UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 Analysis of swamp samples from the heaps of Germignies and Argales (Nord-­‐
Pas-­‐de-­‐Calais, France). Are Thiothrix bacteria present? Senet C1, Konk A1, Billon G3, Gillan DC2* (1) Master student (BBMC) at UMONS, Mons, Belgium -­‐ (2) Proteomic and Microbiology Unit, UMONS, Mons, Belgium -­‐ (3) Université de Lille 1, Sciences & Technologies, Géosystèmes, Villeneuve d'Ascq, France. Abstract Bacteria are good biomarkers to characterize the composition of an environment. In this study, whitish deposits forming on leaves found in a pond at the basis of two heaps of coal (heaps of Germignies and Argales, Nord-­‐Pas-­‐de-­‐Calais, France) were analyzed. The water of the swamps was neutral and was very rich in sulfate. An observation of the whitish deposits under the differential interference contrast microscope revealed that filamentous bacteria were present. These bacteria are Thiothrix-­‐like multicellular bacteria and featured many sulfur inclusions. These sulfur inclusions disappeared after an ethanol treatment. LTH medium was used to try to isolate these Thiothrix-­‐like bacteria on pure culture, but no colonies developped. The bacterium Cupriavidus necator (Ralstonia eutropha) was also searched in the sediments using the PCR approach ans specific primers. However, it was not found. 1. Introduction Thiothrix spp are the most frequent sulfide-­‐oxidizing filamentous bacteria in sulfur-­‐rich environments. The cell, which is 1.2–2.5 µm long with a diameter of 0.7–1.5 µm, form multicellular filaments that move by gliding (Garrity et al. 2005). Thiothrix is aerobic or microaerobic, and is able to form a structure called rosettes. In the presence of sulfide the cell accumulates sulfur granules that can be observed under a microscope (Garrity et al. 2005). The presence of Thiothrix supposes the presence of H2S in the environment, a reduced form of sulfur that is generally produced by anaerobic sulfate-­‐reducing bacteria. 10 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 In this study, we analyzed two coal mining heaps : the heap of “Germignies Nord” (Fig S2) and the heap of “Argales” (Fig S1). The water that accumulates at the basis of the heaps contained high levels of sulfate (G. Billon, pers. com.) as well as white deposits. The aim of this research was to sample these white deposits and look for Thiothrix bacteria. The heap of Germignies is located at Flines-­‐lez-­‐Raches and Marchienne in the Nord-­‐
Pas-­‐de-­‐Calais region. The coal mining is the source of the shale heap which covers 135 hectares. The heap of Argales is one of the biggest pile of the Nord-­‐Pas-­‐de-­‐Calais region located in the township of Rieulay. Mainly composed by coal and others industrial wastes, it extends over 140 hectares of grassland and peat bogs. We sampled in the first site pieces of leaves, water and sediments in the stream with a whitish deposit at the foot of Germignies heap (Fig. S3). In the second site (Fig. S4), just pieces of leaves were sampled in a watering place. Microscopica observations were carried out to detect filamentous bacteria as well as cultivation on Petri dishes to count and isolate Thiothrix bacteria. Then, we performed a DNA extraction followed by a PCR with several primers. 2. Materials and Methods 2.1. Sampling Samples were collected in 2 different places : 12 samples were collected in the swamp at the bottom of the heap of Germignies (Fig S3) and 6 others samples were collected in another swamp at the bottom of the heap of Argales (Fig S4). In the first place, 3 spots have been analyzed. In the first one, samples T1, T2 and T3 were collected and correspond to whitish leaves collected from the swamp. Samples T4, T5, T6 correspond to water collected next to the leaves. In the second spot, samples T7, T8 and T9 correspond to sediments and samples from the third spot T10, T11 and T12 correspond to water of the swamp. In the swamp of Argales, only whitish leaves were collected in 2 different spots corresponding to TA1, TA2, TA3 and TA4, TA5, TA6. A pH meter was used to measure the pH of water samples T7, T9, TA2 and TA6. 11 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 2.2. Viable counts A total of 6 samples were used to make viable counts on medium LB (T1, T2, T3, TA1, TA3, TA4). The whitish deposit was scraped from some leaves and placed into 500 L of filtered NaCl 9 g/L. The tubes were briefly vortexed and sonicated for 15 seconds (0.8 cycle, 80%). These 500 µL were used to make dilutions of 10X, 100X and 1000X. 50 µL of these different dilutions were spread onto Petri dishes and incubated at 37°C for 4 days. 2.3. Differential interference contrast microscopy Bacteria were scraped from leaves and placed into 500 µL of filtered NaCl 9g/L. The tubes were vortexed and analyzed under differential interference contrast microscope (10x, 20x, 40x, 100x). To dissolve some of the sulfur inclusions the same procedure was used but ethanol 70% replaced the NaCl solution. 2.4. DNA extraction DNA was extracted from 6 samples (T3, T7, T9, TA2, two samples of water). For the sediments, samples were centrifuged for 3 minutes at 3000 g. For each sample, 250 mg of sediments were used to extract DNA with a Power Soil Kit (MoBio). Whitish deposits were scraped from leaves and placed into 250 µL of Tris-­‐EDTA. DNA was then extracted using a QIA Amp DNA mini kit. Water samples were filtered using a vacuum pump and a filter of 45 µm to concentrate bacteria. The filter was then scraped and the cells were placed into 250 µL of Tris-­‐EDTA. DNA was extracted using the QIA Amp DNA mini kit. DNA of each sample was then quantified using a Nanodrop. 12 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 2.5. Isolation of Thiohrix on medium LTH Samples of bacteria were spread onto lactate thiosulfate HEPES (LTH) with or without vitamins. The medium usually contains lactate but in this study, lactate was replaced by acetate. This culture medium was composed by 250 mg of (NH4)2SO4, 55 mg of K2HPO4, 42 mg of KH2PO4, 50 mg of MgSO4, 367 mg of CaCl2, 1 mg of FeCl3, 1,5 mg of EDTA, 60 mg of acetic acid 99% and 500 µL of trace elements into 500 mL of MilliQ water. This mixture was placed in two Duran bottles in which 3,75 g of Difco Bacto Agar were previously added. Bottles were sterilized by autoclaving at 121°C for 20 minutes. Then, 1 mL of sodium thiosulfate 0,3 % was filtered (0,45 µm) and added in the two bottles. Vitamins (mainly vitamins of the B group) were also filtered using a filter of 0,45 µm and 1,5 mL were injected in one Duran bottle. The final culture medium was poured in Petri dishes. 2.6. PCR-­‐DGGE A fragment of the 16S rRNA gene (ca. 230 bp long) was amplified using 0.25 µL of primer GM5FCl2 and 0.25 µL of 518R primer (Gillan et al. 2005). The Red’y’Star mix kit (Eurogentec) was then used. A total of 8 samples were used : T7, T9, TA2, T3, two samples of water, a positive and negative control. For T7, T9, T3 and TA2 samples, 2 µL of DNA extracts were used and only 1 µL for water samples and positive control. PCR water were added to reach a final volume of 50 µL. The positive control contained 1 µL of pure culture of Cupriavidus necator DNA, and negative control was prepared without DNA. PCR products were checked on agarose (1.5%) gels using GelRed as a stain. DGGE was performed by the protocol described in Gillan et al. 2005. A fragment of the rpoD gene of C. necator was also amplified by PCR using the environmental DNA extracted in the sediments of the pond. 13 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 3. Results 3.1. Differential interference contrast microscopy In the first site (T1, T2 and T3) filamentous bacteria were observed (Thiothrix-­‐like bacteria) as well as diatoms, nanoflagellates and others microeukarya. For the second site (TA1, TA2 and TA6), the same kind of micro-­‐organisms were observed: Thiothrix-­‐like bacteria, a lot of diatoms, nanoflagellates and Beggiatoa-­‐like bacteria (TA6), a bacterium genus in the order of Thiotrichales. Figure 1. Differential interference contrast microscopy. A–E, Thiothrix filments. On A,D,F, diatoms can also be observed. F, a greenish Beggiatoa-­‐like filament. On B & E, sulfur inclusions can be observed in the filaments. On C & D, rosettes can be observed. 14 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 In order to determine if inclusions seen in bacteria were sulfur inclusions, ethanol was used on samples to dissolve these inclusions. Observations on differential interference contrast microscope revealed bleached bacteria (Fig.2). Figure 2. Differential interference contrast microscope. Bacteria seem to have lost their sulfur inclusions after ethanol treatment. 3.2. Viable counts Dilutions were spread on agar plate with LB medium (Fig. 3). After 72 hours of incubation, colonies were already present. The number of colonies obtained for the 1000X dilution are shown in Table 1. Viable counts were higher for the swamp of Argales than for the swamp of Germignies (Table 1). 15 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 Figure 3. Culture of bacteria on Petri dishes. Colonies after 72h of incubation. Dilution x10. Table 1: Number of colonies per square centimeter of leaves. The third column is calculated according to the following steps: 2cm² of leaves were added to 500µL of NaCl. These 500 µL were used to make dilutions: 10X: 10 µL in 90 µL of NaCl, 100X: 1µL in 99 µL of NaCl and 1000x: 1 µL in 999 µL of NaCl. From each dilution, 50 µL were spread in Petri plates. The formula used is: 12 x 20 (50µL from 1000 µL) x 500 (1 µL from 500 µL) = 120 000 bacteria/2cm² of leaves. Thus, there are 60 000 bacteria/cm² of leaves. Cultures (dil. Number of Colonies/cm2 of 1000X) colonies leaves T1 12 60 000 T2 72 360 000 T3 9 45 000 TA1 157 785 000 TA3 149 745 000 TA4 180 900 000 16 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 3.3. DNA extraction and PCR An extraction of DNA was performed on samples T7, T9, T3, TA2 and two samples of filtered water. Two extraction kits were employed for this analysis (see Materials and methods). The concentration of DNA was then quantified by a Nanodrop Biospec. The results are shown in Table 2. The concentrations range from 15 to 48 ng/µL. The ratio 260/280 was around 1,8 which means that DNA is not too much contaminated. For PCR of the 16S rRNA gene, two controls were performed, a positive control (with DNA of Cupriavidus necator) and a negative control (without DNA) to ensure that the PCR mix is functional and no inhibitor was present. After PCR, samples were placed in wells of an agarose gel (1.5%) to separate DNA amplicons. Unfortunately, no result was obtained, even for positive controls. Table 2: Results of DNA concentration in ng/µL and measures of OD in each sample. [Nucleic acid] ng/µl OD 260/280 OD 260/230 OD 260 OD 280 OD 230 OD 320 T7 T9 T3 TA2 water 15,62 1,86 1,72 0,318 0,173 0,187 0,005 16,1 1,91 1,76 0,331 0,178 0,192 0,009 18,66 1,89 1,89 0,37 0,194 0,194 -­‐0,003 17,94 1,94 1,72 0,352 0,177 0,202 -­‐0,007 24,17 1,7 0,83 0,729 0,53 0,826 0,246 24,75 1,74 0,83 0,746 0,536 0,848 0,251 21,7 1,7 0,27 4,132 3,953 5,296 3,698 21,4 1,1 0,57 2,272 2,232 2,594 1,844 48.69 1.75 0.38 2.773 2.356 4.345 1.799 46.51 2.10 1.28 -­‐0.037 -­‐0.524 -­‐0.239 0.967 3.4. Detection of Cupriavidus necator by PCR A second PCR was conducted to detect the presence of the species Cupriavidus necator. This bacterium can be found in both soil and water and thrives most successfully in the presence of millimolar concentrations of several heavy metals. Only sediments were used for DNA extraction. A PCR was then performed with a specific primer targetting the rpoD gene. No PCR amplicons were detected for the three environmental samples. However, in the samples containing the internal control a band corresponding to C. necator was observed. 17 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 Another band was detected in the positive control, and nothing was detected in the negative control. These two controls ensure that the PCR was not impaired and the positive control also indicate that no inhibitors were present. Following these results, we can confirm that Cupriavidus necator is not present in the studied sediments or that the concentration of his DNA was too low to be detected by PCR. Figure 4. PCR performed on sediments to detect the presence of Cupriavidus necator. The two first lanes are the reference scale. Wells 1,2 and 3 correspond to DNA extracts of sediments without internal control. Wells 4, 5 and 6 correspond to DNA extracts of sediments with internal control (Cupriavidus necator). Well 7 contains the positive control with only DNA of Cupriavidus necator. And well 8 is the negative control in which DNA was replaced by water. 18 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 3.5. Analysis of the pH of the water The analysis of the pH revealed that the water of the swamp at the bottom of the two heaps was close to neutrality (Table 3). Sample pH value T7 7.10 T9 7.05 TA2 6.78 TA6 6.84 Table 3. pH analysis of water of the swamps of Germignies and the Argales. Analyses reveal a pH close to neutrality. Discussion and Conclusion In this study, leaves, sediments and water of two swamps at the bottom the heaps of Germignies and Argales were collected to determine the presence of bacteria of the Thiothrix genus. Analyses under the microscope revealed that Thiothrix-­‐like bacteria were present in the two swamps (Fig. 1). The use of ethanol to dissolve sulfur inclusions confirm their nature (Fig. 2) and thus, we may affirm the presence of sulfide-­‐oxidizing filamentous bacteria of the genus Thiothrix. This is also confirmed by the presence of rosettes, a key feature of the genus. The DNA extraction worked successfully but, the first PCR analysis (16S rRNA gene) failed for all the samples including the positive control and the samples with an internal control. We think that this might be due to a pipetting error which prevented the PCR. The second PCR worked successfully (positive controls and internal controls worked) and we may affirm that C. necator is not present in the swamps or it is present but not in a quantity that can allow a detection using PCR. Culture on LTH medium with and without vitamins did not allow us to isolate the genus Thiothrix. This might be due to an incubation time too short or that the medium was not adapted. 19 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 References Garrity GM, Bell JA, Lilburn T (2005) Order V. Thiotrichales ord. nov. In Brenner, Krieg, Staley and Garrity (Editors), Bergey's Manual of Systematic Bacteriology, 2nd Edition, Volume 2 (The Proteobacteria), Part B (The Gammaproteobacteria), Springer, New York, p.131. Gillan DC, Danis B, Pernet P, Joly G, Dubois P (2005) Structure of the sediment-­‐associated microbial communities along a heavy-­‐metal contamination gradient in the marine environment. Appl. Environ. Microbiol. 71:679-­‐690. Other informations Anon (2016). [online] Available at: http://lafraternellelallinoise.olympe.in/TERRIL.pdf [Accessed 5 May 2016]. PNRSE. (2016). Espace Naturel Sensible -­‐ Terril de Germignies à Flines lez Râches-­‐
Marchiennes. [online] Available at: http://www.pnr-­‐scarpe-­‐escaut.fr/contenu-­‐
standard/espace-­‐naturel-­‐sensible-­‐terril-­‐de-­‐germignies-­‐flines-­‐lez-­‐raches-­‐marchiennes [Accessed 5 May 2016]. https://lenord.fr/jcms/preprd1_145455/le-­‐terril-­‐des-­‐argales Supplementary Figures Fig S1. Geographical map of spoil tip of Argales Fig S2. Geographical map of soil tip of Germignies Fig S3. The swamp at the bottom of the spoil tip of Germignies. Fig S4. The swamp at the bottom of the spoil tip of Argales. 20 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 Fig S1. Geographical map of the heap of Argales 21 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 Fig S2. Geographical map of the heap of Germignies 22 UMons Journal of Environmental Microbiology, May 2016, p. 10–23 Vol. 1 Fig S3. The swamp at the bottom of the heap of Germignies. Fig S4. The swamp at the bottom of the heap of Argales. 23