Rapid adaptation of microalgae to extremely polluted waterbodies from
uranium mining:
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 (IRNASACSIC). PO Box 257. 37071. Salamanca. Spain.
* Corresponding author. E-mail: ecostas@vet.ucm.es
Abstract
An outstanding example of the fast adaptation of microalgae to extreme
anthropogenically-generated environments (i.e. residual waters from U mining
with extremely high levels 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
(with higher content of dissolved 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 singlegene or few genes that confer a large adaptive value under extreme
contamination.
Key words: adaptation, extreme environment, microalgae, mutation, uraniummining, 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 m 3 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 miningeffluent 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% HNO 3 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 (http://www.algaebase.org). 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 in C. reinhardtii and D. chlorelloides using
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 residual waters 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 residual water 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; Nt and N0 are 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. Since the 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. Sixteen independent populations of
each strain as well as sixteen independent mixed-populations comprising both
strains (ChlaA + ChlaB) were grown 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 200mesh 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 high levels 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).
The phytoplankton diversity found in the different waters of U mining
areas was very low (Table 1). Only 4 species were detected in Saelices
evaporation pond, while only 3 species proliferates in Villavieja mining-effluent
pool (Table 1). Chlorophyta species dominates both residual waters. The most
abundant species was the Chlorophyceae Chlamydomonas cf. fonticola. However,
the microalgae biomass was very scarce in these waters (Table 1).
3.2 Adaptation of microalgae to U contamination
When conducting the fluctuation analysis in water from the Saelices
evaporation pond, a massive destruction of sensitive cells was observed in each
experimental culture of sets 1 and 2. Nevertheless, after the incubation during
several weeks, living cells of C. reinhardtii and D. chlorelloides were observed in
some cultures of set 1 experiments (Table 2) as a result of proliferation resistant
cells. By contrast, in all the cultures of set 2 resistant cells were detected living
cells (Table 2).
The fluctuation in the number of resistant cells observed in set 1
experiment significantly exceeded the fluctuation observed in set 2 controls. For
example in C. reinhardtii set 1 cultures fluctuates from more than 105 resistant
cells ml-1 to 0 resistant cells ml-1. In contrast all the C. reinhardtii set 2 cultures
showed around 102 resistant cells ml-1 (Table 2). A similar result was observed
in D. chlorelloides, where the set 1 cultures fluctuates from more than 105
resistant cells ml-1 to 0 resistant cells mL-1 whereas all the set 2 cultures showed
around 105 resistant cells ml-1 (Table 2).
As in set 1 cultures, the variance significantly exceeded the mean
(variance/mean >> 1; P < 0.001, using χ2 as a test of goodness of fit), so it could
be inferred that processes other than sampling error should cause the high
fluctuation found in set 1 cultures. Resistant cells to residual waters from the
Saelices evaporation pond arose by rare, pre-selective spontaneous mutations
rather than by specific physiological acclimatization, adaptive mutants or postselective mutations appearing in response to effect of residual waters of
uranium mining. The resistant mutants appeared randomly and not in response
to residual waters from U mining.
The rates of mutation (μ) from U-sensitivity to U-resistance r were of 3.8
x 10-7 and 1.7 x 10-6 mutants per cell division for C. reinhardtii and D.
chlorelloides, respectively (Table 2).
An U-resistant mutant of D. chlorelloides was isolated and maintained in
BG-11 medium in the absence of the selective agent (i.e. residual waters from U
mining). It was confirmed that the culture was able to retain resistance to
residual waters of U-mining throughout successive generations. Furthermore,
the Malthusian parameter of this U-resistant mutant was significantly lower
than that of the U-sensitive wild type genotype in the absence of the selective
agent (0.34 and 0.91 doubling day-1, respectively).
In contrast, when conducting the fluctuation analysis with residual
waters of Villavieja also a massive destruction of sensitive cells was observed in
each experimental culture of sets 1 and 2, but after the incubation during
several weeks, neither C. reinhardtii nor D. chlorelloides cultures increase in
density again. We were unable to detect proliferation of resistant cells in waters
from mining-effluent pool of Villavieja.
However, the recombination after sexual reproduction was crucial in the
adaptation of Chlamydomonas to mining-effluent pool of Villavieja (Fig. 2).
When the sixteen single-strain cultures without sexual mating were exposed to
water from the Villavieja, the cell density was significantly reduced as a result
of the destruction of sensitive cells by residual waters of U mining. Similar
results were observed in the sixteen mixed-cultures with sexual mating.
However, after further incubation for 30 days, only the mixed cultures with
sexual mating increased its cell density again as a result of growth of resistant
variants. In contrast, the sixteen cultures without sexual mating showed
undetectable growth (Fig. 2).
3.3 Uranium distribution in Chlamydomonas
TEM of ultrathin sections of Chlamydomonas sp. (strain ChlSP) showed the
presence of electron-dense bodies on the cell wall (Fig 3). In addition, electrondense inclusions also appeared inside the cytoplasm (Fig. 3). A TEM–EDS
analysis of these electron-dense bodies on the cell wall as well as within the
cytoplasm showed let to detect the presence of uranium in its composition (Fig.
3).
4. Discussion
Often, eco-toxicological studies assume that the short-term effect of a
pollutant on an organism is a diagnostic of the impacts of this pollutant at the
organism level as well as at the ecological level. But living beings are able to
rapid evolution as a response to anthropogenic global change. Consequently,
interest to study the way in which the microalgae are able to adapt to extreme
contamination is rapidly growing among eco-toxicologists.
One remarkable case is the rapid adaptation to extreme environments. In
contrast with the classic idea that adaptation to extreme environments is slowly
achieved during thousands of years by mean of gradual selection of several
mutations with minor effects (Gould, 2002), adaptation of microalgae residual
waters from uranium mining in Saelices and Villavieja occurred very rapidly –
only fifty two years- since mining activities started in 1960.
The high U concentration in Saelices evaporation pond or Villavieja
mining-effluent pool (25 and 48 mg L-1) indicates that these waters constitute
extreme environments. The normal values of U concentrations in natural waters
do not exceed the order of 1 µg L-1 in water (Tsezos & Volesky, 1982). Even in
areas influenced by mining activities, the usual U concentration is around 3.5
mg L-1 (Dessouki et al., 2005). The limit concentration established by the U Mill
Tailings Remediation Action (UMTRA) in USA is 0.05 mg L-1 (Anderson et al.,
2003). Toxicological tests - Microtox©, inhibition of ɸ (PSII), and inhibition of
growth - show that the residual waters from U mining were extremely toxic.
Consequently, the survival of microalgae in Saelices evaporation pond or
Villavieja mining-effluent pool could only be achieved by a certain kind of
adaptation, which constitutes an astonishing example of the rapid adaptation to
extreme toxicity. Such adaptation is not easy and only a scarce number of
species managed to achieve it (i.e. phytoplankton diversity is so scarce in these
residual waters from U mining in contrast to the high diversity of
phytoplankton that usually occurs in non-contaminated ponds).
Microalgae usually experience extinction in U-polluted waters. US EPA
Ecotox Database indicates a LC50 value around 36,300 μg/L as representative
for microalgae species. As expected, when C. reinhardtii and D. chlorelloides
cultures were treated with water from Saelices evaporation pool or Villavieja
mining-effluent pool, a massive destruction of sensitive cells by the toxic effect
of U occurred after few hours. However, after further incubation for several
weeks, some cultures with water from Saelices evaporation pool increase cell
density again as a result of the growth of cells that were resistant to the toxic
effect of residual waters of U mining. In contrast, there was no adaptation in
any of the cultures using water from Villavieja mining-effluent pool.
The key to understanding adaptation of C. reinhardtii and D. chlorelloides
to the environmental stress of the Saelices evaporation pool waters is to analyse
the rare algal variants that occur after the massive destruction of the sensitive
cells. The large fluctuation in the number of resistant cells observed in the set 1
experiment, in contrast to the scant variation in set 2 controls proves that
resistant cells arose by rare spontaneous mutations. Neither resistance appears
in response to water of Saelices evaporation pool nor residual waters of U
mining stimulate the appearance of resistant cells. It should be noted that rapid
lethal effect of U on phytoplankton prevents the appearance of adaptive
mutations (which are observed only in non-proliferating populations of bacteria
after being incubated on nonlethal selective medium, Foster, 2004).
Mutation at one single gene is enough to enable adaptation of C.
reinhardtii and D. chlorelloides to the hostile environment of Saelices evaporation
pond. Consequently, C. reinhardtii and D. chlorelloides could adapt to residual
waters from U mining much more rapidly than if the ability to survive in
Saelices evaporation pool requires multiple mutations. Recent evidence
suggests that mutations of single-genes that confer a large adaptive value do
happen and, when competing with small-effect mutations, they usually win
(reviewed by Chouard, 2010).
The rate of mutation from sensitivity to resistance against extreme
environments (for example Rio Tinto, Aguas Agrias, Agrio River-Caviahue lake
or Mynydd Parys waters) ranged from 1.1x10-6 to 5.4x10-6 mutants per cell
division in D. chlorelloides (Costas et al., 2008; Lopez-Rodas et al., 2008a, 2008b,
2011). The mutation rate from sensitivity to resistance against residual waters
form uranium mining in D. chlorelloides (1.7 x 10-6 mutants per cell division) was
in the same range.
An Occam’s razor argument can be used to explain the rapid
colonization of the extreme environment of Saelices evaporation pond. New
resistant mutants arise in each generation because of mutation are recurrent
from a wild- type sensitive allele to a resistant mutant. Since the resistant allele
is detrimental to fitness in the absence of residual waters from U mining, then
many of these mutants are eliminated by selection. Thus, at any given time
there will be a certain number of resistant cells that are not yet eliminated. The
balance between mutation rate (µ) and the rate of selective elimination (s) will
determine the average number of resistant mutants:
q = μ / (μ + s) (Kimura & Maruyama, 1966)
where q is the frequency of the resistant mutant allele and s is the coefficient of
selection. Using the Malthusian parameters of resistant allele and wild type
allele measured in non-selective conditions (i.e. BG-11 medium), the values of
fitness were calculated (0.37 and 1.00 for resistant-mutant and wild type
sensitive allele, respectively) and used to estimate the coefficient of selection (s)
and the balance between mutation and selection. An amount of 2.7 resistant
mutants per million D. chlorelloides cells are maintained in the absence of
residual waters from U mining by balance between mutation and selection.
Taking into account the countless cells comprising microalgae populations, it
could be hypothesized that colonization of Saelices evaporation pond was
almost instantaneous, because the microalgae living in the hostile ecosystem of
Saelices evaporation pond could be the descendants of resistant mutants that
arrived fortuitously at this pond in the past. Moreover, this event could still be
happening and may be the explanation of why Saelices evaporation pond was
rapidly colonized.
More and more examples accumulate on rapid adaptation of microalgae to
extreme environments through the selection of rare spontaneous mutations
conferring resistance, which affect only one gene. By means of these
mechanisms, microalgae achieve adaptation to antibiotics and herbicides
(Sager, 1977, 1985; Costas et al., 2001; Lopez-Rodas et al., 2007; Marva et al.,
2009, Gonzalez et al., 2010), algaecides (Garcia-Villada et al., 2004; Costas et al.,
2013), xenobiotics (Garcia-Villada et al., 2002), toxic acid mine spills (Baos et al.,
2002; Lopez-Rodas et al., 2008a, 2008b;) and even volcanic effluents and thermal
waters (Lopez-Rodas et al., 2009b; Costas et al., 2008). However, the high U, the
presence of other heavy metals and the acidity of Saelices evaporation pond
and Villavieja mining-effluent pool indicates that the environmental stress in
these areas might be greater than in other environments where the adjustment
is caused by a single mutation. It is very interesting that a single mutation
allows adaptation to an extreme environment as Saelices evaporation pond. But
the environmental stress in Saelices evaporation pond could be very close to the
upper limit of environmental stress that microalgae can resist by mean of a
single mutation. In fact, when conducting the fluctuation analysis with residual
waters of Villavieja mining-effluent pool (which has higher content of uranium
that Saelices) microalgae were unable for adapting to this water by mean of a
single mutation.
In contrast, the recombination was crucial in the adaptation of
Chlamydomonas to mining-effluent pool of Villavieja. When the cultures were
exposed to water from the Villavieja mining-effluent pool, a massive
destruction of sensitive cells was observed. But, after further incubation for 30
days the mixed cultures with sexual mating increased its cell density again as a
result of growth of resistant variants (in contrast, the cultures without sexual
mating were unable to grow). This suggests that at least two different
mutations are needed for the resistance to waters from mining-effluent pool of
Villavieja. Mutation rates for alleles that enabling resistance to these extreme
environments is in the order of 10-6 (Lopez-Rodas et al., 2009b; Costas et al.,
2007, 2008). If to resist the toxic effects of waters from mining-effluent pool of
Villavieja it just takes a single mutation, resistant mutants should have occurred
in some of the 90 cultures from the fluctuation analysis (each with 105 cells). In
contrast, if survival to water from mining-effluent pool of Villavieja requires at
least 2 different mutations, then the probability that both simultaneously occur
in a single cell is as low as to not be detectable by the fluctuation analysis. But
recombination after sexual reproduction allows that two mutations from
different microalgae finally can appear together in the same cell. This
recombinant cell carrying both mutations can withstand the toxic effects of
water from mining-effluent pool of Villavieja. Adaptation to residual waters of
Villavieja after sexual reproduction provides an excellent example of the
advantage of the reciprocal gene exchange by recombination.
Chlorophyta species seem to be especially capable for rapid adaptation
to residual waters from U mining. Chlorophyta species also were more capable
for rapid adaptation to extreme environments than other microalgae Phyla.
This fact was also observed in several extreme environments such as Aguas
Agrias river (Lopez-Rodas et al., 2008a), Mynydd Parys (Lopez-Rodas et al.,
2008b), Tinto river (Costas et al., 2008), and Agrio river–Caviahue lake (LopezRodas et al., 2011), although the reason is unknown.
We can hypothesize about the cellular basis for the mechanisms involved
in resistance to residual waters from U mining. Microalgae have developed two
strategies to prevent the toxic effects of heavy metals. In particular, they
maintain nontoxic concentrations of heavy metals inside their cells by mean of
metal exclusion, which prevents the indiscriminate entrance of heavy metal
ions into the cell, and by mean of the complexes formation, which prevents
bioavailability of these toxic ions once inside the cell (Perales-Vela et al., 2006).
Apparently, both mechanisms can be observed in TEM of Chlamydomonas cf.
fonticola, which accumulates electron-dense uranium particles on the cell wall as
well as inside the cytoplasm.
Finally, it is tempting to speculate on an amazing natural phenomenon.
Around two billion years ago a deposit of U ore in Oklo (Gabon, Africa)
commenced a self-sustained natural nuclear reactor. During thousands of years
the fission reaction continued off and on until the reactor was shut down
(reviewed by Cowan, 1976). Lovelock (1988) proposed that the microalgae
could have concentrated U in Oklo. After a rapid adaptation microalgae could
be able to accumulate uranium on the cell wall as well as inside the cytoplasm
to defend against the toxic effect of uranium such as occurs in Saelices
evaporation pond.
Acknowledgements
To Lara de Miguel for her excellent technical support. To Pedro
Caravantes for his help in samples preparation and analyses. To Teresa Benito
Criado from Assistant Technical Center Geological Research (Universidad
Complutense de Madrid). Supported by Spanish Secretaría de Estado de
Investigación, Desarrollo e Innovación grant CTM 2012-34757.
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