Website (up tomM
Site (up tomM in June) might be a consequence of acetic acid production by the acidophilic biofilms (Fig.). The excreted acetate could play an ecological role by controlling the growth of chemolithoautotrophs and as a source of electrons for heterotrophic Fe+ reducers like some Alphaproteobacteria, Acidobacterium spp. and Sulfobacillus sppall detected together with the PAM microarray (Fig.). The newly developed Fe+ might be utilized as energy supply for iron oxidizers like L. ferrooxidans plus a. ferrooxidans, closing the iron cycle (Fig. B).nose, pyruvate or acetate. The growth was monitored by iron oxidation and cell counting by using a Newbauer chamber under an optical microscope as previously reported .Environmental RNA extraction and amplificationSamples preserved in RNAlater (Ambion) were centrifuged atg for min and washed in acid water (. M sulfuric acid) for subsequent RNA isolation. Total environmental RNA was extracted and amplified by means of a system according to T RNA polymerase linear amplification as described previously ,Estimation of biodiversity by using a prokaryotic acidophile microarrayMethodsSample collectionSamples made use of in this study have been collected from a permanent spring running beneath a pile of pyrite-containing rocks accumulated by mining activities. Sampling was performed in October and , soon after dry summers and ahead of any autumn rainfall within the location. Biomass from liters of water was recovered by filtration by means of nitrocellulose membranes (. m of pore diameter, Millipore Co.) and filters have been straight away placed in ml of RNAlater solution (Ambion) as outlined by the manufacturer’s protocol. Up to g of filament samples increasing within the exact same sampling site have been collected in ml of RNAlater answer. All samples were frozen on dry ice and kept at – till use.Determination of physicochemical parameters in the sampling sitesThe pH, conductivity, salinity, dissolved oxygen and redox possible were measured in situ with a Multii multiprobe device (WTW GmbH, Weilheim, Germany). The elemental composition and concentration (Table) was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/24248382?dopt=Abstract determined by inductively coupled RIP2 kinase inhibitor 1 biological activity plasma spectrometry (ICP), with an Optima DV instrument (Perkin Elmer) by the Centro de Espectrometr At ica, Departamento de An isis Qu ico Elemental (UCM, Madrid). Total iron, Fe+ and Fe+ have been determined by colorimetric methodsSulfate (SO) was determined by atomic absorption spectroscopy having a Perkin-Elmer instrument by the Centro de Espectrometr At ica, Departamento de An isis Qu ico Elemental (UCM, Madrid). Small organic acids (acetate, formate) had been determined by ion chromatography using a Metrohm Advanced Compact Ion Chromatographer IC (Metrohm AG, Herisau, Switzerland). Proper controls have been run to 
discriminate amongst acetate and glycolate anions (not shown).Strain and culture conditionsThe prokaryotic diversity was determined by a prokaryotic acidophile microarray (PAM) as reported previously , as well as utilized to monitor the prokaryotic diversity in industrial bioleachingThe PAM was developed to monitor the prokaryotic diversity in extremely acidophilic environments with oligonucleotide probes targeting most identified acidophilic microorganisms, such as members from the Alpha, Beta, and Gammaproteobacteria, the Nitrospira phylum, acidobacteria, sulfur lowering bacteria, Actinobacteria, the low G+C Firmicutes group, and Archaea from the Ferroplasma and Thermoplasma genera. The biodiversity was analyzed employing fluorescently-labeled total environmental RNA in the identical samples applied for.