Ecology, adaptation, and function of methane‐sulfidic spring water biofilm microorganisms, including a strain of anaerobic fungus Mucor hiemalis

Abstract Ecological aspects, adaptation, and some functions of a special biofilm and its unique key anaerobic fungus Mucor hiemalis strain EH11 isolated from a pristine spring (Künzing, Bavaria, Germany) are described. The spring's pure nature is characterized by, for example, bubbling methane, marine‐salinity, mild hydrothermal (~19.1°C), sulfidic, and reductive‐anoxic (Eh: −241 to −253 mV, O2: ≤ 0.1 mg/L) conditions. It is geoecologically located at the border zone between Bavarian Forest (crystalline rocky mountains) and the moor‐like Danube River valley, where geological displacements bring the spring's water from the deeper layers of former marine sources up to the surface. In the spring's outflow, a special biofilm with selective microorganisms consisting of archaea, bacteria, protozoa (ciliate), and fungus was found. Typical sulfidic‐spring bryophyta and macrozoobenthos were missing, but many halo‐ and anaerotolerant diatoms and ciliate Vorticella microstoma beside EH11 were identified. Phase contrast and scanning electron microscopy revealed the existence of a stabilizing matrix in the biofilm formed by the sessile fungal hyphae and the exopolysaccharide substance (EPS) structures, which harbors other microorganisms. In response to ecological adaptation pressure caused by methane bubbles, EH11 developed an atypical spring‐like hyphal morphology, similar to the spiral stalk of ciliate V. microstoma, to rise up with methane bubbles. For the first time, it was also demonstrated that under strict anaerobic conditions EH11 changes its asexual reproduction process by forming pseudosporangia via hyphal cell divisions as well as switching its metabolism to chemoautotrophic bacteria‐like anaerobic life using acetate as an e‐donor and ferrihydrite as an e‐acceptor, all without fermentation. EH11 can be suggested to be useful for the microbial community in the Künzing biofilm not only due to its physical stabilization of the biofilm's matrix but also due to its ecological functions in element recycling as well as a remover of toxic metals.


| INTRODUCTION
In contrast to fresh water ecosystems, sulfidic springs at various hydrogeological interfaces present unique microbial ecological habitats because of special physical and chemical conditions, for example, reducing/oxidizing, sulfidic, hydrothermal/cold, anoxic/hypoxic, marinesalinity, and presence of methane and heavy metals. They are mainly fed by deep ground waters of different qualities and are mixed with younger water in various proportions; some are influenced by anthropogenic activities (Heinrichs, Hoque, Wolf, & Stichler, 2000;Hoque & Fritscher, 2016;Hoque, Pflugmacher, Fritscher, & Wolf, 2007).
Selective biofilms and microbial community members of various evolutionary origins grow there, some of which are biotechnologically important (Hoque & Fritscher, 2016;Hoque et al., 2007). The sulfidic Künzing spring is an exceptional example due to bubbling methane of deep origin (Carle 1975). Aquatic fungi of sulfidic springs are filamentous microorganisms embedded in the EPS structures; sometimes mixed with microalgae that adapts itself with unique detoxification functions (Hoque & Fritscher, 2016;Hoque et al., 2007). In the case of Künzing spring, due to flushing away of dissolved oxygen by continuous methane evolution, we can expect a special anaerobic microbial community resistant to bubbling methane and low oxygen levels.
In other sulfidic springs, the biofilm matrices were mainly stabilized by aquatic fungi that offer physical support for other microorganisms essential for their existence and dispersal in this special aquatic ecosystem.
Although fungi can be facultative anaerobic (Deacon, 1984;Kurakov, Lavrent′ev, Nechitailo, Golyshin, & Zvyagintsev, 2008), some of them from the orders Hypocreales, Eurotiales, Mortierellales, and the phylum Zygomycota can live for a month under fermentative anaerobiosis (Kurakov et al., 2008). Strictly anaerobic rumen fungi have been reported (Flint, 1997). Research on anaerobic fungi deserves our attention because they are useful for the bioavailability and nutrient cycling of organic carbon from complex polysaccharides of plant cell walls (Flint, 1997;Gerbi et al., 1996). In this way, they play a significant role in feeding process of ruminants (Chaudhry, 2000;Gordon & Phillips, 1998;Lee, Shin, Kim, Ha, & Han, 1999). They are also useful for many biotechnological applications as they produce noble overexpressed enzymes including diverse cellulase, hemicellulase, chitinase, endoglucanase, and xylanase (Bhat & Bhat, 1997;Flint, 1997;Lee et al., 1999;Yanke, Selinger, Lynn, & Cheng, 1996). They interact with the H 2 -utilizing acetogens beside methanogens or sulfate-reducing bacteria in the microbial community of anaerobic ecosystems (Morvan, Rieu-Lesme, Fonty, & Gouet, 1996). They are apparently significantly responsible for the DOC (Dissolved Organic Carbon) release from complex poorly degradable ligno-cellulosic materials and for carbon cycling in the environment (Flint, 1997). Fungal release of DOC from complex organic materials may have major influences on the microbial community structures that utilize DOC as electron donors, especially in anaerobic environments.
In contrast to anaerobic rumen fungi, we know little about aquatic anaerobic fungi. It was previously reported that some yeasts and filamentous fungi like Fusarium oxysporum, Mucor hiemalis, and Aspergillus fumigatus were able to grow in absence of oxygen by fermenting sugars (Deacon, 1984). There were also several previous reports about some aquatic fungal strains with the ability to live under anaerobiosis, for example, under oxygen-limited conditions of sewage sludges, polluted waters, organic-enriched soils (Tabak & Cooke, 1968), and submerged rice fields (Tonouchi, 2009;Wada, 1976), apparently via fermentation of substrates (Tonouchi, 2009). Aeroaquatic hyphomycetes showed variations in tolerance to anoxic conditions, some of them Helicodendron triglitziense, H. conglomeratum, and H. giganteum as well as Saprolegnialesm species with oospores survived up to 3 months under anaerobic conditions (Field & Webster, 1983). Using various sterilized wood probes in lysimeters, we could show differential affinity of some facultative anaerobic fungi to these wood probes in subsurface at depths ≤5 m (Hoque & Klotz, 2002). Similarly, Krauss et al. (2003) placed sterilized Alnus glutinosa leaf disks in wells and showed the occurrence of aquatic hyphomycetes in subterranean environment, especially in polluted groundwater habitats. It can be assumed that the aquatic hyphomycetes, as well as other facultative anaerobic fungi, play an important role in C cycling from decomposition of litter and woody materials in subsurface waters.
In contrast to subsurface waters and underground soil influenced by rain water, sulfidic springs are mainly fed by deep anaerobic groundwater (Heinrichs et al., 2000) and, as such, are more suitable for the search of fungi living strictly anaerobically. Until now not a single report on the occurrence of strictly anaerobic fungi in groundwater is available that can show their ability to live in anoxic groundwater ecosystems without fermenting sugar. Occurrence of aerobic or facultative anaerobic fungi in subsurface groundwater-sediment systems of varying depths is known (Hoque & Klotz, 2002;Hoque et al., 2007).
One bottle neck of research on anaerobic fungi in groundwater could be the lack of suitable methodologies for cultivation, and morphological and functional analyses of aquatic anaerobic fungi in liquid cultures.
After our thorough search of suitable anaerobic biofilms among sulfidic spring water biofilms, Künzing spring's biofilm offered a good opportunity to look for anaerobic fungi and to develop research methodologies. In order to withstand the pressure and anaerobiosis created by bubbling methane and reducing conditions, the biofilm growing there was adapted to anoxic conditions and stabilized by some anaerobic microorganisms like anaerobic fungi that can build 3D physical structures bound to the rocks. Therefore, the major objectives of our studies are to (1) establish methodologies for a systematic search of strictly anaerobic fungi in groundwater-sediment ecosystems based on both cultivation-dependent and cultivation-independent molecular biological approaches, (2) to unlock the ecological aspects of aquatic anaerobic fungi, (3) to demonstrate the morphology and strictly anaerobic life of a new Mucor hiemalis strain EH11 isolated from nearly anoxic methane-sulfidic spring water, (4) to show the utilization of acetate as an e-donor and ferrihydrite as an electron acceptor with AQDS as an electron shuttle under strictly anoxic conditions, (5) to present the ecological importance and functions of EH11 in removing various toxic metal ions simultaneously, as well as (6) to explore the role of aquatic anaerobic fungi in helping establishment of diverse life at the special deep sea-like terrestrial interface. Germering, Germany) as described (Hoque et al., 2007). Dissolved organic carbon (DOC) concentration was measured following NPOC method as reported (Hoque et al., 2007). The ionic metals (Al, As, Cd, Co, Cr, Cu, Hg, Li, Ni, Pb, Sr, and Zn) after complexation with EDTA were analyzed and quantified by using Dionex 500 system by using inductively coupled plasma-optical emission spectrometry (ICP-OES) for the quantitative and qualitative detection of elements (Hoque et al., 2007). Water age was determined by tritium-(Quantulus 1220, Perkin Elmer, Rodgau, Germany) and 14 C-dating techniques (Heinrichs et al., 2000).

| Biofilm collection and enrichment culture
Fresh biofilms (2-5 g) from submerged rocks in Künzing spring were collected in sterile falcon tubes and immediately placed on dry ice.
The biofilms were transported on dry ice to the laboratory, and immediately centrifuged at 4,000g (5 min) to concentrate as biomass pellets. They were either subjected to DNA extraction (see below) or used as inoculants on sterile malt extract (30 g L −1 )-agar (15 g L −1 ) solid media (Arjmand & Sandermann, 1985) supplemented with 100-ppm streptomycin sulfate. Repeated reinoculations and cultivations led to pure fungal cultures, which were observed under stereo microscopy (MZ16, Leica, Wetzlar, Germany) and phase contrast microscopy (Axiolab, Zeiss, Oberkochen, Germany) after a modified safranin staining (Hoque et al., 2007). The sterilized biofilms of Künzing spring and the biofilms of springs Wildbad Kreuth, Bad Abbach, and Pilzweg devoid of Mucor sp. served as negative controls for comparison.

| Fungal cultivation
The fungal strains of sulfidic springs and ground water sediments (mainly water-saturated bore hole core section from 10.35 m depth, Kellermann et al., 2012) were cultivated in liquid culture medium (see below) and on malt extract agar solid medium supplemented with streptomycin sulfate (100 mg/L) under anaerobic and aerobic/ anaerobic N 2 -flushed conditions, respectively. Temperature range was optimized for optimum growth. A stereo microscope was used for morphological investigation of the fungi and a digital camera was used for documentation.

| Light and electron microscopy as well as elemental analysis
The biofilms were observed under in situ intact conditions in the laboratory by using stereo microscopy (16MZ, Leica). Further morphological details of biofilms and microorganisms (Fungi, Bacteria, Archaea) were detected by light, fluorescence (Axiovert 100, Zeiss), and scanning electron (JSM 630F, Jeol Freising, Germany) microscopy. Prior to light and fluorescence microscope observations, the in situ cell labeling of biofilms and fungal hyphae was performed using modified safranin staining (Hoque et al., 2007) and DAPI (4′,6-diamidino-2phenylindole) staining.
For scanning electron microscope (SEM) observations, samples (biofilms, fungi) from PBS buffer (pH 7.4) suspensions were fixed at first with 1% glutaric aldehyde for 15 min and then with 2% osmium tetroxide in PBS buffer (pH 7.4). The samples were dehydrated in increasing ethanol gradients (see above). Then, uncut samples were sprayed with nanogold particles prior to observations with scanning electron microscopy at 5-15 kV in the secondary electron mode. The EDX elemental analysis of biofilms and EH11 cells was carried out using an eXL EDX system (Oxford Instruments, High Wycombe, UK).
For this purpose, X-ray quanta were detected by a silicium detector and analyzed by an eXL EDX system using Link analytical software (Hoque & Fritscher, 2016).
After incubation, the samples were centrifuged for 3 min at 4000g in order to separate the biological materials from the water. Water was analyzed for residual concentration of metal ions by ion chromatography (Dionex 500 system, see above). For the analysis of arsenics, the samples were treated with HCl (1:1 v/w) and cysteine (1:1 v/w) for 24 h.
The system was calibrated by using 0, 1, 10, 100, 500, and 1000 μg/L concentrations of respective metal standards, whereby the calibration curves with 95% confidence intervals were calculated by linear regression analysis (r 2 = 0.99). The standard deviations of measurements of each data point (n = 3) were maximum ± 5%.

| Statistical analysis
The statistical analysis was performed using nonparametric Mann-Whitney U-tests according to Weber (1986).

| Strain deposit
The pure culture of aquatic M. hiemalis strain EH11 was deposited at the DSMZ (Braunschweig, Germany) under the accession number DSM 16292.

| RESULTS
While exploring occurrence of biofilm and fungi in subsurface sediment samples and in sulfidic spring waters with low oxygen concentrations, we succeeded in discovering an anoxic biofilm along with a unique Mucor hiemalis strain EH11 from a methane-sulfidic salty spring named Künzing spring (Bavaria, Germany). In general, this spring belongs to methane-sulfide-sodium chloride-hydrogen carbonate spring type (spring type 4). It served as a healing thermal spring for the Romans since first century A.D. and was famous in the northern region of the Alps. The history of Künzing dates back 7,000 years.

| Site description and hydrogeochemistry of Künzing spring
Bavarian groundwater landscapes are divided into 29 groundwater regions; some of them around Künzing (EH11 site) were previously visualized in a graphic (Hoque & Fritscher, 2016). Künzing spring is located at the border of groundwater region "Crystalline Rocky Bavarian Forest" near Czech Republic and the "Tertiary Hilly  Table 1). According to tritium measurements, the water of this spring is also the oldest deep ground water among sulfidic springs and is located near all presently known thermal springs of this region.

| Biofilm of Künzing spring and its biodiversity
The ecological environment of ciliates and fungal hyphae (EH11) under low-oxygen methane-bubbling conditions of the sulfidic salty spring Künzing is shown in Figure 1. A sessile ciliate Vorticella microstoma springing up during the rise of a methane gas bubble and springing back after the bubble had risen above the ciliate as well as sessile thread-like fungal hyphae (EH11, F) were observed under in situ intact conditions by 3D microscopy on a rock surface. Due to continuous bubbling of methane, the biofilms of Künzing were compelled to fix themselves to rocks (Figure 1).
To test the hypothesis that microbial community members may live also strictly anaerobically in water-saturated groundwater systems, we developed culture-dependent and culture-independent methods for systematic investigations of fungi under anoxic groundwater-sediment systems. Culture-dependent methods included isolation, cultivation, morphological comparison, and mating behavior. In contrast, culture-independent methods, for example, phase contrast microscope and SEM investigations as well as molecular biological procedures (FISH; PCR, cloning, sequencing, and phylogenetic analysis) were modified and applied for aquatic anaerobic fungal research (Hoque & Fritscher, 2016;Hoque et al., 2007).
In the biofilm matrix of the sulfidic Künzing spring, phase contrast light microscopy revealed coccoidal Archaea arranged like a Chain of Pearls. More details were observed by SEM, as, for example, coccoidal

| Adaptation and spiral morphology of filamentous microorganisms in methane-sulfidic spring of Künzing
The ecological environment of ciliates and EH11 under low-oxygen methane-bubbling conditions of the sulfidic Künzing spring could be described by a sessile ciliate Vorticella microstoma that springs up during the rise of a gas bubble and returns after the gas bubble rises above the ciliate. the corresponding minus strand as previously described (Hoque et al., 2007).

| Effects of salt on Mucor hiemalis strain EH11
The Künzing spring near Passau (Bavaria) shows the highest salt concentration among all the investigated sulfidic spring waters because this spring is fed by a marine molassic bed (

| Accumulation of ionic metals by Künzing's biofilm
In contrast to Marching spring's biofilm with the ability of mercury and zinc removal (Hoque & Fritscher, 2016)

| Search for other anaerobic fungi in contaminated ground water sediments for comparison
We isolated two fungi from 10-to 14-m-depth ground water sediments of a Quaternary sandy aquifer at a former Gasworks (Düsseldorf-Flingern, Germany) by using hollow-stem auger drilling (Anneser et al., 2010). These fungi utilized glucose and cellobiose as an e-donor, and ferrihydrite/AQDS as an e-acceptor under strict anaerobic conditions.
When grown aerobically on solid medium, a consortium of at least two fungi/strains from anaerobic liquid media incubated with cellobiose as an electron donor and ferrihydrite/AQDS as an electron acceptor was shown to exist and were observed by stereomicroscopy. One of these fungi was identified morphologically as Penicillium chrysogenum strain EH31, and used for comparison with EH11 (see below).
The optimum temperature of growth of aquatic fungi under anoxic conditions was detected within ambient temperature (20-30°C).
As, for example, aquatic Mucor hiemalis mycelium grew optimally 10 mm day −1 at this temperature range, but it even grew 1 mm day −1 near freezing temperature (0.3°C). Incubation of Mucor hiemalis strain EH11 in a strictly anaerobic medium containing acetate (50 mmol L −1 ) as an electron donor and ferrihydrite (10 mmol L −1 ) as an electron acceptor with AQDS (10 mmol L −1 ) as an electron shuttle showed that the strain EH11 has the ability to grow chemoautotrophically, without fermentation similar to chemoautotrophic Bacteria under anoxic conditions (see below).
The efficiency of metal accumulation by EH11, except for mercury, was similar to that of the strain EH8, which was isolated from a methane-sulfidic salt spring with similar chemical compositions (Hoque & Fritscher, 2016), but the mean temperature of the Künzing spring is about 8.6°C higher than the other spring.

| Utilization of acetate as an electron donor and ferrihydrite as an electron acceptor with AQDS as an electron shuttle by a new strain EH11 of the fungus Mucor hiemalis
The ferrihydrite reduction [Fe(II) formation] by EH11 (solid line) compared to control Penicillium chrysogenum EH31 (isolated from anaerobic sediments) depending on incubation duration under strict anaerobic conditions is demonstrated in Figure 6 (below). Using acetate (50 mmol L −1 ) as an electron donor and ferrihydrite (10 mmol L −1 ) as an electron acceptor with AQDS (10 mmol L −1 ) as an electron shuttle, it can be shown that the strain EH11 continuously reduced Fe(III) (ferrihydrite) into Fe(II) from the ouset, but after an initial lag period of about 17 days statistically significantly stronger than P. chrysogenum EH31 (Mann-Whitney test, α = 0.05).
These special microbial habitat conditions promoted the selective growth of salt-tolerant micro-and nanosized Archaea, mainly Euryarchaeota and Crenarchaeota, diverse Bacteria and aquatic anaerobic fungus EH11 as shown by FISH analysis (Hoque & Fritscher, 2016;Hoque et al., 2007), and numerous diatoms in the rock-sessile biofilm. Culture-dependent and -independent methods revealed the biofilm's microbial composition and led to characterization and/or F I G U R E 6 Accumulation of ferrihydrite [Fe(III) species] and its reduction by EH11 under strict anaerobic conditions. (a) Fungal filament with ferrihydrite around the hypha grown in an anaerobic liquid medium with cellobiose as an electron donor and ferrihydrite as an electron acceptor with AQDS as an electron shuttle, (b The matched values in biofilms and fungus from selected springs are shown in bold font. The standard deviations of measurements (n = 3) are maximal 5%. *The mean percentages of ionic metal removal from water by bioabsorption are given for each heavy metal cation applied (1,000 μg/L) in a mixture to biofilms (Bf) and corresponding fungal cultures (F: EH11) of Künzing spring in comparison to control spring Teugn and its fungus Mucor hiemalis EH7.
identification of the key players of the biofilm's functions in Künzing spring water.
EH11 and other microorganisms can be suggested to be immigrants to the Künzing spring's biofilm, reaching it by any means of dispersal (wind, surface, and deep marine water), but adapting themselves over years to methane and mild hydrothermal-sulfidic salty marine-like habitat conditions.

| DISCUSSION
Investigations on Künzing spring's biofilm and its community members Protozoa and fungi can grow. The protozoan ciliate V. microstoma and the fungus EH11 were found there. The nearly anoxic igneous oceanic crust circulated by seawater and highly saline hydrothermal fluids was described as the earth's largest fungal habitat (Ivarsson, Bengtson, & Neubeck, 2016). A search of fungal diversity in deep-sea hydrothermal ecosystems also revealed the occurrence of fungi, many of them were unknown and some of them were from higher taxonomic levels like Chytridiomycota, Ascomycota, and Basidiomycota (Calvez, Burgaud, Mahe, Barbier, & Vandenkoornhuyse, 2009). Similarly, the Zygomycete EH11 was isolated from a nearly anoxic sulfidic salt spring fed by marine mollasic bed water, which is comparable to marine habitats of numerous fungi described by Ivarsson et al. (2016). Thus, strictly anaerobic fungi may occur in anoxic deep groundwater-sediment systems. The feasibility of finding many other anaerobic fungi in anoxic-reducing aquifers is high. Occurrence of some other fungi in partially anoxic spring waters was also detected, whereby presence of chytrid-like fungi in a partially anoxic spring water environment was observed by us (Fritscher, 2004 Similar to EH11's hyphal pseudosporangium formation and release, exogenous sporangia formation is regarded as a typical feature of strictly anaerobic polycentric rumen fungi, which could be observed by fluorescence microscopy using 2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole stain (Fliegerova, Hodrova, & Voigt, 2004). Polycentric fungal mycelium shows variations in hyphal forms (tubular and uniform or wide and irregular), sometimes with constricted hyphae at short intervals (bead-like appearance). Monocentric or polycentric thallus, filamentous or bulbous rhizoids, and mono-or polyflagellated zoospores, as well as electron microscope observations of zoospore ultrastructures are often considered as valuable criteria for morphological identification of anaerobic fungi (see above). In contrast to pseudosporangia produced by EH11's hyphal intracellular divisions, chlamydospores from Mucor circinelloides hyphae were oval-subglobose shaped without any subdivisions into sporagiospores, but the sporangia of this fungus were subdivided into sporangiospores (Iwen, Sigler, Noel, & Freifeld, 2007). This micromorphological behavior of Mucor species under anoxic conditions could be of high importance for the assessment of invasion and pathogenicity of some opportunistic fungi under anoxic-hypoxic conditions. It is worth mentioning in connection with this that some anaerobic fungal zoospores were chemoattracted to phenolic acids (Wubah & Kim, 1996), which could be released during degradations of woody materials.
In general, the fungi are considered as key organisms in ecosystems on the basis of their ecological function and the ability to degrade organic matter (Calvez et al., 2009). Nevertheless aquatic fungus also provides physical matrix to harbor Archaea, Bacteria, and other microbial community members in biofilms (Hoque et al., 2007). We could show the unusual metal filtration power of EH11 as one of the ecological roles of this fungus in the natural biofilm under nearly anoxic conditions. Practically, EH11 can also be used in combination with other sulfidic spring's Mucor hiemalis strains for the simultaneous removal of toxic metal ions from groundwater and industrial waste water with high metal and sulfide concentrations (Fritscher, Hoque, & Stöckl, 2006). Thus, it can be suggested that aquatic anaerobic fungi along with other microbial community members, for example, protozoan Vorticella microstoma, play not only a vital role in filtration of toxic ionic metals from water but also in C recycling (Table 2; Shakoori, Rehman, & ul-Haq, 2004). EH11 is not only ecologically important for the Künzing spring's microbial community concerning physical stability, but also for its functions in elimination of toxic metal ions (Table 2). However, many anaerobic fungi possess enzymes essential for the degradation of ligno-cellulosic materials for C-recycling (Chen et al., 2003;Gerbi et al., 1996;Li, Chen, & Ljungdahl, 1997;Yanke et al., 1996). Thus, the ecological role of anaerobic fungi in such an extreme aquatic environment (nearly anoxic sulfidic marine conditions) in the biofilm could be manifold: (1) physical support (Hoque & Fritscher, 2016;Hoque et al., 2007), (2) special enzymes (see above), (3) special chemoautotrophic Bacteria-like metabolism (see results), (4) special asexual reproduction (see results), (5) element recycling (Calvez et al., 2009;Gadd, 2010;Ivarsson et al., 2015Ivarsson et al., , 2016, (6) detoxification of organic toxic substances (Hoque et al., 2007), (7) metal detoxification (Gadd, 2010;Hoque & Fritscher, 2016;Hoque et al., 2007), (8)