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Data for the publication "The role of extracellular polymeric substances of fungal biofilms in mineral attachment and weathering" (https://doi.org/10.1038/s41529-022-00253-1). It includes:
- The Summary of the EPS concentration, EPS sugar components and EPS linkages.
- The Summary of the XPS analysis of freeze-dried biofilm samples of all strains.
- The Summary of the pH, Mg, SI and Fe concentration, biomass and olivine dissolution rate for each time point of all dissolution experiments.
The roles extracellular polymeric substances (EPS) play in mineral attachment and weathering were studied using genetically modified biofilms of the rock-inhabiting fungus Knufia petricola strain A95. Mutants deficient in melanin and/or carotenoid synthesis were grown as air-exposed biofilms. Extracted EPS were quantified and characterised using a combination of analytical techniques. The absence of melanin affected the quantity and composition of the produced EPS: mutants no longer able to form melanin synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, the melanin-producing strains attached more strongly to the mineral olivine and dissolved it at a higher rate. We hypothesise that the pullulan-related linkages, with their known adhesion functionality, enable fungal attachment and weathering. The released phenolic intermediates of melanin synthesis in the Δsdh1 mutant might play a role similar to Fe-chelating siderophores, driving olivine dissolution even further. These data demonstrate the need for careful compositional and quantitative analyses of biofilm-created microenvironments.
Cryomyces antarcticus, a melanized cryptoendolithic fungus endemic to Antarctica, can tolerate environmental conditions as severe as those in space. Particularly, its ability to withstand ionizing radiation has been attributed to the presence of thick and highly melanized cell walls, which—according to a previous investigation—may contain both 1,8‐dihydroxynaphthalene (DHN) and L‐3,4 dihydroxyphenylalanine (L‐DOPA) melanin. The genes putatively involved in the synthesis of DHN melanin were identified in the genome of C. antarcticus. Most important is capks1 encoding a non‐reducing polyketide synthase (PKS) and being the ortholog of the functionally characterized kppks1 from the rock‐inhabiting fungus Knufia petricola. The co‐expression of CaPKS1 or KpPKS1 with a 4′‐phosphopantetheinyl transferase in Saccharomyces cerevisiae resulted in the formation of a yellowish pigment, suggesting that CaPKS1 is the enzyme providing the precursor for DHN melanin. To dissect the composition and function of the melanin layer in the outer cell wall of C. antarcticus, non‐melanized mutants were generated by CRISPR/Cas9‐mediated genome editing. Notwithstanding its slow growth (up to months), three independent non‐melanized Δcapks1 mutants were obtained. The mutants exhibited growth similar to the wild type and a light pinkish pigmentation, which is presumably due to carotenoids. Interestingly, visible light had an adverse effect on growth of both melanized wild‐type and non‐melanized Δcapks1 strains. Further evidence that light can pass the melanized cell walls derives from a mutant expressing a H2B‐GFP fusion protein, which can be detected by fluorescence microscopy. In conclusion, the study reports on the first genetic manipulation of C. antarcticus, resulting in non‐melanized mutants and demonstrating that the melanin is rather of the DHN type. These mutants will allow to elucidate the relevance of melanization for surviving extreme conditions found in the natural habitat as well as in space.
This file contains all the data used for the figures shown in the Dissertation of Ruben Gerrits with the title "An experimental study of fungal olivine weathering".
In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes.
Free-living and (ecto)-mycorrhizal fungi enhance rock weathering. In their roles as mineral weathering agents and mutualistic partners of phototrophs, fungi supply primary producers like plants and phototrophic microorganisms with mineral-derived nutrients. The exact mechanisms behind fungus-induced mineral weathering processes are however not well understood. Progress can be achieved here by reproducible experimental simulations of the natural processes, using well-characterised model organisms and minerals.
In this study, the weathering-affecting, rock-inhabiting fungus, Knufia petricola A95 and the Fe-bearing olivine (Fe0.2Mg1.8SiO4) were selected to investigate fungi-induced effects on mineral dissolution. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, that produced more extracellular polymeric substances (EPS) than the wild type (WT), enabled comparative studies of the role of melanin and EPS in weathering processes.
Three experimental systems, which generate long-term microbiological stability, were developed to study the impact of the WT and ΔKppks on olivine weathering: (1) batch and (2) mixed flow dissolution experiments, and (3) biofilm cultivation experiments. In addition, state-of-the-art analytical techniques were used to monitor changes in the growth medium, as well as of the mineral surface and biofilm-mineral interface.
Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis of the Mg, Si and Fe concentrations in the reacted growth medium was used to quantify olivine dissolution. In abiotic controls, Mg and Si dissolved congruently, while Fe precipitated. The measured olivine dissolution rates at pH 6 were two orders of magnitude lower than previously reported, but similar at acidic pH. X-ray photoelectron spectroscopy (XPS) analyses of the olivine surface confirmed the presence of Fe (oxyhydr)oxide precipitates. Transmission electron microscopy (TEM) imaging of an abiotically reacted polished olivine section from the long-term cultivation experiment showed the presence of an amorphous layer enriched in Fe. All these observations indicate that the precipitation of Fe (oxyhydr)oxides on the olivine surface inhibits olivine dissolution.
Both tested rock-inhabiting fungal strains affect Fe precipitation as well as olivine dissolution. Evaluation of the WT and ΔKppks revealed that the WT formed less biomass but could take up higher amounts of metals (e.g. Fe) and was more efficient in its attachment to olivine. The WT and ΔKppks enhanced olivine dissolution as demonstrated by higher Mg and Si concentration in the reacted growth medium. They furthermore prevented Fe precipitation by binding Fe and retaining it in solution, thereby allowing olivine dissolution to proceed. The WT cells that were attached to the olivine surface were particularly efficient at inhibiting Fe precipitation. By binding Fe directly at the olivine surface, the WT cells removed the inhibition of olivine dissolution almost completely. TEM analysis of polished olivine sections, colonised by a fungal biofilm for seven months, supported this hypothesis. After long-term fungus-olivine interaction, the Fe-enriched, amorphous layer did not develop, and the olivine surface was stronger etched compared to the abiotic control.
To study the effect of mutualism on mineral weathering, K. petricola was grown with the cyanobacterium, Nostoc punctiforme ATCC 29133. Both partners showed an enhanced growth and formed a stratified biofilm which attached more strongly to olivine. Nevertheless, the olivine dissolution rate of the fungus-cyanobacterium consortium was moderate.
Rock weathering simulation systems developed here are promising research instruments. The experimental conditions allow for the alteration of the studied mineral surface, while the clear definition of these conditions delivers a stable growth of microorganisms. The latter makes these systems universally applicable, especially in combination with integrative multidisciplinary analytics. Processes underlying environmental and biological effects on rock weathering, metal corrosion, plastic degradation, or the deterioration of any other substrate can be studied reproducibly and over a long period of time. The chemical and biological complexity of these simulation systems mimics natural rock weathering processes. The mineral dissolution rates generated in this study are therefore relevant to natural ecosystems.
In arid sun-exposed environments, rock-inhabiting fungi dominate microbial communities on rock surfaces. Fungi are known to enhance rock weathering, although the exact mechanisms are not completely understood. These natural processes can be simulated by reproducible experimental systems. Particularly attractive are genetically amenable rock-weathering fungi, where certain traits can be knocked-out to identify weathering-relevant functions. Here, we studied the effect of the rock-inhabiting fungus Knufia petricola A95 and its melanin-deficient mutant (ΔKppks) on the weathering of a Fe-bearing olivine. We examined the olivine dissolution kinetics and looked at the olivine-fungus interface. For the former we inoculated olivine powder with the fungus in batch and mixed flow reactors and analysed the medium solution by ICP-OES, for the latter biofilm-covered olivine sections from flow-through reactors were analysed by FIB-TEM.
In general olivine dissolution was lower when olivine was incubated without fungi: the abiotic olivine dissolution rates were lowest and the surface of the abiotically reacted olivine sections showed no etching. Various chemical analyses show Fe (oxyhydr)oxide precipitation on top of the abiotically reacted olivine, indicating its role in inhibiting olivine dissolution. Both the wild type (WT) and ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe (oxyhydr)oxide precipitation was thus reduced, explaining the enhanced olivine dissolution in their presence. Particularly efficient at preventing Fe precipitation were attached WT cells: the inhibition of olivine dissolution almost completely disappeared. The attachment capacity of the WT is most likely caused by the production of WT-specific extracellular polymeric substances. Our presented experimental systems allowed the precipitation of Fe (oxyhydr)oxides and included a rock-inhabiting fungus and thus simulated weathering processes relevant to natural ecosystems.
Rock-inhabiting fungi are known to colonise air-exposed substrates like minerals, photovoltaic panels building facades and monuments, withstanding the various stresses these extreme habitats are known for. Here we show how both colonisation and stress resistance are linked to the properties of the fungal cell surface. By deleting genes involved in the synthesis of melanin and carotenoid pigments of the model rock-inhabiting fungus Knufia petricola via CRISPR-Cas and comparing the behaviour of the gene-deletion mutants with the wild type (WT), we studied the role of these genes in mineral colonisation and stress sensitivity. The extracellular polymeric substances (EPS) of biofilms of the WT and mutants were extracted, quantified and chemically characterised. We observed that the absence of melanin affected the quantity and composition of the produced EPS: melanin-deficient mutants synthesised more EPS containing fewer pullulan-related glycosidic linkages. Moreover, in mineral dissolution experiments, these mutants showed a lower ability to colonise the mineral olivine. We hypothesise that not melanin, but the pullulan-related linkages enabled the melanin-producing strains to attach more strongly to the mineral. Moreover, ICP-OES analysis of the aqueous mineral-derived solutes showed that biofilms of the K. petricola mutants which could attach were able to dissolve the olivine faster than those that could not. The same mutants were also characterised by their sensitivity to desiccation stress: only the mutant deficient in both melanin and carotenoid synthesis was more sensitive to desiccation compared to the WT, indicating that a combination of both pigments is critical to withstand desiccation. Overall, these results show the critical role of the cell surface in the specific capacities of rock-inhabiting fungi.
Iron is arguably the most essential metal in living organisms. For rock-inhabiting fungi its acquisition might be unconventional as they (1) tend to inhabit iron-deficient, oxygen-rich surfaces like marble monuments and solar panels and (2) produce the black, iron-adsorbing pigment melanin. We used a range of analytical methods, ongoing mineral dissolution experiments and gene deletion mutants of the model rock-inhabiting fungus Knufia petricola to figure out the mechanisms and substrate deteriorating effects of iron uptake by these organisms. To study both siderophore-mediated and reductive iron assimilation (RIA), genes like sidC, encoding a putative siderophore synthetase and ftr1 and fet3 encoding the subunits of an iron permease-oxidase were deleted.
At iron deficient conditions, growth of the wild type (WT) and ΔsidC mutant was similar, whereas growth of the Δftr1-fet3 mutant and the double mutant ΔsidC/Δftr1-fet3 was diminished and absent, respectively. We were not able to detect the siderophore of K. petricola and the WT and mutants were not able to grow at low concentrations of strong iron chelators. However, in a cross-feeding experiment, an overexpression strain of sidC allowed more growth of ΔsidC/Δftr1-fet3 on iron deficient medium than the WT, whereas the ΔsidC mutant could not do so at all. Compared to the WT, the sidC overexpression strain also withstood oxidative stress better and had a shorter lag time and higher growth rate. Combined, these results indicate that K. petricola relies more on RIA than siderophore-mediated uptake as it likely excretes low quantities of a primarily intracellular siderophore. Interestingly, Δftr1-fet3 had a higher iron content than the WT at iron deficient conditions. This difference disappeared upon deletion of melanin synthesis (Δpks1 vs. Δpks1/Δftr1-fet3): melanin-bound iron can likely not be used without RIA. K. petricola’s chelation incapacity implies a habitat free of competition for iron while offering us a mitigation strategy.
Melanins are organic pigments produced by most fungi. These organisms either fix these pigments in their cell wall or secrete them into their extracellular environment to protect themselves against an array of physicochemical stresses (e.g., UV irradiation, desiccation, ...). Melanin can adsorb metals like Fe. How this affects fungal uptake of Fe and deterioration of Fe-containing minerals and metals is however less known. To study this, we use the model fungi Knufia petricola A95, a rock-inhabiting fungus known to deteriorate minerals and have melanised cell walls, and Amorphotheca resinae, able to contaminate fuel tanks, secrete melanin and corrode metals. In K. petricola, we have deleted genes involved in melanin production and Fe uptake using CRISPR/Cas. Through comparison of the geochemical signatures of these gene deletion mutants with those of the wild type (WT), we explore the specific mineral/metal deterioration mechanisms of melanised fungi.
Fe isotope signatures of the biomass of melanin- and Fe uptake-deficient mutants of K. petricola revealed that Fe adsorbed either directly onto melanin or after being reduced by Fe reductases. Importantly, once adsorbed to melanin, Fe could not be mobilised and taken up into the cell: both the WT and its melanin-deficient mutant, previously grown at Fe replete conditions, showed similar growth at Fe deficient conditions.
Olivine dissolution experiments revealed that Fe oxidation inhibits dissolution. K. petricola was able to enhance dissolution when this inhibition is strongest (at pH 6) and prevented dissolution when this inhibition is weakest (at pH 4). The fungus therefore dissolves olivine by interacting with the oxidised Fe at the olivine surface. However, Fe uptake did not seem to be involved: mutants deficient in various Fe uptake mechanism dissolved olivine at the same rate as the WT. This indicates that Fe adsorption onto melanin might play a key role. This is also shown by K. petricola’s ability to enhance olivine dissolution even further if secreting a melanin precursor and A. resinae’s corrosion of carbon steel whilst secreting melanin. Combined, our results imply that the Fe adsorbed to melanin cannot be taken up but enables fungi to deteriorate Fe-containing substrates at a higher rate.
How the rock-inhabiting fungus K. petricola A95 enhances olivine dissolution through attachment
(2020)
Free-living and mycorrhizal fungi are able to enhance the weathering of rock and other solid substrates. Deciphering the exact mechanisms of these natural processes requires their experimental simulation. Moreover, by performing these simulations with genetically amenable rock-weathering fungi, one can knock-out certain fungal traits and consequently identify their weathering-relevant function. Here, the effect of the rock-inhabiting fungus, Knufia petricola A95, on the dissolution kinetics of an Fe-bearing olivine (Mg1.86Fe0.19SiO4) is investigated at 25 °C and pH 6 using reproducible batch and mixed flow experiments. The availability of a melanin-deficient mutant (ΔKppks) of K. petricola A95, which produces more extracellular polymeric substances (EPS) than the wild type (WT), enables the comparative study of the role of melanin and EPS in olivine dissolution. In abiotic dissolution experiments, the olivine dissolution rate decreased considerably over time at pH 6 but not at pH 3.5. This inhibition of abiotic olivine dissolution at pH 6 was most likely caused by the in-situ oxidation of ferrous Fe and/or the precipitation of ferric hydroxides at the olivine surface. In corresponding biotic experiments at pH 6, both the wild type K. petricola and its melanin-deficient mutant ΔKppks solubilised and bound significant amounts of Fe released by olivine dissolution. Fe oxidation and precipitation were thus prevented and olivine dissolution proceeded faster than in the abiotic experiments. By sequestering Fe directly at the olivine surface, the attached wild type K. petricola cells were particularly efficient at preventing the oxidation of Fe at the mineral surface: the slowdown of olivine dissolution almost completely disappeared. The attachment capacity of these wild type cells is most likely mediated by wild type-specific EPS. Our presented experimental systems allow the oxidation of mineral-released Fe and include a rock-inhabiting fungus, thus simulating chemical, physical and biological conditions that set dissolution rates in a way that is relevant to natural ecosystems.