The Testing of the Capacity of an Acidophilic Consortium from an Old Mine Concerning the Elimination of Iron and Manganese from the Underground Waters

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In order to assure the water supply for the localities, the phreatic waters are exploited by different depths drillings, and subjected to some treatment for potability processes (Nitescu, 1977).In treatment plants, the drilling waters are subjected to an elimination of iron and manganese by high aeration and then the water is filtrated throughout the sand filters in order to retain manganese and iron hydroxides precipitates.At the end of treatment processes, the waters are being disinfected in order to take out the microorganisms by chlorination.
The elimination of iron and manganese from the drilling waters in water treatment plants by the mentioned method, has a series of disadvantages, as the filters have to be often washed in order to remove the iron and manganese hydroxides mud.Nowadays, in Germany, the scientists are trying a new elimination of iron and manganese "in situ" method by oxygen injections straight into the underground waters and also a circulation in both ways to avoid the colmation [1 -4].This method is still in the experimental phase and the suspicion of slowly colmation still exists.
In this article we present the experiment by which the capacity of iron and manganese elimination of some underground waters, by acidophilic microorganisms * email: ghermanvasile2000@yahoo.com consortium located on the gelatinous formations from an abandoned mine located in the Banat Mountains from South-West of Romania was tested.This mine was dug in the metamorphic rocks of auriferous sulphides from the Policarpus ore.The microbial consortium is suppllied with a variable flow of the infiltration water.

Experimental part
Materials and methods: The samples for the microbilogical analisys have been taken from a variable flow water stream found in a partially collapsed gallery from Kiesberg mine.
The isolation of acidophilic heterotrophic microorganisms has been realized trough enriched growing on the March medium-pH-2.5[29], and on the G.Y.E. at pH 3 [30].
The isolation and identification of fungi has been realized on the solid Czapeck medium [31,32], 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 [33].
The determination of the microorganisms number 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 [34].The method of counting the colonies on the solid medium was used also for the numerical estimates.
The determination of iron and manganese quantity from the drilling water has been realized by atomic absorption spectroscopy, using the atomic absorbtion spectophotometer Varian Spectr AA 110.
Electronic microscopy observations on the pumice stone inoculated with the microorganisms consortium have been done using S.E.M. (scanning electronic microscopy) Zeiss Gemini 982.The samples have been prepared by the classical SEM method and the observation have been done at an acceleration tension of 15 kV.The work distance was 16 mm.
For the testing of the microorganism consortium capacity of iron and manganese elimination, 3 cilindric plexiglass column pipes, 3 liters each, have been used.These column pipes were made of plastic tubes, which were obturated at their bottom base with plexiglass stopper and every stopper has a orifice with a flexible pipe for the water discharge.
The 3 column pipes have been aerated with a constant and equal air volume.In order to do that we used a filtered air pump and a air distributor with taps.The air distributor has to equalize the air quantity that goes into the 3 column pipes.The flexible pipes are connected between the distributor and the bottom of the 3 column pipes and every flexible pipe has an aeration stone at the end so that little bubbles of air can be formed.
The installation was sterilized with 90 0 alcohol for about 10 min and the pumice stone was sterilized in oven (drying chamber) at 190 0 C for 60 min.
In this experiment, two of the three column pipes were the test pipes (T 1 and T 2 ) that were inoculated with the microorganisms consortium.The first test column pipe (T 1 ) represents the first step of biological treatment, and it was supplied directly with drilling water; the second column pipe (T 2 ) represents the second step of biological treatment, and it was supplied from the first test column pipe.
The witness column pipe (T 0 ) was not inoculated with microorganisms, so it functioned in sterile condition and it was used to compare the obtained results.
The drilling water was sterilized by filtration.
The two test column pipes (T 1 and T 2 ) were inoculated with 500mL microorganisms consortium each, and, after the inoculation, the column pipes were primed.The priming represents the fixation of the active microorganisms consortium on the sterile pumice stone so that the wash of the microorganisms consortium to be avoided when the column pipes are drained.The priming lasted for about 48 h , with alternative 30 min aeration between periods of 10 min aeration stall.
The experiment was realized within 25 min period of time.The sterile drilling water was entered in the first test column pipe (T 1 ) and in the witness column pipe in the same time, and an 25 min aeration was performed in the two column pipes.
In the water treatment plants, the elimination of iron and manganese takes place by a high aeration energy consumer method.Economically speaking, a 25 min aeration period is considered to be profitable and that is the reason why we used this exact period of time, too.
After 25 min of functioning, water samples from the T 1 effluent and T 0 were taken out and the T 1 effluent was introduced in T 2 (the second step of biological treatment).After a 25 min aeration, a sample from the second test

Table 1 THE SPECIES COMPOSITION OF THE TESTED CONSORTIUM AND THEIR NUMBER
column pipe effluent was also taken out.The second step of treatment was used to see whether the iron and manganese concentrations continue to drop, after 25 optimal minutes of treatment in T 1 .If so, we should have prolong the aeration period (over the profitableness limit).
Before the sampling, the test and witness column pipes effluents were put in separate sterile laboratory recipients for about 10 min.These recipients were used for decantation.
Between the testing periods, the column pipes were filled with drilling water and aerated 20 min / h so that the tested consortium was maintained active.
The mentioned testing was realized for 6 days and the iron and manganese concentrations were immediately observed with an atomic absorption spectrometer.
Considering the fact that the drilling water was stored in a closed tank, aside from the T 1 , T 2 , and T 0 samples, a sample from the tank (R) drilling water was also taken every time.

Results and discussions
In table 1 the results of microbiological determinations and the numerical estimation are showed: From table 1, we can observe the fact that 17 strains of microorganisms have been identified .From all the identified strains , the four thiobaccilius together with the actinomyetes represent the bacteria, the rest of the strains represent the fungus: 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.
After 25 min of functioning, water samples from the T0, T1, T2 effluent (and from R Drilling water tank) were taken out in order to spectophotometrically analize the iron and the manganese.
The results of iron and manganese analysis during these 6 days are written in figures 2, 3.
At the end of the experiment, S.E.M. observations were made on the pumice stone from the three column pipes.
In the witness column (fig.4 b), that was not inoculated with the consortium microorganisms, a big porosity of the pumice stone is being observed.The feric hydroxide that was formed by the chemical oxidation of Fe 2+ , is deposited on all the pumice stone structures.
In the test column (fig.4 a), the entire surface of the pumice stone is covered with a very compact microbial biofilm.The Fe 2+ is being oxidized a lot more quickly in the presence of ferooxidant bacteria than by chemical mecanisms.The deposits of the obtained feric hydroxide are done more quickly and more stable in the bacterial extracellular mucoplizaharides.

Conclusions
From the results obtained from the analysis presented in figures 2 and 3 we can withdraw the following conclusions: -during the same experimental conditions, for 25 minutes, the iron is more quickly oxidized than the manganese, by both chemically and combined biochemically processes, and the two elements oxidation continues even after the 25 min period.-the iron and manganese are oxidized more quickly by the combined biological and chemical processes from T 1 and T 2 than by only the chemical process from T 0 .
-this removing of the iron and manganese biological method does not have a great profitableness if applied at an industrial scale, because of the relatively big volume of sublayer (grinding stone or porous ceramic materials) which need to be changed periodical.
However, this experiment brings out an innovating view in the testing of the efficiency of microbial consortium from the acid mine in the elimination of other ions from the drilling or waste waters.On the other hand, the observation of the feric hydroxide precipitation in the bacterial biofilms has a very important role for the understanding of the longitudinal sand filters colmation.These filters are being used for the classic elimination of the iron from the deep drilling waters technology or in the colmation of the underground layers by the elimination of the iron underground technology.

Fig. 2 .Fig. 3 .
Fig. 2. The results of iron analysis during the six days of experiments