The microbiology of acid sulfate soils and sulfidic sediments

Acid sulfate soils and their associated sulfidic sediments present a major hazard to sustainable farming, water security and urban infrastructure. Traditionally these soils are limed in order to neutralise the ‘leachate’ that is a public health hazard and toxic to aquatic organisms. It may be more sustainable to exploit the soil microorganisms capable of sequestering metals to remediate these soils. Until recently, little was known about the microbial ecology of these environments. The soils have a moderately acidic (pH 4) chemistry and a unique ecosystem where the microbial community composition is correlated to bioaccessible metal concentrations. These environments have the potential to provide novel insights into how environmental conditions shape the microbiome that can be exploited for biotechnologies.

Worldwide, the pressure for land to meet food security and urban development needs can lead to the disturbance of waterlogged soils containing iron-sulfide minerals. These minerals are oxidised to form acid sulfate soils (Figure 1); a toxic legacy of rain-induced acidic and metalliferous groundwaters that lead to the corrosion of infrastructure and the severe deterioration of water quality. The associated formation of monosulfides in affected waterways is equally problematic, capable of de-oxygenating the aquatic ecosystem in seconds if disturbed.

Figure 1. Actual acid sulfate soils and associated sulfidic drain sediments.

Current management and treatment practices of acid sulfate soils include minimising the formation of acidity; neutralising acidity and/or turning land back to wetlands. The ongoing cost of these disturbed environments in Queensland alone is estimated at $180 million per year¹.

Up until recently, little was known about the microbial community in coastal acid sulfate soils. Nonetheless; rates of mineral transformation² and mineral transformation products^3,4 cannot be fully explained by abiotic processes, indicating that soil bacteria play an essential, but unknown role in element cycling in this environment. Furthermore, under biotic, redox cycling conditions, the rapid (days to months) transformation of meta-stable minerals to stable minerals, which concomitantly sequesters trace contaminants could be possible³. Thus, understanding the microbial ecology, and particularly the identification of bacteria capable of iron-sulphur transformations, is an essential step towards exploiting the microbial community to remediate these environments.

The microbial ecology was determined using pyrosequencing from samples collected from the model coastal acid sulfate soil site at Blacks Drain, Tweed Valley, NSW, Australia. The sampling strategy is fully detailed elsewhere⁵. This site is considered to be a model setting as it is a pollution hotspot (contaminating adjacent waterways with acidity, Al, Mn and Fe) has undergone detailed geochemical characterisations of the soils and sediments over the past decade⁵.

The acid sulfate soils and sulfidic drain sediments contained an average 186 phylotypes per sample in comparison to non-pyritic soils, which contain 5-fold higher ecosystem complexity, with an average 1,017 phylotypes per sample⁶. Ecosystem complexity is controlled by selection pressures that reduce species diversity⁷. Acid sulfate soils contain a number of selection pressures associated with the seasonal oxidation of the unstable iron minerals; however, in comparison to acid mine drainage environments (pH 2–4) that contain an average of 61 phylotypes per sample⁸, these soils have a 3-fold higher ecosystem complexity.

A total of 23 phyla were identified, of which five phyla dominate (>90%) the community composition, with four common to all environments (Proteobacteria, Acidobacteria, Firmicutes and Chloroflexi), and Bacteriodetes as a major component of sulfidic sediments and Actinobacteria a major component of acid sulfate soil field horizons.

The subdivision of the phyla totalled 48 classes. Focusing on the dominant phyla, drain sediments had two-fold higher abundance of Proteobacteria; and were dominated by δ-proteobacteria. All soil horizons contained δ-proteobacteria but were dominated by β-proteobacteria and α-proteobacteria. Splitting the phylum Chloroflexi into classes revealed that drain sediments were dominated by Anaerolineae in comparison to the soils that were dominated by the Chloroflexi class. The phylum Acidobacteria, also showed that the drain sediments were dominated by the Acidobacteria class and soil horizons dominated by Holophagae. The phylum Firmicutes was dominated by the classes Bacilli and Clostridia across both settings.

Interestingly, there was a low abundance of the class Nitrospira, a group that contains acidophilic Fe(II) oxidising bacteria (Leptospirillum sp.). This is the most important phylotype in acid mine biofilms⁷; is moderately abundant (average 12%) in acid mine drainage sites⁸, but has a low abundance in acid sulfate soils at <3%. The abundance of this group may be determined by pH due to their limited metabolic capacity (derive energy solely from Fe(II) oxidation) thus are adapted to extremely acidic environments (pH <4)⁸ where competing abiotic Fe(II) oxidation kinetics are very slow.

A patchy species distribution and a high proportion of unclassified bacteria characterised acid sulfate soils. Interestingly, these soils lacked an abundance of known acid tolerant Fe(II) oxidisers, of which there are 22 known acidophilic Fe(II) oxidising species across four phyla⁹. Only the Firmicutes, Alicyclobacillus tolerans was detected in these soils. Instead, the microbial community associated with Fe(II) oxidation was the Betaproteobacteria, Sideroxydans lithotrophicus and Sideroxydans paludicola, with a known ability for Fe(II) oxidation over neutrophilic pH range (4–7.5) under micro-aerobic conditions. Another interesting finding was that Chloroflexus was highly abundant in the acid sulfate soil horizons. Little is known about the role of Chloroflexus in iron cycling, but research from microbial mat zones indicate a positive relationship to zones of enriched Fe(II) oxidation¹⁰ and the fully sequenced Chloroflexus aurantiacus contains a candidate Fe(II) trafficking protein (ATCC strain 29364).

Iron reduction is a ubiquitous microbiological mechanism¹¹; but only the neutrophile Anaeromyxobacter dehalogenans was abundant in the acid sulfate soil horizon. Substrate based studies offer the potential to discover the largely unknown Fe(III) reducing the community in these soils as carbon substrates differ between acidophiles and neutrophiles. For example, acetate is key to Geobacter sp.¹²; however, acetate inhibits Fe(III) reduction in acidic conditions and sugars are a key substrate that stimulates Fe(III) reduction by acidophiles¹³.

Few S-cycling bacteria were found in the acid sulfate soils; furthermore, considering there are 220 bacterial species known to be involved in sulfate reduction¹⁴, it was surprising that no sulfate-reducing species were identified at this taxonomic level. This suggests that there is a significant repository of unknown S-cycling bacteria present in these environments.

Unlike the acid sulfate soils, the sulfidic sediments were reasonably well resolved and a conceptual model of microbially mediated iron cycling and sulfate reduction in the sulfidic drain sediments was developed (Figure 2).

Figure 2. Biogeochemistry of sulfidic drain sediments impacted by acid sulfate soil leachates.

The sulfidic drain sediments contained an abundance of Fe(II) oxidising bacteria, for example, Ferrovum. sp; that oxidises Fe(II) aerobically using CO₂. Further, there was an abundance of microbes with capabilities to reduce Fe(III) in the sulfidic sediments which included the acidophile Acidobacterium capsulatum that reduces Fe(III) between pH 2–5, and the neutrophiles Anaeromyxobacter dehalogenans and Geobacter sp. The sulfidic drain sediments contained an abundance of S-compound oxidising bacteria, dominated by Thiomonas delicata. However, similar to the soils, no sulfate reducing species were detected, suggesting that there are many unknown S-cycling bacteria in these environments.

Acid sulfate soil and sulfidic sediments contain highly elevated bioavailable metal(loid) concentrations that are taken up by plants and can be used as biomonitors to isolate pollution hotspots¹⁵. A suite of metal(loid)s (Al, As, Cr, Co, Cu, Fe, Mn, Ni and Zn) were positively correlated to genera abundance (Figure 3), indicating that generally genera in acid sulfate environments have high tolerance capabilities to high metal(loid) bioaccessibility, which shape microbial community composition.

Figure 3. Correlations between genera abundance and bioaccessible metal(loid)s including (a) cobalt, (b) nickel, (c) copper and (d) arsenic.

A key finding was the abundance characteristics of the Acidobacteria and Crenarcheota to bioaccessible Manganese concentrations. Recently, these phyla were found to be enriched in Mn-stimulated microcosms and linked to Mn-dependent organic compound oxidation¹⁶. Thus, this demonstrates the need for high phylogenetic resolution to investigate environmental factors; because only genus level resolution identified this relationship to soil chemistry.

Understanding the biogeochemistry of acid sulfate soils and sulfidic sediments would transform the management and remediation of these environmentally deleterious sites. The microbial ecology has few parallels to geochemically similar environments, with a microbial community composition including both acidophiles and neutrophiles associated with iron and sulphur cycling. Furthermore, acid sulfate soils and sulfidic sediments house a repository of uncharacterised microbes but abundance patterns correlated to soil chemistry. Taken together, acid sulfate soils may be a model environment that can be used to understand the role environmental conditions play on microbial community compositions, and this information could be used to develop novel biotechnologies.