Storage and Distribution of Organic Carbon and Nutrients in Acidic Soils Developed on Sulfidic Sediments: The Roles of Reactive Iron and Macropores.

In a boreal acidic sulfate-rich subsoil (pH 3-4) developing on sulfidic and organic-rich sediments over the past 70 years, extensive brownish-to-yellowish layers have formed on macropores. Our data reveal that these layers ("macropore surfaces") are strongly enriched in 1 M HCl-extractable reactive iron (2-7% dry weight), largely bound to schwertmannite and 2-line ferrihydrite. These reactive iron phases trap large pools of labile organic matter (OM) and HCl-extractable phosphorus, possibly derived from the cultivated layer. Within soil aggregates, the OM is of a different nature from that on the macropore surfaces but similar to that in the underlying sulfidic sediments (C-horizon). This provides evidence that the sedimentary OM in the bulk subsoil has been largely preserved without significant decomposition and/or fractionation, likely due to physiochemical stabilization by the reactive iron phases that also existed abundantly within the aggregates. These findings not only highlight the important yet underappreciated roles of iron oxyhydroxysulfates in OM/nutrient storage and distribution in acidic sulfate-rich and other similar environments but also suggest that boreal acidic sulfate-rich subsoils and other similar soil systems (existing widely on coastal plains worldwide and being increasingly formed in thawing permafrost) may act as global sinks for OM and nutrients in the short run.

Iron­sulfur geochemistry and acidity retention in hydrologically active macropores of boreal acid sulfate soils: Effects of mitigation suspensions of fine-grained calcite and peat.

Acid sulfate soils discharge large amounts of sulfuric acid along with toxic metals, deteriorating water quality and ecosystem health of recipient waterbodies. There is thus an urgent need to develop cost-effective and sustainable measures to mitigate the negative effects of these soils. In this study, we flushed aseptically-prepared MQ water (reference) or mitigation suspensions containing calcite, peat or a combination of both through 15-cm-thick soil cores from an acid sulfate soil field in western Finland, and investigated the geochemistry of Fe and S on the surfaces of macropores and in the solid columnar blocks (interiors) of the soil columns. The macropore surfaces of all soil columns were strongly enriched in total and HCl-extractable Fe and S relative to the interiors, owing to the existence of abundant Fe oxyhydroxysulfates (schwertmannite and partly jarosite) as yellow-to-brownish surface-coatings. The dissolution/hydrolysis of Fe oxyhydroxysulfates (predominantly jarosite) on the macropore surfaces of the reference columns, although being constantly flushed, effectively buffered the permeates at pH close to 4. These results suggest that Fe oxyhydroxysulfates accumulated on the macropore surfaces of boreal acid sulfate soils can act as long-lasting acidification sources. The treatments with mitigation suspensions led to a (near-)complete conversion of jarosite to Fe hydroxides, causing a substantial loss of S. In contrast, we did not observe any recognizable evidence indicating transformation of schwertmannite. However, sulfate sorbed by this mineral might be partially lost through anion-exchange processes during the treatments with calcite. No Fe sulfides were found in the peat-treated columns. Since Fe sulfides can support renewed acidification events, the moderate mineralogical changes induced by peat are desirable. In addition, peat materials can act as toxic-metal scavengers. Thus, the peat materials used here, which is relatively cheap in the boreal zone, is ideal for remediating boreal acid sulfate soils and other similar jarosite-bearing soils.

Dredging and deposition of metal sulfide rich river sediments results in rapid conversion to acid sulfate soil materials.

Sediments along the Baltic Sea coast can contain considerable amounts of metal sulfides that if dredged and the spoils deposited such that they are exposed to air, can release high concentrations of acid and toxic metals into recipient water bodies. Two river estuaries in western Finland were dredged from 2013 to 2018 and the dredge spoils were deposited on land previously covered with agricultural limestone to buffer the pH and mitigate acid and metal release. In this study, the geochemistry and 16S rRNA gene amplicon based bacterial communities were investigated over time to explore whether the application of lime prevented a conversion of the dredge spoils into acid producing and metal releasing soil. The pH of the dredge spoils decreased with time indicating metal sulfide oxidation and resulted in elevated sulfate concentrations along with a concomitant release of metals. However, calculations indicated only approximately 5% of the added lime had been dissolved. The bacterial communities decreased in diversity with the lowering of the pH as taxa most similar to extremely acidophilic sulfur, and in some cases iron, oxidizing Acidithiobacillus species became the dominant characterized genus in the deposited dredge spoils as the oxidation front moved deeper. In addition, other taxa characterized as involved in oxidation of iron or sulfur were identified including Gallionella, Sulfuricurvum, and Sulfurimonas. These data suggest there was a rapid conversion of the dredge spoils to severely acidic soil similar to actual acid sulfate soil and that the lime placed on the land prior to deposition of the spoils, and later ploughed into the dry dredge spoils, was insufficient to halt this process. Hence, future dredging and deposition of dredge spoils containing metal sulfides should not only take into account the amount of lime used for buffering but also its grain size and mixing into the soil.

Metagenomes and metatranscriptomes from boreal potential and actual acid sulfate soil materials.

Natural sulfide rich deposits are common in coastal areas worldwide, including along the Baltic Sea coast. When artificial drainage exposes these deposits to atmospheric oxygen, iron sulfide minerals in the soils are rapidly oxidized. This process turns the potential acid sulfate soils into actual acid sulfate soils and mobilizes large quantities of acidity and leachable toxic metals that cause severe environmental problems. It is known that acidophilic microorganisms living in acid sulfate soils catalyze iron sulfide mineral oxidation. However, only a few studies regarding these communities have been published. In this study, we sampled the oxidized actual acid sulfate soil, the transition zone where oxidation is actively taking place, and the deepest un-oxidized potential acid sulfate soil. Nucleic acids were extracted and 16S rRNA gene amplicons, metagenomes, and metatranscriptomes generated to gain a detailed insight into the communities and their activities. The project will be of great use to microbiologists, environmental biologists, geochemists, and geologists as there is hydrological and geochemical monitoring from the site stretching back for many years.

Comparison of boreal acid sulfate soil microbial communities in oxidative and reductive environments.

Due to land uplift after the last ice age, previously stable Baltic Sea sulfidic sediments are becoming dry land. When these sediments are drained, the sulfide minerals are exposed to air and can release large amounts of metals and acid into the environment. This can cause severe ecological damage such as fish kills in rivers feeding the northern Baltic Sea. In this study, five sites were investigated for the occurrence of acid sulfate soils and their geochemistry and microbiology was identified. The pH and soil chemistry identified three of the areas as having classical acid sulfate soil characteristics and culture independent identification of 16S rRNA genes identified populations related to acidophilic bacteria capable of catalyzing sulfidic mineral dissolution, including species likely adapted to low temperature. These results were compared to an acid sulfate soil area that had been flooded for ten years and showed that the previously oxidized sulfidic materials had an increased pH compared to the unremediated oxidized layers. In addition, the microbiology of the flooded soil had changed such that alkalinity producing ferric and sulfate reducing reactions had likely occurred. This suggested that flooding of acid sulfate soils mitigates their environmental impact.

Indirect DNA extraction method suitable for acidic soil with high clay content.

DNA extraction is an essential procedure when investigating microbial communities in environmental samples by sequencing technologies. High clay soils can be problematic as DNA adsorbs to the clay particles and can thereby be preserved from lysed, non-viable cells for a substantial period of time. In order to accurately estimate the intact and living microbial community in the soil, extracellular DNA from dead, remnant bacterial cells needs to be removed prior to DNA extraction. One possibility is to use a sodium phosphate buffer to release both extracellular DNA and bacterial cells from the clay particles. After removing the extracellular DNA by centrifugation, the remaining viable cells can be harvested and DNA extracted. The described method is a modification of a procedure for separating extracellular DNA and bacterial cells from acidic clay soils. •The modified method increases bacterial cell yields from acidic clay soils, such as acid sulfate soil.•The modified method eliminates some steps from the original method, as only DNA from intact bacterial cells is required.•The indirect DNA extraction method increases the workload compared to standard direct extraction methods, but subsequent downstream analyses will give a more representative picture of the viable microbial community composition in the soil.

Chemical and microbiological evaluation of novel chemical treatment methods for acid sulfate soils.

Naturally occurring sulfide rich deposits are common along the northern Baltic Sea coast that when exposed to air, release large amounts of acid and metals into receiving water bodies. This causes severe environmental implications for agriculture, forestry, and building of infrastructure. In this study, we investigated the efficiency of ultrafine-grained calcium carbonate and peat (both separately and in combination) to mitigate acid and metal release. The experiments were carried out aerobically that mimicked summer conditions when the groundwater level is low and acid sulfate soils are exposed to oxygen, and anaerobically that is similar to autumn to spring conditions. The ultrafine-grained calcium carbonate dissipated well in the soil and its effect alone and when mixed with peat raised the pH and reduced pyrite dissolution while peat alone was similar to the controls and did not halt metal and acid release. High throughput 16S rRNA gene sequencing identified populations most similar to characterized acidophiles in the control and peat treated incubations while the acidophilic like populations were altered in the calcium carbonate alone and calcium carbonate plus peat treated acid sulfate soils. Coupled with the geochemistry data, it was suggested that the acidophiles were inactivated by the high pH in the presence of calcium carbonate but catalyzed pyrite dissolution in the controls and peat incubations. In conclusion, the anaerobic conditions during winter would likely be sufficient to mitigate acid production and metal release from acid sulfate soils and in the summer, treatment with calcium carbonate was the best mitigation method.

In vitro opsonin-independent interactions between cells of smooth and rough phenotypes of Flavobacterium psychrophilum and rainbow trout (Oncorhynchus mykiss) head kidney macrophages.

The cytotoxic activity of smooth and rough phenotypic cells of the fish pathogenic Gram-negative bacterium Flavobacterium psychrophilum to rainbow trout (Oncorhynchus mykiss) head kidney macrophages was investigated in vitro. The cytotoxicity to macrophages was significantly higher for rough cells compared with the smooth cells. The cytotoxic activity increased for both cell types with increasing temperature and the cells retained their cytotoxic nature after metabolical inactivation by heat, suggesting a cell-bound cytotoxic mechanism. The cytotoxicity was significantly reduced in both cell types after treatment with sodium (meta)periodate, indicating that the major bacterial structure involved in the cytotoxicity is of carbohydrate nature. Trypsin treatment further reduced the cytotoxicity in smooth cells, while sialic acid treatment reduced the cytotoxicity in rough cells, suggesting different lysing mechanisms for the two phenotypic variants. The results from the present study therefore suggest that the cytotoxic activity of F. psychrophilum to rainbow trout macrophages in vitro is stronger expressed in the rough phenotype and that it is opsonin-independent and initiated by binding of bacterial surface carbohydrates to lectins on the surface of the macrophages. How the lysis of the macrophages is executed is still unclear but it is suggested to function by different mechanisms in the smooth and the rough cells. The migration of rainbow trout macrophages toward smooth and rough cells of F. psychrophilum was further investigated. The results show that the macrophages were able to recognize both cell types, but the migration rate did not differ between the two phenotypes.

Hemolytic activity in Flavobacterium psychrophilum is a contact-dependent, two-step mechanism and differently expressed in smooth and rough phenotypes.

The hemolytic activity of cells of smooth and rough phenotypic variants of the Gram-negative fish pathogen Flavobacterium psychrophilum was investigated in two different assays, a microplate and an agarose hemolysis assay, using rainbow trout erythrocytes. The smooth cells showed a high and the rough cells a negligible, concentration dependent, hemolytic activity in the microplate assay. Both smooth and rough cells showed a rather weak hemolytic activity, with two distinct hemolytic patterns, in the agarose assay. The hemolytic activity of the cells was not regulated by iron availability and cell-free extracellular products did not show any hemolytic activity. The smooth cells, in contrast to the rough cells, showed a high ability to agglutinate erythrocytes and both hemagglutination and hemolytic activity was impaired by treatment of the cells with sialic acid. The hemolytic activity was furthermore reduced after proteolytic and heat treatment of the cells. The results from the present study suggest that the hemolytic activity in F. psychrophilum is highly expressed in the smooth phenotype, and that it is a contact-dependent and two-step mechanism that is initiated by the binding of the bacterial cells to the erythrocytes through sialic acid-binding lectins and then executed by thermolabile proteinaceous hemolysins.

Phase variation in Flavobacterium psychrophilum: characterization of two distinct colony phenotypes.

Four 'smooth' and 4 'rough' colony phenotypes of the Gram-negative fish pathogen Flavobacterium psychrophilum isolated from rainbow trout Oncorhynchus mykiss were characterized using biochemical, physiological, molecular and virulence tests to better understand the pathogenesis of the bacterium. Biochemically, the 2 cell types did not react significantly differently. Physiologically, the 2 phenotypes had distinct characteristics, and, when grown in broth, the smooth cells were found to be autoagglutinating and able to switch into the non-agglutinating rough phenotype. The rough cells did not switch into the smooth phenotype under any growth conditions tested, indicating that the phase variation from the smooth to rough phenotype is irreversible or that the conditions for the reversible switch are still to be found. Smooth cells were hydrophobic and more adhesive compared to the hydrophilic rough cells, suggesting that the phase variation most probably involves one or several surface structures other than outer membrane proteins and lipopolysaccharides that were found to be similar in both types. Analysis of extracellular products produced by the 2 cell types indicated furthermore that a difference in enzymatic activities could exist. Both cell types were virulent for rainbow trout in an intramuscular challenge; thus, the distinct physiological characteristics of the phenotypes do not seem to be directly associated with virulence, when the body surface of the fish is disregarded. The results suggest that phase variation occurs in F. psychrophilum, but that the importance of the 2 phenotypes for the pathogenesis of the bacterium has still to be investigated.