Microbial Biobanking Cyanobacteria-rich topsoil facilitates mine rehabilitation

Restoration of soils post-mining requires key solutions to complex issues where the disturbance of topsoil incorporating soil microbial communities can result in a modification to ecosystem function. This research was in collaboration with Iluka Resources at Jacinth-Ambrosia (J-A) mineral sand mine located in a semi-arid chenopod shrubland in southern Australia. At J-A, assemblages of microorganisms and microflora inhabit at least half of the soil surfaces and are collectively known as 15 biocrusts. This research encompassed a polyphasic approach to soil microbial community profiling focused on ‘biobanking’ viable cyanobacteria in topsoil stockpiles to facilitate rehabilitation. We found that cyanobacterial communities were compositionally diverse topsoil microbiomes. There was no significant difference in cyanobacterial community structure across soil types. As hypothesised, cyanobacteria were central to soil micro-processes, strongly supported by species richness and diversity. Cyanobacteria were a significant component of all three successional stages with 21 species identified from ten 20 sites. Known nitrogen-fixing cyanobacteria Symploca, Scytonema, Porphyrosiphon, Brasilonema, Nostoc and Gloeocapsa comprised more than 50% of the species richness at each site and 61% of the total community richness. In the first study of its kind, we have described the response of cyanobacteria to topsoil stockpiling at various depths and ages. Cyanobacteria are moderately resilient to stockpiling at depth and over time, with average species richness greatest in the top 10 cm of the stockpiles of all ages and more viable within the first six weeks, indicating potential for biocrust re-establishment. In general, 25 the resilience of cyanobacteria to burial in topsoil stockpiles in both the short and long term was significant, however in an arid environment recolonization and community diversity could be impeded by drought. Biocrust re-establishment during mine rehabilitation relies on the role of cyanobacteria as a means of early soil stabilisation. At J-A mine operations do not threaten the survival of any of the organisms we studied. Increased cyanobacterial biomass is likely to be a good indicator and reliable metric for the reestablishment of soil micro-processes. 30

Discussion of co-occurring nitrogen-fixing species and their role as pioneers: We thank the reviewer for highlighting these findings for us. We have added a section to the discussion to explore the implications of finding these species and their cooccurrence in early stage biocrusts.
Page 11 Lines 10-11: Indeed, the DIVERSE function within PRIMER was used to calculate the diversity values here. This method is mentioned in the corresponding methods section for the genomic profiling. While we have trialed a standalone 5 statistical analyses section, we found it complicated the flow of the manuscript.

Discussion -
Page 13 Line 7: comma added Page 13 second paragraph: we have rewritten this paragraph for clarity Finally, we appreciate the reviewers' comments regarding the difference species involved in crust building (i.e. what we 10 have found in Australia vs. common reports elsewhere) and have added some relevant comments to Section 4.1 (paragraphs 1 and 3).
In response to the reviewers suggestion we also relocated some figures to supplementary material and removed some that were not really necessary. 15 20 25

Introduction
Soil disturbance results in a loss of resources and often has long lasting effects on soil stability, nutrient cycling and surface hydrology (Bowker, 2007;Tongway and Hindley, 2004). Restoration of ecosystem function post disturbance requires appreciation of the dynamic functional status of the landscape prior to disturbance (Tongway and Ludwig, 1996), as well as an understanding of the net accumulative effects of disturbance on the components of the system. Consequently, it is 5 necessary to appreciate the micro-processes that will assist in the restoration of soil function and to monitor recovery along the way.
Microorganisms are critical components of soils that drive micro-processes and impact soil ecosystem function on several levels. Biological soil crusts (biocrusts) are topsoil assemblages of bacteria, fungi, algae, mosses, and lichen which occur 10 where vascular plant growth is restricted, namely arid lands. Highly structured, diverse biocrusts are a significant asset to arid soil ecosystems providing a protective, nutrient rich layer closely integrated into the soil surface (Delgado-Baquerizo et al., 2013;Maestre et al., 2012). As biocrusts develop in structural complexity, the diversity of organisms is regulated by water infiltration, temperature, light and disturbance (Belnap and Eldridge, 2001;Büdel et al., 2009;Elbert et al., 2012).
Early successional biocrusts are mainly composed of bacteria, algae and cyanobacteria whereas late successional biocrusts 15 can incorporate lichens, mosses, liverworts, algae, fungi and bacteria (Büdel et al., 2009). Cyanobacterial re-establishment is a key indicator of early soil surface re-stabilisation, regulation of soil moisture and the balancing of soil carbon and nitrogen (Elbert et al., 2012).
Cyanobacteria in arid landscapes are exceptionally well-adapted to desiccation. Their polysaccharide sheaths and EPS 20 production perform a vital role in maintaining cyanobacterial cell integrity, exchange of information and absorption of water during rehydration (Rossi et al., 2017). EPS has adhesive properties that binds non-aggregated soil particles into a protective encrusted surface that reduces the destructive impacts of wind and water (Eldridge and Leys, 2003;Rossi et al., 2017).
Cyanobacterial biofilms provide stabilisation of initially disturbed surfaces that pave the way for diverse microbial communities, and form bioactive crust-like layers assimilated into the soil (e.g. Büdel et al., 2009;Rossi et al., 2017;Bowker 25 et al., 2014). EPS forms the bulk of the biocrust structure. EPS more than doubles the biocrusts compressive strength and increases cohesiveness by up to six times (Hu et al., 2002).
Physical disturbance can profoundly disrupt biocrust integrity, composition and physiological function where the impact is governed by site characteristics, severity, frequency and timing (Belnap and Eldridge, 2001). Mining disturbance alters the 30 biophysical state of the biocrust community through excavation, crushing, mixing and burial. Biocrust burial within the natural environment as a result of environmental stress and disturbance can have severe negative impacts on biocrust integrity and function. For example, significant declines were recorded in cyanobacterial richness and abundance that was Deleted: from arid ecosystems Deleted: that 35 Deleted: from rainfall linked to a reduction in soil nutrient concentrations (Rao et al., 2012;Williams and Eldridge, 2011). Following disturbance, restoration and regrowth of biocrusts can take place unassisted, seasonally driven and may take many years (Belnap and Eldridge, 2001;Belnap and Gillette, 1998). Alternatively, assisted biocrust restoration places emphasis on the recovery of ecosystem function and necessarily addresses environmental constraints. This incorporates the knowledge of 'potential condition' based on experience with sites of ecological similarity that have undergone disturbance and recovery (Bowker, 5 2007). Biocrust recovery can be altered by dust deposition, fire and climatic conditions (Weber et al., 2016). When biocrusts recover naturally soil properties change. For example, in Southern African and Spanish rangelands an incremental accumulation of soil nutrients, organic matter and a build-up of silt and clay lead to the development of a resilient and multi-functional biocrust (Büdel et al., 2009;Maestre et al., 2012;Weber et al., 2016).

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Mine rehabilitation is a complex process that involves many levels of understanding of difficult issues relating to ecosystem function where the removal or burial of the bioactive soils can have knock-on effects for rehabilitation efforts such as native seedling establishment (Jasper, 2007;Tongway and Ludwig, 1996). Successful ecological restoration of arid mining sites relies on a holistic approach where biocrust recovery to pre-disturbance levels is integral and can serve as an indicator of the integrity of the ecosystem (Tongway, 1990). Research into biocrust disturbance with a focus on recovery post-mining is rare. 15 In the Namaqualand arid lands (Namibia, South Africa) low rainfall and high winds impact the rehabilitation of degraded lands following diamond mining and grazing (Carrick and Krüger, 2007). These researchers found that cyanobacteria and non-vascular plants that form a living and protective surface crust were crucial to surface stabilisation. Jasper, (2007) also recognised the importance of soil microbial communities including cyanobacteria in post-mine rehabilitation in the Jarrah forests of south-western Australia. In the Czech Republic and Germany chrono-sequential studies of old brown coal mine 20 sites found in younger sites that green algal biofilms and a diverse range of cyanobacteria initiated the rehabilitation of the soils (Lukešová, 2001). In serpentinite mine tailings (New South Wales, Australia), McCutcheon et al., (2016) showed filamentous cyanobacteria accelerated carbonate mineral precipitation and stabilised the tailings. They demonstrated cyanobacteria had the capacity to adsorb magnesium while acting as a nucleation site and sequestered carbon. In our current study, preliminary research identified that in undisturbed chenopod shrublands at the edge of the Nullarbor Plain (South 25 Australia) biocrusts cover more than 45% of the soil surfaces between the grass plants and post-mining rehabilitation needs to investigate their role (Doudle et al., 2011). It follows that there is a real need for a focus on practical approaches that contribute to the restoration of soil function and measure relevant aspects of success through soil microbial communities and biocrust reestablishment, especially cyanobacteria (for example: Setyawan et al., 2016;Mazor et al., 1996;Fischer et al., 2014;Chiquoine et al., 2016;Doherty et al., 2015;Harris, 2003;Tongway and Hindley, 2004;Zhao et al., 2014). 30 On the micro-scale, cyanobacterial species richness contributes to soil ecosystem function through micro-processes including carbon fixation through photosynthesis, atmospheric nitrogen fixation in a biological-available form, micro-nutrient breakdown and release, soil particle cohesion, regulation of moisture and soil surface structure (Delgado-Baquerizo et al., 2013;Elbert et al., 2012;Hu et al., 2002;Maestre et al., 2012 and others). In mining, the topsoil is often stripped and stored in stockpiles as a form of "biobanking" for redistribution at the cessation of mining. This process involves in the crushing and burial of biocrusts and results in the inability of crust organisms to photosynthesise due to the lack of light. To our knowledge, the effects of topsoil stockpiling on biocrust organisms such as cyanobacteria, and their recovery time, has not previously been investigated. Should biocrust organisms remain inactive while they are wet, cell death and decomposition 5 may occur (Kidron et al., 2012;Rao et al., 2012). Nevertheless, in dry conditions, cyanobacteria and algae are known to remain desiccated and viable for up to millions of years (Vishnivetskaya et al., 2003).
Our study aimed to examine biocrusts, with a focus on cyanobacteria, within a mining framework to develop informed rehabilitation plans that achieve improved long-term outcomes. Specifically, we sought to determine whether shallow 10 biobanks of cyanobacterial-enriched topsoil would facilitate biocrust recovery on mining disturbed lands. We identified key aspects of biocrust and cyanobacteria ecology that needed to be discerned which directed the establishment of this biocrust research project. The overall goals of the biocrust research program were to: (a) evaluate specific roles of natural, undisturbed biocrusts in ecosystem function at the mine site; (b) determine cyanobacterial community structure in terms of key species that drive early colonisation, biogeochemical cycling and soil stabilisation; and, (c) to investigate the effects of 15 stockpiling topsoil on cyanobacterial survival after burial and subsequent recovery. We hypothesised that cyanobacteria would be essential as ecosystem engineers in soil habitat formation with a clear distinction in community structure between soil types and biocrust successional stages. It was also anticipated that in the topsoil stockpiles there would be a low survival rate especially at depth and over time.

Background and site description
Jacinth-Ambrosia (J-A) heavy mineral sand mine is located on the eastern edge of the Nullarbor Plain, South Australia, across the boundary of two Regional Reserves within the Eucla Basin region. The climate is semi-arid with a mean rainfall of 185 mm, mean maximum temperature of 27.6°C and minimum of 12.1°C (further detail provided in supplementary Figure   S1, www.bom.gov.au). Tertiary sediments deposited in marine and terrestrial settings and the soil distribution of the area 25 reflects the geological history, with at least five marine transgression and regression events depositing 40-50 m of sediments (Hou and Warland, 2005). The landscape is broadly undulating with low open woodlands that have a shrub understorey with chenopod shrub lands as well as dune fields that consist of parallel dunes and inter-dune swales (Doudle et al., 2011;7 The landscape has been characterised into three distinct soil types (Table 1) associated with vegetation communities called soil management units: SMU1deep calcareous yellow sands associated with dune ridges; SMU2shallow calcareous sandy loams; and SMU3deep calcareous sandy loam (Doudle et al., 2011;Hou and Warland, 2005). The biocrusts at J-A had been previously classified into three successional stages representative of the five biocrust types found growing across 5 the landscape (Table S2) (Doudle et al., 2011). Types 1-2 are light coloured, patchy, thin, and fragile cyanobacterial crusts  Table S1). Study locations were selected for: (a) vegetation communities, (b) soil types and 10 underlying geology, (c) biocrust successional types, (d) areas identified for future mine operations and a dry salt lake (Lake Ifould). The dry salt lake would potentially identify cyanobacterial species adapted to saline conditions similar in nature to ground water.
In the process of mining and preparation of the J-A site for future rehabilitation, topsoil (0-100 mm) is stored in low 15 stockpiles, generally less than two metres in depth where stockpiles are comprised of topsoil sourced from only one vegetation type. Later stockpiles will be returned to the surface of mined areas in the rehabilitation process, and the cyanobacterial activity therein must be quantified to enable educated planning and decision making regarding biocrust reestablishment. If low levels of biocrust organisms are detected below the top few centimetres, the addition of propagated biocrust organisms (e.g. cyanobacteria) to returned topsoil may be warranted. Activity and species richness within the 20 stockpiled top soils may also vary with age and this may also influence the establishment of vegetation in the rehabilitation process.

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Preliminary identification of biocrust types had been determined by Doudle et al., (2011) and provided the baseline data for biocrust sampling from Sites 1-10 ( Fig. 1, Table S1). The sites selected encompassed the three SMUs and five crust types (Table S1). All sites are of naturally occurring biocrusts except for Site 6 which is from a two-year-old topsoil stockpile regarded as an early (Type 1) biocrust representing recovery two years post disturbance (Fig. 2). Within each site, eight 10

Biocrust biophysiochemical properties
For each site, duplicate sub-samples were taken from each Petri dish for each biophysiochemical analysis (n=16 per site).
Soil was fine sieved (1.70 mm) and analysed for total C and N and C:N ratio using a high temperature digestion in a vario MACRO Elemental Analyser (Elementar). Soil pH and electrical conductivity (EC) samples were prepared using a 1:5 (soil 5 to water) ratio and shaken for one hour. Following shaking, samples were left to stand for 30 min and EC measured using a Crison Conductivity Meter 525. The sample was mixed again, and pH was measured with a TPS pH meter MC-80 using an ionode IJ44C electrode. Chlorophyll a concentration of the biocrusts were determined following resurrection (by moistening) using a 1:5 ratio of (dry weight) biocrust to Dimethyl sulfoxide (DMSO) (Barnes et al., 1992) with samples placed in a warm bath (65°C) for a two-hour dark extraction, followed by centrifuging for five minutes (5000 g RCF). 10 Chlorophyll a concentration was determined using Wellburn's, (1994) equations.
A pocket penetrometer (8 mm foot) was used to determine the compressive strength (kg cm 2 ) of the dry intact biocrusts samples. Overall the crust thickness was < 0.5 cm. Each sample (10 cm diameter x 2 cm depth) was placed on a solid surface and a total of twelve measurements (3 readings x 4 reps) were taken for each site. The measurement was taken at the point 15 when the crust was broken, and the foot penetrated the crust surface.
Photosynthetic performance (recorded as yield, YII) was measured using pulse-amplitude modulated (PAM) fluorometer (Pocket PAM; Gademann Instruments, Germany). The aim was to demonstrate photosynthetic yield (YII) indicative of active growth of the biocrusts, using the detection of chlorophyll fluorescence from photosystem II (PSII). The sensor was 20 placed onto the biocrust and once started, a series of short pulses of excitation light at high intensity that is amplified resulting in a brief closure of PSII and the measurement of fluorescence yield based on the Genty parameter which is the quantum yield (YII) of the charge separation of PSII (Genty et al., 1989) and recorded on a scale of 0-1 for all photosynthesis. Allowing a short space of time between readings, this process was completed at least six times for each sample (6 x 4 reps per site). 25

Microscopy of biocrust cyanobacteria
Field samples collected in section 2.2 were used to enumerate cyanobacterial richness and diversity and to classify colonies. 30 The dried crust samples were resurrected in the glasshouse for three to five days. For each of the ten sites, twelve replicates were analysed via light microscopy. For each replicate, a minimum of two wet-mount slides incorporating six representative portions of the cyanobacterial colonies were examined (n=144 colonies per site). For the dominant land type, Chenopod shrubland (Site 8), there were an additional 10 x 6-cell multi-well plates. These were treated similarly where two slides were examined from each of the 60 multi-wells (n=120). In total > 2,184 cyanobacterial colonies were examined. Initial inspection of the biocrust and the separation of individual species were made using an Olympus SZH10 microscope at 70 x magnification. Cyanobacterial filaments or colonies were carefully extracted with forceps to recover sufficient material that included important morphological features such as their colour, encasing sheaths as well as cellular structure. Live material 5 was examined by Nomarski differential interference contrast (DIC) microscopy with a Jenaval (Jena Zeiss) and an Olympus BX51 compound microscope (magnifications 400-1000 x). Photomicrographs were taken using an Olympus SC100 digital microscope camera, and morphological measurements of vegetative cells were made from digital images of live material taken at 400 x magnification using Olympus cellSens® digital imaging software.

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Identification was performed to a species level (wherever possible) in the laboratory using the following taxonomic references: Anagnostidis andKomarek, (2005, 2005); Sant'Anna et al., (2011); Skinner and Entwisle, (2002). It was often necessary to record the closest named species as attributes varied somewhat to temperate climate and aquatic specimens described in literature. Nitrogen fixing cyanobacteria were identified based on the three recognised types: (1) heterocystous species (those with specialised N-fixing cells); (2) non-heterocystous species that fix N aerobically and; (3) non-15 heterocystous species that fix N anaerobically (Bergman et al., 1997;Stal, 1995). Using a graticule, abundance was ranked on a scale of 1-8 where the main taxa are ranked in decreasing order of the relative percentage area occupied in a single view (Biggs and Kilroy, 2000). More than one species could be dominant, and all other taxa were ranked in relation to the dominant taxa as abundant, common, occasional and rare. To determine similarities between cyanobacterial communities, cluster analysis, SIMPROF, and non-metric multidimensional scaling (nMDS) were conducted using Primer v6 (Clarke & 20 Gorley 2001).

16S rDNA profiling of native undisturbed biocrust microbiomes
For genomic profiling of naturally occurring successional biocrust communities, a location adjacent to Site 9 was visually determined to contain Bare, Early (Types 1-2) or Late (Types 4-5) stages of development (Table S2). Biocrust successional features were determined by morphological attributes of pigmentation, thickness and surface roughness as well as the 25 presence/absence of lichens and mosses ( Fig. 3) (Chilton et al., 2017). Bare stage was characterised by loose soil particles with no visible biocrust structure. Samples were collected in July 2014. For each successional stage, three replicates were collected that were representative of SMUs 1-3 where a 10 cm 2 plot with 95% coverage of the desired biocrust stage was excised to the depth of the crust and non-aggregated soil discarded (Fig. 4). Samples were processed at UNSW, Sydney. values. ANOVA with post hoc Tukey's tests was used to test for significant differences between stages. Multivariate analyses were performed in Primer upon a Bray-Curtis dissimilarity matrix generated from square-root transformed abundance data. Samples were represented in two and three-dimensional space within a nMDS plot. Pair-wise, a posteriori comparisons of factor Stage were performed using the PERMANOVA function with 9999 Monte Carlo permutations.
Homogeneity of dispersion for each stage was tested using PERMDISP. 10

Cyanobacterial tolerance to stockpiling
Stockpile sampling was carried out in March 2012 with samples sourced from topsoil stockpiled from areas with Acacia papyrocarpa (Western Myall) over-storey (Table S3). Stockpiles established at three different time points (9 months, 20 months and 29 months) as well as corresponding undisturbed sites were sampled in triplicate at six depths (0-2, 2-4, 4-6, 10, 25 and 50 cm). This resulted in 18 replicates for each soil depth for each stockpile time point. Holes were dug to >50 cm 15 depth with a shovel and the exposed profile removed with an ethanol-wiped spatula before taking a soil sample. A second sample at 50 cm was autoclaved to serve as a culturing control. Samples were stored in paper bags and processed at The University of Queensland. For each sample, 20 g of soil was set up in petri dishes with 9 ml of water and sealed with Parafilm ® . Petri dishes were incubated for six weeks at 26°C under a 12-hour photoperiod regime and rotated weekly to prevent site specific effects. Samples were maintained as wet by the addition of sterile water within a laminar flow cabinet. 20 To determine cyanobacterial growth and richness, wet-mounts for each sample were examined under 16 x magnification for cyanobacterial thalli and colony size was estimated via area of coverage of the field of view. Where multiple colonies were present in a slide, the relative abundance of each was used to divide the cover between the colonies accordingly. Where no growth was observed, five soil samples were taken randomly from the sample, mounted and examined under 400 x magnification for the presence of cyanobacterial cells. ANOVAs were performed with post hoc Tukey's tests performed to 25 determine any differences between treatments.

Ecophysiological properties of biocrust cyanobacteria
Soil pH across the three soil management units (SMUs) ranged from pH 8.4-8.6 while the two-year old topsoil stockpile was higher at pH 8.9 (Table 1). Electrical conductivity ranged from 92-140 μS cm -1 . There were no significant differences in pH 30 and EC between field samples and intact samples (data not shown). Total nitrogen was typically <0.1% across all sites and Deleted: morphotypes Deleted: colony

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Deleted: morphotypes total carbon ranged between 1-2% with higher percentages generally found across SMU 2 and 3 ( Table 1). The ratio between carbon and nitrogen was the greatest across SMU 3 and the two-year old topsoil stockpile, also originating from SMU 3.
The penetrometer index of crust compressive strengths across the ten sites showed that Sites 4, 9 and 10 in SMU 1 were significantly different from each other as well as significantly lower than SMU 2, SMU 3 and T2, two-year old topsoil stockpile sites ( Table 2). The means for SMU 1-3 were also significantly different (p < 0.0001), and it was confirmed by a 10 student's t-test that the difference was between SMU 1 and SMU 2 and 3.
The photosynthetic yield measurements showed there was variability between sites (Fig. 5). The yields recorded all fell within the expected range and when data was separated into their respective SMU it provided a clearer picture of the variability. SMU 3 had the lowest mean overall while there were no differences between the others. 15

Cyanobacterial community structure
A total of 21 cyanobacterial species were identified across the ten sites using microscopy (Table 5; Figs. S2-S9). The majority of species richness and abundance was comprised of four filamentous genera: Symploca (18%), Schizothrix (16%), Porphyrosiphon (16%) and Scytonema (16%). Secondary cyanobacteria present variously occupied <1 to 10%, and overall made up 34% of the community. The known nitrogen-fixing cyanobacteria Symploca, Scytonema, Porphyrosiphon, 20 Brasilonema, Nostoc and Gloeocapsa comprised more than 50% of the species richness at each site and formed 61% of the total community richness (Fig. 10). In this study cyanobacterial community structure was tested against the three different soil types to determine whether soil type was influential in determining community structure. There was no significant difference in cyanobacterial community structure across soil types, however the results do suggest some spatial structuring exists across the three SMU (Figs. 11,12). The structural relationship between all samples shows greater similarities rather 25 than dissimilarities, of which there are only a small number of samples that are significantly different (Fig. 12). Soil type did not explain these differences. This suggests that most of the species could potentially be found anywhere across the three zones and their richness and abundance is controlled by other factors. Symploca occurred more frequently and was more abundant in the majority of the samples examined therefore was the most significant contributor to the community (data not shown). Individual sites generally displayed similar trends although there was some variability occurring between sites. Of the 21 species more than half (12 species) of cyanobacteria were identified in SMU 1 where four primary genera made up 75% of the community: Symploca, Schizothrix, Scytonema and Symplocastrum (for more detail see Fig. S10). Cyanobacterial crusts from the dune regions on SMU 1 (deep calcareous yellow sands) were representative of crust types 1-3; patchy, brittle (when dry) early-successional crusts as well as formed dark crusts that were mid to late-successional and included cyanolichens (also see Doudle et al., 2011). 5 Cyanobacterial crusts from the chenopod shrublands and open woodlands in SMU 2 (shallow calcareous sandy loam) represented a broad range of crust types (2-5) but overall could be described as late-successional. Lichens and mosses were highly visible (also see Doudle et al., 2011). There were 21 cyanobacteria recorded: four were primary genera that made up 63% of the community including: Schizothrix; Porphyrosiphon; Scytonema and Symploca (for more detail see Fig. S11). 10 Cyanobacterial crusts from the open woodlands in SMU 3 (deep calcareous sandy loam, Fig. 2c) represented a broad range of crust types (2-5) but like SMU 2 could also be described as late-successional. Lichens and mosses were highly visible (see Doudle et al., 2011). There were nine cyanobacteria recorded of which four were primary genera that made up 85% of the community: Symploca, Porphyrosiphon, Scytonema and Schizothrix (for more detail see Fig S12). Cyanobacteria with 15 the capacity to fix nitrogen contributed to 77% of the community structure.
Cyanobacterial crusts from Site 6 were from the two-year old topsoil stockpile (T2) that had originated from SMU 3 (deep calcareous sandy loam) would be described as early successional crusts with some seasonal mosses. There were eight cyanobacteria recorded of which four were primary genera that made up 84% of the community: Symploca; Symplocastrum; 20 Porphyrosiphon; and Scytonema (also see Fig. S13). It was interesting to note that Symplocastrum was co-dominant with Symploca whereas in the other communities it ranged between 8-13%. Sub-surface species Schizothrix (found in top 5 mm) only contributed to 4% of the richness compared to 10-20% elsewhere. Cyanobacteria with the capacity to fix nitrogen (Symploca, Porphyrosiphon, Scytonema and Brasilonema) contributed to 61% of the community.

16S rDNA profiling of native undisturbed biocrust microbiomes
Microbial community profiling using high through-put sequencing revealed cyanobacteria comprised a significant component of all three stages forming the majority of sequences in Early and Late stages (Fig. 6). There was a diversity of morphotypes observed including simple filamentous, heterocystous and unicellular types (Fig. 7). The most abundant genera identified were Leptolyngbya, Phormidium, Tolypothrix, Nostoc, Brasilonema, Chroococcidiopsis and Acaryochloris. 30 Unclassified Nostocaceae were dominant within Bare soils while Early stages observed a relative even increase in Phormidium, Brasilonema and the unicellular genera (e.g. Chroococcidiopsis, Acaryochloris, Xenococcaceae). Late stage biocrusts showed a slight resurgence of Nostocaceae. There was no significant difference in the richness, evenness or Deleted:

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Formatted: Font color: Auto diversity between the three stages (Table 3). However, there were significant differences in the composition and structure of the communities of each stage. The Bare stage was significantly different to the crusted stages (Fig. 8, Table 4) while resolution between the Early and Late stages was less clear. Ordination of the samples within three dimensions showed Early and Late stages grouped separately however, PERMANOVA failed to identify a significant difference (Table 3).

Cyanobacterial tolerance to stockpiling 5
Examination of stockpile soil samples via microscopy revealed five cyanobacterial genera: Nostoc, Scytonema, Microcoleus, Porphyrosiphon and Leptolyngbya. Average genera richness was highest in stockpiled samples at and above 10 cm depth for all stockpile ages (Fig. 13 a, b). These differences were significant between 50 cm and 0-2 cm in all stockpiles (29-month F(1,5) = 7.00, p= 0.024; 20-month F(1,5) + 8.37, p = 0.016; 9-month F(1,5) = 18.00, p = 0.002). Genera richness at the intermediate depths was not significantly different. Differences were significant between 25 cm and 0-2 cm in the twenty-10 and nine-month old stockpiles (F(1,5) = 32.73, p = 0.000; F = 16.20, p =0.000) respectively. Counterintuitively, after nine and twenty months of stockpiling, average genera richness was higher in stockpiled samples when compared with undisturbed samples except for 20-month old 4-6 cm samples (Fig. 13 a, b) but this difference was only significant in the nine-month old stockpile at 10 cm depths (F(1,5) = 8.27, p = 0.017). High variability between replicates accounted for the lack of significance in the rest. 15 When comparing average genera richness at different depths between stockpiles, richness was greater in material stockpiled for the least amount of time above 10 cm depth but not at or below this level (Fig. 13b). Average genera richness was variable within adjacent undisturbed areas (Fig. 13a). The variability in genera richness within replicates was high (refer to

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Nostoc cf. commune exhibited the best survival response in stockpiling as it had significant coverage in samples from all depths in all stockpile ages. Nostoc had the greatest coverage in the oldest stockpiles, followed up by the most recently created stockpiles. These differences were significant from depths of 6 cm to the surface (4-6 cm F(1,5) = 28.83, p = 0.000;
cf. commune in corresponding undisturbed areas. The Leptolyngbya black morphotype only occurred between 20 and 40 cm depth and were found in only one stockpile samples but were present in six samples from three adjacent areas. Stigonema genus was found in six stockpile samples spanning all ages and in only one adjacent sample in all cases in the upper 10 cm of the soil profile. 5 Nostoc cf. commune, Porphyrosiphon, Microcoleus and Scytonema were the first genera to develop to an identifiable stage.
Filaments of the Stigonema genus were found in low numbers and appeared to be recently formed. It was only present in samples examined in the latter stages of the identification process. The yellow form of Nostoc exhibited a much slower rate of development than Nostoc cf commune and could only be definitively determined as a form of Nostoc when examined after 10 13 weeks of incubation. Samples from undisturbed areas sourced at depths from between 10 cm and 50 cm initially showed no visible signs of growth when examined after six weeks of incubation. Six weeks later cyanobacterial growth was evident yet in many cases had not advanced to the point where morphotypes could be distinguished.

Discussion
In terms of rehabilitation the natural capital in topsoil that has been removed in the mining process is often not recognised or 15 poorly understood. Soils properties are crucial in the re-establishment of a raft of ecosystem services that include fertility, structure, climate regulation and biodiversity (Dominati et al., 2010). Cyanobacteria are ecosystem engineers in that they have the capacity to provide many of these crucial ecosystem services (Jones et al., 1994). This research demonstrated that the cyanobacterial communities in the J-A biocrusts were compositionally diverse topsoil microbiomes that substantially contributed to the Myall-chenopod biomes. We had hypothesised that cyanobacteria would be central to soil micro-processes 20 and this was strongly supported by extensive species richness and diversity. In the first study of its kind we have shown the response of cyanobacteria to topsoil stockpiling at various depths and ages. In this study we have shown cyanobacteria are moderately resilient to stockpiling at depth and over time, providing they are manipulated dry (W. Williams, unpublished data). Cyanobacteria from the top 10 cm were found to be more viable within the first six weeks and showed potential for biocrust re-establishment. We found greater cyanobacterial richness in the nine and 20-month stockpiles compared to 25 undisturbed samples adjacent to the stockpiles. In general, the resilience of cyanobacteria to burial in topsoil stockpiles in the longer term appeared good, however in an arid environment recolonization and community diversity could be impeded by drought (Williams and Büdel, 2012) In this study cyanobacterial community richness and abundance were not related to soil and landform type and this was further supported though the sequenced samples where no significant differences were observed. At J-A any of the 30 cyanobacteria could conceivably occur anywhere across the landscape, with their relative abundance likely determined by microenvironments and microhabitats such as light (sun and shade) and chemical gradients (Stal, 2003), as well as moisture availability (Büdel et al., 2018) and soil particle size (Büdel et al., 2009).

Cyanobacterial community structure
Key cyanobacteria indicating biocrust formation and development were Leptolyngbya, Phormidium, Tolypothrix, Nostoc, Brasilonema, Chroococcidiopsis and Acaryochloris. These genera have consistent morphological traits with those observed 5 via microscopy. Notably, the identification of Brasilonema was supported with sequencing data and had not been previously recorded in Australian soils. Simple filamentous types are often attributed with the primary crust building role, able to span inter-particle gaps within the soil via supra-cellular structures (e.g. Microcoleus, see Garcia-Pichel and Wojciechowski 2009). Sequencing data showed Phormidium was the dominant cyanobacterium for this role and it is likely that Symploca identified though microscopy was the principal Phormidium present. Microcoleus sp. and Porphyrosiphon were also 10 identified as early colonisers however these genera are currently poorly resolved phylogenetically (Garcia-Pichel et al 2013) but share critical morphological features enabling biocrust formation and maintenance. Notwithstanding, the important crustbuilding cyanobacteria in this study appeared to be Symploca that was associated with EPS production, and principal feature of early colonising crust formation (Hu et al., 2002).

15
The cyanobacterial richness at J-A was determined according to their morphological features. In many cases these features (e.g. outer protective sheaths, UV protection, EPS production) provided the basis of attributes that pertained to fundamental survival strategies. Environmentally induced strategies of arid land cyanobacteria reflect their habitat, these survival traits have developed over a long evolutionary history. Many primary (common to abundant) and secondary (uncommon) cyanobacteria recorded at J-A exhibited thick gelatinous sheaths (Porphyrosiphon, Schizothrix, Microcoleus, Nostoc) or 20 were associated with the production of EPS (Symploca, Nostoc, Schizothrix, Leptolyngbya). Filamentous cyanobacteria formed the major part of the J-A crust structure with tufts, webs or creeping masses closely intertwined (e.g.

25
Twenty-one cyanobacteria were recorded from 13 genera. Four species were unicellular and the remaining seventeen were filamentous. Some cyanobacteria found at J-A (Microcoleus paludosus, Nostoc sp., Gloeocapsa) had also been recorded at Lake Gilles (SA) about 400 km southeast of J-A (Ullmann and Büdel, 2001). Although Microcoleus species were recorded at J-A they did not dominate the biocrust compared with many reports from the United States, Asia and elsewhere (e.g. see Belnap and Eldridge, 2001). This infers that the early colonisers such as Microcoleus would not play a dominant role in early 30 stabilisation and colonisation of the soil. At J-A Symploca and Scytonema appeared to be an important colonising Deleted: Cyanobacterial community richness and abundance were not affected by soil type as shown by the similarity between early stage (SMU 1) and late stage (SMU 3), where there were no significant differences.

Moved up [2]
: Contrary to our hypothesis there was not always a clear distinction in community structure between soil types and biocrust successional stages, notably in the early and late stages.

Formatted: Font: Italic
Formatted: Font: Italic cyanobacterium in the biocrusts and have been recorded as playing a key role in carbon sequestration in northern Australian cyanobacterial crusts (Büdel et al., 2018).
The taxonomic status of Brasilonema remained uncertain and may be a variety of Scytonema, however, genomic data supported morphological identification and the type has also been recorded in other terrestrial habitats globally. Due to its 5 similar morphological attributes and genomic data, in this study we called this cyanobacterium Brasilonema (Fiore et al., 2007;Vaccarino and Johansen, 2012). Nostoc commune var. flagelliforme had been recorded at J-A along with Nostoc commune across the shallow and deep sandy loams. Although N. flagelliforme appeared rarely, it had been previously documented from sites in south-western South Australia, Western Australia, Northern Territory (Skinner and Entwisle, 2002) and Victoria (W. Williams, unpublished data). A joint Spanish-Australian study has now shown that both Nostoc 10 commune and N. flagelliforme contain the same genomic markers and cannot be separated, rather the spaghetti-like tubes that are unique ecotype likely associated with aridity (Aboal et al., 2016). This is supported by the semi-arid environment at J-A and it may be more widespread in Australia than previously recorded as it is often only clearly visible following rains.

Cyanobacterial tolerance to stockpiling
Physical disturbance of biocrusts occurs on a large scale at the J-A mine site with the removal and temporary stockpile 15 storage of topsoil. This type of mechanical disturbance results in the burial and translocation of the biocrust. The impacts of burial within the natural environment are rarely studied. In China, artificial sand burial at shallow depths showed there were significant reductions in chlorophyll concentration, UV synthesis, total carbohydrates (EPS) and damage to photosynthetic activity (Rao et al., 2012). In a semi-arid grassland in Australia, wind-borne sand burial of cyanobacterial crusts during a severe drought resulted in a significant reduction in surface dwelling cyanobacteria and significant reductions in biological-20 available nitrogen (Williams and Eldridge, 2011).
Here we have shown that without further disturbance, a proportion of cyanobacteria can survive stockpiling for over two years. This is not surprising due to the recognised ability of cyanobacteria to survive in extreme environments. In previous studies, cyanobacteria have been grown from samples sourced at 18 cm depths in Japanese rice paddy soils (Fujita and Nakahara, 2006), 50 cm in the UK (Esmarch, 1914), and 70 cm depths in the USA (Moore and Karrer, 1919). Yet, the 25 cyanobacteria from the surface depths of the stockpiles (i.e. <10 cm) appeared more resilient in the short-term.
The species sampled at J-A have a proven track record of survival under extreme conditions. Microcoleus and Leptolyngbya have survived and remained viable after up to three million years frozen in lake sediments in permafrost (Vishnivetskaya et al., 2003). Vegetative Nostoc commune material retains viability following several decades of storage in desiccated form (Bristol, 1919;Lipman, 1941). Reactivation of vegetative material after decades of storage was successful but several reflected in the current study where growth was not observed in the undisturbed areas below 10 cm depth for several months.
It may be that the longer the period of inactivity, the longer time taken for reactivation to occur (Billi and Potts, 2002;Williams et al., 2014), or less material is viable therefore it takes longer to rebuild colonies (Agrawal and Singh, 2002).
Akinetes are desiccation resistant cells produced by certain filamentous cyanobacteria that can survive for long periods.
Nostoc and Scytonema produce akinetes (Kaplan-Levy et al., 2010;Tomaselli and Giovannetti, 1993) but many of the other 5 species sampled in this study cannot, therefore alternative survival methods are in action. Heterotrophic growth is also possible for some cyanobacteria (Flores and Herrero, 2010). Cyanobacteria can survive in darkness through utilisation of alternate carbon sources in drinking water systems (Codony et al., 2003) and this may also be true for soil cyanobacteria (Reisser, 2007). Nostoc have the potential to grow at low light in caves and under ice (Dodds et al., 1995) and even in darkness (Huang et al., 1988). Belnap and Gardner, (1993) reported Microcoleus vaginatus sheaths at depths to 10 cm and 10 considered the sheaths to be remnant from a time when the surface was lower than the current day due to a lack of chlorophyll. It is possible that heterotrophic growth was still occurring at these depths for which chlorophyll is unnecessary.
The species richness in taxa at depths in undisturbed areas was like that of surface samples yet with much slower growth.
The fact that these organisms took much longer to grow than those sampled from upper layers would suggest that they have grown from vegetative material that has been photosynthetically inactive for long periods. Long term inactivity of vegetative 15 material can result in long lag times for growth following re-activation (Bristol, 1919;Lipman, 1941;Shaw et al., 2003) and this was observed in species sourced from depths that are incapable of akinete production.
In the context of rehabilitation, it is not practical to store stockpiles at very shallow depths, however in the longer term under optimum conditions a diverse range of cyanobacteria did recover, indicating the depth of burial is an important feature of 20 recovery over time. Gradual removal of the topsoil from the stockpiles at shallow depths may facilitate and provide a greater opportunity for microbial recovery. The timing of topsoil amendments would be crucial in terms of moisture availability for the initial cyanobacterial resurrection and to facilitate enough growth to stabilise the newly laid soil surfaces.

Conclusions 25
Biocrusts and cyanobacteria are a major component of the J-A landscape that protect and enhance soil function. These studies focused on the cyanobacterial community structure at J-A and its recovery following topsoil stockpiling. It was apparent that the top few centimetres of the stockpiles were the most responsive to cyanobacterial regeneration. Detecting increases in key species and shifts of community structure will likely provide more informative and robust verification of desired rehabilitation outcomes. Cyanobacterial species richness is an important measure of biocrusts that incorporate micro-30 processes central to a healthy and functional soil ecosystem. Increased cyanobacterial biomass is likely to also be a good indicator and reliable metric for biocrust reestablishment. Yet, diversity indices derived from sequencing data of the whole bacterial community are poor measures of biocrust formation and development.
Cyanobacteria are well adapted to long periods without water, optimisation of short growing seasons, wet-dry cycles, low water potentials, tolerance of high UV and low light intensities, fluctuating temperatures and in marine type habitats, high 5 salinity. Cyanobacterial strategies central to survival include EPS production, spectral adaptation, nitrogen fixation and motility. Biocrust re-establishment during mining rehabilitation relies on the role of cyanobacteria as a means of early soil stabilisation. Provided there is adequate cyanobacterial inoculum in the topsoil stockpiles their growth and the subsequent crust formation should take place largely unassisted. Nevertheless, even from low-profile stockpiles re-establishment of biocrusts would be dependent on rainfall. It follows that the timing of rehabilitation would be important so as to take 10 advantage of favourable climatic conditions. At J-A microbial biobanks have been created through shallow scraping of topsoil, low-profile topsoil stockpiles and direct-return that facilitate the survival of cyanobacteria we studied. Ongoing monitoring of biocrust recovery is important as it provides an effective means of measuring important soil restoration processes.      Figure 1: Image of biocrust sample sites located within the vegetation associations described in Table S1 (supplied, S. Doudle). Site 11 was initially investigated but later discarded as it was a fourth replicate of the Chenopod Shrubland.  Symploca embedded into EPS that provides a gelatinous and sticky biofilm to create the basis of the biocrust; (c) Schizothrix contained in thick sheaths; (d) biocrusts were typically 1-10 mm thick and could be easily removed in pieces from the soil, white arrow points to tiny black cyanobacterial colony with UV protection in amongst green colonies; (e) gelatinous EPS 5 covering cyanobacterial colonies; (f) a probe is used to lift cyanobacterial crust that is held together by surface and subsurface network of filaments.