plus Environment


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637 638 639

Table 2: Mg Concentration and isotope analysis of the growth solution and biomass

640 641 642 643 644 645 646

Species

Experimental condition

Mg amount in initial BG11 media (µg)

Mg amount in initial biomass (µg)

Dry weight of final biomass (g) ± SDb

Mg amount in final BG11 media (µg) ± SDb

Mg amount in final biomass (µg) ± SDb

Mg concentration in final biomass (µg of Mg/gram of dry cells) ± SDb

Mg isotopic difference (∆26Mgfungi ̶ c solution) ± 2SD

K.petricola

Buffered

1449

0.44

0.03 ± 0.01

1429 ± 10

9.37 ± 0.73

365.28 ± 65.25

0.65 ± 0.14

a

Mutant

Buffered

1449

0.37

0.06 ± 0.03

1400 ± 29

25.48 ± 9.85

401.50 ± 11.48

0.78 ± 0.14

K.petricola

Unbuffered

1556

0.89

0.27 ± 0.03

1497 ± 10

66.83 ± 0.17

245.84 ± 22.46

1.11 ± 0.35

a

Mutant is the melanin-minus mutant of K. petricola. SD represents standard deviation from the triplicate measurements. c 2SD represents the error propagation of analytical uncertainties for individual measurement analyzed by MC-ICPMS or uncertainty derived from the repeatability of three independent experiments, whichever was larger. The δ26MgDSM3 value of BG-11 during the experiments was constant over time, i.e., measured at -4.50 ± 0.1 (2SD, n=11) for the buffered and -4.45 ± 0.1 (2SD, n=3) for the unbuffered experiments. b

647 648 649 650 651 652 653 654 655 656 657 26 ACS Paragon Plus Environment

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658 659

Figures



661 662 663 664 665 666 667 668 669 670 671 672

Figure 1: Analysis of various components of the buffered (left column) and unbuffered (right column) experiment as a function of experimental run time. Open circles (○) represent abiotic experiments, closed circles (●) represent K.petricola experiments, open triangles (∆) represent melanin-minus mutant experiments, closed squares (■) represent Mg isotopic composition of K.petricola biomass and open squares represent (□) Mg isotopic composition of melanin-minus mutant biomass. All error bars represents 2SD. For graphs of number of cells, pH (growth solution), and concentration (Mg, P, K, and Fe of growth solution), the analytical uncertainty or the repeatability of independent experiments was used for interpretation, whichever was larger. ∆26Mgfungi/media ̶ solution is the isotopic difference between either the biomass of fungi or the growth solution at respective sampling time and the initial growth solution. The error on the graph of ∆26Mgfungi/media ̶ solution represent the error propagation of analytical uncertainties for individual measurement analyzed by MC-ICPMS or uncertainty derived from the repeatability of independent experiments, whichever was larger. Detailed information on both types of uncertainties for all data points are given in the supplement (Table S3). For plots where no error bars are shown, the uncertainties are smaller than the symbols.

673 674 675 676

677 678 679 680 681

Page 28 of 29

Figure 2: Simplified model of Mg transport pathways into and and out of the fungal cell. The figure represents cellular magnesium homeostasis, showing the balance between input of Mg into the cell, Mg bonded to the organic molecules ATP and ribosomes, free magesium in the cytoplasm, and efflux of magnesium from the cell. Detailed information of the individual compartments and the corresponding δ-values are given in the main text.

682 683 684 685


Page 29 of 29

686 687 688 689 690 691


Figure 3: The figure shows the dependence of ∆26Mgfungi ̶ solution on the ratio of (Rbonded / Rin) at two assumed equilibrium fractionation factors εribo+ATP ̶ free, i.e., for εribo+ATP ̶ free = 2 (red dashed line) and 4 (black dashed line) in equation 4. Solid horizontal lines represent the measured isotopic difference between fungi and growth solution (∆26Mgfungi ̶ solution) at buffered and unbuffered conditions.

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Mg Isotope Fractionation during Uptake by a Rock ... - ACS Publications

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