Hematite Reduction Buffers Acid Generation and Enhances Nutrient

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Hematite Reduction Buffers Acid Generation and Enhances Nutrient Uptake by a Fermentative Iron Reducing Bacterium, Orenia metallireducens Strain Z6 Yiran Dong, Robert A Sanford, Yun-Juan Chang, Michael J. Mcinerney, and Bruce W. Fouke Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04126 • Publication Date (Web): 12 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016

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Environmental Science & Technology

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Hematite Reduction Buffers Acid Generation and Enhances Nutrient Uptake by a

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Fermentative Iron Reducing Bacterium, Orenia metallireducens Strain Z6

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Yiran Dong*, †, ‡, Robert A. Sanford‡, Yun-juan Chang#, Michael J. McInerney , and Bruce

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W. Fouke†, ‡, #, §

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†

Carl R. Woese Institute for Genomic Biology, ‡ Department of Geology, # Roy J. Carver

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Biotechnology Center, § Department of Microbiology, University of Illinois Urbana-

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Champaign, Urbana, Illinois, 61801, United States, Department of Microbiology and Plant

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Biology, University of Oklahoma, Norman, Oklahoma, 73019, United States

∥

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*Corresponding author email: [email protected];

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Phone: 1 (217) 300-1625; fax: 1 (217) 244-0877

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Mailing address: Carl R. Woese Institute for Genomic Biology and Department of Geology,

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University of Illinois Urbana-Champaign, 1206 W. Gregory Drive, Urbana, IL, 61801, USA

ACS Paragon Plus Environment

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Abstract

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Fermentative iron-reducing organisms have been identified in a variety of environments.

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Instead of coupling iron reduction to respiration, they have been consistently observed to use

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ferric iron minerals as an electron sink for fermentation. In the present study, a fermentative

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iron reducer, Orenia metallireducens strain Z6, was shown to use iron reduction to enhance

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fermentation not only by consuming electron equivalents, but also by generating alkalinity

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that effectively buffers the pH. Fermentation of glucose by this organism in the presence of a

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ferric oxide mineral, hematite (Fe2O3), resulted in enhanced glucose decomposition compared

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with fermentation in the absence of an iron source. Parallel evidence (i.e., genomic

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reconstruction, metabolomics, thermodynamic analyses and calculation of electron transfer)

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suggested hematite reduction as a proton-consuming reaction effectively consumed acid

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produced by fermentation. The buffering effect of hematite was further supported by a

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greater extent of glucose utilization by strain Z6 in media with increasing buffer capacity.

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Such maintenance of a stable pH through hematite reduction for enhanced glucose

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fermentation complements the thermodynamic interpretation of interactions between

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microbial iron reduction and other biogeochemical processes. This newly discovered feature

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of iron reducer metabolism also has significant implications for groundwater management

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and contaminant remediation by providing microbially mediated buffering systems for the

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associated microbial and/or chemical reactions.

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TOC/Abstract Art

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INTRODUCTION

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Microbial iron reduction is an important geochemical process ubiquitous in diverse natural

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environments and plays important roles in controlling carbon cycling. Microbial iron

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reduction can be catalyzed by respiratory iron-reducing organisms that rely on Fe(III)

50

reduction for energy generation. Other organisms such as fermentative iron reducers do not

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conserves a significant fraction of their total energy from iron reduction to support growth1, 2.

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In this process, these organisms ferment organic substrates (e.g. sugars, short-chain fatty

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acids or amino acids) to obtain electron equivalents for reduction of ferric iron1, 3-5. Some of

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the taxa involved in fermentative iron reduction include Clostridium saccarobutylicum, C.

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butyricum, Desulfovibrio alaskensis and Desulfotomaculum reducens6-8. Compared with

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respiratory iron reduction, understanding of the metabolic mechanisms of fermentative iron-

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reducing organisms has been relatively limited.

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Fermentative iron reducers concurrently consume organic substrates (e.g., glucose or

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pyruvate) and produce Fe(II)1, 5-9. The presence of Fe(III) substrates (e.g., Fe(III)-citrate or

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ferrihydrite) significantly enhanced decomposition of the available fermentable substrates

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and increased biomass yields compared to the cultures without Fe(III) amendment. These

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results were hypothesized to be due to enhanced thermodynamic favorability of fermentative

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metabolisms in the presence of iron minerals5, 10. In addition, fermentative iron reduction

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redirects electron and carbon fluxes to less-reduced fermentation products, resulting in faster

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substrate consumption, higher accumulation of fermentation products and greater heat

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generation compared to cultivation without Fe(III) amendments6-8. During fermentative iron

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reduction, however, typically less than 5% of the reducing equivalents from the fermentation

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are transferred to ferric iron. Therefore, it has been widely accepted that iron reduction itself

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only acts as an electron sink and provides limited benefit for the organisms in terms of energy

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and biomass production3-5.


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Almost all of these studies only evaluated soluble Fe(III) complexes (e.g., Fe(III)-

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citrate, Fe(III)-maltol) or amorphous ferrihydrite7. In contrast, the effects of the more

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crystalline iron minerals such as hematite or goethite have been poorly understood11, 12. This

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knowledge gap is important to fill because iron reducers have significantly different capacity

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to transform ferric iron minerals with varied crystallinity, solubility and electrical potential1, 2.

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Some respiratory iron-reducing organisms have been shown to reduce hematite or goethite3,

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13-19

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of reducing hematite in association with glucose fermentation20. These observations, coupled

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with high abundance of these crystalline ferric minerals in different natural environments and

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the fact that they are common weathered products of metastable ferrihydrite21, 22, suggest that

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these substances may also be important natural Fe(III) sources for fermentative iron reducers.

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. Two fermenting species (Clostridium acetobutylicum and C. butyricum) are also capable

In this study, Orenia metallireducens strain Z6 was used as a model organism to

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investigate the mechanism of fermentative iron reduction. This strain was isolated from the

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terrestrial deep subsurface of the Illinois Basin, IL, where quartz arenite coated with ferric

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iron mineral (e.g., hematite and goethite) is ubiquitous and a high concentration of Fe(II) was

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observed in the groundwater (1.18 mM)23, 24. Strain Z6 possessed fermentation and it has

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been the first Orenia species with identified iron-reducing capacity25. It ferments sugars and

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uses H2, a fermentation product, as the sole electron donor for reduction of both amorphous

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and crystalline ferric iron oxides (Dong et al., submitted). These well-defined properties

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enabled us to develop a simplified experimental system to investigate the effect of iron

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reduction on the physiology of strain Z6. Informed by genomic analysis, the reconstruction of

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fermentation-related pathways provided a basis to predict and substantiate intermediates and

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products during growth with glucose. It also allowed calculation of the energy efficiency of

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fermentation with and without the presence of hematite. The kinetics of substrate

95

consumption, concentrations of fermentation products, and biomass yields were


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experimentally compared between the two conditions. The results indicated that iron

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reduction benefited energy usage; however, the biggest benefit of iron reduction appeared to

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be a buffering effect for the acid generated during fermentation that allowed for more glucose

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utilization and higher growth.

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EXPERIMENTAL SECTION

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Culture Experiments. A series of batch culture experiments was developed to investigate

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the impact of different factors on the growth of strain Z6. All amendments were added from

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sterile anaerobic stock solutions. Na2S (200 µM) was added as the reductant to maintain

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anaerobic condition. The cultures were prepared in serum bottles or anaerobic culture tubes

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sealed with butyl rubber stoppers and aluminum seals. Other than shaken manually once a

107

day, they were incubated statically at 42 °C in the dark. For all the culturing conditions,

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replicates were prepared and abiotic controls without cell inoculation were set up to exclude

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potential contributions of abiotic reactions.

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Stoichiometric Analyses of Glucose Fermentation. A set of batch cultures was

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developed to determine the stoichiometry of glucose utilization by strain Z6 under two

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conditions: fermentation alone and fermentation in the presence of hematite. Sterile anaerobic

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basal medium26 was prepared under a N2 headspace. Seventy-two milliliters of basal medium

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was added to 160 mL serum bottles. The medium was buffered with 10 mM piperazine-N,N′-

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bis(2-ethanesulfonic acid) (PIPES) with the final pH 6.5. After autoclaving, 8.0 mL of filter-

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sterilized (0.22 µm) deep subsurface groundwater27 was added to each bottle for the final

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aqueous volume ratio of 10%. The groundwater was obtained from the 2.02 km deep

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subsurface of the Mt. Simon Sandstone Formation of the Illinois Basin, IL and was the

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original source of isolation of strain Z627. Glucose (4.23-4.35 mM) was added as the


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fermentative substrate and the cultures receiving iron oxide were amended with hematite (7

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mM), synthesized as previously reported28.

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Strain Z6 was inoculated at a dilution ratio of 1:50 from a parent culture grown on 5

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mM glucose. At each time point, the cultures were collected for the analyses of different

124

fermentation products and pre-treated for Fe(II) analysis (see Fig. S1 in the Supporting

125

Information (SI)). Basically, one milliliter of headspace gas was withdrawn using a sterile

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syringe and transferred into a sealed 10 mL glass serum vial to analyze gaseous products (e.g.,

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CO2 and H2). Then, a total of 1.7 mL of well-mixed culture was collected with a sterile

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syringe and divided into three parts for the hematite-fed cultures: 0.2 mL was promptly

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acidified with 0.2 mL HCl (1 M) to determine ferrous iron concentration; 0.5 mL was

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injected into a 5 mL sealed glass serum vial containing 100 µL 50 % phosphoric acid to

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quantify dissolved inorganic carbon (i.e., CO2); and the remaining 1 mL culture was placed in

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a microcentrifuge tube and centrifuged at 4,000 ×g for 5 min at 4 °C. The supernatant from

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the centrifuged culture was carefully transferred into a new microcentrifuge tube to quantify

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concentrations of glucose and soluble fermentation products. For the fermentation-alone

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cultures, both gaseous and liquid samples were collected and analyzed in the same manner as

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above except that no portion was collected for ferrous iron quantification. The stoichiometric

137

oxidation-reduction equations without cell synthesis were constructed according to a

138

previously reported method29, 30 as detailed in SI. pH was measured at the end of the

139

experiment using a PHM210 Standard pH meter (Hach Company, CO).

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pH-Adjusted Glucose Fermentation. The culturing conditions were similar to those

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for the stoichiometric analyses as described above with some minor modifications: increased

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glucose concentration (10 mM) and smaller volume (15.0 ml of medium in 27 mL anaerobic

143

culture tubes).


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All the cultures were started with glucose fermentation alone. After growing for 44

145

hours, cell density leveled off (optical density at 600 nm (OD600) 0.16±0.01) with an

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observed pH 5.7±0.19. In order to assess the relative effects of the decrease in pH from the

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initial value (pH 6.5) and accumulation of fermentation products (e.g. H2 and CO2) on

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termination of the fermentation, triplicate cultures were split for each of four different

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treatments at this time point. These included: 1) no amendment; 2) pH was adjusted to 6.5

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with 1M sterile NaOH as needed; 3) headspace was flushed with N2 as needed; and 4) both

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pH adjustment and headspace sparging with N2 were performed as needed. For 2) and 4) pH

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was adjusted to 6.5 and headspace was re-equilibrated with sterile N2 repeatedly according to

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the monitored pH and cell growth of the cultures. In order to evaluate fermentative iron

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reduction with hematite by strain Z6, two additional culturing conditions were created on the

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same pre-fermenting cultures: 5) addition of 10 mM hematite; and 6) addition of 10 mM

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hematite followed by adjustment of pH to 6.5.

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At each time point, 0.2 mL of well-mixed culture was collected using sterile-nitrogen-

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flushed syringes and centrifuged at 4°C and 4000 ×g for 5 minutes. The supernatant was

159

carefully transferred into new microcentrifuge tubes to measure glucose concentrations. For

160

the cultures amended with hematite, an additional 0.2 mL culture was withdrawn and

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promptly acidified by 0.2 mL 1N HCl for ferrous iron analysis. pH was determined at -45, 15,

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67 and 99 hours and NaOH was amended as described above if significant decrease in pH

163

had occurred. Right before each round of pH adjustment, 1-1.5 mL cultures were collected

164

from all the samples and centrifuged. After carefully removing the supernatant, the pellets

165

were washed once with cold PBS buffer solution (pH 7.4) and stored at -20°C for

166

measurement of protein concentrations.

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Effect of pH Buffer Strength on Glucose Fermentation. In order to testify whether pH

168

condition was critical to sustain fermentation and the coexisting hematite reduction acted as a


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microbially mediated buffering system, a series of cultures with different buffer capacity

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were created. Since it was known that acid was produced during fermentation, we

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investigated whether different buffers can control fluctuation of pH and mediate efficiency of

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glucose fermentation. Cultures were grown in media with different buffer strengths to

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evaluate their capacity to maintain stable pH conditions, and to influence the glucose

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metabolism and growth. The buffers tested included: a) 10 mM PIPES; b) 10 mM PIPES and

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20 mM sodium bicarbonate; c) 10 mM 2-(N-morpholino)ethanesulfonic acid (MES); and d)

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10 mM MES and 20 mM sodium bicarbonate. For the media with bicarbonate, they were

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equilibrated by sparging with a N2:CO2 (80:20, v:v) gas mix. The pH was adjusted to 6.5

178

using 1N HCl or NaOH. The buffer capacity was calculated based on:

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= 2.303( + + ∑

( )

).

180

where Kw is water ionization constant; Ka is the dissociation factor of the acid from a specific

181

buffer; and Cbuffer is the concentration of the buffer31.

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Cellular and chemical analyses. OD600 was determined using a SPECTRONIC

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20D+ UV-Vis spectrophotometer (Thermos Scientific, MA). Concentrations of ferrous iron

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were quantified with the ferrozine method32, 33 using a Genesys 20 spectrophotometer

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(Thermo Fisher Scientific Inc., MA) at 562 nm. Total iron concentration was determined by

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reducing ferric iron into ferrous iron with hydroxylamine hydrochloride before the same

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colorimetric analysis34. Concentrations of glucose were determined in the culture supernatant

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using the Glucose (GO) Assay Kit (Sigma-Aldrich Co., MO) following the manufacturer’s

189

instructions. Fermentation products were measured following the methods described in the SI.

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Protein content was measured using a Micro BCATM Protein Assay (Thermo Scientific Pierce

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Protein Biology Products, IL) (SI).

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Genomic and metabolomic analyses. The genome of O. metallireducens strain Z6 (IMG Genome ID 2687453649) was used for reconstruction of fermentation related pathways


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with the aid of IMG/ER pipeline35. Metabolic intermediates of glucose fermentation were

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analyzed on the culture during exponential phase of growth (~0.5 in OD600). The centrifuged

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cell pellets were washed twice with cold PBS buffer (pH = 7.4) before they were promptly

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quenched with 10 mL HPLC-grade pre-chilled methanol (Sigma-Aldrich, MO). The

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quenched samples were submitted to the Metabolomics Services, Roy J. Carver

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Biotechnology Center of UIUC for analyses following the published method36.

200 201

RESULTS

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Fermentation with and without ferric iron. Strain Z6 carried out glucose consumption at

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similar rates via fermentation alone (-0.70±0.23 day-1) and fermentation with hematite (-

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0.58±0.10 day-1) (Fig. 1). Under both growth conditions, substrate consumption activity

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slowed after the first three days. For fermentation alone, 3.06 mM of the glucose (initial

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concentration 4.23±0.32 mM) was consumed; approximately 28% of the glucose remained

207

after the initial rapid decrease and no further consumption occurred afterward. In contrast,

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complete glucose consumption occurred when coupled to active hematite reduction (Fig. 1

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and Table 1). At the end of the growth phase, about 30% (w/w) of the amended hematite was

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reduced, generating 4.3 mM Fe(II). In the abiotic controls under the same conditions without

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inoculation of cells, no glucose consumption or ferrous iron production was observed (Fig. 1).

212

Glucose fermentation products generated by strain Z6 included different

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concentrations of short chain fatty acids, ethanol and H2. Quantitatively, glucose fermentation

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led to generation of H2 (35551±6594 ppmv), acetate (2.05 mM), formate (3.04 mM), ethanol

215

(4.34 mM) and a trace amount of lactate (0.13 mM) (Table 1 and Figs. 1 and S2). In

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comparison, in the presence of hematite, the final ethanol and acetate concentrations

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increased to 5.74 and 2.78 mM, respectively, while similar formate production was observed

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(Table 1). However, significantly lower H2 concentration was observed (12882±2027 ppmv)


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and lactate was below the detection limit under this experimental condition (Table 1, Figs. 1

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and S2).

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The observed glucose fermentation products by strain Z6 were consistent with the

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reconstructed fermentation-related pathways based on its genome. The genome of strain Z6 is

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3.47 Mb and contains 3,269 protein-coding genes and 52 predicted tRNAs (Dong et al.,

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submitted). Metabolic reconstruction by the IMG/ER pipeline35, based on the annotated open

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reading frames (ORFs) from the genome of strain Z6, showed that this organism carries out

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glucose fermentation using glycolysis and pentose phosphate pathway (PPP), from which the

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produced pyruvate is further metabolized to the detected fermentation products (Fig. 2). This

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finding was also substantiated by identification of the corresponding intermediates and

229

products (Table 1, Figs. 1, 2 and S2). The predicted pathways suggested that pyruvate was

230

reduced to lactate or decarboxylated to acetyl-CoA and acetyl phosphate, which were further

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transformed to ethanol and acetate, respectively (Fig. 2). Gene annotation also predicted a

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total of 25 hydrogenase-associated genes in the genome of strain Z6 (Table S1). Among them,

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there are both Fe-only hydrogenases and [Ni-Fe] hydrogenases, which may catalyze the

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reactions producing and consuming molecular hydrogen, respectively37.

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Stoichiometric analysis of the glucose fermentation reactions with and without

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hematite suggested the addition of hematite might provide a potential thermodynamic

237

advantage. Two stoichiometric reactions were generated based on the observed fermentation

238

products (Table 1)30. The observed product concentrations fit well (+/- 11.2 %) with these

239

predicted stoichiometric equations. The Gibbs free energies (∆Gr, 42°C) calculated for the

240

glucose metabolic reactions (Rxns 1 and 2 in Table 1) showed that neither culture condition

241

was thermodynamically constrained at the end of the reactions. Therefore, unidentified

242

factors other than thermodynamic constraints likely accounted for the 30% of glucose

243

remaining in the fermentation-only culture. Possible reasons for incomplete glucose


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utilization were further investigated. Comparison of Reactions 1 and 2 (Table 1) showed that although measured ethanol

246

and acetate concentrations differed under the two conditions, their stoichiometric ratios to the

247

consumed glucose were very similar, independent of the availability of hematite. Meanwhile,

248

in the cultures with hematite, lower concentrations of H2 were observed compared to those

249

with glucose alone (Fig. 1 and Table 1). In contrast to the pH decrease to 5.60±0.06 in the

250

fermentation-alone cultures, relatively stable pH was maintained when hematite was present

251

(Fig. S3).

252

To evaluate the factors limiting glucose fermentation by strain Z6 in the absence of

253

hematite, a suite of growth conditions was created. Briefly, several replicate cultures were

254

grown on glucose until growth stopped at pH reached about 5.7 and when 2.51-3.02 mM

255

glucose was consumed. Although the initial concentrations of glucose for these cultures

256

(9.74±0.48 mM) were about two times as high as that that for the stoichiometric analysis as

257

described above (Table 1 and Fig. 1), the amount of the glucose consumed was similar when

258

the reaction leveled off. At that time, several manipulations of the culture conditions were

259

tested to determine their impact on glucose consumption. The results showed that without

260

increasing the pH, neither the “Glucose” only condition nor repetitive flushing of the

261

headspace to remove CO2 and H2 “Glucose+Flushing” led to further substrate consumption

262

or growth in the cultures. When pH was increased from 5.6 to about 6.4 by adding NaOH

263

(“Glucose+NaOH” and “Glucose +NaOH+Flushing”), glucose fermentation resumed until

264

the pH dropped again (Fig. 3). For the two sets of cultures with pH adjustment, a total of

265

three NaOH additions were required for complete glucose depletion (Fig. 3). The pH

266

measured in non-amended control cultures at various time points (15, 67 and 99 hours)

267

consistently showed that pH values were kept below 5.5 over the course of the experiments,

268

which was significantly lower than the initial pH (Figs. 3e-3f). In addition, for the cultures


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with multiple pH adjustment, growth slowed down when the pH decreased to values less than

270

5.5 (Figs. 3a-3b).

271

Since iron reduction and pH adjustment had both been shown to enhance glucose

272

fermentation, the ability of hematite reduction to buffer the acidity generated during

273

fermentation was evaluated. To test this, the same cultures were pre-grown by glucose

274

fermentation alone and manipulated after glucose consumption stopped. At this point, when

275

the pH dropped to 5.5, hematite was added to the cultures “Glucose+Hematite”, only a slight

276

increase in pH was observed. In contrast, in the replicate glucose-fermenting cultures

277

“Glucose+hematite+NaOH” with both adjusted pH (to 6.4) and hematite addition, no further

278

pH adjustment was needed to maintain the value above 6.0 before all the glucose was

279

consumed (Fig. 4). This was in contrast to the repetitive NaOH additions required in the

280

absence of hematite (Fig. 3). As expected, the “Glucose+hematite+NaOH” cultures also

281

exhibited an increase in Fe(II) concentration (Fig. 4) indicative of active iron reduction

282

shown in Fig. 1.

283

In addition to measuring OD600, cell growth was also quantified by determining

284

protein concentrations at two time points (67 and 99 hours) when glucose consumption

285

leveled off. An increase in biomass was only observed after pH adjustment and when the

286

fermentation activity resumed. In contrast, without pH modification (cultures: Glucose,

287

Glucose+Flushing, and Glucose+Hematite) the protein concentrations remained stable, which

288

was consistent with the observation of no further glucose breakdown (Figs. 3 and 5).

289

Glucose Fermentation under different buffering conditions. In order to confirm

290

that the medium pH was the predominant factor controlling glucose fermentation, strain Z6

291

was grown in media with a gradient of buffer capacities ranging from 0.0049 to 0.0238

292

(Table 2). In the cultures with stronger buffer capacity (PIPES+HCO3- and MES+HCO3-),

293

nearly complete glucose consumption was observed. However, in media with weaker buffer


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capacity (PIPES and MES), only partial glucose fermentation was observed. With stronger

295

buffering capacities (i.e., PIPES+HCO3- and MES+HCO3-), the pH only decreased by 0.5

296

units to about 6.00-6.05 during glucose fermentation. This is in contrast to the pH range 5.29-

297

5.57 at the end of growth for the cultures weakly buffered with PIPES and MES (Table 2).

298 299

DISCUSSION

300

Despite broad awareness that fermentation decreases pH and coexisting microbial iron

301

reduction facilitates fermentation4, 5, 7, 8, 30, 38, 39, the linkage between these two processes has

302

not been addressed. The present study suggests a novel mechanism that enhances

303

fermentation by attenuating acidification through reduction of iron oxides (e.g., hematite).

304

Using strain Z6 as a simplified and defined model, we provide parallel evidence that pH is

305

the driving factor that limits the extent of glucose fermentation. When the pH is controlled by

306

NaOH addition, the presence of stronger buffer, or metabolic iron reduction, this limitation

307

can be alleviated. These observations enabled us to re-assess the previously proposed

308

mechanisms for fermentative iron reduction. To the best of our knowledge, this is the first

309

study elucidating that microbial iron reduction can balance proton generation during

310

fermentation and thus create favorable conditions for sustainable utilization of fermentable

311

substrates.

312

Our experiments showed that pH is the critical factor for achieving sustainable

313

fermentation. With strain Z6, we observed that the pH decreased to the lower limit for its

314

iron-reducing activity (pH 5.5) (Dong et al., submitted) when fermentation ceased (Figs. 3

315

and S3). Continued fermentation and growth were restored when pH was increased to a

316

favorable range (Fig. 3). The pH decrease during fermentation of organic substrates is due to

317

production of organic acids and CO2 (carbonic acid)30. It has been well documented that acid


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318

production is inhibitory to cells40, 41. Thus, strain Z6 exhibits the physiological feature of a

319

typical fermenter in that decreased pH inhibits its fermenting activity.

320

Hematite reduction acted as the natural buffering process for strain Z6, i.e., a stable

321

pH was observed in the fermentative iron-reducing cultures (Figs. 3 and S3). Our

322

interpretation that microbial iron reduction is responsible for generating this alkalinity is

323

based, in part, on the initial physiological characterization. First, the acid produced from the

324

first 25 % of glucose consumed (~ 2.5 mM) was strong enough to inhibit the metabolism of

325

strain Z6 (Figs. 3 and 4). However, when pH was adjusted to 6.4, significantly more glucose

326

(~ 7.5 mM) was consumed in the presence of hematite, and pH remains stable without

327

additional adjustment (Glucose+Hematite+NaOH in Fig. 4). Our results also show that

328

addition of hematite itself does not change pH or extent of fermentation (Glucose+Hematite

329

in Fig. 4). This is consistent with the calculated dissolution of hematite by acid

330

(0.5Fe2O3+3H+

331

experimental conditions (pH 5.5-6.5 and 42 °C). This process would consume 8.9×10-18 -

332

8.9×10-15 mM protons and only change the pH by less than 0.1 units (SI). Thus, the stable pH

333

is mainly maintained by microbial iron reduction catalyzed by strain Z6. Second, when the

334

buffering capacity of the media was simply increased, nearly complete glucose consumption

335

similar to that for fermentative iron reduction could be achieved (Table 2). Third, the

336

stoichiometry and balanced equations show that significantly lower concentration of H2 was

337

produced during fermentative iron reduction than that in fermentation alone, while the

338

stoichiometric coefficients for other fermentation products (e.g., acetate, ethanol, as well as

339

the sum of bicarbonate and CO2) are similar in the presence of hematite (Table 1). The

340

stoichiometric analysis suggests H2 is the only electron equivalent for fermentative iron

341

reduction by strain Z6. This is consistent with our previous examination showing that H2 but

342

not short-chain fatty acids or alcohols from glucose fermentation supported iron reduction by

Fe3++1.5H2O) as an alternative H+ consumption process under the


15


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343

strain Z6 (Dong et al., submitted). These observations are further supported by the presence

344

of hydrogenase genes in the genome of strain Z6, suggesting its capabilities to produce H2

345

from glucose fermentation and use H2 as the electron donor for iron reduction (Fig. 2, Dong

346

et al., submitted). Thus, the overall fermentative iron reduction can be divided into two sub-

347

equations of glucose fermentation with H2 production and H2-driven hematite reduction

348

(Rxns 2, 2-1 and 2-2 in Table 1). Stoichiometric calculations show that fermenting 4.32 mM

349

glucose produces 9.22 mM protons. At the same time, the concomitant reduction of 2.57 mM

350

hematite with hydrogen consumes 10.28 mM protons. Thus, acid formed from glucose

351

fermentation can be completely neutralized during hematite reduction and results in a stable

352

pH for the overall reaction (Rxn 2 in Table 1).

353

Our observations imply that control of pH by the fermentative iron reducers depends

354

on the balance of protons between concomitant fermentation and iron oxide reduction

355

processes. In comparison, reduction of chelated ferric iron does not consume as many protons

356

as that by the reduction of ferric iron oxides42. This may explain why glucose fermentation by

357

Clostridium beijerinckii in the presence of Fe(III)-maltol [Fe(III)-3-hydroxy-2-methyl-4-

358

pyrone] showed significant pH decrease during iron reduction even though the medium was

359

buffered by high concentrations of bicarbonate (40 mM)39. In contrast, our results

360

demonstrate that microbial reduction of iron oxides effectively generates alkalinity that

361

balances acid production during fermentation. Such a mechanism suggests a unique adaptive

362

response of fermentative organisms to counter acid production.

363

Our studies agree with many other previous studies on fermentative iron reducers6-8, 39,

364

enrichment cultures and soil/sediment microbial consortia43, which showed significantly

365

enhanced fermentation, faster growth rates and higher growth yields in the presence of ferric

366

iron compounds compared with fermentation alone4, 5, 7, 8, 38, 39. The widely accepted

367

explanation for these observations has been that ferric iron compounds act as an electron sink


16

Page 17 of 37


368

for fermentation and thus produces more ATP by substrate level phosphorylation coupled to

369

acetate production. This provides an overall thermodynamic benefit. Typically, less than 5 %

370

of the electrons were channeled to ferric iron in these previous studies5, which is consistent

371

with our observation in that only 4.9 % of the electron equivalents from glucose fermentation

372

are used to reduce hematite. Thus, the low percentage of electron transfer suggests that iron

373

reduction itself does not significantly contribute to energy production43. In fact, the free

374

energy changes of glucose fermentation with and without hematite by strain Z6 are

375

thermodynamically favorable when glucose depletion ceases (Table 1). Thus, a

376

thermodynamic interpretation alone is insufficient to explain partial glucose fermentation by

377

strain Z6 in the absence of ferric substrates.

378

Complete glucose consumption by fermentative iron reducers has been frequently

379

observed. In many other studies with fermentative iron reducers, little residual glucose was

380

commonly observed when strongly buffered media were present5, 7, 8, 10, 39. For example, in

381

the presence of ferrihydrite, a fermentative iron reducer strain BS2 related to Clostridium

382

saccarobutylicum consumed 50 mM glucose in a medium buffered with about 60 mM

383

NaHCO3. Therefore, it is challenging to differentiate the relative contribution to pH stability

384

provided by the buffer from that created by the coexisting iron reduction. In the present

385

study, however, we illustrate that without a strong buffer, hematite reduction is responsible

386

for maintaining a stable pH condition and sustains enhanced glucose consumption by

387

fermentative iron reducers.

388

Another factor that may impact the extent of glucose fermentation is end-product

389

inhibition44-50. With strain Z6, however, the presence of 5 mM each of the soluble fermented

390

products (i.e. acetate, lactate, formate and ethanol) or a mixture of these compounds did not

391

inhibit the kinetics or extent of glucose use (data not shown). Also, glucose metabolism by

392

strain Z6 was probably not inhibited by H2 accumulation as repetitive headspace flushing did


17


Page 18 of 37

393

not result in further glucose metabolism (Fig. 3). This is in contrast to pyruvate fermentation

394

by Desulfotomaculum reducens where pyruvate use recommenced when the headspace was

395

sparged with N2, suggesting that H2 accumulation resulted in a thermodynamic constraint6. In

396

our case, the thermodynamic calculations demonstrate that the final Gibb’s free energy was

397

still favorable even at a H2 concentration one order of magnitude higher than the determined

398

value in the fermentation. Thus, neither accumulation of hydrogen nor of soluble

399

fermentation products likely act as the inhibitor for glucose consumption by strain Z6.

400

Putting all the phenotypic, thermodynamic, metabolomic and genomic evidence together, it

401

appears that pH is the critical factor responsible for fermentation efficiency by strain Z6.

402

During glucose fermentation in the presence of iron oxides, the concurrent microbial iron

403

reduction by strain Z6 acts as the internal buffering system that counterbalances acid

404

generation and maintains stable pH for continued glucose uptake.

405 406

Environmental Significance. In this study, iron oxide reduction by O. metallireducens strain

407

Z6 was shown to generate alkalinity that neutralizes acid production created during glucose

408

fermentation, which led to more complete utilization of glucose. This is in contrast to the

409

widely accepted concept that ferric iron reduction acts solely as the electron sink for

410

fermentation of organic substrates by fermentative iron reducers5 and that the benefit of ferric

411

oxide reduction is exclusively a thermodynamic one. Here, we show that the main benefit of

412

ferric oxide reduction may be in maintaining favorable buffering conditions for microbial

413

metabolism.

414

Abundance of ferric iron minerals in natural environments and ubiquitous presence of

415

iron-reducing organisms make microbial iron reduction critical for cycling of carbon and

416

other elements on the Earth2, 3, 15. Previous studies on the impact of microbial iron reduction

417

on other terminal electron-accepting processes (TEAP) have focused on its inhibition on


18

Page 19 of 37


418

sulfate reduction or methanogenesis, mostly due to thermodynamic favorability and

419

competition for electron donors5, 34, 51. However, in addition to the above competitive

420

interactions, there are also cooperative interactions that occur between iron reducers and the

421

organisms that use other TEAP. For example, Jiang et al. (2013) demonstrated that

422

akaganeite [Fe3+O(OH,Cl)] could be cycled by acetate-oxidizing iron reducers (e.g.,

423

Clostridium spp.) and reprecipitation of biogenic Fe(II). This iron-cycling process provided

424

electrons for the coexisting Methanosarcina barkeri for methane production, while methane

425

was not produced in the absence of akaganeite52. In addition, Shewanella oneidensis MR-1

426

can indirectly reduce goethite using the biogenic sulfide via S(0) reduction53. Here, we

427

presented another example that microbial iron reduction creates a favorable pH environment

428

to enhance fermentation of organic substrates.

429

Indirect facilitation of fermentation can be fundamentally important to understand

430

biogeochemical processes and their environmental applications. In the present study, the

431

fermentative iron reducer, strain Z6, carries out concurrent fermentation and iron reduction.

432

However, such pH favorability is not limited for fermentative iron reducers. Instead, multiple

433

organisms with different metabolic capacities may also achieve a similar effect: the

434

buffering of pH mediated by the iron reducers (e.g., dissimilatory iron-respiring organisms)

435

may enable the fermenters to use more of the available fermentable substrates. It is known

436

that iron-reduction typically leads to circumneutral pH conditions in anoxic environments54.

437

Thus, the ubiquitous iron reducers including both fermentative iron reducers and

438

dissimilatory iron-respiring organisms1, 55 would expect to play similar roles in acidity control

439

during hydrolysis and fermentation of complex organic substrates in such systems. In

440

addition, reduction of hematite that generates alkalinity in this study is also applicable for

441

many other naturally occurring ferric iron minerals (e.g., ferrihydrite, lepidocrocite, and

442

goethite) when reduced. More importantly, many practical applications employing microbial


19


Page 20 of 37

443

processes, such as petroleum hydrocarbon degradation in oil fields56, chlorinated solvents

444

remediation57, 58, and fermentation of organic wastes30, share the common feature of acid

445

production during catabolic activity. Carbonic acid, organic acid intermediates, or

446

hydrochloric acid accumulate during these processes and can lower the ambient pH56, 59-61,

447

which may exceed the natural buffering capacity of the environment or engineered systems

448

and inhibit further microbial activity. Thus, organisms capable of reducing ferric iron

449

minerals may have the potential to counterbalance such pH changes and provide a feasible

450

mechanism to enhance chemical and microbial reactions.

451


20

Page 21 of 37

452 453


ACKNOWLEDGEMENTS This project is co-funded by the research grants from the NASA Astrobiology Institute

454

Cooperative Agreement No. NNA13AA91A issued through the Science Mission Directorate,

455

the Institute for Museum and Library Services National Leadership Grant (NLG) No. LG-06-

456

12-0706-12. We appreciate Allan Konopka (Pacific Northwestern National Laboratory),

457

Youneng Tang (Florida A&M University-Florida State University), Roderick I. Mackie

458

(UIUC), Joanne Chee-Sanford (USDA) and Charles J. Werth (The University of Texas at

459

Austin) for insightful discussion and comments. We are grateful to the technical support from

460

the High-Performance Biological Computing, DNA Services and Metabolomics Facilities

461

and Metabolomics Center at Roy J. Carver Biotechnology Center, UIUC. We also thank

462

Michelle Goettge and Jiwon Kim (UIUC) technical assistance as well as Claudia Lutz (UIUC)

463

for manuscript edition.

464


21


Page 22 of 37

465

ASSOCIATED CONTENT

466

Supporting Information Available

467

This information is available free of charge on the ACS Publications website at

468

http://pubs.acs.org.

469

Additional details about chemical and cellular analyses, construction of reaction

470

stoichiometry; discussion about solubility of hematite at experimental temperatures; further

471

details about genetic evidence, pH changes and fermentation products; additional references.

472

(PDF)

473 474

AUTHOR INFORMATION

475 476

* Y. D. phone: 217-300-1625; fax: 217-244-0877; e-mail: [email protected].

477


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478

REFERENCES

479

1.

480

reduction. Adv. Microb. Physiol. 2004, 49, 219-286.

481

2.

482

anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 2006, 4, (10), 752-764.

483

3.

484

(2), 259-287.

485

4.

486

completely oxidize glucose in Fe(III)-reducing sediments. Appl. Environ. Microbiol. 1989, 55,

487

3234-3236.

488

5.

489

iron in anaerobic sediments. Appl. Environ. Microbiol. 1986, 51, (4), 683-689.

490

6.

491

during pyruvate fermentation by Desulfotomaculum reducens strain MI-1. Geobiology 2014,

492

12, (1), 48-61.

493

7.

494

Arbeille, G. M.; Fonty, G., Ferric iron reduction by fermentative strain BS2 isolated from an

495

iron-rich anoxic environment (Lake Pavin, France). Geomicrobiol J 2010, 27, (8), 714-722.

496

8.

497

Park, Y. K.; Chang, H. I., A novel electrochemically active and Fe(III)-reducing bacterium

498

phylogenetically related to Clostridium butyricum Isolated from a microbial fuel cell.

499

Anaerobe 2001, 7, (6), 297-306.

500

9.

501

carbon oxidation coupled to dissimilatory reduction of iron or manganese. Appl. Environ.

502

Microbiol. 1988, 54, (6), 1472-1480.

Lovley, D. R.; Holmes, D. E.; Nevin, K. P., Dissimilatory Fe(III) and Mn(IV)

Weber, K. A.; Achenbach, L. A.; Coates, J. D., Microorganisms pumping iron:

Lovley, D. R., Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 1991, 55,

Lovely, D. R.; Phillips, E. J. P., Requirement for a microbial consortium to

Lovley, D. R.; Phillips, E. J., Organic matter mineralization with reduction of ferric

Dalla Vecchia, E.; Suvorova, E. I.; Maillard, J.; Bernier-Latmani, R., Fe(III) reduction

Lehours, A.-C.; Rabiet, M.; Morel-Desrosiers, N.; Morel, J.-P.; Brigitte, L. J.;

Park, H. S.; Kim, B. H.; Kim, H. S.; Kim, H. J.; Kim, G. T.; Kim, M.; Chang, I. S.;

Lovley, D. R.; Phillips, E. J., Novel mode of microbial energy metabolism: organic


23


Page 24 of 37

503

10.

Lovley, D. R.; Phillips, E. J., Requirement for a microbial consortium to completely

504

oxidize glucose in Fe(III)-reducing sediments. Appl. Environ. Microbiol. 1989, 55, (12),

505

3234-3236.

506

11.

507

Fe(III) oxide reduction. Geomicrobiol. J. 2002, 19, (2), 209-251.

508

12.

509

Dixon, J. B.; Weed, S. B., Eds. Soil Science Society of America: Madison, 1989; Vol. 1, pp

510

379–438.

511

13.

512

akaganeite, by an anaerobic Fe(III)-reducing bacterium (Shewanella alga) isolated from

513

marine environment. Geosciences J. 2003, 7, (3), 217-226.

514

14.

515

colloids strongly enhance microbial iron reduction. Appl. Environ. Microbiol. 2010, 76, (1),

516

184-189.

517

15.

518

1995, 33, (3), 365-381.

519

16.

520

of biological reduction of hematite by electron shuttling and Fe(II) complexation. Environ.

521

Sci. Technol. 2002, 36, (9), 1939-1946.

522

17.

523

reductive dissolution of hematite by ferrous iron. Environ. Sci. Technol. 2004, 38, (1), 187-

524

193.

525

18.

526

A. S.; Lower, B. H., Bioreduction of hematite nanoparticles by the dissimilatory iron

Roden, E. E.; Urrutia, M. M., Influence of biogenic Fe(II) on bacterial crystalline

Schwertmann, U.; Taylor, R. M., Iron oxides. In Minerals in Soil Environments,

Lee, S. H.; Lee, I.; Roh, Y., Biomineralization of a poorly crystalline Fe(III) oxide,

Bosch, J.; Heister, K.; Hofmann, T.; Meckenstock, R. U., Nanosized iron oxide

Lovley, D. R.; Chapelle, F. H., Deep subsurface microbial processes. Rev. Geophy.

Royer, R. A.; Burgos, W. D.; Fisher, A. S.; Unz, R. F.; Dempsey, B. A., Enhancement

Royer, R. A.; Dempsey, B. A.; Jeon, B. H.; Burgos, W. D., Inhibition of biological

Bose, S.; Hochella, M. F.; Gorby, Y. A.; Kennedy, D. W.; McCready, D. E.; Madden,


24

Page 25 of 37


527

reducing bacterium Shewanella oneidensis MR-1. Geochim. Cosmochim. Acta 2009, 73, (4),

528

962–976.

529

19.

530

analysis of the bacterial reduction of goethite. Environ Sci Technol 2001, 35, (12), 2482-2490.

531

20.

532

Limitations to the reductive dissolution of Al-substituted goethites by Clostridium butyricum.

533

Soil Biol. Biochem. 2002, 34, (8), 1147-1155.

534

21.

535

from ferrihydrite. Clays Clay Miner. 1983, 31, (4), 277-284.

536

22.

537

transformation to goethite via the Fe(II) pathway. Am. Mineral. 2006, 91, (1), 92-96.

538

23.

539

O.; Flynn, T. M.; Sanford, R. A.; Krapac, I. G.; Locke, R. A.; Hong, P.-Y.; Tamaki, H.; Liu,

540

W.-T.; Mackie, R. I.; Hernandez, A. G.; Wright, C. L.; Mikel, M. A.; Walker, J. L.; Sivaguru,

541

M.; Fried, G.; Yannarell, A. C.; Fouke, B. W., Halomonas sulfidaeris-dominated microbial

542

community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir. Environ.

543

Microbiol. 2014, 16, (6), 1695-1708.

544

24.

545

Medina, C. R.; Rupp, J., Depositional and diagenetic variability within the Cambrain Mount

546

Simon Sandstone: Implications for carbon dioxide sequestration. Environ. Geosci. 2010, 18,

547

(2), 69-89.

548

25.

549

E. J.; Chang, Y. J.; Locke, R. A., 2nd; Weber, J. R.; Egan, S. M.; Mackie, R. I.; Cann, I.;

550

Fouke, B. W., Orenia metallireducens sp. nov. strain Z6, a novel metal-reducing Firmicute

551

from the deep subsurface. Appl. Environ. Microbiol. 2016, 82, (21), 6440-6453.

Liu, C. X.; Kota, S.; Zachara, J. M.; Fredrickson, J. K.; Brinkman, C. K., Kinetic

Dominik, P.; Pohl, H. N.; Bousserrhine, N.; Berthelin, J.; Kaupenjohann, M.,

Schwertmann, U.; Murad, E., Effect of pH on the formation of goethite and hematite

Yee, N.; Shaw, S.; Benning, L. G.; Nguyen, T. H., The rate of ferrihydrite

Dong, Y.; Kumar, C. G.; Chia, N.; Kim, P.-J.; Miller, P. A.; Price, N. D.; Cann, I. K.

Bowen, B. B.; Ochoa, R.; Wilkens, N. D.; Brophy, J.; Lovell, T. R.; Fischietto, N.;

Dong, Y.; Sanford, R. A.; Boyanov, M. I.; Kemner, K. M.; Flynn, T. M.; O'Loughlin,


25


Page 26 of 37

552

26.

Roh, Y.; Liu, S. V.; Li, G.; Huang, H.; Phelps, T. J.; Zhou, J., Isolation and

553

characterization of metal-reducing Thermoanaerobacter strains from deep subsurface

554

environments of the Piceance Basin, Colorado. Appl. Environ. Microbiol. 2002, 68, (12),

555

6013-6020.

556

27.

557

oxide grain coatings support bacterial Fe-reducing metabolisms in 1.7-2.0 km-deep

558

subsurface quartz arenite sandstone reservoirs of the Illinois Basin (USA). Front. Microbiol.

559

2014, 5, 511.

560

28.

561

Characterization. 2nd ed.; Wiley-VCH Verlag GmbH: Weinheim, 2000; p 188.

562

29.

563

Applications. McGraw-Hill: New York, 2001.

564

30.

565

production in glucose fermentation. Environ. Sci. Technol. 2008, 42, (7), 2401-2407.

566

31.

Hulanicki, A., Reactions of Acids and Bases. PWN & Ellis Horwood: 1989.

567

32.

Gibbs, C. R., Characterization and application of ferrozine iron reagent as a ferrous

568

iron indicator. Anal. Chem. 1976, 48, (8), 1197-1200.

569

33.

570

1970, 42, (7), 779.

571

34.

572

aquatic sediments. Appl. Environ. Microbiol. 1987, 53, (7), 1536-1540.

573

35.

574

Ratner, A.; Jacob, B.; Huang, J.; Williams, P.; Huntemann, M.; Anderson, I.; Mavromatis,

575

K.; Ivanova, N. N.; Kyrpides, N. C., IMG: the Integrated Microbial Genomes database and

576

comparative analysis system. Nucleic Acids Res. 2012, 40, D115-122.

Dong, Y.; Sanford, R. A.; Locke, R. A.; Cann, I. K.; Mackie, R. I.; Fouke, B. W., Fe-

Schwertmann, U.; Cornell, R. M., Iron Oxides in the Laboratory: Preparation and

Rittmann, B. E.; McCarty, P. L., Environmental Biotechnology: Principles and

Lee, H. S.; Salerno, M. B.; Rittmann, B. E., Thermodynamic evaluation on H2

Stookey, L. L., Ferrozine - a new spectrophotometric reagent for iron. Anal. Chem.

Lovley, D. R.; Phillips, E. J., Rapid assay for microbially reducible ferric iron in

Markowitz, V. M.; Chen, I. M.; Palaniappan, K.; Chu, K.; Szeto, E.; Grechkin, Y.;


26

Page 27 of 37


577

36.

Singh, A. K.; Ulanov, A. V.; Li, Z.; Jayaswal, R. K.; Wilkinson, B. J., Metabolomes

578

of the psychrotolerant bacterium Listeria monocytogenes 10403S grown at 37 degrees C and

579

8 degrees C. Int. J. Food Microbiol. 2011, 148, (2), 107-114.

580

37.

581

Biol. 1999, 9, (6), 670-676.

582

38.

583

acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of

584

Fe(III) to the oxidation of glucose. Appl. Environ. Microbiol. 1999, 65, (8), 3633-3640.

585

39.

586

K.; Richardson, D. J., Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated

587

from freshwater sediment using Fe(III) maltol enrichment. FEMS Microbiol. Lett. 1999, 176,

588

(1), 131-138.

589

40.

590

Limits Growth of and Acetogenesis by Clostridium-Thermoaceticum. Appl. Environ.

591

Microbiol. 1984, 48, (6), 1134-1139.

592

41.

593

Clostridium sporogenes MD1 via a mechanism involving a decline in intracellular glutamate

594

rather than protonmotive force. Microbiol. 2006, 152, 2619-2624.

595

42.

596

Thermodynamic Ladder in Geomicrobiology. Am. J. Sci. 2011, 311, (3), 183-210.

597

43.

598

T. P., Quantifying inorganic sources of geochemical energy in hydrothermal ecosystems,

599

Yellowstone National Park, USA. Geochim. Cosmochim. Ac. 2010, 74, (14), 4005-4043.

Peters, J. W., Structure and mechanism of iron-only hydrogenases. Curr. Opin. Struc.

Kusel, K.; Dorsch, T.; Acker, G.; Stackebrandt, E., Microbial reduction of Fe(III) in

Dobbin, P. S.; Carter, J. P.; Garcia-Salamanca San Juan, C.; von Hobe, M.; Powell, A.

Baronofsky, J. J.; Schreurs, W. J. A.; Kashket, E. R., Uncoupling by Acetic-Acid

Flythe, M. D.; Russell, J. B., Fermentation acids inhibit amino acid deamination by

Bethke, C. M.; Sanford, R. A.; Kirk, M. F.; Jin, Q. S.; Flynn, T. M., The

Shock, E. L.; Holland, M.; Meyer-Dombard, D.; Amend, J. P.; Osburn, G. R.; Fischer,


27


Page 28 of 37

600

44.

Zoetemeyer, R. J.; Matthijsen, A. J. C. M.; Cohen, A.; Boelhouwer, C., Product

601

Inhibition in the Acid Forming Stage of the Anaerobic-Digestion Process. Water Res. 1982,

602

16, (5), 633-639.

603

45.

604

Dissimilation of Glucose in Anaerobic Continuous Cultures by Free Butyric-Acid. Appl

605

Microbiol Biot 1988, 29, (1), 89-94.

606

46.

607

inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl. Environ.

608

Microbiol. 1990, 56, (4), 1004-1011.

609

47.

610

production by fermentation. Int. J. Hydrogen Energy 1998, 23, (7), 559-563.

611

48.

612

bacteria, facilitated by H2 removal: bioenergetics and H2 production. Appl. Environ.

613

Microbiol. 2006, 72, (2), 1079-1085.

614

49.

615

new, extreme thermophilic, anaerobic bacterium. Arch. Microbiol. 1981, 128, (4), 343-348.

616

50.

617

hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus.

618

Biotechnol. Bioeng. 2002, 81, (3), 255-262.

619

51.

620

microbial respiration across spatial and temporal gradients in saltmarsh sediments.

621

Biogeochemistry 2002, 60, (1), 49-76.

622

52.

623

J.; Hur, H. G., Methanogenesis facilitated by geobiochemical iron cycle in a novel syntrophic

624

methanogenic microbial community. Environ. Sci. Technol. 2013, 47, (17), 10078-10084.

Vandenheuvel, J. C.; Beeftink, H. H.; Verschuren, P. G., Inhibition of the Acidogenic

Luli, G. W.; Strohl, W. R., Comparison of growth, acetate production, and acetate

Tanisho, S.; Kuromoto, M.; Kadokura, N., Effect of CO2 removal on hydrogen

Adams, C. J.; Redmond, M. C.; Valentine, D. L., Pure-culture growth of fermentative

Wiegel, J.; Ljungdahl, L. G., Thermoanaerobacter ethanolicus gen. nov., spec. nov., a

van Niel, E. W.; Claassen, P. A.; Stams, A. J., Substrate and product inhibition of

Kostka, J. E.; Roychoudhury, A.; Van Cappellen, P., Rates and controls of anaerobic

Jiang, S.; Park, S.; Yoon, Y.; Lee, J. H.; Wu, W. M.; Phuoc Dan, N.; Sadowsky, M.


28

Page 29 of 37


625

53.

Flynn, T. M.; O'Loughlin, E. J.; Mishra, B.; DiChristina, T. J.; Kemner, K. M.,

626

Sulfur-mediated electron shuttling during bacterial iron reduction. Science 2014, 344, (6187),

627

1039-1042.

628

54.

629

Academic Press, Inc.: New York, 1972; Vol. 24, pp 29-96.

630

55.

631

review. Geomicrobiol. J. 1987, 5, (3-4), 375-399.

632

56.

633

Physical, and Chemical Processes. CRC Press LLC: Boca Raton, FL, 1998.

634

57.

635

Environmental Applications. Kluwer Academic Publishers: New York, 2003.

636

58.

637

transformation of trichloroethylene by iron sulfide and iron metal. Environ. Sci. Technol.

638

2001, 35, (19), 3884-3891.

639

59.

640

dechlorination of hexachloroethane by iron sulfide. Environ. Sci. Technol. 1998, 32, (9),

641

1276–1284.

642

60.

643

during enhanced reductive dechlorination using a simplified reactive transport model. Adv. in

644

Water Resour. 2012, 43, 14-27.

645

61.

646

hexavalent chromium by dissolved ferrous iron in poorly buffered aqueous systems. Water.

647

Res. 2001, 35, (6), 1534-1546.

Ponnamperuma, F. N., The chemistry of submerged soils. In Advances in Agronomy,

Lovley, D. R., Organic-matter mineralization with the reduction of ferric iron - a

Rinser-Roberts, E., Remediation of Petroleum Contaminated Soils: Biological,

Häggblom, M. M.; Bossert, I. D., Dehalogenation: Microbial Processes and

Butler, E. C.; Hayes, K. F., Factors influencing rates and products in the

Butler, E. C.; Hayes, K. F., Effects of solution composition and pH on the reductive

Brovelli, A.; Barry, D. A.; Robinson, C.; Gerhard, J. I., Analysis of acidity production

Schlautman, M. A.; Han, I., Effects of pH and dissolved oxygen on the reduction of

648


29


649 650

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Table 1. Mass balance, theoretical and measured product concentrations, and thermodynamics for fermentation by strain Z6 under different conditions. Fermentation alone C6H12O6 + (32/39) H2O (4/3)C2H5OH + (2/3)CH3COO- + (1)HCOO- + (2/13)HCO3- + (11/13)CO2+(1/2)H2 + (71/39)H+ Glucose (mM) Ethanol (mM) Acetate (mM) Formate (mM) H2 (ppmv) H+ (mM) a Theoretical -3.04 4.05 2.03 3.04 34048 5.53 Measured -3.04±0.18 4.34±0.30 2.05±0.02 3.04±0.14 35551±6594 ∆G0’ (298K, pH 7): -157.0 (kJ/reaction)

(Rnx. 1)

ΔG (315K, experimental pH)b Initial: -362.9 kJ/reaction End: -251.2 kJ/reaction Fermentation in the presence of hematitec C6H12O6 + (3/5)Fe2O3+(4/15)H+(4/3)C2H5OH + (2/3)CH3COO- + (2/3)HCOO-+(4/5)HCO3-+(8/15)CO2+(1/3)H2O+(6/5)Fe2+ +(1/15)H2 (Rxn. 2) + d C6H12O6 + (22/15)H2O(4/3)CH3CH2OH + (2/3)CH3COO + (2/3)HCOO + (4/5)HCO3 +(8/15)CO2+ (2/3)H2 + (32/15)H (Rxn. 2-1) + Glucose (mM) Ethanol (mM) Acetate (mM) Formate (mM) H2 (ppmv) H (mM) Theoretical -4.32 5.76 2.88 2.88 70272 9.22 Measured -4.32±0.10 5.14±0.01 2.91±0.20 2.78±0.31 + 2+ d Fe2O3+4H +H22Fe +3H2O (Rxn. 2-2) + H2 (ppmv) H (mM) Fe(II) (mM) ∆H2 (ppmv) Theoretical -57568 -10.28 5.14 12704 Measured 5.14 12882±2027 ∆G0’ (298K, pH 7): -213.8 kJ/reaction

∆G (315K, experimental pH)b Initial: -370.3 kJ/reaction

651 652 653 654 655 656

a.

End: -205.0 kJ/reaction The theoretical mass balance for fermentation alone was calculated based on the consumed glucose concentration. b.Experimental temperature (315 K) and changes of pH

along the reactions were considered in calculation of ΔG. The concentrations of initial product and final substrate concentrations were assumed to be 2 µM if they were below detection limits. c.The theoretical mass balance for fermentation in the presence of hematite was based on the consumed glucose concentration and production of reduced Fe(II). d.Due to the simplicity of the nutrient requirement for strain Z6, parallel fermentation and iron reduction (Rxn. 2) was divided into two sub-equations of fermentation (Rxn. 2-1) and hematite reduction (Rxn. 2-2). Proton balance under the two conditions was highlighted with gray shade.

30 ACS Paragon Plus Environment

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Fermentation enhanced by microbial iron reduction 657 658

659 660 661 662 663 664

Table 2. Summary of glucose fermentation by strain Z6 in the cultures under different buffering conditionsa Fraction of glucose c reacted (%)

Protein concentration c (µ µg/mL)

5.29±0.02

20.8±2.8

28.9±1.8

5.57±0.26 6.05±0.02

56.8±5.0 100d

78.69±0.34 92±16

Buffer capacity (β β )b

Final pH

PIPES

0.0049

MES PIPES+HCO3-

0.0101 0.0204

c

MES+HCO30.0238 6.00±0.09 98.64±0.09 94.48±0.06 The cultures were amended with 10 mM glucose and incubated at 42 °C; the headspace for the cultures was N2 for PIPES and MES versus N2:CO2 (80:20, v:v) for PIPES+HCO3- and MES+ HCO3-, respectively. bBuffer capacity was calculated considering commercial buffers (PIPES or MES), bicarbonate and phosphate in the media. cThe analyses were conducted after 34 hours of incubation when the OD600 under all the conditions leveled off. dThe residual glucose concentration was below detection limit (0.055 mM). a


31


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Fermentation enhanced by microbial iron reduction

60000

H2Glucose+Hematite Glucose+Hematite H2 50000

H2 Conc. (ppmv)

80 Glucose Glucose+Hematite

60

Glucose control Glucose+Hematite control

40

6

Fe(II) Glucose+Hematite control Fe(II) Glucose+Hematite

5

40000 4 30000 3 20000 2

20

10000

0

1

0 0

665 666 667 668 669 670 671 672 673

7

(b)

H2 H2Glucose Glucose

2

4

6

8

Fe(II) Conc. (mM)

Remaining Glucose Fraction (%)

(a) 100

0 0

2

Time (days)

4

6

8

Time (days)

Figure 1. Glucose decomposition (a), hydrogen production (solid lines) and Fe(II) generation (dashed lines) (b) during fermentation alone and fermentative iron reduction by strain Z6. The plots show average of triplicate samples and error bars indicate standard deviation of the replicates. The controls were prepared under the same conditions but without cell inoculation. The hydrogen generation in the control samples was below detection limit and thus was not shown.

674


32

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Fermentation enhanced by microbial iron reduction

Glc ADP Gluconolactone -6P

Gluconate-6P NADP+

ATP

NADPH+

G6P NADP+

NADPH++CO2

PPP

Ru-5-P

F6P

R-5-P ADP GADP

ATP

X-5-P

F1,6BP

Ery-7P

DHAP GADP NADH+

Glycolysis

Sedoheptulo se-7P

NAD+ 1,3-BPG

ATP

Fe(III) Reduction

Hematite (Fe2O3)

ADP 3PG

(NADH+) H++ H2 H2O

Fe2+

Fdox 2PG

Fdred (+NAD+)

PEP ATP ADP Pyruvate CO2 EOH

675 676 677 678 679 680 681 682 683 684 685 686 687

MECHO

+ Fdred

Lactate

Formate

Acetyl-CoA ATP ADP Acetate

CO2 H2

Fe(III) reduction

Pyruvate Metabolism

Figure 2. Pathways related to glucose fermentation by strain Z6 predicted from its genome. The pathways with the appropriate functional enzymes predicted by genome annotation were illustrated in red arrows. The metabolomic intermediates identified were highlighted with gray background. The fermentation products identified were highlighted with peach background. Multiple pathways predicted for hydrogen production and subsequent iron reduction were shown in dashed lines. Glc: glucose; G6P: glucose-6-phosphate; F6P: Dfructose 6-phosphate; F1,6BP: Fructose-1,6-biphosphate; GADP: D-glyceraldehyde-3phosphate; DHAP: hydroxyacetone-phosphate; 1,3-BPG: D-1,3-biphosphglycerate; 3PG: 3phosphoglycerate; 2PG: 2-phosphoglycerate; PEP: phosphoenolpyruvate; Ru-5-P: ribulose-5phosphate; X-5-P: xylulose-5-phosphate; Ery-7P: Erythrose-4-phosphate; MECHO: acetylaldehyde; EOH: ethanol.


33


Page 34 of 37

Fermentation enhanced by microbial iron reduction 0.45

Glucose+NaOH

0.36 0.27

*

0.18

Glucose+Flushing

*

*

(b)

*

0.09 0 -45

-15

15

45 75 Time (hours)

105

135

-45

(c)

100 80 60

*

40

*

20

-15

*

15

45 75 Time (hours)

105

135

(d)

120 Remaining Glucose (%)

120 Remaining Glucose (%)

*

0.27

0

*

*

*

100 80

*

60

*

40 20

*

0

0 -45

-15

15

45 Time (hours)

75

105

7

-45

135

-15

15

45 75 Time (hours)

105

7

(e)

6.5

6.5

6

6

pH

pH

*

Glucose+NaOH+Flushing

0.18

0.09

5.5

5.5

5

5

135

(f)

4.5

4.5 -45

688 689 690 691 692 693 694 695 696 697 698 699 700 701

Control

0.36 OD600

OD 600

*

*

Glucose

*

0.45

(a)

Control

-15

15

45

75

105

135

-45

-15

Time (hours)

15

45

75

105

135

Time (hours)

Figure 3. OD600, glucose consumption and pH for the strain Z6 cultures under the conditions of fermentation alone (a, c, e) and fermentation with flushed headspace (b, d, f) with different amendments. All the cultures were started from fermentation of about 10 mM glucose for 44 hours. When fermentation of glucose alone leveled off, different conditions were created by the amendments at the time points marked with asterisks. The red asterisks and arrows indicate addition of 1M NaOH to raise the significantly decreased pH (t-test, p

Hematite Reduction Buffers Acid Generation and Enhances Nutrient

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