Guillaume Cailleau

Fungi in Biogeochemical Cycles: The oxalate–carbonate pathway in soil carbon storage: the role of fungi and oxalotrophic bacteria

Introduction Although fungi are generally disregarded in the biogeochemical literature, they undoubtedly constitute crucial biogeochemical factors in many elemental cycles. This fact, combined with their abundance in the soil warrants greater detailed study into their geoecological impact. The network formed by fungal filaments can represent 10 000 km of thread-like mycelia in 1m of fertile soil. Their mass is evaluated at 3500 kg ha 1 at a depth of 20 cm in an average continental soil, i.e. taking into account all the different terrestrial environments on the Earth (Gobat et al., 2004). In comparison, bacteria and algae would represent 1500 and 10–1000kgha 1 respectively, in the same virtual average soil. Fungi are not only biologically important as saprophytes in the recycling of organic matter, but also play a geological role by excreting notable amounts of organic acids, among which oxalic acid is particularly important (Gadd, 1999), contributing to continental weathering as well as to mineral neogenesis (Verrecchia & Dumont, 1996; Verrecchia, 2000; Burford et al., 2003 a, b). The first fossil fungi have been identified in rocks dated from the Ordovician, i.e. 460 to 455Ma ago (Redecker et al., 2000). However, molecular clock estimates for the evolution of fungi have suggested a Late Precambrian (600Ma) colonization on land (Berbee & Taylor 2000). Recent molecular studies, based on protein sequence analysis, indicate that fungi were present on continents 1 billion years ago and possibly affected (together with plants) the evolution of Earth’s atmosphere and climate since 700Ma (Heckman et al., 2001). Therefore, if fungi have been present on the Earth’s surface for such a long time, producing large amounts of oxalic acid able to precipitate as metal oxalates, why is there no evidence of oxalate accumulation in paleosols?

Fungi, bacteria and soil pH: the oxalate-carbonate pathway as a model for metabolic interaction.

The oxalate-carbonate pathway involves the oxidation of calcium oxalate to low-magnesium calcite and represents a potential long-term terrestrial sink for atmospheric CO(2). In this pathway, bacterial oxalate degradation is associated with a strong local alkalinization and subsequent carbonate precipitation. In order to test whether this process occurs in soil, the role of bacteria, fungi and calcium oxalate amendments was studied using microcosms. In a model system with sterile soil amended with laboratory cultures of oxalotrophic bacteria and fungi, the addition of calcium oxalate induced a distinct pH shift and led to the final precipitation of calcite. However, the simultaneous presence of bacteria and fungi was essential to drive this pH shift. Growth of both oxalotrophic bacteria and fungi was confirmed by qPCR on the frc (oxalotrophic bacteria) and 16S rRNA genes, and the quantification of ergosterol (active fungal biomass) respectively. The experiment was replicated in microcosms with non-sterilized soil. In this case, the bacterial and fungal contribution to oxalate degradation was evaluated by treatments with specific biocides (cycloheximide and bronopol). Results showed that the autochthonous microflora oxidized calcium oxalate and induced a significant soil alkalinization. Moreover, data confirmed the results from the model soil showing that bacteria are essentially responsible for the pH shift, but require the presence of fungi for their oxalotrophic activity. The combined results highlight that the interaction between bacteria and fungi is essential to drive metabolic processes in complex environments such as soil.

An Ultrastructural Approach to Analogies between Fungal Structures and Needle Fiber Calcite

Needle fiber calcite (NFC) is an ubiquitous terrestrial secondary calcium carbonate mineral often associated with calcitic nanofibers. NFCs origin has been debated for a long time and a fungal origin is often proposed. Fungi are known to be involved in mineral weathering and production of metal oxalate, but little information exists regarding the genesis of other minerals, such as calcite. In this study, a comparison of similar ultrastructural characteristics of fungal hyphae and NFC has been performed to highlight analogies between both features. These analogies clearly demonstrate the probable close relationship between fungal filaments (hyphae and rhizomorphs) and NFC and its associated nanofibers.

Calcitic nanofibres in soils and caves: a putative fungal contribution to carbonatogenesis

Abstract The origin of soil mineralized nanofibres remains controversial. It is attributed to either biogenic factors or physicochemical processes. Scanning electron microscope and transmission electron microscope observations show that nanofibres could originate from the breakdown of fungal hyphae, especially its cell wall. It is hypothesized that during the decay of organic matter, cell wall microfibrils are released in the soil where they are exposed to mineralizing pore fluids, leading to their calcitic pseudomorphosis and/or are used as a template for calcite precipitation. When associated with needle fibre calcite bundles, nanofibres could indicate the relict of an organic sheath in which calcite has precipitated. This paper emphasizes the important roles of both organic matter and fungi in carbonatogenesis, and consequently in the soil carbon cycle.

MICROBIOLOGICAL ACTIVITIES IN MOONMILK MONITORED USING ISOTHERMAL MICROCALORIMETRY (CAVE OF VERS CHEZ LE BRANDT, NEUCHATEL, SWITZERLAND)

Studies of the influence of microbial communities on calcium carbonate deposits mostly rely on classical or molecular microbiology, isotopic analyses, and microscopy. Using these techniques, it is difficult to infer microbial activities in such deposits. In this context, we used isothermal microcalorimetry, a sensitive and non- destructive tool, to measure microbial activities associated with moonmilk ex-situ. Upon the addition of diluted LB medium and other carbon sources to fresh moonmilk samples, we estimated the number of colony forming units per gram of moonmilk to be 4.8 3 10 5 6 0.2 3 10 5 . This number was close to the classical plate counts, but one cannot assume that all active cells producing metabolic heat were culturable. Using a similar approach, we estimated the overall growth rate and generation time of the microbial community associated with the moonmilk upon addition of various carbon sources. The range of apparent growth rates of the chemoheterotrophic microbial community observed was between 0.025 and 0.067 h 21 and generation times were between 10 and 27 hours. The highest growth rates were observed for citrate and diluted LB medium, while the highest carbon-source consumption rates were observed for low molecular weight organic acids (oxalate and acetate) and glycerol. Considering the rapid degradation of organic acids, glucose, and other carbon sources observed in the moonmilk, it is obvious that upon addition of nutrients during snow melting or rainfall these communities can have high overall activities comparable to those observed in some soils. Such communities can influence the physico-chemical conditions and participate directly or indirectly to the formation of moonmilk.

Reliability of stable carbon and oxygen isotope compositions of pedogenic needle fibre calcite as environmental indicators: examples from Western Europe.

Stable carbon and oxygen isotope analyses were conducted on pedogenic needle fibre calcite (NFC) from seven sites in areas with roughly similar temperate climates in Western Europe, including the Swiss Jura Mountains, eastern and southern France, northern Wales, and north-eastern Spain. The δ13C values (−12.5 to−6.8 ‰ Vienna Pee Dee Belemnite (VPDB)) record the predominant C3 vegetation cover at the sites. A good correlation was found between mean monthly climatic parameters (air temperature, number of frost days, humidity, and precipitation) and δ18O values (−7.8 to−3.4‰ VPDB) of all the NFC. Similar seasonal variations of δ18O values for monthly NFC samples from the Swiss sites and those of mean monthly δ18O values of local precipitation and meteorological data point out precipitation and preferential growth/or recrystallisation of the pedogenic needle calcite during dry seasons. These covariations indicate the potential of stable isotope compositions of preserved NFC in fossil soil horizons as a promising tool for palaeoenvironmental reconstructions.

Identification of active oxalotrophic bacteria by Bromodeoxyuridine DNA labeling in a microcosm soil experiments

The oxalate-carbonate pathway (OCP) leads to a potential carbon sink in terrestrial environments. This process is linked to the activity of oxalotrophic bacteria. Although isolation and molecular characterizations are used to study oxalotrophic bacteria, these approaches do not give information on the active oxalotrophs present in soil undergoing the OCP. The aim of this study was to assess the diversity of active oxalotrophic bacteria in soil microcosms using the Bromodeoxyuridine (BrdU) DNA labeling technique. Soil was collected near an oxalogenic tree (Milicia excelsa). Different concentrations of calcium oxalate (0.5%, 1%, and 4% w/w) were added to the soil microcosms and compared with an untreated control. After 12 days of incubation, a maximal pH of 7.7 was measured for microcosms with oxalate (initial pH 6.4). At this time point, a DGGE profile of the frc gene was performed from BrdU-labeled soil DNA and unlabeled soil DNA. Actinobacteria (Streptomyces- and Kribbella-like sequences), Gammaproteobacteria and Betaproteobacteria were found as the main active oxalotrophic bacterial groups. This study highlights the relevance of Actinobacteria as members of the active bacterial community and the identification of novel uncultured oxalotrophic groups (i.e. Kribbella) active in soils.