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8/3/2019 21.7 Microbial Community AMD
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Microbial Community Composition and Ecology of an Acidic
Aquatic Environment: The Tinto River, Spain
A.I. Lopez-Archilla, I. Marin, R. Amils
Centro de Biologa Molecular, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Received: 7 December 1999; Accepted: 12 April 2000; Online Publication: 16 November 2000
A B S T R A C T
We studied the correlation between physicochemical and biological characteristics of an acidic river,
the Tinto River, in Southwestern Spain. The Tinto River is an extreme environment characterized
by its low pH (mean of 2.2) and high concentrations of heavy metals (Fe 2.3 g/L, Zn 0.22 g/L, Cu
0.11 g/L). These extreme conditions are the product of the metabolic activity of chemolithotrophic
microorganisms, including iron- and sulfur-oxidizing bacteria, that can be found in high concen-
trations in its waters. The food chain in the river is very constrained and exclusively microbial.
Primary productivity in the Tinto River is the sum of photosynthetic and chemolithotrophicactivity. Heterotrophic bacteria and fungi are the major decomposers and protists are the major
predators. A correlation analysis including the physicochemical and biological variables suggested a
close relationship between the acidic pH values and abundance of both chemolithotrophic bacteria
and filamentous fungi. Chemolithotrophic bacteria correlated with the heavy metals found in the
river. A principal component analysis of the biotic and abiotic variables suggested that the Tinto
River ecosystem can be described as a function of three main groups of variables: pH values, metal
concentrations, and biological productivity.
Introduction
Extremophiles, organisms capable of thriving under extreme
conditions, have become of interest from both an academic
and biotechnology perspective because of their interesting
ecology and physiology. Understanding the microbial ecol-
ogy of extreme environments may provide insight into the
limits of life and its possible origin. Extremophilic microor-
ganisms have also important industrial and environmental
applications, which include processes for metal extraction
from naturally occurring ores or industrial waste [9, 50],
microbial desulfurization of coal [6, 27], and bioremediation
processes [8, 45, 46].
Proton concentration (pH) is an important physiological
factor. In general, microorganisms cannot thrive at very high
(basic) or low (acidic) pH values. In these conditions, ex-
posed microbial cell components can be hydrolyzed or pro-
Present address: Departamento de Ecologa, Universidad Autonoma de
Madrid, Cantoblanco, 28049 Madrid, Spain.
Correspondence to: R. Amils; Fax: (34) 91 397 8087; Email: ramils@
cbm.uam.es
MICROBIALECOLOGY
Microb Ecol (2001) 41:2035
DOI: 10.1007/s002480000044
2001 Springer-Verlag New York Inc.
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8/3/2019 21.7 Microbial Community AMD
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teins denatured. Dissociation and solubility of many mol-
ecules that directly or indirectly affect microorganisms are
also strongly influenced by the pH. For instance, metal ions
that are toxic at high concentrations are much more solubleat low pH, thus generating additional physiological stresses
[2].
The Tinto River, a 92-km river in Southwestern Spain, is
an example of such an extreme biotope, exhibiting a con-
stant very low pH and high concentration of heavy metals.
This river has its origin at Pena de Hierro (Iron Mountain)
and flows through the copper mining district of Riotinto,
where it acquires its special characteristics [33].
This study presents a report of the microbial communi-
ties found in the Tinto River and describes the main phys-icochemical and biological features of this extreme habitat.
Materials and Methods
Study Sites, Sampling Characteristics, and Estimation of
Microbial Abundance
Samples in triplicate were collected from different stations along
the river, mine effluents, and water reservoirs (Fig. 1). Sampling site
E1 corresponds to a small water reservoir located near the rivers
source, with a pH close to 7 and very low metal concentrations; this
was considered a neutral pH reference site for this work. Water
samples were collected in February, May, August, and November of
1993. Samples for chemical analysis were collected in 100 ml poly-
propylene bottles. Samples for microbial isolation were taken in
sterile 20 ml tubes. Samples for enumeration of microorganisms
and biomass estimation in the riverbed were collected using 50 ml
sterile syringes. These samples were fixed with formaldehyde (2%
v/v) and homogenized in a Braun Labsonic V apparatus at 20 kHz
for 1.5 min. Cells were stained with a mixture of acridine orange
(AO) and 6-diamidino-2-phenylindole (DAPI) (100 mg/L and 5
mg/L, respectively) on black Nuclepore filters with a pore size of 0.5
m, and then washed with citrate buffer pH 4. Quantitative mi-
croscopic observations were done according to the method de-
scribed by Fry [19], except that dilutions were done with sterile
water at pH 2, in order to prevent metal precipitation. The number
and size of microorganisms were determined by direct observation
using a ZEISS Axioskop microscope under UV light, with an in-terference filter (bandpass 450 to 490 nm). Cell volume was esti-
mated by comparing shapes to known geometric forms and direct
measurement of the cell dimensions. Chemolithotrophic bacteria
were quantified by the Most Probable Number (MPN) using Col-
lins method with five dilution series [12].
Isolation of Microorganisms
Samples were plated onto different media containing 1.5% agar:
medium A (9K mineral medium [48] supplemented with 1% (w/v)
glucose, and 1% (w/v) yeast extract); medium I (9K medium
supplemented with 0.1% (w/v) bactotryptone, 1% (w/v) malt ex-
Fig. 1. Geographic position of
the Tinto River. The location of
the sampling sites and the min-
ing region are indicated.
Microbial Community Composition and Ecology of an Acidic Aquatic Environment 21
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tract, 1% (w/v) glucose, 0.5% (w/v) yeast extract, and 0.5% (w/v)
sucrose); medium J (9K medium supplemented with 0.1% (w/v)
casamino acids, 0.1% (w/v) bactopeptone, 0.5% (w/v) yeast extract,
and 0.5% (w/v) sucrose); medium F (1 mM KH2PO
4, 1mM MgCl
2,
1.5 mM (NH4)2SO
4, 0.5% (w/v) glucose, 0.05% (w/v) malt extract,
0.5% (v/v) trace metals [1]). Final pH, adjusted with concentrated
H2SO
4, was 3 for all solid media and 2.5 for liquid media. Different
media for chemolithotrophic bacteria enrichments were obtainedby supplementing 9K medium with ferrous iron (44.8 g/L FeSO
4
7H2O), tetrathionate (100 mM), elemental sulfur (10g/L), or metal
sulfides (200 g/L of Fe, Cu, or Zn concentrates). Chemolithotropic
bacteria were subsequently isolated from single colonies growing
on agarose plates [43] and identified by their phenotypic properties
[22, 23, 25, 43]. Gram-positive bacteria were characterized using
API 50CH and API 20E systems and additional antibiotic sensitivity
and halotolerance tests. The identification of yeasts was carried out
using physiological and biochemical criteria [5, 28, 29, 34]. Iden-
tification of filamentous fungi, algae, and heterotrophic protists
was carried out by direct microscopic observation using different
phenotypic characteristics [7, 14, 18, 24, 30, 31, 36, 41, 42, 51, 52].
Analysis of Physicochemical Parameters
Total content of Fe, Cu, Zn, and Mg was measured by atomic
absorption spectrophotometry using a Perkin Elmer 1100B instru-
ment. Ca, As, K, and Ni concentrations were measured by X-ray
fluorescence reflection with a Rich Seifert & Co. model Extra II
instrument. Sulfate concentration was determined by a turbidimet-
ric method [15] and ferrous iron by a colorimetric method using a
Metrohm 662 photometer [16]. Conductivity, pH, oxygen, and
redox potential values were measured in situ using specific elec-trodes. Redox potential and pH values were determined with a
Crison 506 pH/mV-meter bearing an Orion-9778SC electrode.
Conductivity values were estimated with an Orion-122 conductim-
eter. Oxygen concentration and water temperature were deter-
mined with an Orion-810 oxymeter.
Genomic Analysis
Pulsed field gel electrophoresis of intact DNA prepared from dif-
ferent microorganisms was performed as described in [21, 38].
Statistical Analysis
All physicochemical and biological parameters for each sampling
site were arranged in a single matrix. Statistical analysis was per-
formed by principal component analysis (PCA), which was carried
out using the computer program SYSTAT, version 5.0 (Systat,
Inc.). PCA simultaneously considers many correlated variables and
identifies the lowest number needed to accurately represent the
structure of the data set. These variables are then linearly combined
with the eigenvectors of the correlation matrix to generate a prin-
cipal component axis. The first principal component axis (AI) is
formed from the original variables with the greatest variance. All
subsequent principal components (AII, AIII, AIV, etc.) are based
on the original (high variance) variables that are uncorrelated with
the previously defined components. Since each additional principal
component has a lower variance than the previous one, most of the
variance in the sample data can be accounted for within two or
three axes.
Correlation analysis was also applied to the data using the
Spearman test for nonparametric variables (iron- and sulfur-oxidizing bacteria, unicellular chlorophytes, and euglenas) or the
Pearson correlation test for parametric variables (rest of variables).
It was deemed that those variables whose correlation values (p)
were lower than 0.05 (significance level of 95%) were correlated.
The computer program used for this analysis was STATGRAPH-
ICS, version 2.1, by Statistical Graphics Corporation.
Results
Geomorphological and Physicochemical Parameters
The basin of the Tinto River covers an area of 1676 km2 in
the province of Huelva (southwest region of Spain). From its
source at Pena de Hierro (altitude 500 m), it has a course of
92 km until reaching the Atlantic Ocean in Huelva. The
slope of the river is gentle, with an average value of 0.59%.
The resultant gentle flow facilitates the settlement of a dense
microbial community on the riverbed. The river flow is ex-
tremely variable depending on the season. The highest flow
values are reached in January or February (8.1 m
3
/s) and thelowest in August (0.07 m3/s) [37]. These fluctuations are due
to the regional climatology. The river is subject to a Medi-
terranean type regime, with an average annual temperature
of 17.9C and an accumulated precipitation of 750 L/m2
(data corresponding to 1993).
Values of the main physicochemical parameters mea-
sured in the Tinto River are shown in Table 1. Some pa-
rameters, namely pH (with an annual mean value of 2.2), the
concentration of some heavy metalstotal Fe (2.26 g/L of
mean), Cu (0.11 g/L) or Zn (0.235 g/L)or the concentra-
tion of some anions, mainly sulfate (average 10.11 g/L),
showed very atypical values when compared to those found
in nearby rivers and the reference sampling site E1 (Table 2).
It is important to point out that extremely low pH values,
between 1.7 and 3.1, were measured along the entire length
of river. The pH remained low year-round, regardless of the
temperature and the volume of the water flow [33]. The
concentration of metallic ions as well as the concentration of
sulfate showed a relative decrease from the source to the
mouth of the river, but they were still consistently high at the
end of the river at sampling site 11.
22 A.I. Lopez-Archilla et al.
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Table 1. Values of physico-chemical parameters measured in the different seasons; (Cond.) conductivity in mS; (Rex) Redox potential in
mV; (O2) Oxygen in ppm; rest of parameters in mg l-1
Samplingsite
pH t Cond. Rex. O2
Zn SO4
2
Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD
Winter E1 6.67 0.06 6 0.2 0.79 0.11 169 12 12.0 1.62 10 0.01 500 0,35
1 2.66 0.03 15 0.1 9.49 0.25 382 58 10.0 0.28 160 0.1 13,800 1,1
2 2.77 0.01 15 0.1 9.50 0.27 350 11 2.5 0.31 210 0.1 12,120 1,0
3 1.97 0.08 10 0.2 11.62 0.16 456 25 1.1 0.08 160 2.0 14,800 1,3
5 2.45 0.04 2 0.3 3.97 0.09 478 35 11.4 0.92 80 2.2 5,410 0,5
6 2.15 0.08 11 0.3 12.59 0.36 423 31 8.7 0.55 230 3.0 29,220 2,2
m3 1.93 0.03 12 0.2 13.97 0.28 424 41 7.0 0.63 160 1.1 30,350 1,5
m4 2.22 0.07 20 0.3 13.80 0.32 414 26 3.9 0.14 760 4.0 16,900 1,2
sw1 2.67 0.06 6 0.4 2.67 0.10 167 18 10.7 0.82 10 0.1 1,470 0,3
7 2.04 0.05 11 0.1 11.66 0.38 408 32 8.0 0.51 420 1.5 14,200 1,0
8 2.39 0.06 11 0.1 6.85 0.22 450 26 3.7 0.20 250 0.9 15,240 1,1
11 2.35 0.07 13 0.2 5.27 0.20 511 34 11.4 0.32 120 0.1 8,900 0,9
Spring E1 4.30 0.05 26 0.1 0.58 0.09 316 18 7.6 0.53 0 0.00 314 0,26
1 2.50 0.05 31 0.1 9.37 0.31 416 29 5.3 0.26 100 0.2 6,780 0,8
2 2.60 0.04 29 0.1 7.91 0.39 340 31 3.2 0.29 100 0.5 6,760 0,8
3 2.00 0.02 33 0.5 16.5 0.52 443 26 1.1 0.15 50 0.9 5,900 0,7
5 2.30 0.04 15 0.4 4.42 0.42 506 45 7.1 0.08 220 1.0 6,500 0,7
6 2.00 0.02 28 0.2 19.39 0.45 446 37 5.4 0.18 130 0.5 9,220 0,9
m3 2.10 0.02 26 0.2 7.92 0.32 449 34 6.3 0.20 300 2.1 11,900 1,1
m4 1.80 0.03 22 0.1 25.20 0.59 390 28 5.3 0.17 1,500 18.9 7,800 0,5
sw1 2.67 0.05 21 0.3 3.07 0.13 362 27 7.4 0.50 10 0.1 1,600 0,2
7 2.00 0.02 24 0.4 14.67 0.12 412 34 4.7 0.24 560 3.5 10,010 1,1
8 2.40 0.02 28 0.1 4.12 0.08 518 42 8.2 0.19 110 1.5 7,210 1,0
11 2.20 0.01 28 0.3 3.45 0.02 535 49 11.3 0.27 50 0.1 4,050 0,8
Summer E1 7.10 0.06 25 0.1 0.44 0.07 295 12 13.3 0.92 50 0.05 10 0,0
1 2.55 0.04 19 0.2 11.89 0.12 368 27 7.4 0.05 150 0.5 12,750 1,1
2 3.10 0.08 21 0.1 9.90 0.24 346 29 8.5 0.21 210 0.8 12,220 1,2
3 2.26 0.03 22 0.1 16.53 0.35 455 32 6.5 0.24 190 1.0 25,950 2,85 2.65 0.02 19 0.2 10.46 0.38 455 38 10.5 0.32 230 1.3 13,230 1,5
6 2.60 0.05 26 0.3 13.67 0.41 402 41 5.5 0.16 250 1.1 21,750 2,0
m3 2.41 0.02 26 0.3 15.39 0.46 419 29 6.6 0.25 200 0.6 22,920 2,2
m4 2.30 0.02 24 0.4 19.76 0.35 386 30 6.5 0.30 370 1.1 21,600 2,1
sw1 2.88 0.01 21 0.1 3.67 0.12 356 28 13.9 0.24 5 0.0 4,650 0,9
7 2.62 0.02 24 0.1 9.60 0.29 413 34 8.7 0.18 290 0.3 21,770 1,9
8 2.53 0.01 20 0.2 11.21 0.32 456 32 2.1 0.04 350 1.8 33,880 2,5
11 2.88 0.01 30 0.3 46.10 0.81 445 36 17.9 0.71 10 0.1 4,120 0,8
Autumn E1 6.62 0.05 18 0.2 0.36 0.09 110 9 9.0 0.26 5 0.0 100 0,1
1 2.00 0.03 24 0.2 9.55 0.27 442 35 9.6 0.15 150 1.2 9,350 1,0
2 2.60 0.05 22 0.1 5.16 0.35 360 29 4.3 0.16 90 0.1 4,240 0,8
3 1.67 0.02 22 0.1 31.50 0.56 413 38 6.5 0.28 100 1.1 2,450 0,7
5 1.85 0.03 12 0.3 15.15 0.24 443 41 10.5 0.30 130 1.3 7,100 0,96 1.45 0.02 21 0.3 56.70 0.82 427 49 10.0 0.27 450 3.4 3,390 0,7
m3 1.99 0.02 20 0.4 11.40 0.34 434 40 7.7 0.25 100 0.9 5,440 0,5
m4 1.87 0.02 24 0.5 24.10 0.46 383 42 4.4 0.08 1,420 16.3 5,380 0,6
sw1 3.05 0.08 16 0.1 2.14 0.12 376 38 9.7 0.12 10 0.1 1,490 0,3
7 1.96 0.06 19 0.2 14.88 0.25 406 39 8.6 0.16 280 1.0 5,960 1,0
8 2.42 0.03 18 0.2 3.74 0.22 420 45 9.3 0.20 50 0.5 2,760 0,5
11 2.70 0.02 22 0.2 1.54 0.20 442 28 12.3 0.34 20 0.1 1,840 0,3
E1: external sampling station; sw1: sewage water from Riotinto village that is incorporated to the river; m3: water mine reservoir; m4: water from the internal
mine; TFe: total iron concentration
Microbial Community Composition and Ecology of an Acidic Aquatic Environment 23
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Table 1. (Continued).
NO3
Cu Fe2+ TFe Mg As Ca K Ni
Mean SD Mean SD Mean SD Mean SD
5.70 0.2 0 0.0 0.62 0.1 30 0.5 20 0 36.87 4.83 0.03
5.70 0.2 30 0.3 5.79 0.8 2,260 190 340 0.42 438.47 20.88 1.69
4.95 0.1 70 0.1 242 3.6 2,350 250 290 0 325.42 3.27 1.12
6.52 0.3 50 0.4 9.60 0.6 3,430 426 320 6.62 321.57 4.62 1.54
4.76 0.2 20 0.2 2.70 0.2 730 28 250 0.22 249.66 1.24 0.86
5.32 0.2 270 2.0 33 1.3 2,510 182 540 18.22 172.78 3.18 9.33
8.40 0.4 200 1.2 859 4.2 3,290 220 440 32.10 161.76 5.64 6.97
5.01 0.2 170 0.9 373 2.6 3,750 203 580 13.69 262.79 0 2.11
17.3 0.9 90 0.5 0.70 0.1 3,280 254 160 0.04 112.30 7.55 0.12
9.15 0.7 0 0.0 662 3.3 2,710 158 n.d. 12.22 216.79 5.62 2.41
6.15 0.3 90 0.6 4.50 0.2 700 65 240 8.54 153.32 4.18 1.89
8.43 0.3 40 0.5 2 0.1 660 24 170 1.77 163.98 1.16 0.99
8.68 0.7 0 0.0 0 0.0 0 0 10 0 33.80 6.72 0.06
5.58 0.2 20 0.2 650 3.3 2,000 98 280 1.38 285.97 2.66 0.97
4.96 0.2 50 0.3 259 3.0 2,020 119 250 0 311.04 2.64 1.29
8.06 0.3 40 0.2 560 2.9 3,390 298 220 6.62 185.13 4.09 0.73
4.96 0.1 20 0.1 1.60 0.2 850 54 150 1.08 136.75 1.01 0.55
9.92 0.3 370 1.1 250 1.0 2,670 157 280 104.22 125.13 4.68 10.20
6.20 0.2 110 0.8 77 0.8 1,910 100 210 12.17 96.17 4.85 3.25
0.92 0.1 160 0.5 560 2.5 5,200 367 680 25.56 253.30 13.07 3.68
16.12 0.9 0 0.0 60 0.8 3,730 245 200 0.06 140.84 7.92 0.21
7.44 0.3 160 0.5 446 2.0 2,170 212 380 18.20 208.99 0 2.68
4.96 0.1 60 0.2 2.20 0.1 410 39 130 2.45 78.33 2.91 0.84
5.58 0.2 30 0.1 1.40 0.1 240 27 70 0.75 70.87 1.78 0.49
3.72 0.1 5 0.0 0 0.0 15 0.2 10 0 33.62 4.30 0.02
6.82 0.3 35 0.2 1,100 56 1,630 166 420 1.15 412.82 4.85 2.06
4.96 0.1 110 0.4 1,130 48 2,500 210 270 0 410.33 3.89 1.88
8.06 0.4 70 0.3 34 0.5 3,700 245 400 9.27 371.51 6.67 1.73
4.96 0.1 30 0.2 90 0.6 1,140 170 840 0.26 489.26 3.60 3.56
4.96 0.1 360 1.5 2,490 80 3,350 294 680 9.15 189.83 4.11 11.03
8.06 0.3 280 1.1 760 6.5 3,600 276 400 36.48 188.03 8.28 6.30
8.68 0.3 100 0.5 3,790 110 6,050 421 540 20.06 305.89 6.67 6.75
9.30 0.3 0 0.0 390 24 2,200 113 180 0 119.39 6.66 0.14
6.82 0.2 130 0.3 630 38 50 3 460 6.08 230.39 6.06 2.25
6.82 0.2 180 0.4 38 0.2 600 41 540 18.56 305.12 6.49 3.14
390.6 2.8 5 0.0 45 0.2 0 0 1040 0 211.78 117.10 0.45
4.34 0.1 5 0.0 0 0.0 15 0.1 0 0 29.42 3.67 0.03
6.20 0.3 65 0.4 2,690 57 3,500 200 190 2.54 123.19 3.04 0.43
4.34 0.1 20 0.1 2,360 68 2,750 159 150 0 195.92 2.49 0.64
25.42 1.0 55 0.2 3,000 97 5,600 324 160 11.33 154.81 8.47 0.87
11.16 0.8 135 0.8 12 0.1 1,900 101 360 38.24 100.21 3.89 2.6875.02 1.8 695 2.5 1,800 29 6,100 337 1080 379.05 112.65 11.35 14.88
13.64 0.9 165 0.9 47 0.8 1,850 118 300 40.23 99.39 3.96 3.06
12.20 0.9 490 1.6 5,210 258 8,100 410 800 34.26 307.08 11.23 4.40
15.50 0.9 10 0.1 13 0.1 3,350 199 110 0 82.99 5.76 0.12
13.02 0.9 195 0.5 1,430 95 60 12 300 25.36 167.10 4.62 2.39
6.82 0.3 45 0.3 3 0.0 40 3 80 2.03 46.44 2.15 0.58
6.82 0.3 15 0.1 68 0.5 20 0.9 10 0.05 33.50 2.50 0.10
24 A.I. Lopez-Archilla et al.
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Microbial Community of the Tinto River
The Tinto River ecosystem is unique for a river in that the
biological community is exclusively microbial. Higher eu-
karyotes have not been detected in any of the sampling sta-
tions of the river. Most of the biomass was localized on the
riverbed forming dense and compact biofilms, composed
mainly of filamentous algae, fungi, and bacteria, in which
heterotrophic protists could also be found. Significant min-
eral precipitation was normally observed on the surface of
the biofilms.
In order to elucidate the degree of diversity of this envi-
ronment, we used classic methods for the isolation and char-
acterization of microorganisms. To date, we have identified
and characterized fungi, heterotrophic protists, algae, and
bacteria from the Tinto River. A summary of our findings is
presented in Table 3.The different microbial populations found in the Tinto
River can be grouped according to their ecological role as
primary producers (photosynthetic algae and chemo-
lithotrophic bacteria), decomposers (heterotrophic bacteria
and fungi), and consumers (heterotrophic protists).
Primary Producers. Algae accounted for the greatest propor-
tion of biomass (65%) as number of cells ml1, in the Tinto
River. Because of their photosynthetic abilities they consti-
tute, together with the chemolithoautotrophic bacteria, the
primary producers. Some strains of the Chlorophyta and
Rhodophyta phyla have been isolated. Members of the
Euglenophyta and Bacillariophyta were also observed under
the light microscope.
Diatoms (phylum Bacillariophyta) were variable through
the year (Table 4). They displayed the highest population
during the summer, probably due to faster growth at warmer
temperatures. This interpretation was supported by the posi-
tive correlation obtained between diatom concentration and
water temperature in the statistical analysis (see below). The
relative abundance of diatoms in the Tinto River together
with their large volume made them a major contributor to
the algal biomass (41%).
The second highest proportion of algal biomass (32%) in
the Tinto River corresponded to the Euglenophytes. Some of
the Euglenophytes observed have been identified as Euglena
mutabilis, and their concentration was estimated at differentseasons (Table 4).
Some of the Chlorophyta (green algae) from Tinto River
were filamentous algae of the order Ulotrichales, probably
belonging to the genus Klebsormidium, with filaments larger
than 23 m diameter with parietal chloroplasts occupying
one-half of the cellular periphery and uninucleate cells with
chloroplasts containing only one pyrenoid. Representatives
of another filamentous genus, Zygnema (class Conjugato-
phyceae), were sporadically observed during the autumn.
However, the most ubiquitous algae were unicellular Chlo-rophyta. They were found at almost all sampling stations
throughout the year, although they corresponded to only
11% of the algal biomass. During winter and spring some of
the unicellular Chlorophyta observed were flagellated. Some
of them may be identified as Chlamydomonas acidophila.
Others may have been zoospores of filamentous algae. Uni-
cellular nonflagellated Chlorophyta were observed and iso-
lated all year long. Their phenotypic characteristics re-
sembled those of the genus Chlorella. Comparison of the
pigment absorption spectra between 12 isolated strains and
C. vulgaris CCAP 211/2 revealed little variability between the
isolated strains, although two absorption maxima at 536 and
412 nm exhibited by all the Tinto isolates were not observed
in the reference C. vulgaris spectrum (data not shown). Re-
gardless of their similar phenotypes, several genomic poly-
morphisms (different chromosome numbers and sizes) were
observed using pulsed-field gel electrophoresis (PFGE), sug-
gesting the existence of different strains or even different
species [32].
Some of the unicellular and spherical algal isolates from
the river belong to the phylum Rhodophyta (red algae). They
Table 2. Comparison of some physicochemical parameters of Tinto River with several rivers of the same area; concentration in mg L-1
pH Fe Zn Cu Mg Ni K SO4
2 NO3
References
Guadiamar 7.8 12.1 0.97 0.07 nd 0.045 6.63 312.5 0.83 40a
Rocina 7.1 7.39 0.35 0.025 nd 0.02 2.73 72.45 0.48 40a
Partido 7.8 24.8 0.11 0.135 nd 0.026 58.4 89.75 1.15 40a
Agrio 4.3 50 33.97 1.97 nd nd nd nd nd 48a
Sampling E1(mean) 6.2 15 6.1 2.5 10 0.035 4.88 81.2 5.61 This workTinto (mean) 2.2 2,261 225 109 337.4 2.6 7.42 10,110 9.33 This work
nd: not determined.
Microbial Community Composition and Ecology of an Acidic Aquatic Environment 25
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8/3/2019 21.7 Microbial Community AMD
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were identified as thermoacidophilic microalgae of the genus
Galdieria. Two of these isolated strains may constitute new
species within this genus, as suggested by their phenotypic
characteristics and a PFGE karyotypic analysis when com-
pared to type collection reference strains [38].
In addition to the photosynthetic algae, chemolithotro-
phic bacteria were another type of abundant primary pro-ducer in the Tinto system (Table 4). Most iron-oxidizing
bacteria (IOB) isolated were Gram-negative, aerobic rods
with characteristics similar to those of Thiobacillus ferrooxi-
dans. However, macrorestriction analysis of their genomic
DNA revealed several polymorphisms in relation to the T.
ferrooxidans type collection strains, suggesting that the Ro
Tinto isolates may be related but distinct species [21]. The
rest of the isolated IOB exhibited a curved shape, progressing
in old cultures to a spirilla-like structure, a typical charac-
teristic ofLeptospirillum ferrooxidans [4]. Fourteen strains ofsulfur-oxidizing bacteria (SOB) were also isolated, for which
the pH, growth temperature range, and spectrum of energy
sources were analyzed (Table 5).
Chemolithoautotrophic organisms corresponding to the
domain Archaea were previously isolated from the Rio Tinto
mining area [20] and were detected using molecular ecology
techniques (Gonzalez-Toril and Amils, manuscript in prepa-
ration), but they were not isolated in the aquatic ecosystem
during this study.
Decomposers. Bacteria accounted for the most important
proportion of decomposers. Large amounts of heterotrophic
bacteria were detected year round and accounted for 23% of
the total biomass. A high number of heterotrophic bacteria
were isolated initially from enrichment cultures, but many of
them did not grow after the second or third culture transfer,
probably because some component of the original inoculum
was diluted out, affecting their growth. A total of 124 strains
were isolated (45 from the summer samples, 31 from the
autumn samples, 31 from the winter samples, and 18 from
the spring samples). Some of the isolated strains corre-
sponded to the genus Acidiphilium. Members of this genus
have been shown to be frequently associated with chemo-
lithoautotrophic bacteria, especially iron oxidizers [22, 25].
Many bacterial isolates were Gram-positive bacilli, aero-
bic spore former of the genus Bacillus. These bacilli grew
optimally at relatively neutral pH and formed resistant
spores, so we were unsure if they actually grew in the river or
were resistant forms of bacteria from surrounding environ-
ments. In order to determine the proportion of microorgan-
isms actively growing in the river, samples were subjected to
a heat shock, 85C during 12 min, to inactivate the vegetative
sensitive bacteria [39]. The number of colonies recovered
from the heat treated samples was much lower (average of 17
colonies/ml) than the untreated ones (mean 183 colonies/
ml), suggesting that most bacilli found in the river corre-
sponded to vegetative forms. The identified bacilli strains
corresponded to five different species: B. megaterium, B.amyloliquefaciens, B. stearothermophilus, B. cereus, and B.
subtilis.
Within the decomposers, fungi showed a high abundance
and diversity, including yeast and filamentous forms. A high
percentage (43%) of the hyphomycete isolates (274 strains)
were able to grow in the Tinto water conditions. Some of the
yeast species isolated from the Tinto River can be also found
in other less extreme aquatic environments (Lopez-Archilla
et al., manuscript in preparation). But, the isolated dema-
tiaceous seem to be specific to this kind of habitat, since theyare rarely present in neutral freshwaters (pH near 7 and low
metal concentration).
Among the eukaryotes, heterotrophic protists constitute
the major consumer group in the Tinto ecosystem. They
were scarce in fresh samples, but after storage of biofilms in
the laboratory in acidic conditions, their proportion was
notably increased, probably as a consequence of the distur-
bance of these complex structures. We observed different
flagellates (phylum Zoomastigina), amoeba of the class Lo-
bosea (phylum Rhizopoda), some representatives of classHeliozoa (phylum Actinopoda), and ciliates (phylum Cili-
ophora).
Statistical Analysis
In order to establish the relationship between environmental
and biological variables, we conducted a statistical study of
their correlations. Data from this analysis are shown in Table
6. Because of the lack of information on IOB and SOB in the
winter sampling, and taking in consideration that the total
number of chemolithotrophic bacteria were similar in all
seasons, the average numbers of IOB and SOB for the mea-
sured seasons (spring, summer, and autumn) were used for
the statistical analysis. As expected, metal variables (total Fe
(FeT), Fe2+, Cu, Zn, and Mg concentration) and conductiv-
ity were positively correlated. Also, some biological variables,
such as number of filamentous fungi, total bacteria, IOB,
and SOB, were positively correlated with each other and with
the group of metal variables. pH values correlated negatively
with metal and a cluster of biological variables (IOB, SOB,
and filamentous fungi). Sulfate concentration correlated
26 A.I. Lopez-Archilla et al.
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8/3/2019 21.7 Microbial Community AMD
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Table 3. Taxonomic classification of different microbial groups detected in the Tinto River
Domain Phylum Class Order Family or group Genus Species
Eukarya Bacillariophyta Bacillariophyceae
Euglenophyta Euglenophyceae Euglenales Euglenaceae Euglena E. mutabilis
Chlorophyta Chlorophyceae Chlamydomonadales Chlamydomonadaceae Chlamydomonas C acidophila
Chlorococcales Oocystaceae Chlorella Chlorella sp.
Ulvophyceae Ulotrichales Ulotrichaceae Klebsormidium Klebsormidium spRhodophyta Rhodophyceae Porphyridiales Cyniaceae Galdieria G. sulphurarin
Conjugatophyta Conjugatophyceae Zygnemales Zygnemataceae Zygnema Zygnema sp
Ciliophora Spirotricha Stichotrichida Strongylidiidae Strongylidium Strongylidium sp.
Rhizopoda Lobosea Amoebida
Actinopoda Heliozoa
Mastigophora Amebomastigotaothers (biflagelates)
Zygomycetes Mucorales Mortierella Mortierella sp.
Deuteromycetes Demateaceous Scytalidium S. acidophilum
S. lignicola
S. termophilum
Scytalidium sp.Bahusacala B. cookei
B. olivaceonigra
Bahusakala sp.
Phoma P. pomorum
Phoma sp.
Heteroconium H. chaetospira
Moniliales Penicillium P. atramentosum
P. brasilianum (serie)
P. canescens (serie)
P. cremeo-griseum
P diversum
P. frecuentans
P grancanariaeP. glaucolanosum
P. griseum-azureum
P. lignorum
P moldavicum
P montanense
P. purpurescens
P. sartoryi
P. spinulosum
P. verruculosum
Penicillum sp.
Lecytophora L. hoffmannii
Cryptococcaceae Rhodotorula R. aurantiaca
R. glutinisR. minuta
R. rubra
Cryptococcus C. albidus
C. elinovii
C. flavus
C. gastricus
Candida C. auricularia
C. citrea
C. dendrica
C. fluviatilis
C. krusei
C. muscorum
C. scotii
Microbial Community Composition and Ecology of an Acidic Aquatic Environment 27
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8/3/2019 21.7 Microbial Community AMD
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positively with the biological variables cluster and the
Euglena cell number. Oxygen concentration correlated nega-
tively with the metal variables and positively with pH.In addition, a principal component analysis (PCA) was
conducted in order to obtain information on the environ-
mental biological variables cluster. PCA allows the number
of relevant variables to be reduced, so that the visualization
of the data is considerably simplified. Figure 2 shows the
distribution of the variables in the space formed by the first
two components, which explain 45% of the variance. The
first axis contributes 27% and axis II explains 18% of the
variance. The positive zone of both axes comprised the phys-
icochemical variables (principally those related to metals),
and the IOB and SOB concentrations. The rest of biological
parameters were located in the space formed by the positive
zone of PI and the negative zone of PII. The negative zone of
both axes was occupied by the pH and oxygen concentra-
tion.
Discussion
The objective of this study was to document seasonal
changes in the occurrence of different microbial groups
found in a unique highly acidic ecosystem and relate occur-
rence to physical and chemical parameters.
The particular geology and climatology of the region fa-vors the creation of the Tinto Rivers special environment,
which provides the base on which the biological communi-
ties establish and proliferate. The river rises in the Iberian
Pyritic Belt, one of the worlds richest complex polymetallic
sulfide deposits. The sulfide minerals provide the necessary
substrate for the development of chemolithotrophic bacte-
ria. The high water table, which has been a serious hindrance
to the exploitation of the mines in the past [3], maintains the
river flow during the summer, in the virtual absence of rain
and with a high rate of evaporation. The particular pluvicusregime of this region prevents an excessive dilution of the
river even during the rainy seasons (spring and autumn),
which is important for the maintenance of the constant
physicochemical characteristics of the river.
The abundance of sulfides, especially pyrite and chalcopy-
rite, facilitates the development of high concentrations of
chemolithotrophic bacteria. The total concentration of SOB
was higher than the concentration of IOB in the seasons
measured (Table 4). This appears reasonable, since most
lithotrophic bacteria (including various IOB) are able to
Table 3. (Continued).
Domain Phylum Class Order Family or group Genus Species
Basisiomycetes Tremellaceae Tremella T. encephala
T. fuciformis
T. subanomala
Holtermannia H. corniformis
teliospore-forming Leucosporidium L. antarticumL. stokesii
Ascomycetes Saccharomycetoideae Hansenula H. saturnus
Bacteria Proteobacteria Undeterminated Gram heterotrophic bacteria group
Thiobacillus T. ferrooxidans
T. f. related
T. thiooxidans
T. t related
Thermophilic
Thiobacillus sp.
group Acidiphilium Acidiphilium sp.
Other lithotrophic bacteria Leptospirillum L. ferrooxidans
Gram positives Low G + C group Bacillus B. megateriumB. subtilis
B amyloliquefaciens
B. stearothermophilus
B. cereus
High G + C group
Actinomycetes
28 A.I. Lopez-Archilla et al.
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Table 4. Seasonality of microorganism populations at in different sampling sites from the Tinto River (cells ml-1)
Samplingsites Winter Spring Summer
Autumn
UA E1 9.75 105 1.65 104
1 1.93 106 1.03 106 1.54 108 1.14 107
2 9.42 107 1.16 106 8.70 107 5.07 107
3 1.47 103 2.75 105 1.00 106 2.32 106
4 4.37 105 5.50 105
5 4.75 104 2.75 105 1.02 108 2.62 105
6 5.00 105 9.87 104 6.87 107 3.32 105
m4 1.12 105 7.50 105 8.3 105
Swl 1.55 105 9.50 103 5.25 105 5.6 104
7 2.83 105 4.50 106 5.00 105
8 1.00 105 8.20 104 3.50 106 1.13 105
11 6.20 105 9.27 105 9.27 106 1.38 106
Diatoms E1 2.47 105 1.27 106
1 4.10 105 7.50 104 3.70 107 1.97 107
2 1.62 106
3 9.60 104 4.00 106 7.50 105
4 2.50 104 1.12 105
5 7.62 104 6.50 105 7.50 107 8.75 104
6 6.75 106 1.65 105
m4 1.20 106 1.00 105 5.50 106 4.15 106
Swl 1.42 106 1.12 105
7 1.25 105 9.50 106
8 8.20 104 8.75 105
11 8.25 105 1.50 104
Euglena E1
1 9.54 105 2.25 106 2.07 106
2
3 7.35 102
4
5 3.82 104 9.50 104
6 1.90 105 3.25 106
m4 8.30 105
Swl 9.50 103
7 9.60 104
8 1.00 105 8.20 104 8.75 105
11 8.25 104
HF E1 4.10 103 1.65 104
1 4.10 105 1.90 104 5.50 106 1.00 106
2 6.78 105 9.60 103 2.45 106
3
4 1.25 104 1.92 104 5.50 105
5 3.82 104 1.87 105 5.00 106
6 3.75 105 4.50 106 1.65 105
m4 8.50 105 1.66 106
Swl 9.50 10
3
5.75 10
4
1.12 10
5
7 2.50 105 9.60 104 3.33 105
8 1.00 105
11 1.26 106 4.75 104 6.90 105
TB E1 4.10 103 1.65 104
1 4.10 105 1.90 104 5.50 106 1.00 106
2 6.78 105 9.60 103 2.45 106
3
4 1.25 104 1.92 104 5.50 105
5 3.82 104 1.87 105 5.00 106
6 3.75 105 4.50 106 1.65 105
m4 8.50 105 1.66 106
Swl 9.50 103 5.75 104 1.12 105
7 2.50 10
5
9.60 10
4
3.33 10
5
8 1.00 105
11 1.26 106 4.75 104 6.90 105
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8/3/2019 21.7 Microbial Community AMD
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oxidize inorganic reduced forms of sulfur, whereas only afew are able to oxidize ferrous iron. However, in some
places, the abundance of IOB was higher than that of SOB,
indicating that, under special conditions, bacteria such as L.
ferrooxidans that are unable to oxidize reduced sulfur com-
pounds can develop very successfully in the river [23, 26,
44].
The high numbers of IOB and SOB in the water column,
together with their constant presence all along the river,
contribute greatly to explain the extreme characteristics of
this peculiar environment. The metal sulfides from the py-
ritic belt in the headwaters of the river are subject to exten-
sive microbial oxidation. During this process, the microbial
activity generates different forms of oxidized sulfur, mainly
sulfate, ferric iron, and protons. These products create
strong oxidizing conditions, leading to further oxidation of
other metal containing minerals [17].
Iron and sulfur have a fundamental role in this fluvial
ecosystem. They are extraordinarily abundant in their oxi-
dized forms, which raises the question of how the chemo-
lithotrophic bacteria (IOB and SOB) can sustain their ener-
getic metabolism along the 90 km river in the absence of
appropriate substrates. One possibility could be that the che-molithotrophic bacteria present in the river are those washed
out from a large underground chemolithotrophic region in
the Pyritic Belt [33]. In this case their concentration down-
stream should be reduced because of the correspondent di-
lution factor (up to two orders of magnitude) produced by
the different neutral tributaries. However, bacterial chemo-
lithotrophs maintain a rather constant concentration all
along the river. Another plausible explanation is that the
oxidized forms of iron and sulfur are reduced by different
microbial activities making them available for the chemo-
lithotrophic bacteria. In fact, reduced forms of sulfur can be
obtained from sulfate both by limited-scale assimilatory pro-
cesses and by dissimilatory sulfate reduction [11, 47]. Ferric
iron may also be microbiologically reduced to ferrous iron.
Dissimilatory ferric iron respiration may be carried out by
both strictly anaerobic and facultative anaerobic bacteria
[17]. Some autotrophs can also use iron as terminal electron
acceptor. T. thiooxidans and T. ferrooxidans can reduce ferric
iron using elemental sulfur as electron donor. T. thiooxidans
can perform this reduction aerobically, whereas T. ferrooxi-
dans forms Fe2+ only anaerobically, reoxidizing it under
Table 4. (Continued).
Samplingsites Winter Spring Summer
Autumn
IOB E1 nd
1 nd 1.8 106 2.0 105 1.8 106
2 nd 1.8 106 2.5 105 9.0 104
3 nd 4.0 10
4
3.0 10
6
1.8 10
8
4 nd 6.0 103 3.0 105 2.0 106
5 nd 7.0 104 1.7 105 3.0 106
6 nd 1.4 105 2.0 106 1.1 107
m4 nd 4.0 104 4.0 105 3.0 106
Swl nd 4.0 104 2.5 104 2.5 105
7 nd 2.5 104 1.7 106 0.9 106
8 nd 1.4 105 1.6 107 3.0 105
11 nd 2.5 104 1.6 102 1.7 105
SOB E1 nd 0.2 10 0.2 10
1 nd 5.0 107 3.0 105 3.0 107
2 nd 2.5 106 1.7 106 2.5 105
3 nd 6.0 104 1.7 105 3.5 105
4 nd 3.5 10
5
9.0 10
6
1.4 10
6
5 nd 9.0 103 2.5 106 1.2 106
6 nd 7.0 103 6.0 106 1.4 106
m4 nd 6.0 107 3.0 105 2.0 106
Swl nd 1.6 104 3.0 106 9.5 105
7 nd 2.0 105 1.6 107 3.0 105
8 nd 2.5 105 6.0 106 9.0 105
11 nd 4.0 105 8.0 104 5.0 104
UA: unicellular algae; HF: hyphomycete fungi; TB: total bacteria; IOB: iron oxidizing bacteria; SOB: sulfur oxidizing bacteria; nd: not determined; : not
found
30 A.I. Lopez-Archilla et al.
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8/3/2019 21.7 Microbial Community AMD
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Ta
ble
5.
Phenotypicpropertiesofdiffer
entSOBisolatedfromTintoRiver
pHrange
Optimal
pH
Size(m)
Flagellum
Optimal
T
NaCl
2%
NaCl
5
%
NaCl
10%
Catalase
Gram
Fe2+
S0
G+Ye
TTT
THI
CCu
CZn
CFe
A1
17
3
1.60.5
3037
+++
+++
+/
AB
11.5
1.5
1.40.5
3037
+
+
+++
++
AC
0.57
2.5
10.25
+s
3037
++
+
+
+++
++
+/
AE
14
2.5
1.40.6
+
3037
+
nd
nd
++
+++
+++
AF
0.53
1.5
0.90.5
+s
3037
++
+
+
+/
+++
+++
+/
+/
+/
AG
0.53
1.5
1.50.6
3037
+++
+
+
+/
+++
++
+/
AH
14.5
2.5
1.30.22
+
3037
+++
+
+
n
d
+++
nd
+++
nd
nd
nd
nd
A
0.57
2.5
1.90.5
3037
+++
+
+
+++
++
A
0.57
3.5
1.10.2
+s
3037
+++
+
+
+++
++
+/
AK
0.57
3
1.00.2
+s
3037
+++
+
+
+++
++
AM
1.56.5
3
1.10.4
3037
nd
nd
n
d
+++
nd
+++
nd
nd
nd
nd
AN
1.57
3
10.3
+s
3037
++
+
+
++
++
AO
16.5
3.5
1.10.3
+s
3037
++
nd
nd
++
+++
AP
16.5
2
1.30.5
3037
++
nd
nd
++
+++
(nd
)notdetermined;()nogrowthdetected;
(s)several;G+Ye:9Kmediumsupplemen
tedwith0.1mlofasolutionat10%(w/v)ofglucoseandyeastextract;TTT:tetrathion
ate;THI:thiosulfate;Ccu:
me
talsulfide(Cuconcentrate);CZn:metalsulfide;(Znconcentrate);CFe:metalsulfide(
Feconcentrate).(+);(++);(+++):differentialgrowthyield
Microbial Community Composition and Ecology of an Acidic Aquatic Environment 31
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8/3/2019 21.7 Microbial Community AMD
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Ta
ble
6.
Resultsofthecorrelationanalysisa
pH
Ta
SO4
2
NO3
Cond
Rex
O2
Cu
Zn
TFe
Fe2+
Mg
Ca
IOB
SOB
TB
UA
DiatomsEuglenaYeasts
HF
pH
0.0110.3070.281
0.6870.487
0.3630.5740.574
0.6940.405
0.4030.1010.51
10.215
0.169
0.212
0.011
0.202
0.0710.336
Ta
0.937
0.400
0.08
0.141
0.1570.189
0.0760.011
0.120
0.323
0.0550.004
.14
80.103
0.161
0.380
0.382
0.1470.027
0.039
SO
42
0.0
35
0.816
0.166
0.079
0.2720.371
0.543
0.675
0.182
0.349
0.363
0.480
0.43
4
0.452
0.555
0.248
0.235
0.328
0.202
0.567
NO
1
0.053
0.953
0.254
0.341
0.034
0.153
0.1250.010
0.095
0.194
0.1460.173
0.29
60.195
0.0290.103
0.002
0.008
0.061
0.031
Co
nd
0.0
00
0.412
0.646
0.0
19
0.212
0.064
0.660
0.625
0.616
0.654
0.792
0.505
0.48
2
0.248
0.396
0.188
0.251
0.108
0.114
0.369
Rex
0.0
08
0.362
0.108
0.812
0.214
0.020
0.210
0.248
0.1210.141
0.213
0.163
0.20
3
0.179
0.0370.088
0.050
0.2360.122
0.136
O2
0.0
12
0.269
0.0
26
0.294
0.709
0.906
0.4080.488
0.3950.384
0.0390.3370.23
10.066
0.202
0.042
0.033
0.2240.0170.111
Cu
0.0
01
0.600
0.0
00
0.391
0.0
00
0.150
0.0
05
0.700
0.630
0.449
0.619
0.233
0.60
1
0.3670417
0.159
0.091
0.273
0.033
0.378
Zn
0.0
01
0.941
0.0
00
0.490
0.0
00
0.089
0.0
08
0.0
00
0.688
0.522
0.688
0.480
0.45
4
0.416
0.457
0.142
0.154
0.197
0.077
0.449
TF
e
0.0
00
0.485
0.289
0.515
0.0
00
0.483
0.0
17
0.0
00
0.0
00
0.732
0.485
0.325
0.52
3
0.307
0.479
0.142
0.268
0.194
0.147
0.446
Fe2+
0.0
05
0.260
0.0
16
0.183
0.0
00
0.332
0.0
08
O.0
02
0.0
00
0.0
00
0.527
0.455
0.49
3
0.415
0.673
0.481
0.404
0.054
0.382
0.361
Mg
0.0
05
0.749
0.300
0.315
0.0
00
0.213
0.822
0.0
02
0.0
00
0.0
03
0.0
00
0.494
0.45
2
0.465
0.399
0.223
0.303
0.136
0.202
0.499
Ca
0.483
0.977
0.0
01
0.234
0.0
00
0.267
0.0
18
0.109
0.0
01
0.0
24
0.0
018
0.0
04
0.38
9
0.451
0.513
0.493
0.310
0.085
0.225
0.325
IOB
0.0
02
0.379
0.0
10
0.080
0.0
04
0.228
0.171
0.0
00
0.0
07
0.0
20
0.0
03
0.0
07
0.0
21
0.591
0.599
0.317
0.304
0.1590.002
0.440
SO
B
0.224
0.541
0.0
07
0.457
0.141
0.639
0.694
0.029
0.138
0.069
0.0
14
0.0
05
0.0
07
0.00
0
0.463
0.310
0.354
0.020
0.069
0.422
TB
0.245
0.269
0.0
00
0.840
0.060
0.794
0.168
0.0
04
0.0
01
0.0
01
0.0
00
0.0
06
0.0
02
0.00
0
0.0
6
0.658
0.558
0.187
0.413
0.541
UA
0.146
0.0
09
0.881
0.477
0.196
0.544
0.773
0.175
0.772
0.330
0.0
01
0.125
0.0
03
0.06
0.66
0.0
00
0.471
0.126
0.391
0.132
Diatoms0.941
0.0
08
0.107
0.987
0.084
0.727
0.821
0.531
0.290
0.066
0.0
05
0.0
37
0.066
0.71
0.36
0.0
00
0.0
01
0.199
0.248
0.449
Euglena0.166
0.314
0.0
24
0.953
0.456
0.105
0.124
0.060
0.176
0.321
0.710
0.348
0.611
0.34
4
0.905
0.197
0.387
0.171
0.141
0.229
Yeasts
0.627
0.849
0.166
0.176
0.431
0.399
0.902
0.818
0.593
0.313
0.0
08
0.165
0.182
0.98
6
0.679
0.0
04
0.0
07
0.0
88
0.334
0.289
HF
0.0
21
0.787
0.0
00
0.833
0.0
11
0.351
0.442
0.0
09
0.0
02
0.0
02
0.0
13
0.0
01
0.054
0.00
9
0.0
16
0.0
00
0.363
0.0
02
0.0
45
0.0
47
aT
heupperpartofthetablegivesthecorrelationcoefficient(r)andthelowerpartgives
significancelevel(p