The Sulphur and Iron Metabolism in Acidophilic Microbial Gelatinous Formations from an Auriferous Sulphides Mine from the South-West of Romania

This study concerns the iron and sulphur metabolism in the gelatinous formations found in the Kiesberg gallery which is a part of an old mine located in the South-West of Romania which was dug in the metamorphic rocks. The gelatinous formations from the Kiesberg mine represent a complex assemblage formed by 1. chemoautotrophic acidophilic microorganisms (water pH 2.8) and 2. by heterotrophic microorganisms. The oxidative metabolism of the chemolitotrophs is very intense, oxidizing from pyrite: 1. Fe 2+ and has, as a result Fe(OH) 3 which is deposited at the base of the gelatinous formations, 2. sulphur compounds from which important quantities of SO 42-.

This study refers to an old mine (the Kiesberg Mine) located in the South-West of Romania.This mine was worked until the end of the XIX th century and it was dug in the auriferous sulphides metamorphic rocks of a Policarpus ore.
In this mine, the presence of some microbiological gelatinous formations: stalactites (snotites), leakings, was noticed.The microorganisms presence in the gelatinous formations is evidenced by the significant large biomass accumulations (the gelatinous deposits).
This type of gelatinous formations are not characteristic only for this kind of cavity, they are also often found in the ferous and sulphurous artificial (mines) or natural cavities (caves).
According to the specialized literature, the most well known and often found types of gelatinous formations are the ones from the hydrothermal cavities.These types of gelatinous formations are supplied with thermal and mezothermal waters with temperatures that vary between 18 and 50 0 C. Sometimes, these waters come from active volcanic areas that generally contain great quantities of sulphur or sulphuretted hydrogen.
Here are some examples of thermal and mezothermal cavities where the gelatinous formations were found: the Cueva de Villa Luz from the Plateau Tabasco in Mexic [14]; the Frassassi Cave from Italy [26]; the Movile Cave from Dobrogea, Romania [25].
In Wales, UK, there is an artificial cavity, a pyrite mine, closed more than 70 years ago -the "Cae Coch" Mine [2,4,6] -which is similar to Kiesberg mine that we studied about.
The gelatinous formations of microorganisms' consortium can be differently formed, depending on several environmental factors: the cavity climate, the temperature of the water in which they are found, the * email: ghermanvasile2000@yahoo.com chemical composition of the rock that forms the cavity and the chemical composition of the waters that drain various minerals [2].
The geological characteristics of the area where the Kiesberg mine is located have induced the existence of a particular kind of medium and of microbial associations that form a series of characteristical gelatinous formations.The number of the microorganisms from these microbial associations varies quite a lot, depending on the rain water quantities during the year.The metabolism of the gelatinous microbial association from the Kiesberg mine is based on the oxidation of the minerals from the cavity walls.Compounds such as sulphate and ferric hydroxide are the result of this oxidation.In this study, 1. the identification of microbial associations from characteristical gelatinous formations from the Kiesberg mine 2. the description of the oxidative microbial metabolisms types from the gelatinous formations is suggested.
The numerical estimations that have shown that the number of the microorganisms depends on the rainfall quantities are also a part of this study.
Microbiological determinations and numerical estimates have been carried out in years 2004 and 2005, for two hydrological different seasons, a wet and a dry one.Estimates of populations from different physiological of microorganisms groups have been also calculated.

Experimental part
Materials and methods: The chemical water analysis in which the gelatinous formations are found have been done by using: the electro-chemical method, for the pH; the iodic-chemical method, for the dissolved O 2 , the solvents extraction and the measurement by spectrophotometry for the phosfats, azotates; the turbidization-precipitation method with BaCl 2 and the spectrophotometry for the sulphides and the atomic absorbtion -spectrophotometryspectrophotometer GBC 932 plus, for the metals.
The isolation of acidophilic heterotrophic microorganisms has been realized trough enriched growing on the March medium-pH-2.5[28], and on the G.Y.E. at pH 3 [5].
The isolation and identification of fungus has been realized on the solid Czapeck medium [19,12], starting from the serial dilutions of the enriched growing realized from samples in the liquid March medium.
The indirect determination of Acidithiobacillus strains by the measurement of pH medium was achieved by seeding the test samples on the 9K, S 5 and S 6 , and also on the W 6 medium.The growing was determined by the quantity of oxidized thiosulphate.The Acidithiobacillus is estimated by the final pH value after 28 days [3].
The determination of the most probable number of the microorganisms was done by performing decimal or serial dilution, with 3 repetitions/dilution, while the observation of the number of positive tubes, the numerical value, was established from the table [20].The method of counting the colonies on the solid medium was used also for the numerical estimates.
Observations in S.E.M. and in T.E.M. on the gelatinous formations from the Kiesberg mine have also been done.The samples for the electronic microscopy have been done by the standard method [13].
In (SEM), for the dehydration of the samples the hexametil-disilazan method were used.The aparratus used was a Zeiss Gemini 982 microscop.The observation took place at a acceleration tension of 15 kV and at a work distance of 16mm.The obtained images are digitals with a resolution of 1021 X 1024 pixels.
In (TEM), the aparratus used was an ultramicrotom Ultracut E Reichert and the observations were done on two aparratus: JEOL 1010 and JEOL 1230 at an acceleration tension of 80 kV.
In 2004, the measurements were realized in March, when the total rainfall value was 29.9 mm (the dry season) and in July, when the total rainfall value was 141,8 mm (the rainy season).In 2005, the measurements were realized in November, when the total rainfall value was 37.4 mm (the dry season) and in April, when the total rainfall value was 226.4 mm (the rainy season).From a hydrological point of view 2004 was a drier year (875 mm) than 2005 (1202 mm).

Results and discussion
The results of the microbiological analysis made on the gelatinous formations from Kiesberg mine are shown in table 1.The fact that 16 strains of microorganisms were identified in 2004 can be noticed from table 1.For the next year, 2005, one more strain -Scytalidium (Dematiaceae Family) -was identified beside the 16 ones.
The identified microorganisms strains are intimately associated by extra-cellular mucopolysaccarides, forming an association or a consortium.From all the identified strains, the four Acidithiobacillus together with the actinomycetes represent the bacteria, the rest of the strains represent the fungus.A. neapolitanus, A. thiooxidans and A. thioparus represent acidophilic chemoautotrophic bacteria which realize the oxidation of the reduced sulphur compounds.These compounds stem from the chemical and biological oxidation of pyrite from the Kiesberg gallery walls.In the consortium, the Acidihiobacillus represent the primary organic substance producers.A. intermedius is a strain with a mixotrophic metabolism; it is a facultative heterotroph.The autochthonous organic substance, from the primary producers (the Acidothiobacillus), together with allochthonous organic substances stemming with the water from other parts of the cavity, is scavenged by the heterotrophic microorganisms from this consortium.The heterotrophs are represented by actinomycetes, whose strains, unfortunately, haven't been identified yet, and by the following strains of fungus [12]: Fam.Moniliaceae: Aspergillus nidulans, Aspergillus thomii, Penicillium steckii, P. expansum, P. verucosum, P. griseo-fulvus, P. implicatum, P. verucosum var.corybiferum ; Fam.Cryptococaceae: Rhodotorula sp.; Fam.Dematiaceae: Cladosporium herbarum.Unidentified Protists in the gelatinous formations were also noticed by microscopy.
The bacterial strains are largely dominating, with 90 % of the population, the fungus strains representing only 10 %.
During the rainy season 2005, the dominant strains were the chemoautotrophic Acidithiobacillus (68 %) among which A. intermedius is 10 %.The heterotrophs represented 32% with 21 % actinomycetes, and 11% of fungus strains (of the total population).During the dry season 2005, as well as during the rainy one, the chemoautotrophic Acidithiobacillus strains were dominant (66 %), among which A. intermedius (24 %).The fungus strains represent 10% of the total population (fig.1). ( The chemolitotrophic bacteria near the pyrite crystals take over the Fe 2+ resulted from the pyrite oxidized by the attached bacteria (2). (2) The resulted Fe 3+ causes then the chemical oxidation of the residual pyrite (3).In this process, the quantity of Fe 2+ that is available to the chemolitotrophic microorganisms of the consortium grows: (3) As a consequence of the oxidation of Fe 2+ from the surface of the pyrite crystals, the Fe 3+ deposits itself passively and actively in the microorganisms biofilms as ferric hydroxide (4).(4) In the very fine sections from the gelatinous consortium a big number of alive microorganisms are found.These microorganisms are surrounded by fine acicular crystals of ferric hydroxide that are found at the cells exterior (Fig.

3.).
Besides oxidizing the Fe 2+ from pyrite, the chemolitotrophic bacteria also oxidizes the sulphur from the sulphide.The majority of the acidithiobacils oxidize the sulphide to sulphate, no matter what the pressure level of oxygen is (Acidithiobacillus thiooxidans) [1].
In this case, from the chemical analysis done on samples from the water in which gelatinous formations are found, a great quantity of sulphate as a result of this metabolism was observed (2810.5 mg/l).
In the areas in which the pressure of the oxygen is lower (below saturation) the internal pH of A. thioparus In the Cae Coch Mine from Wales, the same as in the mine that we studied, the development of gelatinous microbial communities is based on the metallic sulphides oxidation from the rock mass [6].The main chemoautotrophic strains from this mine community belong to the Acidithiobacillus type and the specific hetrotrophs are the heterotrophic eubacteria, the actinomycetes, a series of eukaryotes (like fungus and Protists strains) and, very rarely, micro-unvertebrated (such as Nematods) [2].
From the biodiversity point of view, the gelatinous formations from the Kiesberg Mine that we studied, and also the ones from the Cae Coch Mine, are less rich in strains compared to the gelatinous formations from the hydrothermal cavities.Even so, the biomass formed by microorganisms in the mezothermal mines is quite large.In our case, the gelatinous formations biovolume is a few cubic meters, but in the Cae Coch Mine the biovolume is beyond 100m 3 [2,6].
The water course in which the gelatinous formations are developed is quite variable during the entire years, depending on the rainfall quantities from the exterior.
The substratum that supplies with energy these microorganisms consortium is pyrite which is found in great quantities within the ore (1) (fig.2.).becomes slightly alkaline and it can produce S 0 from the sulphide (5) [22]. ( In the areas in which the oxygen is at a high level, A. thioparus will oxidize sulphide to the sulphate (6) [24].(6) Acidithiobacillus ferrooxidans has the capacity to anaerobically oxidize S 0 using Fe 3+ as a final electrons acceptor [17,18].
The sulphite can be further oxidized by the chemolitotrophic bacteria from the gelatinous formations.
Within the consortium, thiosulphate is also oxidized to the sulphate (7,8).(7) The sulphite is then oxidized to sulphate: (8) The sulphur is also oxidized via sulphite (9): (9) Pronk et al. (1990) [15] sum up the proof of a case in which A. ferrooxidans, A. thiooxidans and A. acidophilus oxidize the tiosulphate by forming the tetrathionate during an initial phase (10): (10) followed by a series of hydrolytic and oxidative phases by which the tetrationate is transformed in sulphate with passing accumulations of intermediate sulphur from the sulphan-monosulfonic acids (polithionates) [1].
As a results of all these biochemical reactions that we suppose they take place in the gelatinous formations we obtain important quantities of sulphate ( 3g/L -1 ).

Conclusions
The gelatinous formations represent a complex association formed by heterotrophic, autotrophic (chemolitotrophs) acidophilic microorganisms, which interact very closely.
The oxidative metabolism of chemolitotrophs is very intense, oxidizing from pyrite: 1. Fe 2+ from which Fe(OH) 3 results; 2. Fe(OH) 3 is deposited in the extracellular mucopolisaccharides; 3. sulphur compounds from which significant quantities of SO 4 2-results.

Fig. 1 .
Fig. 1.The ratio of physiological groups of microorganisms from the gelatinous formations, during the rainy and dry seasons of 2004 and 2005 Fig. 2a,b.General view with one pyrite crystal covered by a compact biofilm.E.D.S. specter on the pyrite crystal

Table 1
MICROORGANISMS IDENTIFIED IN THE GELATINOUS FORMATIONS IN KIESBERG MINE AND THEIR MOST PROBABLE NUMBER IN THE TWO SEASONS DURING THE YEARS OF 2004 AND 2005 REV. CHIM.(Bucureºti) ♦ 59 ♦ Nr.10 ♦ 2008 1166