Rapid adaptation of microalgae to extremely polluted waterbodiesfrom uraniummining: an explanation of how the mesophilic organisms can rapidly colonize extremely toxic environments

C. García-Balboaa, B. Baselga-Cerveraa, A. García-Sanchezb, J.M. Igualb, & E. Costas*a

a. Genetica. Facultad de Veterinaria. Universidad Complutense de Madrid. 28040. Madrid. 28040. Spain.

b Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC). PO Box 257. 37071. Salamanca. Spain.

* Corresponding author. E-mail:

Abstract

An outstanding example of the fast adaptation of microalgae to extreme anthropogenically-generated environments (i.e. residual waters from U mining with extremely highlevels U contamination, severe acidity and elevated conductivity)has been discovered in a huge evaporation pond at Saelices mining area as well as in a mining-effluent pool from Villavieja mine (Salamanca. Spain). Although it is usually assumed that extremophile species inhabit these extreme environments, all the microalgae living in these ponds are mesophile species that have developed a very fast adaptation to extreme waters (fifty years, the period from uranium-mining works started in 1960 to the sampling time in 2012 is very short from a bio-geologycal point of view). Experiments have proven that only a single, rare, spontaneous mutation is necessary to produce the adaptation to the extreme contamination in Saelices evaporation pond. In contrast, adaptation to a Villavieja mining effluent pool (withhigher content ofdissolved uranium) was only possible after the recombination subsequent to sexual mating, because adaptation requires more than one mutation. Microalgae living in the extreme ponds of residual waters from U mining could be the descendants of mutants with changes on a single-gene or few genes that confer a large adaptive value under extreme contamination.

Key words: adaptation, extreme environment, microalgae, mutation, uranium-mining, recombination.

1. Introduction

Through photosynthesis, phytoplankton produces around half of the Earth's atmosphere oxygen, driving the 'biological pump' that fixes 100 million tonnes of carbon dioxide per day (Falkowski & Raven, 1997; Schiermeier, 2010). As a consequence of human activities the quantity of phytoplankton on Earth has decreased since 1950 (Boyce et al., 2010). Microalgae biomass concentration apparently went down to 40%, probably in as a consequence to global change (Boyce et al., 2010). Behrenfeld et al. (2006) also observed a significant reduction in phytoplankton productivity due to anthropogenic activities. Understanding the causes of phytoplankton decline is a relevant topic.

Water pollution is an important cause of the phytoplankton decline. Consequently, the effect of anthropogenic contaminants on microalgae and cyanobacteria has been studied in detail (e.g. Ramakrishman et al., 2010).

An alternative approach is to study the mechanisms that allow adaptation of phytoplankton to anthropogenic pollution. Adaptability of microalgae to contaminated environments is very relevant to understand the evolutionary ecology of phytoplankton under anthropogenic global change.

Accordingly, here we studied adaptation of microalgae in two extremely polluted ponds of residual waters from uranium mining in Salamanca (Spain), which have low pH, high concentrations of dissolved uranium and other heavy metals. From the Bronze Age mining has been a major cause of anthropogenic environmental pollution at this site.

Uranium is a ubiquitous element considered hazardous owing to its radioactivity and toxicity as heavy metal (Markich, 2002). Dissolved U can be found in some natural waters due to the legacy of nuclear accidents, detonation of nuclear weapon, nuclear-fuel production, radioactive waste storage, nuclear industry, mining and also some cases due to natural causes. Concentrations up to 1 µgL-1 U may be detected in the surrounding of mining areas (Kalin et al., 2005).

The main U deposits in Spain occur in fracture areas in shale and schist of the pre-ordovician schist-greywacke complex (Arribas, 1987), that forms part of the paleozoic basement of the Hesperian Massif. The mines of Saelices and Villavieja (Salamanca province) are the most important U deposits with a total volume of 25 million cubic meters, and an ore grade ranging from 400 to 800 mg kg-1 of U. From the end of the last glacial period (approximately 10,000 years ago) to the beginning of mining activities, the natural U background concentration in surface waters of this region was low. The mining activities, including static and dynamic acid lixiviation, were carried out between 1960 and 2000. As a result of these mining activities there are several ponds with a total extension of 30 ha and an average volume of 1 million m3 of polluted water. The U contamination of these ponds is caused by U lixiviation from the mining areas, and waste of shale, schist and mud with U contents up to 200 mg kg-1. These ponds are extreme ecosystems with an intense gradient of U contamination, acidity and radioactivity. Astonishingly, these ponds have abundant microalgae biomass, which provides a fascinating example of rapid adaptation of current mesophilic microalgae species to extreme contamination.

Extreme environments often support microalgae communities living at the limits of their tolerance (Seckbach & Oren, 2007; Costas et al., 2008). For example, in Rio Tinto (Spain), an extremely acidic environment (pH 1.7–2.5) with a very high metal content (> 20 g L–1), eukaryotic microalgae closely related to mesophilic species rather than extremophile lineages contributed at least 60% of the total biomass in the Rio Tinto (Amaral Zettler et al., 2002). It was proposed that adaptation of microalgae to Río Tinto must have occurred (Amaral Zettler et al., 2002; Costas et al., 2007).

Little is known about the mechanisms that allow rapid adaptation of microalgae to these extreme environments. Microalgae are able to survive to short-term unpredictable environmental stress by means of physiological acclimatization as a result of the modification of gene expression (Bradshaw and Hardwick, 1989; Fogg, 2001; Costas et al., 2008). However, when environmental stress exceeds physiological limits, only the occurrence of mutations that into confers resistance can allow adaptation (Sniegowski and Lenski, 1995; Lopez-Rodas et al., 2001; Costas et al., 2001; Flores-Moya et al., 2005; Sniegowski, 2005).

However, it is commonly accepted that genetic adaptation to extreme environments is achieved by selection of several mutations with minor effects following a gradual and slow process involving thousands of years (Gould, 2002). The idea that evolution acts only on long temporal scales has marked the mindset of biologists ever since the Charles Darwin’s axiom ‘natura non facit saltum’ (nature does not take leaps).

In contrast, adaptation of microalgae to acidic uranium and heavy metal polluted waters from uranium mining in Salamanca (Spain), could change this preconception, because adaptation took place rapidly since mining activities started in 1960. There are few known cases where microalgae are able to quickly colonize an extremely polluted environment.

2. Material & Methods

2.1 Sampling sites

A huge evaporation pond at the Saelices mining area and a mining-effluent pool at the Villavieja mining area (Salamanca province, Spain) were studied. Samples were collected during March and April 2012. In each location two different samples were taken from each one of the sites: one of them for physicochemical analysis and the other for the biological research. Water samples were collected in sterile bottles and stored at 4ºC in darkness. Laboratory analysis and phytoplankton identification were performed 4 hours after sampling.

2.2 Chemical analysis

The values of conductivity and pH in the evaporation pond at the Saelices mining area as well as in the mining-effluent pool at the Villavieja mining area were determined using a pH meter and conductimeter (Crison Instruments). Dose equivalent measurements of radiation were taken in situ with a Geiger counter (Lamse, Mod. Eris1R, Madrid, Spain).

The U concentration was measured by means of an Inductively Coupled Plasma-Mass Spectrometry equipment (ICP-MS VARIAN RedTop). The detection limit was 0.1 ng/mL. Calibration was performed by preparing solutions from a commercial solution of 1000 ppm uranium. Tantalum was used as internal standard. Uranium isotope patterns were used as control. Prior to the chemical analysis, the water samples were acidified with 2% HNO3 and maintained in a dark emplacement at 4ºC. U results were obtained by averaging the data obtained in three replicates.

The metals were analyzed by means of a Graphite Furnace Atomic Absorption Spectrometry (GFAAS).

2.3 Phytoplankton identification and biodiversity

Phytoplankton species living in the evaporation pond at the Saelices and in the mining-effluent pool at the Villavieja were directly identified from fresh sample, and cell density was counted on fixed samples(4% formalin) in settling chambers using an inverted microscope (Axiovert 35, Zeiss, Oberkochen, Germany). Identification of algae was carried out in accordance with algae database ( Phytoplankton biodiversity in each water sample was estimated by mean of Margalef index:

D = (S-1) / loge N (Margalef, 1957; 1969)

where: D = Margalef biodiversity index, S = number of phytoplankton species and N = total number of phytoplankton individuals.

2.4 Experimental organisms and culture conditions

Wild-type mesophilic strains of the Chlorophyceae Chlamydomonas reinhardtii Dangeard (strains ChlaA and ChlaB) and Dictyosphaerium chlorelloides (Naumann) Komárek and Perman (strain DC1M) from algae culture collection of UCM (which had been isolated from pH=8 waters without heavy contamination nor radiation) were grown in 100 mL cell culture flasks (Greiner, Bio-One Inc., Longwood, NJ, USA) with 20 mL BG-11 medium (Sigma-Aldrich Chemie, Taufkirchen, Germany), at 22 °C under continuous light of 80 μmol m-2 s-1 over the waveband 400-700 nm. Prior to the experiments, the cultures were re-cloned by isolating a single cell, to avoid previous spontaneous mutants accumulated in the culture.

In addition, a strain of Chlamydomonas cf. fonticola (strain ChlSP) was isolated using micropipettes from the waters of Saelices evaporation pond (SP) and grown in filtered water from Saelices U pond under the aforementioned conditions. Cultures were maintained axenically in mid-log exponential asexual growth by serial transfers of subcultures to fresh medium once every fifteen days.

2.5 Toxicity tests: inhibition of effective quantum yield, cell growth and Microtox©

As a complementary trial for testing the toxicity of the residual waters from U mining a Microtox© test according to manufacturer recommendations was used (Microtox© Model 500 Analyser (AZUR Environmental, Carlsbad, CA, USA). Samples of 5% of waters from evaporation pond at the Saelices mining area (SP) as well as mining-effluent pool at the Villavieja (VP) were prepared in the solvent supplied by the manufacturer. Ten replicates of SP and VP were measured after 5 minutes exposure.

Additionally, the toxicity of the water samples was also estimated through photosynthetic performance. The changes in effective quantum yield of photosystem II (ΦPSII) were measured inC. reinhardtii and D. chlorelloidesusing a pulse-amplitude modulated fluorimeter ToxYPAM (Walz, Effeltrich, Germany) after 24 hours of exposure to the pollutant. Maximum fluorescence of light-acclimatized microalgae (F´m) was determined after a saturating-white pulse of ca. 10,000 μmol m-2 s-1 PAR for 0.8 s, which makes it possible to assume that all PSII reaction centers of PSII system are fully closed (Altamirano et al., 2004, Peña et al., 2010). The inhibition of F´m was used as estimator of the toxic effect of residual waters from U mining. The percentage of inhibition was calculated as:

Inhibition (%) = 100 - [100 x (F´m)U/(F´m)control]

where(F´m)U corresponds to the maximum fluorescence after 24 hours U exposure and (F´m)control is the maximum fluorescence of the control. The value of F´m was determined in the two samples selected in this study.

Finally, the toxic effect of the residualwaters from U mining on the growth rates of the two wild-type phytoplankton species was determined as follows: 105 cells from mid-log, exponentially-growing cultures of wild-type strains of C. reinhardtii and D. chlorelloides were placed in experimental tubes containing 2 mL of each residualwater at 22ºC. Triplicates of each sample and unexposed controls (containing only BG-11 medium) were prepared. Cells were counted blind (i.e. the person counting the test did not know the identity of the tested sample) using the inverted microscope. Growth rate (m) was calculated according to:

m = Loge (Nt / N0)/t (Crow and Kimura, 1970)

(t = 5 days; Ntand N0are the cell numbers at the end and at the start of the experiment, respectively).

2.6 Adaptation of microalgae to residual waters from U mining

Usually, microalgae should die in the residual waters from U mining. However, some microalgae could survive in these contaminated waters as a result of physiological acclimatization by modification of gene expression, rare spontaneous pre-selective mutations generating new resistant alleles, or environmental-induced post-adaptive mutations in response to environmental selection (reviewed by Sniegowski, 2005). In a seminal paper, the Nobel Prize Luria and Delbrück (1943) presented fluctuation analysis as a combined set of experiments and statistical method to distinguish between resistant cells arising by rare spontaneous mutations, and the other causes. Sincethe pioneering studies of Sager (1954, 1962), fluctuation analysis was early employed to study adaptation of microalgae to toxic stress. In the present study we used a fluctuation analysis to investigate the nature of the adaptation to residual waters from U mining. Methodological details are given in previous work using fluctuation analysis to know how microalgae achieve adaptation to antibiotics and herbicides (Sager et al., 1977; Costas et al., 2001; Lopez-Rodas et al., 2007; Marva et al., 2010, Gonzalez et al., 2012), xenobiotics (Garcia-Villada et al., 2002), heavy metals (Garcia-Villada et al., 2004; Sanchez-Fortun et al., 2009); toxic spills (Baos et al., 2002, Costas et al., 2007, Lopez-Rodas et al., 2008a, 2008b, Carrera Martinez et al., 2010, 2011, Romero et al., 2012); and extreme environments (Lopez-Rodas et al., 2009b; 2011; Costas et al., 2007; 2008). In short: Two different sets (experiments and controls) were prepared for each species. In each set 1 experiment 90 culture flasks were inoculated with N0 = 102 cells (a number small enough to reasonably ensure the absence of pre-existing mutants in the inoculum) and propagated under non-selective conditions until Nt = 1· 105 cells. Then water from evaporation pond at the Saelices mining area (SP) or the mining-effluent pool at the Villavieja (VP) was added. In each set 2 control 45 culture flask containing water from SP or VP were inoculated with Nt = 1· 105 cells from the same parental culture for sampling the variance of the parental population. All the cultures were kept for 75 days to ensure that if a mutant occurred, its progeny would be large enough to be detected. Three independent observers counted the resistant cells.

Two different results can be attained in the set 1 experiments each of them as the independent consequence of two distinct adaptation mechanisms: i) if resistant cells arise by random mutations that occur spontaneously during the period in which the cultures reached Nt from N0 before the exposure to residual waters from U mining, then the inter-culture (flask-to-flask) variation would be high, not consistent with the Poisson model (variance/mean) (Fig. 1, set 1A) ; ii) on the contrary, if the occurrence of resistant cells is induced by the residual waters from U mining, every cell is then likely to have the same possibility of developing resistance and the inter-culture (flask-to-flask) variation will be very low following the Poisson model (variance/mean = 1) (Fig. 1, set 1B).The set 2 is a control for the experimental error (Fig. 1) and hence variance is expected to be low because set 2 tracks the variance due to the parental population.

In addition, mutation rates can be estimated from the proportion of set 1 cultures showing non-resistant cells (P0 estimator) after exposure to residual waters from U mining as follows:

μ = −logeP0/(Nt− N0)Luria & Delbrück (1943)

where μ = mutation rate (in mutants per cell division); P0 = proportion of cultures with non resistant cells in the fluctuation analysis; N0 = initial cell number; Nt = final cell number.

In addition, C. reinhardtii cultures (strains ChlaA and ChlaB) were used to evaluate the contribution of recombination (after sexual mating) in the adaptation process to U contamination. Sixteenindependent populations of each strain as well as sixteen independent mixed-populations comprising both strains (ChlaA + ChlaB) weregrown in 20 ml BG-11 medium, 22ºC under continuous light of 60 μmol m-2 s-1 until reaching a mass populations of 5· 105 cells mL-1, a number of cells high enough as to ensure that U-resistant mutants would occur. Sexual mating had effect in the mixed-populations (morphologically checked). Then water from VP was added in each culture and obviously, the cell density was significantly reduced by the toxic effect of U. After further incubation for 30 days, the cells were microscopically observed in order to count the U-resistant cells. The results of cultures with and without sexual mating were compared.

2.7 Electron microscopy

In an attempt to find out what happened with the uranium in the microalgae that colonized residual waters from uranium mining, and infer possible mechanisms that allow them to resist this pollution, Chlamydomonas cf.fonticola cells growing in the uranium polluted waters from Saelices evaporation pond (SP) were rinsed three times in saline phosphate buffer (to eliminate all the SP water), preserved in EM fixative (2.5% glutaraldehyde in sodium cacodylate-saccharose buffer, pH 7.2 for 24h at 4ºC), post fixed in 2% buffered OsO4 for 1h at 4º C, dehydrated in a series of acetone, embedded in Spurr, sectioned at 80 nm thick (in a LKB 2088 ultramicrotome) and collected on 200-mesh copper grids. Uranium containing compounds (i.e. uranyl acetate) were not used in the post-processing of the samples to assure that any uranium present in the cells come from the uranium captured previously during their growth in water of Saelices evaporation pond. TEM images were obtained using a JEOL JEM-2010 transmission electron microscope (Jeol Ltd., Tokyo, Japan) operated at 100 kV. The microscope is equipped with X-ray energy dispersive spectroscopy (XEDS) with a resolution of 133 eV at 5.39 keV, which was applied to detect uranium in cell structures of Chlamydomonas. All the reagents were from Sigma-Aldrich Chemical Co. St. Louis, MO, USA.

3. Results

3.1 Environmental conditions, toxicity and phytoplankton community of residual waters from U mining

Residual waters from U mining from Saelices evaporation pond and Villavieja mining-effluent pool showed extremely highlevels U contamination, severe acidity and elevated conductivity (Table 1). Arsenic was also detected (0.15 and 1.2 mg L-1 in Saelices and Villavieja, respectively) and had a minor presence in compared with uranium. Traces of copper, zinc and lead were also detected. Radioactivity, measured as equivalent dose at 1 m high was of 4 μSv h-1.

As expected these residual waters from U mining are extremely toxic(Table 1). Solutions containing a 5% of waters from Saelices evaporation pond or Villavieja mining-effluent pool produced complete inhibition of Microtox© test (Table 1). Accordingly, the water samples from the Saelices evaporation pond and Villavieja mining-effluent pond completely inhibited photosynthesis at 24h (100% inhibition with respect to unexposed controls). Finally, the residual waters from U mining completely inhibited the growth rates of C. reinhardtii and D. chlorelloides (Table 1).