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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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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



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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



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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.



7/16

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



8/16

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



9/16

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


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



10/16

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


11/16

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


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



12/16

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



13/16

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

21.7 microbial community amd

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