New cultivation medium for “Ferrovum” and Gallionella-related strains

New cultivation medium for “Ferrovum” and Gallionella-related strains

Journal of Microbiological Methods 95 (2013) 138–144 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: ...

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Journal of Microbiological Methods 95 (2013) 138–144

Contents lists available at ScienceDirect

Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth

New cultivation medium for “Ferrovum” and Gallionella-related strains Judith S. Tischler a,⁎, Rawaa Jaffer Jwair a, Nadja Gelhaar a, Anna Drechsel a, Anne-Marie Skirl a, Claudia Wiacek a, Eberhard Janneck b, Michael Schlömann a a b

Interdisciplinary Ecological Center, Institute of Biosciences, TU Bergakademie Freiberg, Leipziger Straße 29, 09599 Freiberg, Germany Department of Biotechnology, G.E.O.S. Freiberg Ingenieurgesellschaft mbH, Gewerbepark “Schwarze Kiefern”, 09633 Halsbrücke, Germany

a r t i c l e

i n f o

Article history: Received 3 May 2013 Received in revised form 30 July 2013 Accepted 31 July 2013 Available online 13 August 2013 Keywords: Acid mine water “Ferrovum” Gallionella relatives Iron-oxidizing bacteria Isolation Overlay plates

a b s t r a c t Since the first isolation of the well-known iron oxidizer Acidithiobacillus ferrooxidans various media and techniques have been developed to isolate new species of acidophilic iron-oxidizing bacteria. A successful strategy in many cases was the use of iFeo medium in double-layer plates with a heterotrophic strain in the underlayer. However, even with samples which had been shown by molecular techniques to be dominated by “Ferrovum myxofaciens” and Gallionella-related bacteria, these bacteria were isolated considerably less frequently than Acidithiobacillus spp. on iFeo. Therefore, a new medium was designed which corresponded largely to the chemical composition of the mine water in a treatment plant dominated by the bacterial groups mentioned and was called artificial pilot-plant water (APPW). The analyses of approximately 500 colonies obtained from mine waters of two different sampling sites by PCR with primers specific for Acidithiobacillus spp., “Ferrovum” spp., Gallionella relatives, and Acidiphilium spp. revealed higher abundances of “Ferrovum” spp. and Gallionella relatives on the newly designed APPW medium than on iFeo which favored Acidithiobacillus spp. Molecular analysis of the colonies obtained indicated the occurrence of at least two species of iron-oxidizing bacteria and/or the heterotrophic Acidiphilium spp. in most of the colonies. Furthermore, the influence on the isolation of the concentrations of iron, phosphate, and ammonium of APPW, in levels of the iFeo medium previously described was studied. © 2013 Elsevier B.V. All rights reserved.

1. Introduction One common habitat of acidophilic iron-oxidizing bacteria is acid mine waters formed by oxidative dissolution of exposed sulfide minerals (Banks et al., 1997; Johnson and Hallberg, 2003). The first acidophilic iron-oxidizing bacterium was isolated from acid mine drainage of a bituminous coal mine in 1949 and named Thiobacillus ferrooxidans (today known as Acidithiobacillus ferrooxidans) (Colmer et al., 1950; Kelly and Wood, 2000; Temple and Colmer, 1951). At the beginning of the 1990s only three other species of acidophilic iron-oxidizing bacteria have been isolated indicating the difficulty to isolate acidophilic iron oxidizers (Brierley, 1978; Clark and Norris, 1996; Golovacheva et al., 1992; Hippe, 2000; Markosyan, 1972). Two main factors complicate the isolation of acidophilic iron oxidizers. An optimal gelling agent had to be established, since many ironoxidizing bacteria are autotrophic and sensitive to organic compounds (e.g. pyruvate, galacturonic acid, citric acid, primary alcohols, peptone, yeast extract, and sugars) (Frattini et al., 2000; Harrison, 1984; Kelly, 1971). The concentration of organic compounds occurring in agar was minimized by purification and separate sterilization of agar (Manning, 1975) or by substitution of agar by alternative gelling agents like agarose, silica gel, or gelrite (Harrison, 1984; Khalid et al., 1993; Leathen et al., ⁎ Corresponding author. Tel.: +49 3731 394015; fax: +49 3731 393012. E-mail address: [email protected] (J.S. Tischler). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.07.027

1956; Mishra et al., 1983). The presence of acidophilic heterotrophic bacteria facilitates the growth of A. ferrooxidans as well, since such bacteria metabolize organic compounds toxic for acidophilic iron oxidizers (Harrison, 1984). Butler and Kempton (1987) observed an enhanced growth of A. ferrooxidans on solid ISP (iron–salts–purified agar) medium by inoculating it with a heterotrophic acidophile. The plating efficiency was increased further by the overlay-plate technique described by Johnson and McGinness (1991). In contrast to previously described approaches, apart from A. ferrooxidans also L. ferrooxidans grew on overlay plates (Johnson and McGinness, 1991). Secondly, the chemical composition of the medium used had to fit to growth conditions of the iron oxidizers. Since A. ferrooxidans was isolated for the first time using sterilized natural acidic mine water (Colmer et al., 1950), the chemical composition of the media had been adapted to the chemistry of mine waters (Leathen et al., 1951). Leathen et al. (1951) proposed a base medium containing (NH4)2SO4, KCl, K2HPO4, MgSO4, Ca(NO3)2, and FeSO4 and A. ferrooxidans was isolated using this medium (Leathen et al., 1956). By increasing the salt and iron concentrations Silverman and Lundgren (1959) designed the 9 K medium, which since then has extensively been used for the isolation and cultivation of A. ferrooxidans and L. ferrooxidans (Markosyan, 1972; Silverman and Lundgren, 1959). Johnson and Hallberg (2007) designed the iFeo medium by adding Na2SO4 to the basic compounds of 9 K and by reducing the compound concentrations, except of MgSO4 and Ca(NO3)2. Besides A. ferrooxidans and L. ferrooxidans various new acidophilic iron-

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oxidizing bacteria (e.g. “Ferrovum myxofaciens”, Ferromicrobium acidiphilum, Ferrithrix thermotolerans, L. ferriphilum) have been isolated in recent years using the iFeo medium and the overlay-plate technique (Hallberg et al., 2006; Hedrich et al., 2009; Johnson et al., 2009; Johnson and Hallberg, 2007; Liu et al., 2007; Zhang et al., 2010). However, the distribution of taxa isolated on iFeo medium did not correlate with the composition of the microbial community of the water sample (Hedrich et al., 2009). Although several methods and media have been proposed, the isolation of some acidophilic iron-oxidizing bacteria is still difficult. For instance, the isolation of the recently detected iron-oxidizing bacterium “F. myxofaciens” (formerly: “Ferribacter polymyxa”) has only been reported twice (Hallberg et al., 2006; Hedrich et al., 2009). Using cultureindependent methods, besides “F. myxofaciens” relatives of Gallionella were detected in high percentages in mine-impacted waters, but the isolation of the Gallionella relatives has not yet been successful (Bruneel et al., 2006; Hallberg et al., 2006; Heinzel et al., 2009a; Kimura et al., 2011; Tan et al., 2009). Since Gallionella spp. are known so far as neutrophilic, microaerophilic iron-oxidizing bacteria (Emerson and Moyer, 1997; Hanert, 1968), Hallberg (2010) suggested the Gallionella relatives detected in mine waters as a new species of acidophilic iron-oxidizing bacteria. Furthermore, presumed “isolated” iron-oxidizing bacteria often turned out to be contaminated with acidophilic heterotrophs or other iron oxidizers (Arnold, 2010; Harrison, 1981; Harrison, et al., 1980; Johnson and Kelso, 1983; Lobos et al., 1986). The majority of known 16S rRNA gene sequences of “Ferrovum” spp. and Gallionella relatives originated from culture-independent studies (e.g. García-Moyano et al., 2012; Hallberg et al., 2006; Hao et al., 2010; Heinzel et al., 2009a; Kimura et al., 2011; Tan et al., 2009; Ziegler et al., 2009). To characterize these putative new species of acidophilic ironoxidizing bacteria, the isolation of representatives of the species is required. Since relatives of “F. myxofaciens” and of Gallionella dominate the microbial community of a mine water treatment plant located at the opencast pit Nochten (Lusatia, Germany) (Heinzel et al., 2009a, 2009b), it was attempted in this study to isolate relatives of both taxa from mine water of the treatment plant using a new cultivation medium. A portion of this work has been presented previously (Kipry et al., 2013). 2. Material and methods 2.1. Sampling Samples were taken from a mine water treatment plant located at the opencast pit Nochten in 2011 (April, August) and in 2012 (February, May) (Glombitza et al., 2007). A second sample site was a puddle in the area of Wilhelm Stehender Nord in the mine Reiche Zeche (Freiberg, Germany). Detailed positions of the sampling sites and chemical parameters of the water samples are given in the Supplementary material (Fig. A1, Table A1). 2.2. Media Isolation of iron-oxidizing bacteria was performed using the overlayplate technique according to Johnson and McGinness (1991). As cultivation media iFeo, described by Johnson and Hallberg (2007), and artificial pilot-plant water (APPW) were used (Table 1). APPW medium was designed based on the chemical composition of the original groundwater of the treatment plant and contained 0.022 g/l Na2SO4, 0.024 g/l K2SO4, 3.24 g/l MgSO4 ∗ 7 H2O, 0.515 g/l CaSO4 ∗ 2 H2O, 0.058 g/l NaHCO3, 0.010 g/l NH4Cl, 0.014 g/l Al2(SO4)3 ∗ 18 H2O, 1.344 g/l FeSO4 ∗ 7 H2O, 0.023 g/l MnCl2 ∗ 4 H2O, and 0.0004 g/l ZnCl2. The pH of both media used was 3.0. The concentration of ferrous iron, phosphate, and ammonium was adjusted in three separate modifications of APPW to those of iFeo. The two water samples that originated from the treatment plant in 2011 were only plated on APPW and incubated at 20 °C. Approximately

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Table 1 Detailed composition of the cultivation media iFeo and APPW. Ion

3+

Al BO3− 3 Ca2+ − Cl Co2+ Cr3+ Cu2+ Fe2+ HCO− 3 K+ 2+ Mg Mn2+ MoO4− Na+ NH+ 4 Ni2+ − NO3 PO3− 4 SeO2− 4 SO42− VO− 3 WO2− 4 2+ Zn

Concentration [mM] in iFeo

APPW

0 0.01 0.06 0.67 0.004 0.002 0.004 25 0 1.04 2.03 0.004 0.002 0.94 6.80 0.004 0.06 0.37 0.003 31 0.001 0.0003 0.04

0.04 0 3 0.43 0 0 0 5 0.96 0.28 13.2 0.12 0 1 0.19 0 0 0 0 21.4 0 0 0.003

200 colonies were picked after 10 to 20 days. Representatives of regrown colonies were analyzed by PCR using specific primer pairs and 16S rRNA genes were sequenced. The handling of the water samples that originated from the treatment plant in 2012 was done by two persons. 100 μl of the water samples was plated onto overlay plates of each medium and incubated at 20 °C. For both water samples approximately 100 colonies of each medium and per person were picked after 10 to 20 days. Depending on growth of the colonies after picking 14 to 95 colonies per person were analyzed by PCR using specific primer pairs. The detailed results of each person are given in the Supplementary material. To test the colonies for contamination by heterotrophic bacteria, material from the colonies was cultivated on fructose–yeast extract-medium (FYe) and medium used for the cultivation of Acidiphilium strain SJH (called herein: medium SJH) (Johnson and Hallberg, 2007). The water sample of the second sampling site, Wilhelm Stehender Nord (mine Reiche Zeche), was only plated on APPW and iFeo and incubated at 20 °C. 47 colonies of each medium were analyzed as described above by one person.

2.3. Identification 2.3.1. PCR with specific primers A lysate of each colony was prepared as DNA template for the subsequent PCRs to identify the various colonies according to Okibe et al. (2003). The lysates were tested by amplification of the bacterial 16S rRNA gene using universal bacterial primers 27f and 1387r (Eurofins MWG Operon, Table A2 of the Supplementary data). Afterwards the lysates were investigated by PCR with primers specific for “Ferrovum” spp., Gallionella relatives, Acidithiobacillus spp., and Acidiphilium spp. (Eurofins MWG Operon, Table A2 of the Supplementary data). Primers for Acidithiobacillus spp. theoretically detected A. ferrooxidans, A. ferrivorans, A. thiooxidans, A. caldus, A. albertensis, and A. cuprithermicus according to Escobar et al. (2008) and own sequence comparisons (data not shown). However, no reduced sulfur compounds or elementary sulfur was added to the media and thus only A. ferrooxidans and A. ferrivorans should be able to grow on media used. The concentration of the components needed for the different PCRs and the corresponding PCR program are described in the Supplementary material (Table A3).

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2.3.2. T-RFLP and DNA sequencing The composition of the microbial community of water samples plated and of selected colonies was analyzed by T-RFLP. 16S rRNA genes of cultures obtained from water samples of 2011 were sequenced. For both approaches the bacterial 16S rRNA gene was amplified in three separate PCR reactions using the primers 27f and 1387r or Fmy_1494r (Eurofins MWG Operon, 5′-CTTCACCCCAGTCATGAAT-3′, Mühling, pers. communication) according to Section 2.3.1. For T-RFLP application Cy5-labeled 27f-primer (Eurofins MWG Operon) was used instead of unlabeled 27f-primer in the PCR mixture for amplification of the bacterial 16S rRNA gene. PCR products were pooled and purified using the SureClean Plus kit (Bioline). For T-RFLP analyses purified 16S rRNA gene (120 ng) was digested using 1 U of the restriction enzymes AluI, HaeIII, or HhaI (Fermentas), respectively, at 37 °C over night. Fragments were separated and analyzed by the Beckman Coulter CEQ8000 genetic analysis system as described by Heinzel et al. (2009b). TRFs detected were assigned using the TRF database of Heinzel et al. (2009b). The relative abundance of TRF signals was calculated according to Hallberg et al. (2006). 16S rRNA genes were sequenced using the GenomeLab™ DTCS Quick Start Kit (Beckman Coulter, Inc.). For that, purified PCR product (125 ng) was incubated at 96 °C for 1 min. Afterwards the mixture was stored on ice and 0.5 μM primer (27f, 1387r, 643f, or Fmy_1494r, respectively) and 0.4× Quick Start MasterMix (Beckman Coulter, Inc.) was added. For sequencing the following PCR program was used: 30 cycles 20 s at 94 °C, 20 s. at 55 °C and 4 min at 60 °C. The PCR product was purified using the Agencourt CleanSEQ® kit (Beckman Coulter, Inc.) according the product information and analyzed with the Beckman Coulter CEQ8000 genetic analysis system. Sequences obtained for the forward and reverse primers were combined using the Staden Package software and subsequently compared with sequences available in the GenBank database using BLAST (http://blast.ncbi.nlm.nih.gov/Blast. cgi). 16S rRNA gene sequences obtained have been deposited in the GenBank database under accession numbers KC677641 to KC677661. 2.4. Phylogenetic analysis 16S rRNA gene sequences obtained in this study and 16S rRNA gene sequences of nearest relatives obtained from the GenBank database were analyzed phylogenetically using the software MEGA5 (Tamura et al., 2011). Based on the alignment of 16S rRNA gene sequences performed with ClustalW the phylogenetic tree was calculated with the Maximum-Likelihood method. Phylogeny was tested by the Bootstrap method using 1000 replications. 3. Results 3.1. Microbial community of water samples The different water samples taken were investigated before plating concerning the composition of the respective microbial community. T-RFLP analyses revealed the dominance of “Ferrovum” spp. in all five samples. In the water sample of the Reiche Zeche “Ferrovum” spp. was detected as the only iron-oxidizing bacterium. The microbial community of the water samples taken from the treatment plant consisted mainly of “Ferrovum” spp. (79–99%). In the water samples of April 2011 and February 2012 16% and 19% Gallionella relatives, respectively were detected. 5% Acidithiobacillus spp. were found in the water sample of May 2012. In four of the five samples up to 5% of the peak areas in T-RFLP could not be assigned to a known bacterium (Table 2).

Table 2 Composition of the microbial community of the mine water of the oxidation basin of the pilot plant and of the area Wilhem Stehender Nord (mine Reiche Zeche) before plating. Sampling site

Date of plating

Pilot plant Pilot plant Pilot plant Pilot plant Reiche Zeche

11.04.11 25.08.11 10.02.12 31.05.12 26.04.12

Composition of microbial communitya (%) “Ferrovum” spp.

Gallionella relatives

Acidithiobacillus spp.

Not identified

84 99 79 90 95

16 ± 1 – 19 ± 0 – –

– – – 5±5 –

– 1 3 5 5

± ± ± ± ±

1 1 3 0 3

± ± ± ±

1 3 5 3

a The microbial community was analyzed by T-RFLP using the restriction enzymes AluI, HaeIII, and HhaI. The relative abundance is shown as the mean values obtained with the three enzymes; ± designates deviation obtained by use of the different enzymes.

Acidiphilium spp. Molecular analyses revealed the presence of at least two genera in most of the colonies obtained. To get information about the dominating taxa on the various media, the occurrence of each taxon in the colonies investigated from the various media was summed up (Table 3). Presumed pure cultures were transferred from the solid media to the corresponding liquid media to obtain biomass to test the cultures for contamination with heterotrophs by using the media FYe and SJH. Unfortunately, not all cultures grew in liquid media after the transfer from the solid media and thus some could not be tested regarding a heterotrophic contamination. All cultures inoculated in the media for heterotrophs showed turbidity after few days indicating a contamination of these cultures with heterotrophic acidophiles. 3.3. Growth on APPW and iFeo Comparison of growth of the iron-oxidizing Acidithiobacillus spp., “Ferrovum” spp., and Gallionella relatives on the two different solid media iFeo and APPW showed that the microbial growth was favored on APPW in general and thus more colonies were obtained from APPW (Table 3). Molecular analyses revealed that the growth of Acidithiobacillus spp. was favored on iFeo. Up to 100% of analyzed colonies from this medium contained iron-oxidizing Acidithiobacillus spp. independent if the water sample originated from the pilot plant or from the Reiche Zeche. In colonies obtained on iFeo from the water samples of the pilot plant “Ferrovum” spp. (16–63%) and Gallionella relatives (8%) were detected as well (Table 3). In contrast, the majority of colonies obtained on APPW plates inoculated with water samples of the pilot plant contained “Ferrovum” spp. (78%) and Gallionella relatives (up to 33%), whereas Acidithiobacillus spp. were found in only 10–34% of colonies from APPW (Table 3). Thus, the dominance of “F. myxofaciens”-related species in the mine water of the treatment plant as detected by T-RFLP was reflected in the distribution of taxa occurring in colonies obtained on APPW plates inoculated with mine water of the treatment plant. The observation of Gallionella relatives and Acidithiobacillus spp. on both media used confirmed the presence of these species in the water samples, although they were not found or found in minor percentages by T-RFLP (Table 2). Cultures originating from colonies on APPW plates inoculated with water of Reiche Zeche were dominated by iron-oxidizing Acidithiobacillus spp. (85%). “Ferrovum” spp. and Gallionella relatives were detected in 55% and 2%, respectively, of colonies obtained on this medium. Neither “Ferrovum” spp. nor Gallionella relatives were found in colonies obtained from this source on iFeo plates (Table 3). 3.4. Effect of iron, phosphate, and ammonium concentrations

3.2. Analyses of colonies Colonies picked from the various media inoculated with water samples of 2012 were analyzed by PCR with primers specific for Acidithiobacillus spp., “Ferrovum” spp., Gallionella relatives, and

The iron concentration of APPW was increased to 25 mM (giving APPW-Fe), i.e. the iron concentration of iFeo, to investigate the influence of iron on the growth of the iron-oxidizing bacteria. The analyses of colonies obtained on APPW-Fe revealed relatives of “F. myxofaciens”

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Table 3 Taxonomic composition of all colonies obtained according the analysis using PCR and T-RFLP. The water samples originated from the pilot plant in February 2012 (PP1) and May 2012 (PP2) and from the area Wilhelm Stehender Nord (mine Reiche Zeche — RZ). The percentages of colonies of this respective composition of all cultures obtained [%] are shown. Disaggregated results of each person who performed the analysis are given in the Supplementary material. Taxa occurring in colony

Percentage of the colonies with respective taxonomic composition on plates with iFeo

Presumable pure cultures “Ferrovum” Gallionella rel. Acidithiob. Acidiphilium Colonies containing two taxa “Ferrovum” Gallionella rel. “Ferrovum” “Ferrovum” Gallionella rel. Gallionella rel.

Acidithiob. Acidiphilium Acidithiob. Acidithiob.

Colonies containing three or four taxa “Ferrovum” Gallionella rel. “Ferrovum” Acidithiob. Gallionella rel. Acidithiob. “Ferrovum” Gallionella rel. Acidithiob. No taxon identified “Ferrovum” containing colonies overall Gallionella relatives containing colonies overall Acidithiobacillus containing colonies overall Acidiphilium containing colonies overall Total number of colonies investigated

Acidiphilium Acidiphilium

Acidiphilium Acidiphilium Acidiphilium Acidiphilium

APPW-NH4

APPW-PO4

PP1

PP2

RZ

PP1

APPW PP2

RZ

PP1

APPW-Fe PP2

PP1

PP2

PP1

PP2

– – 23 –

1 – 2 10

– – – –

– 1 1 9

1 – – 11

– – – –

1 – – 25

13 – 4 19

2 – – 35

3 1 3 3

– 2 – 52

– – – 16

– – – – – 61

– – 39 – 2 24

– – – – – 100

– – 47 – 1 3

– – 47 – 12 –

– – 15 – – 45

– – 67 – – 1

– 1 38 – 1 5

– – 48 1 1 –

3 – 28 1 3 10

– – 2 – 17 12

– – 14 – 14 7

– 16 – – – 16 – 100 77 31

– 17 – 6 – 63 8 49 97 119

– – – – – – – 100 100 47

1 29 – 1 8 78 4 34 90 156

19 8 – 2 – 77 33 10 99 144

– 38 – 2 – 55 2 85 100 47

– 4 – – 2 72 – 5 97 139

– 15 – – 5 67 1 25 78 120

– 6 – 1 7 57 3 8 91 107

5 30 – – 12 69 13 44 81 115

– 2 2 – 10 4 21 16 88 42

44 3 – 1 – 62 59 11 100 86

Data of February 2012 were presented in previous work by Kipry et al. (2013).

in approximately 70% and iron-oxidizing Acidithiobacillus spp. in 5–25% of analyzed colonies. Gallionella relatives were found in only 1% of colonies originating from this modified APPW medium (Table 3). In addition, the influence of the essential nutrient phosphate on the growth of the iron-oxidizing bacteria was investigated. For that, the phosphate concentration of APPW was adapted to the phosphate concentration of iFeo (giving APPW-PO4). In contrast to the other media used, microbial growth was inhibited by the elevated phosphate concentration on APPW-PO4 in general and thus only 42 and 86 colonies, respectively, could be analyzed (Table 3). As on APPW relatives of “F. myxofaciens” and of Gallionella were the dominating iron-oxidizing bacteria in cultures originating from colonies from APPW-PO4 plates. 21–59% of colonies obtained from APPW-PO4 contained Gallionella relatives and “Ferrovum” spp. were detected in 4–62% of colonies obtained from this medium. Acidithiobacillus spp. was found in 11–16% of colonies obtained on APPW-PO4 (Table 3). An essential nutrient required by many iron-oxidizing bacteria is ammonium (Tuovinen et al., 1971, 1979). To investigate a possible limitation by ammonium on APPW the ammonium concentration was increased to 6.8 mM (APPW-NH4) as used in iFeo. Colonies which obtained from APPW-NH4 contained “Ferrovum” spp., relatives of Gallionella, and iron-oxidizing Acidithiobacillus spp. Relatives of “F. myxofaciens” were dominating and were detected in 57–69% of colonies obtained from APPW-NH4. Gallionella relatives and Acidithiobacillus spp. were found in 3–13% and 8–44%, respectively, of colonies originated from APPW-NH4 (Table 3). 3.5. Phylogenetic analysis In the isolation studies of 2011, 21 colonies were obtained from APPW plates inoculated with water samples of the pilot plant. After initial PCR-analyses using taxon-specific primers (Table A6 of the Supplementary data), the bacterial 16S rRNA genes were sequenced. The phylogenetic analysis of the sequences revealed “F. myxofaciens” P3G as the closest cultivated relative of 20 cultures (16S rRNA gene

sequence identity: 95–99%) and G. ferruginea and G. capsiferriformans ES-2 as the closest cultivated relatives of one culture (16S rRNA gene sequence identity: 96%). Based on the distance matrix 16S rRNA gene sequences related to “F. myxofaciens” P3G were clustered, if the distance was less than 0.03. So, five clusters within the genus “Ferrovum” were identified (Fig. 1).

4. Discussion To improve the isolation and cultivation of relatives of the novel iron-oxidizing bacterium “F. myxofaciens” and of acidophilic Gallionella relatives the new medium APPW was designed based on the chemical composition of mine waters of the open pit Nochten. Iron-oxidizing bacteria and heterotrophic acidophiles grew on this medium resulting in cultures of “Ferrovum” spp., Gallionella relatives, Acidithiobacillus spp., and/or Acidiphilium spp. (Table 3, Fig. A2 and Fig. A3 of the Supplementary data). Simultaneous appearance of various ironoxidizing bacteria and/or heterotrophic acidophiles in one culture as well as changes in the microbial composition of cultures have been reported previously (Arnold, 2010; Bhattacharyya et al., 1992; Das et al., 1989; Lobos et al., 1986; Mackintosh, 1978). A function of acidophilic heterotrophic bacteria, in particular Acidiphilium spp., is obviously that in culture they detoxify the environment for iron oxidizers due to the metabolism of organic compounds toxic for acidophilic iron-oxidizing bacteria (Harrison, 1984). Heterotrophs may also increase the local availability of CO2 and thus enhance the growth of iron-oxidizing bacteria (Kermer et al., 2012; Mosler et al., 2012). Due to the detection of acidophilic heterotrophs in acid mine waters (Hallberg, 2010; Heinzel et al., 2009a; Johnson and Hallberg, 2003) and observed simultaneous growth of iron oxidizers and heterotrophs on agar(ose) plates (Fig. A2 and Fig. A3 of the Supplementary data; Battaglia et al., 1994; Johnson et al., 1987) the observed contaminations could originate from the water sample used. However, at least in some cases the origin of the contamination of iron-oxidizing bacteria with

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Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences of cultures obtained in this study from APPW, i.e. entries starting with JA or PN-J, in comparison to entries of GenBank database. 16S rRNA gene sequences obtained in this study are in bold. 16S rRNA gene sequences originating from samples of the treatment plant are indicated by an asterisk. The tree was calculated using the Maximum-Likelihood algorithm and the phylogeny was tested by bootstrapping and the percentage of trees in which the associated taxa clustered together in the Bootstrap test (1000 replicates) are shown. 16S rRNA gene sequences of A. ferrooxidans ATCC 23270 and A. ferrivorans NO-37 were used as outgroup. GenBank accession numbers are in parentheses.

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Acidiphilium spp. could also be Acidiphilium strain SJH used in the underlayer of the overlay plates. Relatives of “F. myxofaciens” and of Gallionella were detected in higher abundances on APPW than on iFeo obviously resulting from the chemical composition of the medium, which is similar to the one of the water of the treatment plant. Consequently, the medium mimics the “natural” conditions of “F. myxofaciens” and Gallionella relatives dominating the microbial community of the treatment plant (Table 2; Heinzel et al., 2009a, 2009b). The chemical composition of APPW seems to be more suitable for the growth requirements of “Ferrovum” spp. and Gallionella relatives in general, since colonies originating from APPW inoculated with water taken at the second sampling site (area Wilhelm Stehender Nord, mine Reiche Zeche) contained “Ferrovum” spp. and Gallionella relatives as well (Table 3). The occurrence of “Ferrovum” spp. and Gallionella relatives in minor percentages on iFeo suggested an advantage of Acidithiobacillus spp. or inhibitory or missing components of iFeo for “Ferrovum” spp. or Gallionella relatives, respectively. Comparison of the chemical composition of iFeo and APPW revealed obvious differences in the chemistry of both media used (Table 1). The ammonium concentration is significantly higher in iFeo (6.8 mM) than in APPW (0.2 mM) hypothesizing an ammonium starvation of bacterial growth on APPW. However, traces of ammonium, which are present in distilled water and in chemicals used or absorbed by the acidic medium from the atmosphere, are sufficient to ensure some iron oxidation (Tuovinen et al., 1971, 1979). Iron oxidation of A. ferrooxidans has been reported to be limited at ammonium concentrations below 0.2 mM (Tuovinen et al., 1979). Thus, the ammonium concentration of APPW and a similar relative abundance of iron oxidizers in colonies obtained on APPW and on APPW-NH4 indicated no limitation of the growth of the iron-oxidizing bacteria on APPW by ammonium. One significant difference in the chemical composition of APPW and iFeo is the ferrous iron concentration, which is fivefold higher in iFeo than in APPW. Growth of iron-oxidizing Acidithiobacillus spp. including A. ferrooxidans is obviously stimulated by 25 mM ferrous iron applied in iFeo and APPW-Fe, since Johnson et al. (1987) reported a ferrous iron concentration of 36 mM as optimal concentration for growth of A. ferrooxidans on solid medium. Lower abundances of “Ferrovum” spp. on APPW-Fe compared to APPW indicated a slight inhibition of this species at elevated iron concentrations. The significant inhibition of growth of Gallionella relatives on APPW-Fe and iFeo is probably caused by the increased ferrous iron concentration, since neutrophilic species of Gallionella like G. ferruginea are typically found in habitats characterized by low concentration of iron (5–25mg/l) (Hanert, 1992). Phosphate, an essential nutrient for all living organisms, was not added intentionally to APPW thus probably resulting in a limitation of microorganisms by phosphate on APPW. Gallionella relatives seem to be limited by phosphate on APPW, since they were detected in highest percentages in colonies obtained on APPW-PO4 (phosphate concentration 0.4 mM) compared to the other media used in this study. 0.5 mM phosphate has been proposed as optimal phosphate concentration for growth and iron oxidation of neutrophilic Gallionella spp. (Hanert, 1992). In contrast, iron-oxidizing Acidithiobacillus spp. and “Ferrovum” spp. were obviously inhibited by addition of phosphate to APPW plates. Inhibited growth of A. ferrooxidans on plates with elevated phosphate concentrations has been observed previously (Johnson et al., 1987; Manning, 1975; Visca et al., 1989). Manning (1975) and Mishra et al. (1983) assumed that the phosphate content added by impurities of gelling agent used is sufficient for microbial growth. However, the inhibition observed is not trivial, since it is known that in liquid cultures additional phosphate enhanced the growth of A. ferrooxidans and “F. myxofaciens” (Seeger and Jerez, 1993; Tischler et al., submitted for publication; Tuovinen et al., 1971). These differing observations on solid and liquid media indicate a different effect of elevated phosphate concentrations on the growth of iron-oxidizing bacteria resulting from the incubation conditions, but it is not clear so far what the exact reason

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is. One explanation could be a different precipitation of iron-phosphates in solid and liquid media, since the iron-phosphate formation is determined thermodynamically and kinetically. Besides the chemical differences of APPW and iFeo investigated experimentally, also other ion concentrations varied between both media. For instance, iFeo contains 0.06 mM calcium, while 3 mM calcium is used in APPW. Growth studies performed by Tuovinen et al. (1971) revealed no influence of the calcium concentration on the growth of A. ferrooxidans. In contrast, the low calcium concentration of iFeo may have caused the poor growth of Gallionella relatives on iFeo, since Hallbeck and Pedersen (2005) proposed an essential calcium concentration for G. ferruginea of 0.6 mM. Consequently, Gallionella relatives may be limited by the calcium concentration of iFeo. The influence of the calcium concentration on growth of “F. myxofaciens” has not been investigated up to now. Furthermore, APPW and iFeo differ in the concentration of trace elements like copper, chromium, manganese, molybdenum, or nickel. However, concentrations used are lower than the minimum inhibitory concentrations for “F. myxofaciens” and A. ferrooxidans (Cabrera et al., 2005; Hedrich, 2011) and thus an inhibitory effect of trace elements used in iFeo should be negligible for the growth of “Ferrovum” spp. on this medium. The detailed phylogenetic analysis of the 16S rRNA gene sequences related to those of “F. myxofaciens” indicated the existence of five putative species within the genus “Ferrovum” (Fig. 1). In the mine water treatment plant at least three species of “Ferrovum” (cluster 1, 2, 4) are present (Fig. 1; Hedrich et al., 2009). First physiological analyses between the designated type strain “F. myxofaciens” P3G and strain EHS6 (both cluster 1) on the one hand, and strain JA12 (cluster 4) on the other hand suggested that the differences on the 16S rRNA level correlate with physiological differences (data not shown). Although we did not succeed to get pure cultures of “Ferrovum” spp. and Gallionella relatives in this study, a medium was established, which prevents an overgrowth of both taxa mentioned by Acidithiobacillus spp. as observed on iFeo. Using APPW, strains related to two different “Ferrovum” clusters and strains of acidophilic Gallionella relatives were cultivated on overlay plates for the first time and first indications for cultivation conditions of these species were obtained. In future, further characterization studies of the new “Ferrovum” strains and of the Gallionella relatives are necessary to confirm the assumption of new species of “Ferrovum” and Gallionella. Acknowledgment We thank the BMBF for funding the research projects SURFTRAP (number: 03G0714B) and SURFTRAPII (number: 03G0821B) within the R&D-program GEOTECHNOLOGIEN. R. J. Jwair is grateful to the Federal State of Saxony for a PhD scholarship. We are thankful to Klaus Grund for the support with sampling in the mine Reiche Zeche. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2013.07.027. References Arnold, J., 2010. Screening for genes and corresponding proteins. Ferrovum myxofaciens"related strains using genetic and proteomic methods. Diploma thesis, TU Bergakademie Freiberg, Freiberg. Banks, D., Younger, P., Amesen, R.-T., Iversen, E., Banks, S.B., 1997. Mine-water chemistry: the good, the bad and the ugly. Environ. Geol. 32 (3), 157–174. Battaglia, F., Morin, D., Garcia, J.-L., Ollivier, P., 1994. Isolation and study of two strains of Leptospirillum-like bacteria from a natural mixed population cultured on a cobaltiferous pyrite substrate. Antonie Van Leeuwenhoek 66 (4), 295–302. Bhattacharyya, S., Das, A., Chakrabarti, B.K., Banerjee, P.C., 1992. A comparative study of characteristic properties of Thiobacillus ferrooxidans strains. Folia Microbiol. 37 (3), 169–175.

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