Aquatic hyphomycetes and leaf decomposition in ...

Arch. Hydrobiol.

162

3

417–429

Stuttgart, March 2005

Aquatic hyphomycetes and leaf decomposition in contaminated groundwater wells in Central Germany Gudrun Krauss1, K. R. Sridhar2 and Felix Bärlocher3 UFZ Centre for Environmental Research in the Helmholtz Association, Leipzig-Halle With 2 figures and 2 tables

Abstract: Six heavy metal contaminated groundwater wells near abandoned copper shale mines of Mansfelder Land in Central Germany yielded 13 aquatic hyphomycete species (3–7 per site) on immersed Alnus glutinosa leaves. Anguillospora sp. 2, Cylindrocarpon sp. and Tetracladium marchalianum were the three top conidial producers. Mass loss rates and release of conidia were below those of leaves exposed in surface streams with similar or higher heavy metal loads, while ergosterol levels were comparable. In food choice experiments, Gammarus fossarum distinguished between leaves conditioned in the six wells. Consumption rates correlated with numbers of released conidia but not with ergosterol levels of the substrate. Key words: Aquatic hyphomycetes, groundwater, heavy metal pollution, leaf decomeschweizerbartxxx position, conditioning. Gudrun Krauss es_2005_1003

Introduction Aquatic hyphomycetes belong to a group of anamorphic fungi, whose growth and reproduction is strongly associated with the presence of deciduous leaf litter in well-aerated surface water (Bärlocher 1992, Suberkropp 1997, Gessner & Van Ryckegem 2002). Both dissolved and particulate organic matter can infiltrate the stream bed and enter the hyporheic zone, which connects 1

Authors’ addresses: Department Groundwater Microbiology, UFZ Centre for Environmental Research, Leipzig-Halle, Theodor-Lieser-Straße 4, 06120 Halle/Saale, Germany; E-mail: [email protected] 2 Department of Biosciences, Mangalore University, Mangalagangotri, Mangalore 574 199, Karnataka, India. 3 Department of Biology, Mount Allison University, Sackville, N. B., Canada E4L 1G7. DOI: 10.1127/0003-9136/2005/0162-0417

0003-9136/05/0162-0417 $ 3.25

 2005 E. Schweizerbart’sche Verlagsbuchhandlung, D-70176 Stuttgart

418

G. Krauss, K. R. Sridhar and F. Bärlocher

to the true groundwater (Orghidan 1959, Schwoerbel 1961, Williams & Hynes 1974). Similarly, aquatic hyphomycete conidia, some germinated, have been reported from glass beads buried 10 cm deep in stream sediment (Bärlocher & Murdoch 1989). Provided that oxygen supply is adequate, such fungi may be involved in breaking down particulate matter in subterranean habitats (Storey et al. 1999), and several studies have indeed reported the occurrence of unidentified fungal hyphae in the hyporheal and aquifers (Sinclair & Ghiorse 1989, Hirsch et al. 1992, Madsen & Ghiorse 1993, Ellis et al. 1998). Leaves incubated in microcosms fed by groundwater springs lost mass and supported microbial respiration (Eichem et al. 1993); however, no distinction was made between bacterial and fungal contributions. Similar results were reported from leaves or wood exposed in cave streams (Galas et al. 1996, Simon & Benfield 2001), which are generally fed by subsurface drainage networks dominated by groundwater (White 1988). While aquatic hyphomycetes prefer well-aerated, pristine waters, they survive and remain active when exposed to various types of pollution (e. g. organic pollution: Au et al. 1992, Raviraja et al. 1998, Sridhar & Raviraja 2001; heavy metal pollution: Bermingham et al. 1996, Krauss et al. 1998, 2001, 2003 a, 2004, Maltby & Booth 1991, Sridhar et al. 2000, 2001). Due to a long history of copper mining in the Mansfeld district in Central Germany, many surface and groundwater habitats are extensively contaminated with a variety of heavy metals and metalloids. We nevertheless observed up to 20 fungal species and substantial leaf decomposition rates in surface waters (Sridhar et al. 2000, 2001). Sterile leaves exposed in 11 groundwater wells were also colonized by aquatic hyphomycetes, but despite lower levels of heavy metal contamination, fungal species richness and spore production were considerably lower than in surface water (Krauss et al. 2003 b, 2004). The objective of the current study was to evaluate various decomposition parameters by exposing sterile leaves in six of these wells. We measured decomposition rates, and accumulation of fungal biomass and spore production. We expected these values to be lower than in surface streams, and possibly insufficient to effectively condition leaves for invertebrate consumption (Bärlocher 1992, Suberkropp 1992, Gessner & Van Ryckegem 2002).

Materials and methods Study sites and water analyses Six groundwater observation wells were investigated (P1, P6, P6B, P10, P11A, P12; Table 1; Krauss et al. 2003 b). They are installed downstream of contaminated areas in the former industrial area around the Helbra copper shale smelter in the Mansfelder Land, Saxony-Anhalt. The final depths of the observation wells range from 2.9 m to

Aquatic hyphomycetes in contaminated groundwater

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Table 1. Physicochemical parameters of 6 groundwater wells. Coordinates according to the Transverse Mercator (Bessel System). Measurements, generally in triplicate, were taken on April 19, May 17 and 31, and June 14, 2000. Averages and range. P1 Coordinates x: 4465001 y: 5711759 Temperature 12.0 (˚C) (8.0 – 14.1) pH 6.9 (6.8 – 7.1) Conductivity 3.9 (mS/cm) (3.7 – 4.1) O2 2.6 (mg/l) (2.1 – 3.0) DOC 14.7 (mg/l) (13 – 17) TOC 14.9 (mg/l) (13 – 18) Phosphate < 0.1 (mg/l) Sulfate 2.1 (mg/l) (2.0 – 2.2) Ammonium < 0.1 (mg/l) Nitrate 9 (mg/l) (9.0 – 9.3)

P6

P6B

P10

P11A

P12

x: 4465418 y: 5712139 10.3 (9.1 – 11.2) 7.1 (7.1 – 7.2) 3.7 (3.6 – 3.8) 3.5 (2.9 – 5.0) 5.3 (1.9 – 9.4) 3.6 (2.5 – 5.0) 0.2 (0.1 – 0.3) 2.2 (2.1 – 2.3) < 0.1

x: 4465419 y: 5712135 7.7 (4.2 – 9.7) 7.2 (7.0 – 7.4) 2.8 (2.7 – 2.9) 3.8 (2.6 – 5.0) 3.6 (0.4 – 8.3) 4.3 (0.1 – 8.3) < 0.1

x: 4465782 y: 5712267 10.8 (8.3 – 12.3) 6.9 (6.8 – 7.0) 2.8 (2.6 – 2.9) 3.9 (2.6 – 6.6) 4.9 (2.9 – 6.9) 6.4 (3.3 – 9.2) < 0.1

x: 4465950 y: 5712473 10.5 (7.8 – 12.7) 7.0 (6.8 – 7.1) 3.8 (3.6 – 3.9) 4.0 (3.5 – 4.8) 3.0 (1.3 – 4.6) 4.8 (3.7 – 6.2) < 0.1

x: 4465560 y: 5712448 9.6 (7.1 – 13.6) 7.2 (6.9 – 7.4) 4.1 (4.0 – 4.2) 3.9 (2.2 – 6.8) 5.0 (4.5 – 5.7) 5.1 (4.3 – 6.2) < 0.1

1.3 (1.2 – 1.4) < 0.1

1.4 (1.2 – 1.5) < 0.1

1.6 (1.5 – 1.7) < 0.1

2.3 (2.2 – 2.4) < 0.1

49 (34 – 64)

30 (29 – 32)

19 (13 – 24)

41 (38 – 43)

63 (60 – 67)

34.4 m. They monitor three aquifers in the Lower Bunter (Buntsandstein) formation, which is developed as silty sandstone (aquitard) with oolithic limestone intercalations (“Rogenstein” aquifer). Groundwater observation wells P1, P6, and P6B are situated south, south-east and north-east, respectively, of the major Theisen sludge storage basin (Pond 10) and cover the outflow of leachates of this deposit. Observation wells west of Pond 10 (P10) have been installed to monitor the dispersal of leachates from the former industrial complex. The newly installed wells P11A, and P12 provide data on pollutant migration in the groundwater to the east and south-east, following the Glume river. The groundwater is contaminated with various heavy metals and other pollutants (Krauss et al. 2003 b). Water temperature, pH, conductivity, and oxygen were measured in the field (Universal Taschenmessgerät MultiLine P4-WTW, Germany). For additional analyses, water was collected in sterilized dark-brown Schott-Duran glass flasks and transported back to the lab at 4 ˚C. All measurements were done in triplicate on each sampling date. Ion chromatography (DX 100 Ion Chromatograph, Dionex) was used to determine nitrate and sulfate. Ammonium and phosphate were measured photometrically (EPOS Analyzer 5060, Eppendorf). Dissolved (DOC) and total organic carbon (TOC; TOC-5050 Shimadzu) were determined according to the German standard for the examination of water, wastewater, and sludge (DIN 38409 H3-1).

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Concentrations of selected heavy metals and metalloids were reported in Krauss et al. (2003 b). Few individual values exceed European guidelines for drinking water (European Council Directive 98/83/EC), but a relatively high level of Pb was found P12 (up to 0.09 mg/l), while Mn was above recommended levels for drinking water in P1, P6, and P6B (up to 0.9 mg/l). All groundwater wells tested in this area inhibited pollen tube growth by 20 – 50 % (Krauss et al. 2003 b), indicating significant cytotoxicological effects.

Leaf collection and preparation Naturally shed leaves were collected from a single alder tree (Alnus glutinosa Gaertner) in the Botanical Garden of the Martin-Luther-University Halle-Wittenberg in late September and early October 1999. They were soaked in tap water, cut into 1.5 cm disks, air-dried and stored until used. Leaf disks were weighed, autoclaved and placed in nylon mesh bags (25 disks, 10 × 10 cm, 1-mm mesh). To determine initial ash-free dry mass, 5 replicates of 5 air-dried disks each were exposed to 550 ˚C for 4 h and weighed again. The bags were introduced into the groundwater wells on 20 April, 2000 (immersion depth 0.7– 4.6 m). Samples were recovered at 2-week intervals.

Mass loss On each sampling date, the contents of 4 randomly selected litter bags were gently rinsed in tap water, dried (80 ˚C, 24 h) and weighed. To determine ash-free dry mass, the disks were exposed to 550 ˚C for 4 h and weighed again. Exponential decay rates were estimated by non-linear curve-fitting procedures and compared with an F-test, using SYSTAT 5.3.1 for Macintosh computers (SYSTAT Inc., Evanston, IL; http:// www.curvefit.com).

Fungal analyses Colonization by aquatic hyphomycetes was assessed by counting and identifying spores released from leaf disks, as described in Krauss et al. (2003 b). Disks were aerated in 150 ml distilled water for 2 days at 20 ˚C, and the suspension filtered through 8-µm pore membrane filters (Millipore). Spores trapped on the filters were stained (aniline blue in lactophenol) and identified. The disks were dried (overnight, 80 ˚C) and weighed to calculate spore production per unit weight. Three replicates, each consisting of 5 disks from one bag, were evaluated. Estimates of fungal biomass were based on ergosterol measurements, modified from Newell et al. (1988). For extraction, 4 to 6 freeze-dried leaf disks were homogenized in 15 ml of methanol with an IKA Ultra Turrax T25 (21000 rpm, ice bath). After the addition of another 5 ml of methanol and centrifugation, the supernatant was supplemented with 5 ml of a methanolic KOH solution (4 % KOH, 95 % methanol) and saponified (30 min, 80 ˚C). After cooling to room temperature, 10 ml of water was added and lipids were partitioned into pentane by 3 washings. The pooled pentane fractions were evaporated to dryness and redissolved in 3 ml of methylene chloride, again evaporated to dryness and redissolved in 1ml of methanol. After filtering through a 0.2 µm


421

filter, 20 µl aliquots were analyzed with HPLC (La Chrom D-7000, Merck) with a 5 µm RP 18 LiChrospher 100 column (250 × 4.6 mm). The mobile phase was 100 % methanol at a flow rate of 1.5 ml/min. Ergosterol was identified by comparing UV absorption spectra and retention time with those of a pure standard (Sridhar et al. 2001). Averages were based on 5 replicate measurements, each based on 4 – 6 leaf disks.

Food selection experiment Mature specimens of Gammarus fossarum Koch were collected from a clean stream near Ballenstedt (Saxony-Anhalt) and maintained in stream water at 20 ˚C. Animals were given a choice of disks that had been exposed in groundwater for 8 weeks. A treatment replicate consisted of a glass bowl with 200 ml sterile tap water, a total of 12 freeze-dried, preweighed leaf disks (2 from each well, marked with various notches) and 4 specimens of G. fossarum. The amphipods were allowed to feed for 24 h at room temperature under natural light conditions. The disks were then collected and dried (80 ˚C, 24 h), and their remaining mass was determined. Mass loss due to leaching was measured in 5 control bowls without amphipods. Food preference data were analyzed as described in Sridhar et al. (2001), using Resampling Stats 5.0 for Macintosh (www.statistics.com), and with Friedman’s test, followed by Dunn’s multiple comparison test (InStat, www.graphpad.com). Consumption rates of the leaves were regressed against numbers of fungal species, released conidia and ergosterol levels, with p values adjusted for multiple comparisons (Bonferroni adjustment; SYSTAT 5.3.1 for Macintosh computers).

Results Physicochemical parameters of the six groundwater wells are summarized in Table 1. Ammonium and phosphate were consistently below 0.1mg/l, with the exception of phosphate in P6, which on average was 0.2 mg/l. Sulfate (1.3 – 2.3 mg/l) and nitrate (9 – 63 mg/l) levels were high in all sites, as were conductivity, DOC and TOC levels, while oxygen concentrations were always well below saturation. Remaining mass of leaf disks as function of time in the six wells is presented in Fig. 1 A. Daily decay coefficients ranged from 0.005 in P1 to 0.009 in P10, respectively, but did not differ significantly from each other (F-test, p = 0.17). Ergosterol levels were measured after 4 and 8 weeks (Fig. 1 B). On both dates, they were highest in P12 (62 and 124 µg/g, respectively), followed by P10 (47 and 35, respectively). Conidial output during 2 days of aeration was high from leaf disks that had been incubated for two weeks in P10 (99 conidia/mg ash-free dry mass; Fig. 1C) or P12 (54 conidia/mg). They were generally lower by 2 – 4 orders of magnitude on disks from the other sites. The conidia were assigned to a total

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Fig. 1. Alder leaf disks incubated in six groundwater wells. A: Remaining ash-free dry mass (AFDM), averages of 5 replicates, ± SEM. B: Ergosterol content in µg/g AFDM of leaf litter, averages of 5 replicates, ± SEM. C: Conidia released g –1 AFDM d –1 (log scale), averages of 3 replicates. D: Total number of aquatic hyphomycete species identified from released conidia in the three replicates.

of 13 taxa belonging to 11 genera of aquatic hyphomycetes (Table 2); at any given sampling date, species numbers varied between 1 and 6 (Fig. 1 D). Only three taxa were found at P6, while a maximum of seven was identified at P10. Anguillospora sp. 2 and Heliscus lugdunensis occurred at all sites. Anguillospora sp. 2 was the dominant conidial producer at four sites; it was replaced by Cylindrocarpon sp. at P10 and by Tetracladium marchalianum at P6B (Table 2). Ergosterol levels, sporulation rates and species numbers did not correlate with any of the stream parameters listed in Table 1, or concentrations of metals (reported in Krauss et al. 2003 b). A resampling test (Sridhar et al. 2001) and Friedman’s test both revealed significant differences in the consumption rates when Gammarus fossarum was offered a choice between leaf disks incubated for 8 weeks in the six groundwater wells (Fig. 2). Highest values for Pearson’s correlation coefficients were found between consumption rate and number of released conidia (R = 0.81, p = 0.05) and ergosterol (R = 0.74, p = 0.09).


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Table 2. Aquatic hyphomycete species identified from conidia released from disks exposed in six groundwater wells and their contributions in % to total conidium production (data of all three samples combined; + equals < 1 %). P1

P6 P6B P10 P11A P12

Alatospora flagellata (Gönczöl) Marvanová + + Anguillospora sp. 1 5.5 Anguillospora sp. 2 50.0 82.3 12.1 41.8 94.1 Cylindrocarpon sp. 50.0 Flagellospora sp. 11.1 + 3.6 Heliscus lugdunensis Sacc. et Thérry 16.7 14.7 + 4.0 5.1 Lambdasporium sp. + Mycocentrospora acerina (Hartig) Deighton 16.7 2.9 Tetracladium marchalianum De Wild. 75.5 + Tricellula sp. Tricladium angulatum Ingold 10.6 + 0.7 Ypsilina graminea (Ingold, Dann et McDougall) Descals, J. Webster et Marvanová Ypsilina sp. Total species

5

3

6

7

4

93.9

1.0

1.8 2.4 + 5

Fig. 2. Consumption by G. fossarum offered a choice of leaf disks exposed for 8 weeks in six groundwater wells, averages of 5 replicates, ± SD. Consumption rates covered by the same line are not significantly different (p < 0.05; Friedman’s test, followed by Dunn’s multiple comparisons test).

Discussion Small streams are often characterized as being heterotrophic, i. e., much of the energy sustaining food webs is imported from riparian vegetation in the form of leaves, needles, twigs and dissolved organic matter (Allan 1995). This de-

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pendence on allochthonous matter is likely to increase as we move from surface water down to hyporheic and groundwater habitats (Boulton et al. 1998). Phototrophy and lithotrophy are restricted in these subterranean zones, and most nutrients must be provided through infiltration (Madsen & Ghiorse 1993, Brunke & Gonser 1997). Particulate (leaves or leaf fragments) and dissolved organic matter can travel deep into the sediments where they may nourish invertebrates (Williams & Hynes 1974, Boulton 1993). While oxygen is often plentiful in downwelling water, it may rapidly be used up through respiration by microbes and invertebrates feeding on organic nutrients (Boulton & Foster 1998). Depending on water flow and porosity, and on the balance between nutrients and consumers, hyporheic and groundwater habitats will consist of a mosaic of oxygenated, hypoxic and anoxic microsites, which will widely differ in their suitability for invertebrate and microbial life (Fischer et al. 1996, Pusch 1996, Storey et al. 1999, Danielopol et al. 2000). Due to microbial respiration and no direct access to the atmosphere, groundwater often has low oxygen and high carbon dioxide levels (Madsen & Ghiorse 1993). In the presence of limestone, large amount of Ca and bicarbonate may be liberated. This can result in large solution channels, or even underground streams, often connected to the surface by cracks or larger openings in the soil. Surface and subsurface drainage systems are therefore strongly linked in karst regions and may both be characterized by turbulent flow, which facilitates replenishing oxygen levels in the water (White 1988, Gibert et al. 1990). Cave streams, though fed by water that has percolated through the soil and bedrock, therefore share many characteristics with surface streams. A major difference is the scarcity of organic matter and invertebrates in the former. Decomposition of Alnus incana leaves was nevertheless slower in a cave stream (k = 0.0014 day –1; Galas et al. 1996) than in a comparable mountain stream (k = 0.0025 day –1; Galas 1996). On the other hand, decay rates of Quercus alba leaves in several cave streams spanned the range of values reported from surface streams (k = 0.003 – 0.026; Simon & Benfield 2001), and leaves and wood rapidly accumulated fungal biomass. Decomposition of Quercus macrocarpa and Ulmus americana leaves in microcosms fed by groundwater springs depended crucially on the water replacement rate; at low water flow, anoxic microzones formed within leaves even though the water was well oxygenated (Eichem et al. 1993). None of these studies identified fungi or bacteria involved in decomposition. In the current study, oxygen levels in all six sites were reduced to 24 – 46 % of saturation (Table 1), and the water in the wells was essentially stagnant. Even though nitrate levels were high, daily exponential decay rates (k = 0.005 – 0.009) were at the low end of values reported for A. glutinosa (e. g., 0.015 – 0.023: Bermingham et al. 1996; 0.0065: Chauvet 1987; 0.03: Gessner & Chauvet 1994). They were considerably less than the rate measured in


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a stream site H9 with a moderate load of heavy metals (k = 0.055), but approached the one in a neighbouring stream site H4 with an extremely high load of Zn and other metals (k = 0.009; Sridhar et al. 2001). Thus, moderate heavy metal contamination, especially when it has existed for long periods of time (in the Mansfelder District presumably for centuries) may inhibit leaf degradation to a lesser extent than other water parameters. These may include oxygen concentration, current flow, and microbial inocula. In surface streams, aquatic hyphomycetes dominate leaf decomposition. In an earlier study, Krauss et al. (2003 b) demonstrated the occurrence of aquatic hyphomycetes in some groundwater wells, and their potential to colonize introduced leaves. We confirmed these observations in the current study. Maximum ergosterol levels on leaves in the six wells varied between 16 and 124 µg/g AFDM (Fig. 1 B) corresponding to a fungal contribution of 0.27 to 2.1 % to total detrital mass (using conversion factors by Gessner & Chauvet 1993). This compares to 0.36 % and 1.1 % in the surface streams H4 and H9, respectively (Sridhar et al. 2001), and up to 17 % reported from non-polluted streams (Gesssner 1997). Conidium production varied between 0.1 and 100 mg –1 day –1 (Fig. 1 C), compared to 2.5 in H4 and 1200 in H9, respectively (Sridhar et al. 2001) and up to 8000 in pristine streams (Gessner 1997). If the fungal community in our wells was dominated by aquatic hyphomycetes, our results indicate that their reproduction is more readily disrupted than growth (which has in fact been demonstrated; Abel & Bärlocher 1984). In addition, the possibility exists that other fungi play a larger role under these conditions; even in streams, molecular techniques have revealed the presence of many fungal taxa not belonging to aquatic hypomycetes (Nikolcheva & Bärlocher 2004). During the 56 days of leaf decomposition, we distinguished between 3 and 7 taxa per site, with a median value of 5. This compares with a median of 24 species reported from 6 more heavily polluted surface waters in the same region (Sridhar et al. 2000). Based on spore production, the two most common taxa were assigned to Anguillospora (Anguillospora sp. 2; we have thus far not been able to isolate it into pure culture) and Heliscus lugdunensis. Tetracladium marchalianum and Tricladium angulatum were common in two of the sites; they are generally dominant in surface waters of the same region. It is unclear whether this reflects taxon-specific physiological or ecological preadaptations or is due to chance. Despite the low number of species and release of conidia, which may suggest limited involvement of aquatic hyphomycetes, stream exposure of alder leaves clearly improved their palatability to a detritus-feeding invertebrate (Fig. 2). The three preferred leaves, from sites P10, P11A, P12, also had highest ergosterol and sporulation levels, though their consumption was not significantly different from that of the next two leaves (P6, P6B). This agrees

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with the general conclusion that palatability increases with colonization by fungi but is not a linear function of their accumulated biomass (Suberkropp 1992). This study confirms that aquatic hyphomycetes do occur in polluted groundwaters of the Mansfelder Land area and colonize introduced leaves. However, species numbers and reproductive activities, as well as mass loss rates of the leaves, are considerably less than in surface waters of the same region. This suggests that these groundwaters represent a marginal habitat for aquatic hyphomycetes. Possibly, other fungal taxa replace or supplement their involvement in leaf breakdown and conditioning. Acknowledgements Part of this work was supported by the Canadian-German Scientific and Technology Research cooperation (BMBF/WTZ No. ENV 99/1). We thank Klaus Birger and Simon Kuckert, MDSE GmbH Bitterfeld, Neutraanlage Helbra, Barbara Krause, Dept. Groundwater Microbiology, Silke Leider and Gabriele Strenge, Dept. Hydrogeology, Jürgen Steffen, Dept. Analytical Chemistry, UFZ Leipzig-Halle for their technical assistance and Rainer Wennrich for helpful cooperation. KRS is grateful to Mangalore University for granting a leave during the tenure of this investigation and to the UFZ Leipzig-Halle GmbH for a fellowship.

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