Biogeochemical cycling of iron: Implications for biocementation and slope stabilisation

Journal Pre-proof Biogeochemical cycling of iron: Implications for biocementation and slope stabilisation

Alan Levett, Emma J. Gagen, Paulo M. Vasconcelos, Yitian Zhao, Anat Paz, Gordon Southam PII:

S0048-9697(19)36124-8

DOI:

https://doi.org/10.1016/j.scitotenv.2019.136128

Reference:

STOTEN 136128

To appear in:

Science of the Total Environment

Received date:

11 August 2019

Revised date:

4 November 2019

Accepted date:

13 December 2019

Please cite this article as: A. Levett, E.J. Gagen, P.M. Vasconcelos, et al., Biogeochemical cycling of iron: Implications for biocementation and slope stabilisation, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.136128

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© 2019 Published by Elsevier.

Journal Pre-proof Biogeochemical cycling of iron: implications for biocementation and slope stabilisation

Alan Levetta*, Emma J. Gagena, Paulo M. Vasconcelosa, Yitian Zhaob, Anat Paza, Gordon Southama

School of Earth and Environmental Sciences, University of Queensland, St. Lucia, QLD, Australia

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School of Mechanical and Mining Engineering, University of Queensland, St. Lucia, QLD,

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Australia

Manuscript in preparation for submission to Science of the Total Environment

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*author to whom correspondence should be directed. [email protected]

Journal Pre-proof Abstract Microbial biofilms growing in iron-rich seeps surrounding Lake Violão, Carajás, Brazil serve as a superb natural system to study the role of iron cycling in producing secondary iron cements. These seeps flow across iron duricrusts (referred to as canga in Brazil) into hydraulically restricted lakes in northern Brazil. Canga caps all of the iron ore deposits in Brazil, protecting them from being destroyed by erosion in this active weathering environment. Biofilm samples collected from these seeps demonstrated heightened

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biogeochemical iron cycling, contributing to the relatively rapid, seasonal formation of ironrich cements. The seeps support iron-oxidising lineages including Sideroxydans, Gallionella,

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and an Azoarcus species revealed by 16S rRNA gene sequencing. In contrast, a low relative

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abundance of putative iron reducers; for example, Geobacter species (<5% of total sequences in any sample), corresponds to carbon limitation in this canga-associated ecosystem. This

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carbon limitation is likely to restrict anoxic niches to within biofilms. Examination of a canga

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rock sample collected from the edge of Lake Violão revealed an array of well- to poorlypreserved microbial fossils in secondary iron cements. These heterogeneous cements

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preserved bacterial cell envelopes and possibly extracellular polymeric substances within the

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microfossil iron-rich cements (termed biocements). Bacterial iron reduction initiates the sequence, and intuitively is the rate-limiting step in this broadly aerobic environment. The organic framework of the active- and paleo-biofilms appear to provide a scaffold for the formation of some cements within canga and likely expedites cement formation. The accelerated development of these resilient iron-rich biocements in the lake edge environment compared with surroundings duricrust-associated environments may provide insights into new approaches to remediate mined land, aiding to stabilise slopes, reduce erosion, restore functional hydrogeology and provide a substrate akin to natural canga for revegetation using endemic canga plant species, which have adapted to grow on iron-rich substrates.

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Journal Pre-proof Keywords: biocements, iron reduction, iron precipitation, organic scaffold, biofilm, iron seep 1. INTRODUCTION Some of the world’s longest-lived landforms resist erosion because they are iron-cemented (Monteiro et al., 2014; Shuster et al., 2005). To strengthen these erosion-resistant landforms, physical weathering and erosion must be combatted by the formation of new cements. These cements form as a result of the geochemical cycling of cations, particularly iron, aluminium

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and silicon, from the micro- to the macroscale. Geochemical cycling of cations at the macroscale promotes weathering and cementation at distinct landscape positions. For example, during the weathering of banded iron formations (BIFs) in semi-arid environments,

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silicon may be leached from the BIF and transported to lower horizons where it redeposits,

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stabilising these lower landforms (Morris, 1983). In contrast, the physiochemical conditions

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that influence the solubility of iron (both ferrous and ferric) in tropical climates are more complex, leading to short- and long-range iron transport (Yamaguchi et al., 2007). Biological

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processes that influence redox conditions, including availability of complexing agents and

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reductants, alter the geochemical cycling of iron (Colombo et al., 2014). For short-range geochemical cycling of iron, these erosion-resistant horizons must contain distinct

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microenvironments; some that promote mineral dissolution and others conducive to precipitation (Yamaguchi et al., 2007). Landscapes degraded by anthropogenic activity, including land cleared for road construction, building and mining, may, in principle, be stabilised by iron cements. Understanding these natural systems, where weathering and subsequent re-precipitation of iron minerals contributes to long-term stabilisation of the landscape, provides valuable insights from which to glean strategies for effective surface stabilisation. Microbially-promoted stabilisation techniques typically target calcium carbonate precipitation for cement formation (DeJong et al., 2010); however, iron biogeochemical

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Journal Pre-proof cycling may provide a more chemically resistant alternate for landform stabilisation. Ironreducing microorganisms utilise a variety of mechanisms including outer membrane c-type cytochromes (Lovley et al., 2004; Richardson, 2000), nanowire production (Reguera et al., 2005) and soluble redox-active small molecules (for example, flavins) (Breuer et al., 2015) to shuttle electrons to iron (III) minerals, placing ferrous iron into solution. In circumneutral environments, ferrous iron is relatively soluble compared with ferric iron. In oxic solutions, ferrous iron is rapidly oxidised to ferric iron, which is unstable and precipitates as hydrous

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ferric oxides (Rentz et al., 2007). Therefore, iron-reducing microorganisms and changes in oxidation potential at a constant circumneutral pH can promote the geochemical cycling of

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iron between solution and mineral precipitates at the microscale (Roden et al., 2004).

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In the present study, we sought to understand the contributions of natural biogeochemical iron cycling to the cementation of ferruginous (iron cemented) duricrusts in the iron ore

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ridges at Serra Sul (S11D), Serra dos Carajás, Pará, Brazil. We focused on lake edge

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the present day.

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environments as biological ‘hotspots’ where iron dissolution and re-precipitation is active in

2. GEOLOGICAL AND ENVIRONMENTAL SETTING

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Surface water runoff into local hydrologically restricted dissolution (karstic) depressions within the ferruginous duricrust in Serra Sul contributes to the formation of lake environments such as Lake Violão and Lake Amendoim (Fig. 1). In some locations, seeps that flow into the lake support thick biofilms interspersed with fresh iron oxide precipitates (Fig. 2). The biofilms and the surrounding rocks suggests that these microbial ‘hotspots’ promote the biogeochemical cycling of iron. Lake Violão and Lake Amendoim sit at an elevation between approximately 700 - 720 m. Sahoo et al. (2017) recently characterised the geochemistry of the lakes and Silva et al. (2018) described the local geomorphology, which have developed over lateritic ferruginous

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Journal Pre-proof duricrusts that cap high grade iron ore. Lake Violão ranges from oligotrophic in the dry season (June to October) towards eutrophic in the wet season (November to May) whereas Lake Amendoim is classified as ultra- to slightly-oligotrophic throughout the year (Sahoo et al., 2017). Lake Violão has a surface area of approximately 0.27 km2, 7.77 km circumference and a catchment area of approximately 1.83 km2, whereas Lake Amendoim is slightly smaller; surface area, perimeter and basin catchment area are 0.126 km2, 6.89 km and 1.20 km2, respectively (Silva et al., 2018). The climate in the Serra Sul is tropical, with average

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daily-high temperatures of ~26 °C year round and an annual rainfall between 1800 – 2300 mm, predominately (approximately 80%) falling in the wet season (Silva et al., 2018). The

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average water temperatures for the lakes is typically ~ 27 C; pH (6.3 – 7.9), dissolved

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oxygen (7.1 – 7.9 mg/L), electrical conductivity (6.1 – 6.9 s/cm) and oxidation-reduction

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potential (259 – 389 mV) do not vary significantly between wet and dry seasons and are not stratified throughout the major lake bodies (Sahoo et al., 2016; Sahoo et al., 2015). The lakes

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are currently protected by Brazilian Law restricting mining (Silva et al., 2018) but the nature of the ore body and recent developments in iron ore mining technologies that reduce

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environmental impact have allowed the development of the world’s largest iron ore mine

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(S11D) in close proximity to the protracted lakes (Fig. 1). The ferruginous duricrusts predominately support shrubby and herbaceous rupestrian plant species with less vegetation (Fig. 1) compared with the surrounding montane and Capão Island forests (Nunes et al., 2015). Each lake has steeply dipping sides, typically greater than 20 and occasionally approaching 50 (Silva et al., 2018). The steeply dipping slopes along the lake edges are primarily composed of detrital fragments of hematite cemented together by thin (mm-scale) authigenic goethite. The low cement:fragment ratios in these rocks are consistent with Dorr’s (1964) description of a rock called ‘canga rica.’ The thin section of the canga sample from the edge

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Journal Pre-proof of Lake Violão highlights the low cement:fragment ratio (Fig. 3A). The cements are heterogeneous and deceptively robust: a well-directed, firm strike with a rock hammer is required to fracture the rock along planes of weakness in the cements. The canga rica preferentially fractures unevenly into sheet-like rock samples, typically less than 5 cm thick. The steeply dipping canga rica that forms at the edge of Lakes Violão and Amendoim is of particular interest to this study as it highlights that ferruginous cements can be formed relatively quickly, stabilising loose fragments on the slope that would otherwise be eroded

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into the lake. The water chemistry and microbial populations of streams that flow into the lakes (Fig. 2) as well as the canga rica substrate that forms the lake edge (Fig. 1B) were

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examined at the microscale to understand the role that microorganisms may play in the

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3. METHODS AND MATERIALS

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formation and evolution of these ferruginous cements.

3.1. Locations for sample collection

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Sampling locations for biological ‘hotspots’ of iron cycling, was guided by the presence of

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aqueous ferrous iron. To indicate where aqueous ferrous iron in solution exceeded 1 mg L-1, approximately 0.1 mL of water was pipetted into 1 mL of 1 g L-1 ferrozine, a modification of

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the ferrozine assay developed by Stookey (1970). Positive ferrozine assays indicating ferrous iron concentration greater than 1 mg L-1 were recorded for seeps at Lake Amendoim (ASGB; Fig. 2A and AS-BlB; Fig. 2B), the surface and bottom of an ephemeral pool (VP-SB and VP-BB) and two of the three seeps sampled at Lake Violão (VS-OB; Fig. 2D and VS-FeS; Fig. 2F). Two sample locations, including one of the seeps at Lake Violão (VS-RB; Fig. 2C) and ‘foam’ that formed by wind blowing across the lake (LV-F; Fig. 2E) did not produce a strong ferrozine reaction, indicating ferrous iron in solution was less than 1 mg L-1. Note that these data were only used to target hotspots of iron-cycling and a complete set of samples for water chemistry, microscopy (light and electron microscopy; data not shown) and DNA

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Journal Pre-proof sequencing were subsequently collected. Biofilm samples were collected from the field into sterile Falcon tubes (15 and 50 mL), with DNA preserved by adding LifeGuardTM to a final concentration of approximately 10% (aq). All samples were kept cool (~ 4 C) in the field (less than eight hours) until they could be frozen. To collected corresponding samples for water chemistry, approximately 10 mL of water was filtered through a 0.22 m syringe filter into sealed serum vials containing nitrogen gas phase (and using a vent needle to avoid

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pressurisation during filtration) in the field to minimise ferrous iron oxidation and precipitation out of solution.

Where water flowed down the slope away from the orange-coloured biofilms, additional

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water samples were collected into a nitrogen filled vial to monitor the iron in solution. In

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addition, where water appeared relatively stagnant (for example, an ephemeral pool within

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the duricrust) a dissolved oxygen profile was taken using a FireSting GO2 (Optical Oxygen Meter, Pyroscience GmbH, Aachen, Germany) with a robust probe (3 mm diameter). The

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water pH was measured in the field for locations using MColorpHastTM pH strips 4.0 – 7.0 (Merck KGa, Darmstadt, Germany). A rock sample from the edge of Lake Violão (Fig. 1B)

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was also collected for scanning electron microscopy (Figs. 3 – 4).

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3.2. Scanning electron microscopy

A representative canga rica rock sample fractured from the lake edge around Lake Violão was dehydrated in a 40 °C oven overnight and embedded in an epoxy resin (EpoxiCure 2) to produce a petrographic thin section, which was polished to a thickness of 100 m. A final 0.25 m diamond abrasive was used to achieve a submicron polish for examination using a scanning electron microscope (SEM). A JEOL7100 SEM in backscattered electron mode with an accelerating voltage of 15 kV was used to examine the petrographic thin section. The sample was degassed at 50 °C overnight and coated with 10 nm iridium using a Quorum Q150T sputter coater prior to examination. ImageJ was used to estimate the surface area

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Journal Pre-proof cement:fragment ratio for a representative portion of the cross-section (Fig. 3A; black rectangle). 3.3. Nanoindentation Nanoindentation tests were used to characterise the mechanical properties of various cements and a hematite-rich fragment throughout the canga thin section using A TI-900 Hysitron Triboindentor® equipped with a diamond Berkovich tip (radius ca. 100 nm). A load-control

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function with loading, holding and unloading times of 10 s, 10 s and 15 s, respectively. An indentation load of 5 mN was used to determine the hardness (H) and reduced modulus (Er), which were calculated based on the load-displacement (P-h) curves. Two textures of

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goethite-rich lamella cements were tested: ‘abiotic’ mineral precipitates (goethite cements)

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and those that appear to be influenced by microorganisms (microbial cements). For each of

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these textures, 4 × 4 indentation arrays with 10 m spacing between each indent were used. Dissolution pitting within the hematite fragment required indent locations to be selected

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individually on polished surfaces, with each indentation location more than 10 m apart. The heterogeneous nature of the sample at a scale of 100 nm occasionally resulted in individual

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loading curves being unreproducible and these were subsequently removed for statistical

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analysis. A two-tailed t-test assuming equal variance indicated there was no statistical difference between the hardness (p = 0.37) and the reduced modulus (p = 0.94) for the different arrays of microfossil cements tested; therefore, these data were combined for statistical analysis. 3.4. Water chemistry Water samples were kept at room temperature (~ 25 C) and transferred to the laboratory, where they were acidified to a final HNO3 concentration of 7% (aq) using concentrated (70% (aq))

HNO3. Water samples were transferred to Teflon tubes and digested in a MARS Xpress

microwave at 160 °C for 10 min and 170 °C for a further 10 min. Digested samples were

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Journal Pre-proof diluted to a final concentration of 5% (aq) HNO3 and analysed using a Perkin Elmer Optima 7300DV inductively coupled plasma optical emission spectroscopy with an argon plasma gas (15 L min-1). Samples were analysed for the following soluble elements (detection limit in ppm provided in parentheses): aluminium (0.0012), barium (0.00004), calcium (0.0005), iron (0.0003), potassium (0.0003), magnesium (0.0001), sodium (0.0002), sulphur (0.0002) and zinc (0.0002).

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3.5. DNA extraction, sequencing and sequence analysis To extract DNA, samples were thawed and centrifuged at 10,000×g for 10 min, the supernatant was removed and the remaining biomass was collected for DNA extraction

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following the DNeasy Powersoil Kit (Qiagen, Hilden, Germany).

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The V6 – V8 portion of the 16S rRNA gene was amplified from the extracted DNA using the universal primers 926f and 1392r, modified to contain Illumina specific adapter sequence;

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926F: 5’-

3’ and 1392wR: 5’-

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TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAACTYAAAKGAATTGRCGG-

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GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGGGCGGTGWGTRC-3’.

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Libraries were prepared using the workflow outlined by Illumina (#15044223 Rev.B), with the exception that the NEBNext® UltraTM II Q5 Mastermix (New England Biolabs #M0544) was used in standard PCR conditions. Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used to purify the resulting PCR amplicons before the purified DNA was indexed using the Illumina Nextera XT 834 sample Index Kit A-D (Illumina FC-131-1002, San Diego, CA, USA) in standard PCR conditions (aforementioned PCR mastermix) to assign unique 8-bp barcodes. Indexed amplicons were pooled in equimolar concentrations and sequenced using a MiSeq Sequencing System (Illumina) with paired end sequencing (V3

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Journal Pre-proof 300-bp chemistry) in accordance with the manufacture’s protocol at the Australian Centre for Ecogenomics, The University of Queensland. Sequences were processed using MOTHUR as per the MiSEQ standard operating procedures (https://www.mothur.org/wiki/MiSeq_SOP, accessed on 20th April, 2019) with minor alterations to allow for processing forward reads only. Briefly, forward reads were trimmed on quality using a quality average of 35 across a sliding window size of 50 nt before the primer was removed. Reads were subsequently trimmed to 250 nt and ambiguous bases and

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homopolymeric structures (8 nt repeats) were removed before alignment using the SILVA SSU Ref NR 99 v132 reference database (Pruesse et al., 2007). Putative chimeras identified

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using the silva132.gold alignment database as a reference were removed and the remaining

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sequences were classified using the SILVA SSU Ref NR 99 v132 database. Sequences classified as Eukaryota, Chloroplast, Mitochondria or unknown were also removed.

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Sequences were clustered into Operational Taxonomic Units (OTUs) at a distance of ≤ 0.03

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for further processing. Libraries were subsampled to the smallest number of unique OTUs (8128 OTUs) and sample coverage, species richness (Sobs indicator), Shannon-Weaver

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diversity index and Shannon evenness index were determined using the calculators in

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MOTHUR. Singletons (OTUs that only occur once in the entire dataset) were then removed before generating a heatmap to visualise shared OTUs. Nucleotide basic local alignment search tool (BLASTn) (Altschul et al., 1990), using the NCBI and non-redundant nucleotide collection, was used to compare the major OTUs with publicly available sequences. Sequences were initially compared by excluding uncultured and environmental samples. OTUs that demonstrated less than 97% identity with a named species were re-analysed including all publicly available sequences. Sequences have been submitted to the NCBI Sequence Read Archive under BioProject number PRJNA554967.

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Journal Pre-proof 4. RESULTS 4.1. Characterisation of canga rica from the edge of Lake Violão The canga that formed at the edge of Lake Violão was very competent (non-friable), with microscale fragments and secondary goethite-rich precipitates cementing large hematite-rich fragments, ranging from less than 1 cm to greater than 5 cm (Fig. 3A). The canga was primarily composed of the detrital fragments (surface area of approximately 77%) with the

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cement material only accounting for approximately 23% of the surface area of the sample (Fig. 3A).

Bacteriomorphic structures resembling previously characterised canga-associated

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microfossils (Levett et al., 2016; Levett et al., 2019) were commonly identified throughout

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the goethite-rich cements in the canga (Fig. 3). Fewer, relatively large microfossils (ranging

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from 3 – 5 µm) are interpreted as relicts of microbial eukaryotes; for example, algae or fungi (Fig. 3). Smaller, approximately 1 µm, microbial fossils are also evident in the pore spaces in

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proximity to the larger microfossils (Fig. 3; white arrows). Encrusted cell envelopes are preserved throughout the cement portion of the canga rica (Fig.

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4A; white arrows). These bacteriomorphic cell envelope structures are relatively sparse

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compared with other biofilms identified in canga (Levett et al., 2016) but are frequently identified and give much of the cement throughout the canga rica sample a ‘biologicaltexture.’ For example, microbial fossils are frequently identified in the iron cements between the large detrital fragments (Fig. 4B; within the black portion of the black-and-yellow line traces). Lamellae are also common throughout the biologically-textured cements (Fig. 4B; white arrows). The fragment has also undergone weathering, evidenced by some of the smaller portions of the fragment maintaining limited connections with original fragment (Fig. 4B; dashed black line).

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Journal Pre-proof Spherical, rod-shaped and elongated structures are remnants of cocci, bacilli and filamentous structures that are preserved throughout the iron-rich cements within the canga (Fig. 5A), which can form large (Fig. 5B) and relatively robust structures that cement large fragments (Fig. 3). The average hardness of abiotic cements (goethite cements = 7.5 ± 0.6 GPa) was significantly (p < 0.001) harder than cements influenced by microorganisms (microfossil cements = 1.4 ± 0.4 GPa; Fig. 5B). As a comparison, the average hardness for the hematite-

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rich fragment was 13.9 ± 0.8 GPa and the resin was 0.21 ± 0.0017 GPa (Fig. 5B). 4.2. Water chemistry

Aqueous iron concentrations, presumably as ferrous iron given the circumneutral pH

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conditions (pH 5 – 6) and a strong reaction with ferrozine in the field, ranged from 15.8 ppm

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to below detection limit (Table 1). Soluble iron was below detection limit at the perimeter of

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Lake Violão associated with the foam (Fig. 2E). Similarly, the seep (Fig. 2C) that flowed through the pore spaces in canga (for example, Fig. 3A) had no detectable soluble iron;

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however, the biofilm and the rock surface at the overflow contained fresh iron oxide precipitates (red), indicating oxidation of aqueous ferrous iron and subsequent precipitation.

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The highest soluble iron concentrations were typically recorded in association with well-

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developed mm-scale microbial biofilms. Soluble iron decreased with increasing distance from the well-developed microbial biofilms (Table 1; VS-OB samples and VS-FeS samples). Soluble iron concentrations did not appear to change with water depth in the stream pool or in the oxidised ephemeral pool that was sampled, indicating effective mixing of solutions within this water body (Table 1; AS-GB samples and VS-SB – VS-BB). Dissolved oxygen concentrations were relatively high in this small water body, maintaining 20% air saturation even at a depth of approximately 15 cm within an ephemeral pool (Table 1; sample 21E). The alkaline-earth (magnesium and calcium) and alkaline (sodium and potassium) metals were enriched within the ephemeral pool compared with all other sample locations.

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Journal Pre-proof 4.2. Microbial community structures Proteobacteria were a dominant phylum in all samples, accounting for 26 – 72% of all sequences per sample (Fig. 6). Biofilms supported by the surface streams and seeps that flowed into Lake Violão (VS-RB, VS-OB and VS-FeS) also contained a relatively high proportion of Patescibacteria (11 – 31%) and bacteria unclassifiable below the phylum level (7 – 16%), with sample VS-RB also containing a relatively high proportion of Firmicutes (10%; Fig. 6). Aside from Proteobacteria, Planctomycetes (22%), Armatimonadetes (20%)

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and Actinobacteria (10%) were also dominant in Lake Violão (LV-F; Fig. 6). Other major phyla at the surface (VP-SB) and bottom (VP-BB) of the ephemeral pool samples were

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Actinobacteria (15%) and Planctomycetes (11%; Fig. 6). Strong similarities in the microbial

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community structure at the surface and bottom of the sampled ephemeral pool were consistent with mixing solutions and the absence of broad anoxic conditions (VP-SB and VP-

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BB; Fig. 6). In contrast, the black biofilm associated with the surface stream at Lake

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Amendoim (AS-BlB) contained abundant Acidobacteria (10%), while Plantomycetes (11%) and Firmicutes (10%) accounted for large proportions of the total sequences in the globular

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biofilm (Fig. 6).

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biofilm (AS-GB), which was sampled approximately 10 m downstream from the black

Sequencing coverage of the samples ranged from 61 to 98% (Supplementary Information). The Shannon-Weaver diversity index ranged from 3.98 for iron foam in Lake Violao to >7 for an iron seep flowing into the same lake (Supplementary Information). The Shannon evenness index reflected the same trend, with the least even microbial community the lake foam (0.58) and the most even microbial community the iron seep (0.89; Supplementary Information). Of the eukaryotic sequences that were recovered with the primers used in this study (926f and 1392r; see Section 3.5.) the greatest proportion of eukaryotes to the total community was in the surface biofilm of the ephemeral pool (VP-SB; 17%). The foam from

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Journal Pre-proof Lake Violão, the bottom biofilm of the ephemeral pool at Lake Violao and the globular biofilm from an iron seep flowing into Lake Amendoim had between 5 and 9% eukaryotesin the total sequences. All other biofilms associated with iron seeps contained fewer than 5% eukaryotic sequences (Supplementary Information). Microbial lineages affiliated with well-known iron-oxidising microorganisms were present throughout water stream samples from Lake Amendoim (AS-GB and AS-BlB) and Lake Violão (VS-RB, VS-OB and VS-FeS; Fig. 7). For example, OTU008 demonstrated 100%

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identity across the sequenced region of the 16S rRNA gene to a well-characterised neutrophilic iron-oxidising microorganism, Sideroxydans lithotrophicus (Weiss et al., 2007)

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and was present in all surface stream samples (Fig. 7). OTU034, OTU038 and OTU040

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present in the surface stream (LS and VS) samples, also appear to be members of a welldescribed family of iron- and sulphur-oxidisers, the Gallionellaceae (Fig. 7). OTU038 was

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also detected in the ephemeral pool (VP) samples. OTU 010 and OTU 042 demonstrated 98%

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identity across the sequenced region of the 16S rRNA gene to Burkholderiales bacterium GJE10, a recently described chemolithotrophic iron-oxidising microorganism (Fukushima et al.,

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2015). OTU011, a dominant lineage from a surface stream at Lake Violão (sample VS-FeS;

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Fig. 7), may represent a novel iron-oxidising lineage. There is genomic evidence that the nearest named isolate (95% identity across the sequenced 16S rRNA gene region to Azoarcus sp. CIB CP011072) has iron oxidation capabilities. Specifically, the genome of Azoarcus sp. CIB encodes three decaheme cytochromes that demonstrate very high homology to MtoA ctype cytochromes (e-value = 1e-126 to MtoA Gallionella capsiferriformans ES-2) and each of these genes are followed by genes encoding proteins demonstrating high homology with MtoD (e-value = 2e-34 to Sideroxydans lithotrophicus MtoD). MtoA and MtoD are the key enzymes in electron transport for iron oxidation (Liu et al., 2018a).

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Journal Pre-proof Known iron-reducing lineages in canga-associated surface waters were less abundant than putative iron oxidisers. OTUs 025 and 049, present in the surface streams (AS-GB, AS-BlB, VS-RB, VS-OB and VS-FeS) but absent from the ephemeral pool (VP-SB and VP-BB) and the Lake Violão (LV-F) samples, demonstrated strong (>99%) 16S rRNA gene identity to the known iron-reducing lineage Geobacter (Fig. 7). Well-described iron-cycling microorganisms were less prevalent in the sample collected from the boundary of Lake

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Violão (LV-F) and the oxidised ephemeral pool (VP-SB and VP-BB). 5. DISCUSSION

The iron oxide cements that consolidate the ferruginous duricrust at the edge of Lake Violão

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appear to form relatively quickly, possibly within decades to millennia rather than on a scale

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of millions of years as demonstrated for typical canga horizons (Monteiro et al., 2014). The

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steeply dipping slopes at the lakes’ edges together with monsoonal rainfall during wet seasons would quickly erode loose detrital fragments into the lakes if cements were not

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formed relatively rapidly. The low cement:fragment ratio of the canga rica (approximately 1

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part cement to 3 parts fragment) at the lake edge also supports a relatively rapid formation of cements. In addition, the canga rica that forms at the lakes’ edges preferentially fractures in

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sheet-like rock samples (less than 5 cm thick), indicating that these surface rocks had been conglomerated more recently and independently from the underlying duricrust. Microorganisms appear to play an important role in relatively rapid formation of these cements at the edges of both Lakes Amendoim and Violão. An array of well- to poorlypreserved microbial fossils, commonly observed throughout the goethitic cements, highlights the involvement of microorganisms in the cementation of canga at the lake edge (Figs. 3 – 5). The microbial biofilms that grow throughout the seeps and streams at the lakes’ edges appear to aggregate detrital fragments and provide an organic framework for the nucleation of authigenic hydrous ferric oxide minerals. As the microbes become fossilised, they contribute

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Journal Pre-proof to the formation of robust cements within the canga. These organic frameworks appear to expedite the formation of cements throughout canga rica; in their absence, the soluble iron in solution would simply precipitate out to coat fragments and form ferruginous pisolith structures instead of robust cements. The formation of ferruginised microbial cements within the canga is likely to be limited by the reduction of iron oxide minerals (Gagen et al., 2018). Microbial biofilms in the streams and seeps that supplement Lakes Violão and Amendoim coincide with increased soluble iron

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concentrations. Up to 15 ppm of iron in solution was recorded in association with extensive biofilm, indicating rapid iron cycling. In comparison, the average dissolved iron in the lakes

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is less than 0.5 ppm (Sahoo et al., 2017). The gentle flow of surface water and the small size

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of the seeps are also likely to allow for the development of extensive biofilms compared to within the open lake. These extensive biofilms likely create anoxic niches that accommodate

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and promote microbial-induced iron reduction. The low relative abundance of sequences

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affiliated with well-known iron-reducing microorganisms in the biofilms appears to be inconsistent with the elevated soluble iron concentrations (Fig. 7). However, the

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stoichiometry of the microbial iron reduction equation highlights that a single hydrated

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carbon molecule can account for up to four ferrous iron cations being placed into solution (Reaction 1). Therefore, a low concentration of hydrated carbon may support a relatively small but consistent population of iron-reducing microorganisms. Inorganic and organic acids generated by microbial processes may also promote the reductive dissolution of iron (III) oxide minerals (Schwertmann, 1991). 𝐶𝐻2 𝑂 + 4𝐹𝑒 3+ + 𝐻2 𝑂 → 4𝐹𝑒 2+ + 𝐶𝑂2 + 4𝐻 +

(1)

Together, the electron microscopy of the rock samples, the water chemistry and microbial community analysis highlight that microorganisms play important roles in the weathering of iron oxide minerals in canga, placing iron into solution. Microbial cell envelopes provide

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Journal Pre-proof sites for the nucleation of iron oxide minerals to form stable ferruginous microbial cements. Each of these processes takes place within microenvironments within the canga horizon, creating a highly resistant, ‘self-healing’ duricrust (Monteiro et al., 2014). 5.1. Ferruginous biocement formation: industrial applications By understanding these natural microbial processes that contribute to the relatively rapid cementation, it is possible to develop new biotechnologies that target the biogeochemical

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cycling of iron to chemically and physically stabilise degraded landscapes. The redox-based biogeochemical cycling of iron that occurs throughout canga is accelerated within the lake edge environments. Within canga lake edge environments, surface water and carbon-rich

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nutrients from nearby plants (for example, Fig. 2F) support the enhanced growth of diverse

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microbial biofilms compared with the rest of the canga horizon. These microbial hotspots

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create an environment that accelerates iron cycling. Respiration of carbon sources from the nearby plants depletes dissolved oxygen and creates anoxic microenvironments, potentially

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isolated to within the biofilm (Table 1), accelerating the rate-limiting dissolution of iron oxide minerals (Gagen et al., 2018). Harnessing these natural process will contribute to the

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development of new biotechnologies with various applications. For example, these novel

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biocements may be used to physically and chemically stabilise hillslopes and create mineralised surfaces that restore the natural hydrogeology of landscapes degraded by anthropogenic activities. Given the chemical stability of goethitic cements in canga over millions of years (Monteiro et al., 2014), these biotechnologies also provide alternative solutions for protecting sulphide-based waste piles, reducing the generation of the acid mine drainage (Liu et al., 2018b). Accelerating the formation of long-term hydrogeochemically stable ferruginous biocements may provide effective chemical and physical barriers to protect sulfidic waste piles.

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Journal Pre-proof 5.2. Limitations of natural iron reduction Trace element analysis demonstrates that the environments that contributed to canga formation and evolution are overall oxidising (Monteiro, 2017). Relatively high oxygen concentrations in the seeps studied here may indicate that available organic carbon is a limitation for the generation of broadly anoxic condition that are required for microbiallyinduced iron reduction. Carbon limitations for iron reduction are also supported by increased iron and aluminium in solution during the wet season compared with the dry season (Sahoo et

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al., 2017). In the natural environment, carbon limitations may be important for the reoxidation of iron, preventing its loss through weathering channels. Gagen et al. (2018)

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postulated that carbon limitations and overall oxic conditions with iron-rich duricrust

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environments are critical for the net generation of iron oxide minerals. Therefore, broadly oxic conditions and carbon-limitations appear to be essential for the short-range

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biogeochemical cycling of iron that occurs in canga horizons and contributes to their

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extraordinary resistance to physical erosion (Monteiro et al., 2018). For industrial applications, these limitations may be overcome by isolating microbial iron reduction to

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optimised bioreactors before releasing the soluble iron into the broadly oxic environment to

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create chemically and physically stable surfaces. 5.3. Contributors to biocement formation: iron oxidation and microbial fossilisation A number of putative iron-oxidising microorganisms, including Sideroxydans, Gallionella, and an Azoarcus species were identified in the biofilms. These putative iron-oxidising microbial lineages may be underrepresented in the microbial fossils preserved within the cements of the canga (Figs. 3 – 4). Chemolithotrophic microorganisms that enzymatically oxidise iron as an energy source have typically developed strategies to prevent becoming encrusted in iron oxide minerals (Chan et al., 2011; Hegler et al., 2010; Miot et al., 2009). Gallionella species commonly produce extracellular stalks as a nucleation site for hydrous

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Journal Pre-proof ferric oxides (Chan et al., 2011). The poorly crystalline iron (III) oxide precipitates that may be produced by iron-oxidising microorganisms may also be readily reduced by iron-reducing bacteria; for example, Geobacter species within the biofilm (Roden, 2003; Roden, 2012; Roden et al., 2004). Therefore, microbially induced iron oxide minerals are unlikely to contribute to biocement formation in canga unless they are no longer viable or inactive. Instead, the microbial fossils preserved within the canga are likely to be non-iron-oxidising lineages within the microbial community that have not evolved mechanisms to avoid

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encrustation (Loiselle et al., 2018). Microbial cell envelopes have a net negative charge, forming electrostatic interactions with iron in solution, which together with the low solubility

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of iron in circumneutral environments, provide sites for the nucleation of iron (III) minerals

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(Ferris et al., 1988; Li et al., 2013). These electrostatic interactions are passive but can metal binding competes with protons in metabolising cells (for example, protons from membrane-

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induced proton motive force) (Mera et al., 1992). Therefore, dead cells may initially bind

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metals faster than metabolising cells. Quantitatively, cell envelope structures are likely to represent the most important and active sites for biologically influenced mineralisation (Fein

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et al., 1997). Aluminium in solution also plays a role in the preservation of these

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biosignatures (Levett et al., 2019), binding irreversibly with the cell envelope structures while iron is enriched within the intracellular void (for example, Fig. 3 insert). A continuous supply of soluble iron can completely fossilise and permineralise entire biofilms (Levett et al., 2016), producing new iron-rich minerals that can aggregate and cement fragments. In the cements investigated here, the microbial fossils were relatively poorly preserved and the cell population density appears to be low (Figs. 3 – 5). While direct biosignatures (for example, microfossils presented in Fig. 4B) are relatively sparse, the surrounding iron oxide minerals are extremely heterogeneous (Fig. 4B), suggesting their precipitation was influenced by the microbial biofilm. Biofilms in the streams are extensive

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Journal Pre-proof and closely associated with the canga substrates (Figs. 1B – 2). The EPS matrix, which can account for 90% of the dry mass of a biofilm (Flemming and Wingender, 2010), appears to be completely replaced by the iron oxide minerals with only the resistant cell envelope structures preserved as direct biosignatures within the heterogeneous cements. The nonuniform mineral formation and element distribution, as indicated by the backscattered electron micrographs of many of the authigenic iron oxides (for example, Fig. 4), gives much of the cement a biological texture (Fig. 4B).

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Lamellae in the biologically-textured cements (Fig. 4B) highlight that the cementation is generational, incrementally developing through time. These lamellae bands may be indicative

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of wet and dry seasons, where microbial activity and biofilm formation are less well

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developed during dry seasons. It is interesting to note that Sahoo et al. (2017) recorded an approximate 10-fold increase in the dissolved iron and aluminium in Lakes Amendoim and

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Violão during the wet season (November to May) compared with the dry season. These data

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are consistent with our hypothesis that the microbial processes that promote iron oxide mineral dissolution are accelerated during the wet seasons, when biologically available

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organic matter (washed from the surrounding vegetation) would be more readily available,

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aiding microbial respiration and the development of anoxic niches. As iron mineralised biofilms are exposed to dehydrating conditions during the dry season, they may come fossilised and contribute to the formation of new iron-rich biocements in these erosion resistant horizons. Quantifying the portions of cements in the canga rica sample that are biologically influenced during formation is difficult due to the low density of direct biosignatures. If we assume the biologically-textured cements represent the fossilisation of entire biofilms over multiple generations, a significant portion of the cement in the canga rica that forms on the lakes’

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Journal Pre-proof edges can be considered to have been influenced, and accelerated, by the growth of microbial biofilms. 6. CONCLUSIONS Iron-rich cement formation within ferruginous duricrusts that cap iron ore deposits in Brazil appears to be accelerated by ‘hotspots’ of microbiological activity. A relatively low but consistent population of iron-reducing microorganism (Geobacter lineages) that survive

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within microscale anoxic niches within well-developed biofilms appear to be responsible for driving the reductive dissolution of iron oxide minerals. These iron-reducing microorganisms support the growth of abundant and relatively diverse iron-oxidising populations, which

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contribute to the precipitation of fresh iron oxide minerals. Cements with canga are formed

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when ferrous iron in solutions is oxidised and subsequently nucleates on nearby cell envelope

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structures, fossilising microorganisms and possibly the extracellular polymeric substances associated with the biofilms. The biofilms provide an important framework to guide the

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formation of iron-rich cements within the canga. Understanding these natural processes may

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contribute to new technologies enabling enhanced physical and chemical stabilisation of degraded land sites and the generation of protective barriers for sulfidic mine waste,

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potentially reducing acid mine drainage. Acknowledgements

We acknowledge support from the Vale S.A.-UQ Geomicrobiology initiative and the Australian Research Council Linkage Program (LP140100805) to G. Southam and P. Vasconcelos. The authors acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy and Microanalysis Research Facility at the Centre of Microscopy and Microanalysis, at the University of Queensland. Alan Levett acknowledges the support from the Australian Government Research Training Program.

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Journal Pre-proof Figure Legends Fig. 1. (A) Satellite (Google Earth) image highlighting the canga plateaus of the Serra Sul (S11 regions) de Carajás Mineral Province, Pará, Brazil. Within the canga plateau are Lakes Violão and Amendoim (sample locations indicated for each), with the S11D mine towards the East. (B) Photograph of the edge of Lake Violão highlights the red fresh iron oxide precipitates where streams flow over the canga into the lake. Fig. 2. Photographs of microbial biofilms that grew at the edges of Lake Amendoim (A and

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B) and Lake Violão (C – F). (A) Microbial ‘globular’ biofilms form along grass roots that grow next to the stream within a relatively stagnant pool. (B) Fresh dark (black) biofilm next

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to the stream was sampled. Dried, flaky biofilm coated the canga above the stream. (C) An

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aqueous seep that flowed through the canga (karst-like feature) and fresh iron oxide precipitates were observed. No iron in solution was detected here. (D) Microbial biofilms

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were interspersed with fresh iron oxide precipitates where organic matter accumulated within

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a stream that flowed in Lake Violão. (E) Foam that accumulated at the edge of Lake Violão. Sample included some foam and lake water. (F) A microbial biofilm associated with fresh

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iron oxide precipitates (red) within a stream.

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Fig. 3. (A) Photograph of the polished section of the canga that formed at the edge of Lake Violão. The black rectangle highlights the representative region used to calculate cement (~23%) to fragment (~77%) surface area ratio. The low cement to fragment ratio is consistent with Dorr’s (1964) description of a rock called ‘canga rica.’ (B) Backscattered electron micrograph of large microbial fossils and small encrusted cell envelopes within the pore spaces (white arrows) that contribute to the formation of cements within the canga rica. (*) indicates region used for fragment hardness (see Fig. 5). Fig. 4. (A) Backscattered scanning electron micrograph revealing limited numbers of sporadically emplaced cell envelope structures within heterogeneous cements. (B) Low

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Journal Pre-proof magnification backscattered scanning electron micrograph of cements forming next to a large, weathered fragment within the canga. The inner black portion of the black-and-yellow line traces approximately outlines the heterogeneous cements that contain microbial remnants and are considered to have a ‘biological texture’. Fig. 5. Backscattered scanning electron micrograph of iron-rich cements within the canga rica sample. (A) Poorly preserved cocci, baccili and possible remnants of filamentous microfossils contribute to the formation of the ferruginous cement. (B) Low magnification

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micrograph highlighting the heterogeneous nature of the biologically-textured cements. Nanoindentation statistical report for the microbial cements, neighbouring goethite cements,

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hematite-rich fragment (highlighted in Fig. 3A) and resin (n – number of tests, x̅H – average

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hardness, 𝜎H – standard deviation for hardness, x̅Er – average reduced modulus, 𝜎H – standard deviation for reduced modulus).

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Fig. 6. Phylum level classification of unique 16S rRNA sequences associated with microbial

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communities and biofilms collected from steams and seeps around Lake Amendoim (AS-GB and AS-BlB), the surface (VP-SB) and bottom (VP-BB) of an oxidised ephemeral pool

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surrounding Lake Violão, at the edge of Lake Violão (LV-F) and streams and seeps that

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flowed into Lake Violão (VS-RB, VS-OB and VS-FeS). Phyla below 1.5% relative abundance are grouped into other including, Altiarchaeota, Archaea_unclassified, BRC1, Chlamydiae, Cloacimonetes, Crenarchaeota, Cyanobacteria, Deferribacteres, DeinococcusThermus, Dependentiae, Diapherotrites, Elusimicrobia, Epsilonbacteraeota, Euryarchaeota, FBP, FCPU426, Fibrobacteres, Firestonebacteria, Fusobacteria, GAL15, Gemmatimonadetes, Hydrogenedentes, Kiritimatiellaeota, Latescibacteria, Lentisphaerae, Margulisbacteria, Nitrospinae, Nitrospirae, Omnitrophicaeota, Rokubacteria, Spirochaetes, Tenericutes, Thaumarchaeota, WOR-1, WPS-2, WS1, WS4, Zixibacteria.

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Journal Pre-proof Fig. 7. Heatmap analysis of 16S rRNA gene sequences showing the most abundant OTUs (OTU clustered at a distance ≤ 0.03) in each of the biofilms that formed in the streams that feed Lake Amedoim and Lake Violão and within Lake Violão. The scale bar represents relative abundance within sample, from red (most abundant) to black (least abundant), with white indicating undetected OTUs. Relative abundances above 3% are marked in the figure. The nearest named representative in the public domain and its accession number are provided for each OTU, with blue text used to highlight putative iron-oxidising lineages and red for

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putative iron-reducing lineages.

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Table 1. Water chemistry of all sample locations. Soluble ion concentrations presented in mg/L (ppm). Dissolved oxygen (D.O.) presented as a relative percentage of air. (-) indicates no sample was taken. D.O.

Al

Ba

Ca

Fe

K

Mg

Na

S

Zn

1.68 1.79 1.60 1.41

1.77 1.60 0.68 2.72

Lake Amendoim Seep - Globular Biofilm (AS- GB; Fig. 2A) 95 78 71 48

0.05 0.03 0.39 0.11

0.04 0.03 0.40 0.02

1.89 2.49 0.30 1.73

7.85 8.15 8.42 8.02

0.09 0.03 0.04 0.03

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Surface seep 'pool' 2 cm below surface 6 cm below surface 8 cm below surface

0.26 0.26 0.19 0.13

0.49 0.56 0.56 0.66

Lake Amendoim Seep - Black Biofilm (AS-BlB; Fig. 2B) Surface seep 'pool'

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0.07 0.60 0.42

0.695

0.16 0.05 0.67 0.88 0.36

Top (0.5 cm) of pool (VP-SB) 2 cm below surface 5 cm below surface

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Lake Violão Ephemeral Pool - Surface Biofilm (VP-SB) and Bottom Biofilm (VP-BB) 0.11 0.09 4.09

5.81

3.34 0.85 1.65 1.79 0.83

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0.10 0.05 2.42 0.23 0.57 1.30

5.44 5.13

3.23 0.95 2.64 1.76 1.03 3.18 0.85 1.62 1.71 0.55

12 cm below surface

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0.37 0.55 1.40

5.74

3.50 0.84 1.61 1.50 0.37

15 cm below surface (VP-BB)

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0.05 0.18 1.16

1.956

1.47 0.54 2.28 1.01 0.45

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Water flowing out of porous canga

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Lake Violão Seep - Red Biofilm (VS-RB; Fig. 2C) -

0.00 0.03 0.10

0.0

0.06 0.01 0.35 0.21 0.02

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Lake Violão Seep - Organic Biofilm (VS-OB; Fig. 2D)

Surface seep 'pool' with organic matter 1 m downstream 2 m downstream 3 m downstream 4 m downstream 8 m downstream Surface seep enters Lake Violão

-

0.03 0.82 0.18

3.39

0.07 0.12 0.73 1.59 0.74

-

0.55 0.10 0.12 0.02 0.02

2.17 4.39 0.23 0.46 0.24

1.112 1.284 0.799 0.528 0.586

0.10 0.00 0.05 0.20 0.00

-

0.02 0.69 0.31

0.658

0.07 0.08 0.78 1.49 0.43

0.08 0.03 0.82 0.64 0.66

0.18 0.05 0.16 0.15 0.17

0.78 0.63 0.78 1.00 0.91

1.60 1.77 1.84 1.54 1.68

2.25 3.45 0.72 0.63 0.49

Lake Violão Seep - Foam (VS-F; Fig. 2E) Lake Violão - foam

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0.37 0.01 0.81

0.0

0.15 0.05 0.38 0.27 0.40

Lake Violão Seep - Iron Seep (VS-FeS; Fig. 2F) Iron seep

-

0.81 0.77 0.28

15.790

0.23 0.26 0.97 2.06 0.98

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0.00 0.05 0.71 0.11

0.03 0.65 0.03 0.52

2.22 0.53 2.35 0.64

4.89 0.568 2.86 1.261

0.18 0.27 0.16 0.13

0.18 0.26 0.13 0.20

0.82 1.02 0.85 0.79

1.68 1.56 1.74 1.42

4.46 0.45 2.58 0.51

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1 m downstream 3 m downstream 7 m downstream 10 m downstream

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Journal Pre-proof Highlights

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1. Microorganisms accelerate short-range biogeochemical cycling of iron within duricrusts 2. Organic scaffold (biofilm) essential for robust cement generation 3. Enhancing biogeochemical cycling of iron for degraded land remediation

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7