MICROBIAL ECOLOGY

Microb Ecol (2001) 41:2035 DOI: 10.1007/s002480000044 2001 Springer-Verlag New York Inc.

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

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 concentrations 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 chemolithotrophic activity. 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-

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

ogy of extreme environments may provide insight into the limits of life and its possible origin. Extremophilic microorganisms 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, exposed microbial cell components can be hydrolyzed or pro-

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Fig. 1. Geographic position of the Tinto River. The location of the sampling sites and the mining region are indicated.

teins denatured. Dissociation and solubility of many molecules 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 soluble at 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 constant 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 communities found in the Tinto River and describes the main physicochemical and biological features of this extreme habitat.

Materials and Methods

Study Sites, Sampling Characteristics, and Estimation of Microbial Abundance

samples were collected in February, May, August, and November of 1993. Samples for chemical analysis were collected in 100 ml polypropylene 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 microscopic observations were done according to the method described 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 interference filter (bandpass 450 to 490 nm). Cell volume was estimated 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 Collins method with five dilution series [12].

Isolation of Microorganisms
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 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-

22 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 KH2PO4, 1mM MgCl2, 1.5 mM (NH4)2SO4, 0.5% (w/v) glucose, 0.05% (w/v) malt extract, 0.5% (v/v) trace metals [1]). Final pH, adjusted with concentrated H2SO4, was 3 for all solid media and 2.5 for liquid media. Different media for chemolithotrophic bacteria enrichments were obtained by supplementing 9K medium with ferrous iron (44.8 g/L FeSO4 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]. Identification 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].

A.I. Lopez-Archilla et al. 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 sulfuroxidizing 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 STATGRAPHICS, 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 extremely variable depending on the season. The highest flow values are reached in January or February (8.1 m3/s) and the lowest in August (0.07 m3/s) [37]. These fluctuations are due to the regional climatology. The river is subject to a Mediterranean 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 measured in the Tinto River are shown in Table 1. Some parameters, 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 concentration 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.

Analysis of Physicochemical Parameters

Total content of Fe, Cu, Zn, and Mg was measured by atomic absorption spectrophotometry using a Perkin Elmer 1100B instrument. 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 turbidimetric 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 electrodes. 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 conductimeter. Oxygen concentration and water temperature were determined with an Orion-810 oxymeter.

Genomic Analysis
Pulsed field gel electrophoresis of intact DNA prepared from different 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 performed 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 principal component axis. The first principal component axis (AI) is formed from the original variables with the greatest variance. All

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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 Sampling site Winter E1 1 2 3 5 6 m3 m4 sw1 7 8 11 E1 1 2 3 5 6 m3 m4 sw1 7 8 11 E1 1 2 3 5 6 m3 m4 sw1 7 8 11 E1 1 2 3 5 6 m3 m4 sw1 7 8 11 pH Mean 6.67 2.66 2.77 1.97 2.45 2.15 1.93 2.22 2.67 2.04 2.39 2.35 4.30 2.50 2.60 2.00 2.30 2.00 2.10 1.80 2.67 2.00 2.40 2.20 7.10 2.55 3.10 2.26 2.65 2.60 2.41 2.30 2.88 2.62 2.53 2.88 6.62 2.00 2.60 1.67 1.85 1.45 1.99 1.87 3.05 1.96 2.42 2.70 SD 0.06 0.03 0.01 0.08 0.04 0.08 0.03 0.07 0.06 0.05 0.06 0.07 0.05 0.05 0.04 0.02 0.04 0.02 0.02 0.03 0.05 0.02 0.02 0.01 0.06 0.04 0.08 0.03 0.02 0.05 0.02 0.02 0.01 0.02 0.01 0.01 0.05 0.03 0.05 0.02 0.03 0.02 0.02 0.02 0.08 0.06 0.03 0.02 Mean 6 15 15 10 2 11 12 20 6 11 11 13 26 31 29 33 15 28 26 22 21 24 28 28 25 19 21 22 19 26 26 24 21 24 20 30 18 24 22 22 12 21 20 24 16 19 18 22 t SD 0.2 0.1 0.1 0.2 0.3 0.3 0.2 0.3 0.4 0.1 0.1 0.2 0.1 0.1 0.1 0.5 0.4 0.2 0.2 0.1 0.3 0.4 0.1 0.3 0.1 0.2 0.1 0.1 0.2 0.3 0.3 0.4 0.1 0.1 0.2 0.3 0.2 0.2 0.1 0.1 0.3 0.3 0.4 0.5 0.1 0.2 0.2 0.2 Cond. Mean 0.79 9.49 9.50 11.62 3.97 12.59 13.97 13.80 2.67 11.66 6.85 5.27 0.58 9.37 7.91 16.5 4.42 19.39 7.92 25.20 3.07 14.67 4.12 3.45 0.44 11.89 9.90 16.53 10.46 13.67 15.39 19.76 3.67 9.60 11.21 46.10 0.36 9.55 5.16 31.50 15.15 56.70 11.40 24.10 2.14 14.88 3.74 1.54 SD 0.11 0.25 0.27 0.16 0.09 0.36 0.28 0.32 0.10 0.38 0.22 0.20 0.09 0.31 0.39 0.52 0.42 0.45 0.32 0.59 0.13 0.12 0.08 0.02 0.07 0.12 0.24 0.35 0.38 0.41 0.46 0.35 0.12 0.29 0.32 0.81 0.09 0.27 0.35 0.56 0.24 0.82 0.34 0.46 0.12 0.25 0.22 0.20 Rex. Mean 169 382 350 456 478 423 424 414 167 408 450 511 316 416 340 443 506 446 449 390 362 412 518 535 295 368 346 455 455 402 419 386 356 413 456 445 110 442 360 413 443 427 434 383 376 406 420 442 SD 12 58 11 25 35 31 41 26 18 32 26 34 18 29 31 26 45 37 34 28 27 34 42 49 12 27 29 32 38 41 29 30 28 34 32 36 9 35 29 38 41 49 40 42 38 39 45 28 O2 Mean 12.0 10.0 2.5 1.1 11.4 8.7 7.0 3.9 10.7 8.0 3.7 11.4 7.6 5.3 3.2 1.1 7.1 5.4 6.3 5.3 7.4 4.7 8.2 11.3 13.3 7.4 8.5 6.5 10.5 5.5 6.6 6.5 13.9 8.7 2.1 17.9 9.0 9.6 4.3 6.5 10.5 10.0 7.7 4.4 9.7 8.6 9.3 12.3 SD 1.62 0.28 0.31 0.08 0.92 0.55 0.63 0.14 0.82 0.51 0.20 0.32 0.53 0.26 0.29 0.15 0.08 0.18 0.20 0.17 0.50 0.24 0.19 0.27 0.92 0.05 0.21 0.24 0.32 0.16 0.25 0.30 0.24 0.18 0.04 0.71 0.26 0.15 0.16 0.28 0.30 0.27 0.25 0.08 0.12 0.16 0.20 0.34 Mean 10 160 210 160 80 230 160 760 10 420 250 120 0 100 100 50 220 130 300 1,500 10 560 110 50 50 150 210 190 230 250 200 370 5 290 350 10 5 150 90 100 130 450 100 1,420 10 280 50 20 Zn SD 0.01 0.1 0.1 2.0 2.2 3.0 1.1 4.0 0.1 1.5 0.9 0.1 0.00 0.2 0.5 0.9 1.0 0.5 2.1 18.9 0.1 3.5 1.5 0.1 0.05 0.5 0.8 1.0 1.3 1.1 0.6 1.1 0.0 0.3 1.8 0.1 0.0 1.2 0.1 1.1 1.3 3.4 0.9 16.3 0.1 1.0 0.5 0.1 SO42 Mean 500 13,800 12,120 14,800 5,410 29,220 30,350 16,900 1,470 14,200 15,240 8,900 314 6,780 6,760 5,900 6,500 9,220 11,900 7,800 1,600 10,010 7,210 4,050 10 12,750 12,220 25,950 13,230 21,750 22,920 21,600 4,650 21,770 33,880 4,120 100 9,350 4,240 2,450 7,100 3,390 5,440 5,380 1,490 5,960 2,760 1,840 SD 0,35 1,1 1,0 1,3 0,5 2,2 1,5 1,2 0,3 1,0 1,1 0,9 0,26 0,8 0,8 0,7 0,7 0,9 1,1 0,5 0,2 1,1 1,0 0,8 0,0 1,1 1,2 2,8 1,5 2,0 2,2 2,1 0,9 1,9 2,5 0,8 0,1 1,0 0,8 0,7 0,9 0,7 0,5 0,6 0,3 1,0 0,5 0,3

Spring

Summer

Autumn

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 Mean 5.70 5.70 4.95 6.52 4.76 5.32 8.40 5.01 17.3 9.15 6.15 8.43 8.68 5.58 4.96 8.06 4.96 9.92 6.20 0.92 16.12 7.44 4.96 5.58 3.72 6.82 4.96 8.06 4.96 4.96 8.06 8.68 9.30 6.82 6.82 390.6 4.34 6.20 4.34 25.42 11.16 75.02 13.64 12.20 15.50 13.02 6.82 6.82 SD 0.2 0.2 0.1 0.3 0.2 0.2 0.4 0.2 0.9 0.7 0.3 0.3 0.7 0.2 0.2 0.3 0.1 0.3 0.2 0.1 0.9 0.3 0.1 0.2 0.1 0.3 0.1 0.4 0.1 0.1 0.3 0.3 0.3 0.2 0.2 2.8 0.1 0.3 0.1 1.0 0.8 1.8 0.9 0.9 0.9 0.9 0.3 0.3 Mean 0 30 70 50 20 270 200 170 90 0 90 40 0 20 50 40 20 370 110 160 0 160 60 30 5 35 110 70 30 360 280 100 0 130 180 5 5 65 20 55 135 695 165 490 10 195 45 15 Cu SD 0.0 0.3 0.1 0.4 0.2 2.0 1.2 0.9 0.5 0.0 0.6 0.5 0.0 0.2 0.3 0.2 0.1 1.1 0.8 0.5 0.0 0.5 0.2 0.1 0.0 0.2 0.4 0.3 0.2 1.5 1.1 0.5 0.0 0.3 0.4 0.0 0.0 0.4 0.1 0.2 0.8 2.5 0.9 1.6 0.1 0.5 0.3 0.1 Mean 0.62 5.79 242 9.60 2.70 33 859 373 0.70 662 4.50 2 0 650 259 560 1.60 250 77 560 60 446 2.20 1.40 0 1,100 1,130 34 90 2,490 760 3,790 390 630 38 45 0 2,690 2,360 3,000 12 1,800 47 5,210 13 1,430 3 68 Fe2+ SD 0.1 0.8 3.6 0.6 0.2 1.3 4.2 2.6 0.1 3.3 0.2 0.1 0.0 3.3 3.0 2.9 0.2 1.0 0.8 2.5 0.8 2.0 0.1 0.1 0.0 56 48 0.5 0.6 80 6.5 110 24 38 0.2 0.2 0.0 57 68 97 0.1 29 0.8 258 0.1 95 0.0 0.5 Mean 30 2,260 2,350 3,430 730 2,510 3,290 3,750 3,280 2,710 700 660 0 2,000 2,020 3,390 850 2,670 1,910 5,200 3,730 2,170 410 240 15 1,630 2,500 3,700 1,140 3,350 3,600 6,050 2,200 50 600 0 15 3,500 2,750 5,600 1,900 6,100 1,850 8,100 3,350 60 40 20 TFe SD 0.5 190 250 426 28 182 220 203 254 158 65 24 0 98 119 298 54 157 100 367 245 212 39 27 0.2 166 210 245 170 294 276 421 113 3 41 0 0.1 200 159 324 101 337 118 410 199 12 3 0.9 20 340 290 320 250 540 440 580 160 n.d. 240 170 10 280 250 220 150 280 210 680 200 380 130 70 10 420 270 400 840 680 400 540 180 460 540 1040 0 190 150 160 360 1080 300 800 110 300 80 10 0 0.42 0 6.62 0.22 18.22 32.10 13.69 0.04 12.22 8.54 1.77 0 1.38 0 6.62 1.08 104.22 12.17 25.56 0.06 18.20 2.45 0.75 0 1.15 0 9.27 0.26 9.15 36.48 20.06 0 6.08 18.56 0 0 2.54 0 11.33 38.24 379.05 40.23 34.26 0 25.36 2.03 0.05 36.87 438.47 325.42 321.57 249.66 172.78 161.76 262.79 112.30 216.79 153.32 163.98 33.80 285.97 311.04 185.13 136.75 125.13 96.17 253.30 140.84 208.99 78.33 70.87 33.62 412.82 410.33 371.51 489.26 189.83 188.03 305.89 119.39 230.39 305.12 211.78 29.42 123.19 195.92 154.81 100.21 112.65 99.39 307.08 82.99 167.10 46.44 33.50 4.83 20.88 3.27 4.62 1.24 3.18 5.64 0 7.55 5.62 4.18 1.16 6.72 2.66 2.64 4.09 1.01 4.68 4.85 13.07 7.92 0 2.91 1.78 4.30 4.85 3.89 6.67 3.60 4.11 8.28 6.67 6.66 6.06 6.49 117.10 3.67 3.04 2.49 8.47 3.89 11.35 3.96 11.23 5.76 4.62 2.15 2.50 0.03 1.69 1.12 1.54 0.86 9.33 6.97 2.11 0.12 2.41 1.89 0.99 0.06 0.97 1.29 0.73 0.55 10.20 3.25 3.68 0.21 2.68 0.84 0.49 0.02 2.06 1.88 1.73 3.56 11.03 6.30 6.75 0.14 2.25 3.14 0.45 0.03 0.43 0.64 0.87 2.68 14.88 3.06 4.40 0.12 2.39 0.58 0.10 Mg As Ca K Ni

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Table 2. Comparison of some physicochemical parameters of Tinto River with several rivers of the same area; concentration in mg L-1 pH Guadiamar Rocina Partido Agrio Sampling E1(mean) Tinto (mean)
nd: not determined.

Fe 12.1 7.39 24.8 50 15 2,261

Zn 0.97 0.35 0.11 33.97 6.1 225

Cu 0.07 0.025 0.135 1.97 2.5 109

Mg nd nd nd nd 10 337.4

Ni 0.045 0.02 0.026 nd 0.035 2.6

K 6.63 2.73 58.4 nd 4.88 7.42

SO42 312.5 72.45 89.75 nd 81.2 10,110

NO3 0.83 0.48 1.15 nd 5.61 9.33

References 40a 40a 40a 48a This work This work

7.8 7.1 7.8 4.3 6.2 2.2

Microbial Community of the Tinto River The Tinto River ecosystem is unique for a river in that the biological community is exclusively microbial. Higher eukaryotes have not been detected in any of the sampling stations 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 mineral precipitation was normally observed on the surface of the biofilms. In order to elucidate the degree of diversity of this environment, we used classic methods for the isolation and characterization 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 chemolithotrophic bacteria), decomposers (heterotrophic bacteria and fungi), and consumers (heterotrophic protists). Primary Producers. Algae accounted for the greatest proportion of biomass (65%) as number of cells ml1, in the Tinto River. Because of their photosynthetic abilities they constitute, 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 positive 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 different seasons (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 Conjugatophyceae), were sporadically observed during the autumn. However, the most ubiquitous algae were unicellular Chlorophyta. 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. Unicellular nonflagellated Chlorophyta were observed and isolated all year long. Their phenotypic characteristics resembled 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). Regardless of their similar phenotypes, several genomic polymorphisms (different chromosome numbers and sizes) were observed using pulsed-field gel electrophoresis (PFGE), suggesting 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

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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 compared to type collection reference strains [38]. In addition to the photosynthetic algae, chemolithotrophic bacteria were another type of abundant primary producer in the Tinto system (Table 4). Most iron-oxidizing bacteria (IOB) isolated were Gram-negative, aerobic rods with characteristics similar to those of Thiobacillus ferrooxidans. 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 characteristic of Leptospirillum ferrooxidans [4]. Fourteen strains of sulfur-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 preparation), 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 corresponded to the genus Acidiphilium. Members of this genus have been shown to be frequently associated with chemolithoautotrophic bacteria, especially iron oxidizers [22, 25]. Many bacterial isolates were Gram-positive bacilli, aerobic 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 environments. In order to determine the proportion of microorganisms 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 corresponded 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 dematiaceous seem to be specific to this kind of habitat, since they are 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 disturbance of these complex structures. We observed different flagellates (phylum Zoomastigina), amoeba of the class Lobosea (phylum Rhizopoda), some representatives of class Heliozoa (phylum Actinopoda), and ciliates (phylum Ciliophora).

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 measured 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 conductivity 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

Microbial Community Composition and Ecology of an Acidic Aquatic Environment Table 3. Taxonomic classification of different microbial groups detected in the Tinto River Domain Phylum Class Bacillariophyceae Euglenophyceae Chlorophyceae Order Family or group Genus Species

27

Eukarya Bacillariophyta Euglenophyta Chlorophyta

Ulvophyceae Rhodophyta Rhodophyceae Conjugatophyta Conjugatophyceae Ciliophora Rhizopoda Actinopoda Mastigophora Zygomycetes Deuteromycetes

Euglenales Chlamydomonadales Chlorococcales Ulotrichales Porphyridiales Zygnemales

Euglenaceae Chlamydomonadaceae Oocystaceae Ulotrichaceae Cyniaceae Zygnemataceae Strongylidiidae

Euglena Chlamydomonas Chlorella Klebsormidium Galdieria Zygnema Strongylidium

E. mutabilis C acidophila Chlorella sp. Klebsormidium sp G. sulphurarin Zygnema sp Strongylidium sp.

Spirotricha Stichotrichida Lobosea Amoebida Heliozoa Amebomastigota others (biflagelates)

Mucorales Demateaceous

Mortierella Scytalidium

Bahusacala

Phoma Heteroconium Penicillium

Moniliales

Cryptococcaceae

Lecytophora Rhodotorula

Cryptococcus

Candida

Mortierella sp. S. acidophilum S. lignicola S. termophilum Scytalidium sp. B. cookei B. olivaceonigra Bahusakala sp. P. pomorum Phoma sp. H. chaetospira P. atramentosum P. brasilianum (serie) P. canescens (serie) P. cremeo-griseum P diversum P. frecuentans P grancanariae P. glaucolanosum P. griseum-azureum P. lignorum P moldavicum P montanense P. purpurescens P. sartoryi P. spinulosum P. verruculosum Penicillum sp. L. hoffmannii R. aurantiaca R. glutinis R. minuta R. rubra C. albidus C. elinovii C. flavus C. gastricus C. auricularia C. citrea C. dendrica C. fluviatilis C. krusei C. muscorum C. scotii

28 Table 3. (Continued). Domain Phylum Basisiomycetes Class Order Family or group Tremellaceae Genus Tremella

A.I. Lopez-Archilla et al.

Species T. encephala T. fuciformis T. subanomala H. corniformis L. antarticum L. stokesii H. saturnus T. ferrooxidans T. f. related T. thiooxidans T. t related Thermophilic Thiobacillus sp. Acidiphilium sp. L. ferrooxidans B. megaterium B. subtilis B amyloliquefaciens B. stearothermophilus B. cereus

teliospore-forming Ascomycetes Bacteria Proteobacteria Undeterminated Gram Saccharomycetoideae heterotrophic bacteria group

Holtermannia Leucosporidium Hansenula Thiobacillus

group Other lithotrophic bacteria Gram positives Low G + C group

Acidiphilium Leptospirillum Bacillus

High G + C group Actinomycetes

positively with the biological variables cluster and the Euglena cell number. Oxygen concentration correlated negatively with the metal variables and positively with pH. In addition, a principal component analysis (PCA) was conducted in order to obtain information on the environmental 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 physicochemical 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 concentration.

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 occurrence to physical and chemical parameters. The particular geology and climatology of the region favors the creation of the Tinto Rivers special environment, which provides the base on which the biological communities 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 bacteria. 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 pluvicus regime 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 chalcopyrite, 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 4. Seasonality of microorganism populations at in different sampling sites from the Tinto River (cells ml-1) Sampling sites UA E1 1 2 3 4 5 6 m4 Swl 7 8 11 E1 1 2 3 4 5 6 m4 Swl 7 8 11 E1 1 2 3 4 5 6 m4 Swl 7 8 11 E1 1 2 3 4 5 6 m4 Swl 7 8 11 E1 1 2 3 4 5 6 m4 Swl 7 8 11 Autumn Winter 1.93 106 9.42 107 1.47 103 4.37 105 4.75 104 5.00 105 1.12 105 1.55 105 1.00 105 6.20 105 4.10 105 2.50 104 7.62 104 1.20 106 1.25 105 9.54 105 7.35 102 3.82 104 1.00 105 4.10 105 6.78 105 1.25 104 3.82 104 3.75 105 2.50 105 1.00 105 1.26 106 4.10 105 6.78 105 1.25 104 3.82 104 3.75 105 2.50 105 1.00 105 1.26 106 Spring 1.03 1.16 2.75 2.75 9.87 9.50 2.83 8.20 9.27 2.47 7.50 9.60 6.50 1.00 8.20 8.25 9.50 1.90 9.50 9.60 8.20 8.25 4.10 1.90 9.60 1.92 1.87 9.50 9.60 4.10 1.90 9.60 1.92 1.87 9.50 9.60
6

Summer 9.75 1.54 8.70 1.00 5.50 1.02 6.87 7.50 5.25 4.50 3.50 9.27 1.27 3.70 4.00 1.12 7.50 6.75 5.50 1.42 9.50 8.75 1.50 2.25 3.25 8.75 5.50 5.50 5.00 4.50 8.50 5.75 4.75 5.50 5.50 5.00 4.50 8.50 5.75 4.75 105 108 107 106 105 108 107 105 105 106 106 106 106 107 106 105 107 106 106 106 106 105 104 106 1.65 104 1.14 107 5.07 107 2.32 106 2.62 105 3.32 105 8.3 105 5.6 104 5.00 105 1.13 105 1.38 106 1.97 107 1.62 106 7.50 105 8.75 104 1.65 105 4.15 106 1.12 105 2.07 106 8.30 105 1.65 104 1.00 106 2.45 106 1.65 105 1.66 106 1.12 105 3.33 105 6.90 105 1.65 104 1.00 106 2.45 106 1.65 105 1.66 106 1.12 105 3.33 105 6.90 105

10 106 105 105 104 103 105 104 105 105 104 104 105 105

Diatoms

104 105

Euglena

104 105 103 104 104 104 103 104 103 104 105

106

105

HF

106

103 104

105 106 106 105 104

104 106

TB

10 104 103 104 105

103 104

105 106 106 105 104

104

30 Table 4. (Continued). Sampling sites IOB E1 1 2 3 4 5 6 m4 Swl 7 8 11 E1 1 2 3 4 5 6 m4 Swl 7 8 11

A.I. Lopez-Archilla et al.

Autumn Winter nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd Spring 1.8 106 1.8 106 4.0 104 6.0 103 7.0 104 1.4 105 4.0 104 4.0 104 2.5 104 1.4 105 2.5 104 0.2 10 5.0 107 2.5 106 6.0 104 3.5 105 9.0 103 7.0 103 6.0 107 1.6 104 2.0 105 2.5 105 4.0 105 Summer 2.0 105 2.5 105 3.0 106 3.0 105 1.7 105 2.0 106 4.0 105 2.5 104 1.7 106 1.6 107 1.6 102 0.2 10 3.0 105 1.7 106 1.7 105 9.0 106 2.5 106 6.0 106 3.0 105 3.0 106 1.6 107 6.0 106 8.0 104 106 104 108 106 106 107 106 105 106 105 105 3.0 107 2.5 105 3.5 105 1.4 106 1.2 106 1.4 106 2.0 106 9.5 105 3.0 105 9.0 105 5.0 104 1.8 9.0 1.8 2.0 3.0 1.1 3.0 2.5 0.9 3.0 1.7

SOB

UA: unicellular algae; HF: hyphomycete fungi; TB: total bacteria; IOB: iron oxidizing bacteria; SOB: sulfur oxidizing bacteria; nd: not determined; : not found

oxidize inorganic reduced forms of sulfur, whereas only a few 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 compounds 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 pyritic belt in the headwaters of the river are subject to extensive 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 oxidized forms, which raises the question of how the chemolithotrophic bacteria (IOB and SOB) can sustain their energetic metabolism along the 90 km river in the absence of

appropriate substrates. One possibility could be that the chemolithotrophic 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 downstream should be reduced because of the correspondent dilution factor (up to two orders of magnitude) produced by the different neutral tributaries. However, bacterial chemolithotrophs 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 chemolithotrophic bacteria. In fact, reduced forms of sulfur can be obtained from sulfate both by limited-scale assimilatory processes 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. ferrooxidans forms Fe2+ only anaerobically, reoxidizing it under

Table 5. Phenotypic properties of different SOB isolated from Tinto River Flagellum G + Ye +++ nd nd +s + +s + +s +s +s +s 3037 3037 3037 3037 3037 3037 3037 3037 3037 3037 3037 3037 3037 3037 + ++ + ++ +++ +++ +++ +++ +++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ +/ +/ nd nd nd nd nd nd nd nd nd nd +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ Optimal T Catalase Gram Fe2+ S0 NaCl 2% NaCl 5% NaCl 10% TTT +++ ++ ++ +++ +++ ++ +++ ++ ++ ++ +++ ++ +++ +++ THI +/ +/ nd +/ nd CCu +/ +/ nd nd CZn +/ +/ nd nd CFe nd nd

Microbial Community Composition and Ecology of an Acidic Aquatic Environment

pH range

Optimal pH

Size (m)

A1 AB AC AE AF AG AH A A AK AM AN AO AP

17 11.5 0.57 14 0.53 0.53 14.5 0.57 0.57 0.57 1.56.5 1.57 16.5 16.5

3 1.5 2.5 2.5 1.5 1.5 2.5 2.5 3.5 3 3 3 3.5 2

1.6 0.5 1.4 0.5 1 0.25 1.4 0.6 0.9 0.5 1.5 0.6 1.3 0.22 1.9 0.5 1.1 0.2 1.0 0.2 1.1 0.4 1 0.3 1.1 0.3 1.3 0.5

(nd) not determined; () no growth detected; (s) several; G + Ye: 9K medium supplemented with 0.1 ml of a solution at 10% (w/v) of glucose and yeast extract; TTT: tetrathionate; THI: thiosulfate; Ccu: metal sulfide (Cu concentrate); CZn: metal sulfide; (Zn concentrate); CFe: metal sulfide (Fe concentrate). (+); (++); (+++): differential growth yield

31

32

Table 6. Results of the correlation analysisa Rex 0.574 0.694 0.405 0.403 0.101 0.511 0.011 0.120 0.323 0.055 0.004 .148 0.675 0.182 0.349 0.363 0.480 0.434 0.010 0.095 0.194 0.146 0.173 0.296 0.625 0.616 0.654 0.792 0.505 0.482 0.248 0.121 0.141 0.213 0.163 0.203 0.488 0.395 0.384 0.039 0.337 0.231 0.700 0.630 0.449 0.619 0.233 0.601 0.688 0.522 0.688 0.480 0.454 0.000 0.732 0.485 0.325 0.523 0.000 0.000 0.527 0.455 0.493 0.000 0.003 0.000 0.494 0.452 0.001 0.024 0.0018 0.004 0.389 0.007 0.020 0.003 0.007 0.021 0.138 0.069 0.014 0.005 0.007 0.000 0.001 0.001 0.000 0.006 0.002 0.000 0.772 0.330 0.001 0.125 0.003 0.06 0.290 0.066 0.005 0.037 0.066 0.71 0.176 0.321 0.710 0.348 0.611 0.344 0.593 0.313 0.008 0.165 0.182 0.986 0.002 0.002 0.013 0.001 0.054 0.009 O2 Cu Zn TFe Fe2+ Mg Ca IOB SOB TB UA Diatoms Euglena Yeasts 0.202 0.147 0.328 0.008 0.108 0.236 0.224 0.273 0.197 0.194 0.054 0.136 0.085 0.159 0.020 0.187 0.126 0.199 0.334 0.045 HF 0.071 0.336 0.027 0.039 0.202 0.567 0.061 0.031 0.114 0.369 0.122 0.136 0.017 0.111 0.033 0.378 0.077 0.449 0.147 0.446 0.382 0.361 0.202 0.499 0.225 0.325 0.002 0.440 0.069 0.422 0.413 0.541 0.391 0.132 0.248 0.449 0.141 0.229 0.289 0.047

pH

Ta

SO42 NO3 Cond

pH Ta SO42 NO1 Cond Rex O2 Cu Zn TFe Fe2+ Mg Ca IOB SOB TB UA Diatoms Euglena Yeasts HF

0.011 0.307 0.281 0.687 0.487 0.363 0.574 0.937 0.400 0.08 0.141 0.157 0.189 0.076 0.035 0.816 0.166 0.079 0.272 0.371 0.543 0.053 0.953 0.254 0.341 0.034 0.153 0.125 0.000 0.412 0.646 0.019 0.212 0.064 0.660 0.008 0.362 0.108 0.812 0.214 0.020 0.210 0.012 0.269 0.026 0.294 0.709 0.906 0.408 0.001 0.600 0.000 0.391 0.000 0.150 0.005 0.001 0.941 0.000 0.490 0.000 0.089 0.008 0.000 0.000 0.485 0.289 0.515 0.000 0.483 0.017 0.000 0.005 0.260 0.016 0.183 0.000 0.332 0.008 O.002 0.005 0.749 0.300 0.315 0.000 0.213 0.822 0.002 0.483 0.977 0.001 0.234 0.000 0.267 0.018 0.109 0.002 0.379 0.010 0.080 0.004 0.228 0.171 0.000 0.224 0.541 0.007 0.457 0.141 0.639 0.694 0.029 0.245 0.269 0.000 0.840 0.060 0.794 0.168 0.004 0.146 0.009 0.881 0.477 0.196 0.544 0.773 0.175 0.941 0.008 0.107 0.987 0.084 0.727 0.821 0.531 0.166 0.314 0.024 0.953 0.456 0.105 0.124 0.060 0.627 0.849 0.166 0.176 0.431 0.399 0.902 0.818 0.021 0.787 0.000 0.833 0.011 0.351 0.442 0.009

0.215 0.169 0.212 0.011 0.103 0.161 0.380 0.382 0.452 0.555 0.248 0.235 0.195 0.029 0.103 0.002 0.248 0.396 0.188 0.251 0.179 0.037 0.088 0.050 0.066 0.202 0.042 0.033 0.367 0417 0.159 0.091 0.416 0.457 0.142 0.154 0.307 0.479 0.142 0.268 0.415 0.673 0.481 0.404 0.465 0.399 0.223 0.303 0.451 0.513 0.493 0.310 0.591 0.599 0.317 0.304 0.463 0.310 0.354 0.06 0.658 0.558 0.66 0.000 0.471 0.36 0.000 0.001 0.905 0.197 0.387 0.171 0.679 0.004 0.007 0.088 0.016 0.000 0.363 0.002

The upper part of the table gives the correlation coefficient (r) and the lower part gives significance level (p < 0.05). Abbreviatures are as in Tables 1 and 4.

A.I. Lopez-Archilla et al.

Microbial Community Composition and Ecology of an Acidic Aquatic Environment

33

Fig. 2. Distribution of the variables analyzed in the space formed by the two first components (AI and AII). Variables are abbreviated as in Table 4.

aerobic conditions [10, 40]. From the pathways to produce reduced inorganic compounds outlined above, sulfate reduction is the most important in a great number of artificial and natural environments. It requires anaerobic conditions and neutral pH. These are not typical characteristic of the Tinto River, but recently the isolation of acidic sulfate reducers associated with acid main drainage have been reported [15]. The search for sulfate-reduction activity in the Tinto has been unsuccessful to date (Amils et al., personal communication), except in the estuary, where the ocean water mixes with the river, increasing the pH and inducing a massive metal precipitation. Important concentrations of T. ferrooxidans can be found all along the river, which could cause the production of reduced iron. Nevertheless, the microbial communities of Tinto River are rather complex and biofilms thrive on the surface of the rocks and in the riverbed. It is possible that within these biofilms, anaerobic microniches exist [13] in which sulfate and iron reduction are carried out by assimilatory and dissimilatory microbial activities, facilitating the continuous recycling of these elements. The exploration of these possibilities requires a further analysis. In the Tinto River, the organic matter is generated by photosynthetic as well as by chemolithoautotrophic activity. This organic matter may be subsequently used by heterotro-

phic bacteria, fungi, and heterotrophic protists. The food chain in the river is short and exclusively microbial. Primary producers also need some inorganic compounds and elements (S, Ca, K, Mn, Fe, Cu, Zn, or Mg); these can be found in the river in concentrations that exceed their requirements (Table 1). Nitrate enters the river from municipal wastes and in fertilizer runoff from adjacent cultivated hillsides. In addition, T. ferrooxidans can fix atmospheric N2 [49, 35], as possibly do closely related microorganisms, providing the system with an additional source of nitrogen. The correlation analysis performed showed how the environmental and biological parameters are related in the Tinto system. The negative correlation observed for SOB and IOB with the pH is a direct consequence of the chemolithotrophic metabolism of these microorganisms. In addition, the presence of high concentrations of ferric iron, together with the low pH, generates strong oxidizing conditions that can promote the oxidation of different metals present in the abundant complex polymetalic sulfide minerals of the Pyritic Belt. This might explain the positive correlation found between the lithotrophic bacteria and the heavy metals detected in the river. We cannot rule out the possibility of a direct biooxidation of some of these metals from their mineral sources [17]. The principal component analysis also supports a direct

34

A.I. Lopez-Archilla et al.

relationship between chemolithotrophic bacteria, pH values, and heavy metal concentrations in the river waters. In this analysis (Fig. 2), heavy metals and chemolithotrophic bacteria clustered in the positive quadrant, while pH appeared in the opposite negative quadrant. Chemolithotrophic bacteria remain separated from the rest of the biological parameters, which were located in the lower right quadrant. Although the concentration of hyphomycetes fungi also correlated with low pH values, with metal concentrations, and with IOB and SOB, it formed a cluster with the rest of biological variables very close to diatom and euglenophyte values. A possible explanation is that as the hyphomycetes are decomposers, their proliferation must be dependent on the biomass available, mainly constituted by this type of algae. The PCA reveals that much of the variation in the Tinto River ecosystem can be described by three main groups of variables: pH metal concentrations, and biological productivity. The Tinto River is an interesting and probably unique model of an extreme environment in which the extreme conditions are of biological origin. The chemolithotrophic bacteria, which have specifically adapted to grow at low pH, produce large amounts of sulfuric acid because of the oxidation of reduced sulfur compounds. Together with ferric iron, the acid can produce the leaching of other metals included in the minerals. Ferric iron at low pH can also promote the oxidation of reduced metals, thus increasing the concentration of heavy metals in the river. These microorganisms contribute to the so-called extreme characteristics of this habitat. The high level of biodiversity found in this system underlines an interesting level of adaptation of different prokaryotic and eukaryotic microorganisms to low pH and high concentrations of heavy metals, which is not found in equivalent geophysically imposed extreme conditions (hot springs, hot acid springs, soda lakes). Further characterization of the Tinto system should give us insight not only into the role of the different microorganisms living there, but also into their unusual physiology and their possible biotechnological applications.

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We thank D. Moreira, P. Lopez-Garca, E. Zettler, and L. Amaral for discussion, as well as the two anonymous reviewers whose comments enabled us to improve the manuscript. This work was supported by grants from the CICYT, BIO990184, from the CAM 07M/0023/1999, and an institutional grant to the CBMSO from the Fundacion Areces.

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