Contribution of microorganisms to the oxidation of pyrite

G.J.M.W. Arkesteyn

Research output: Thesisinternal PhD, WU


Optimum conditions for the accumulation of substantial amounts of pyrite (FeS <sub>2</sub> ) in the sediment are found in estuarine areas, especially in the tropics. In such areas anaerobic conditions prevail owing to continuous saturation with water. There is an abundant supply of organic matter, in the tropics mainly derived from extensive mangrove forests. Continuous supply of sulphate takes place from the see by the tidal movement. The complex organic material is decomposed by anaerobic bacteria into low-molecular compounds which in turn can be utilized as energy and/or carbon source by sulphate-reducing bacteria. Sulphate is utilized as electron-acceptor by these bacteria and reduced to hydrogen sulphide. Part of the latter compound is fixed as iron sulphide from which pyrite can be formed. Iron is mostly present in such amounts that accumulation up to 10% pyrite is possible. As a result of the strong tidal movement, partial oxidation of sulphide to elemental sulphur (S <sup>0</sup> ) is possible and there is a constant removal of alkaline compounds like HCO <sub>3</sub><sup>-</sup> which is formed during sulphate reduction. S <sup>0</sup> is a necessary intermediate in the formation of pyrite, whilst the continuous removal of the bicarbonate results in a decreased pH which is kinetically favourable for the formation of pyrite.<br/>Drainage of the sediment leads to cracking of the unripened clay through which air can penetrate into the soil. Pyrite is then oxidized partly to sulphuric acid that causes a pH drop of the soil near neutral to 4 or even lower. Such soil is useless for agricultural purposes as a result of the low pH. Besides sulphuric acid, the straw-yellow mineral jarosite, KFe <sub>3</sub> (SO <sub>4</sub> ) <sub>2</sub> (OH) <sub>6</sub> , is formed from which the name "cat clay" originates.<br/>The oxidation of pyrite at a pH below 4, is mainly a microbial process. When starting the experiment it was unknown in what way the initial acidification of the sediment from near neutral to pH 4 proceeds.<br/>Chapter 2 contains the results of a study of microbial processes involved in the formation of acid sulphate soils. Special attention was given to the initial drop of pH from 7 to 4. It appeared that the initial acidification is a non-biologically catalysed process.<br/><em>Thiobacillus thioparus, Thiobacillus thiooxidans</em> and heterotrophic thiosulphate-oxidizing bacteria were isolated from acidifying potentially acid sulphate soil. However, these bacteria were not able to oxidize pyrite. The oxidation of the mineral in synthetic media inoculated with <em>Thiobacillus thioparus, Thiobacillus thiooxidans, Thiobacillus intermedius</em> or <em>Thiobacillus perometabolis</em> did not proceed faster than that in sterile media. Nevertheless, the numbers of cells in the inoculated media increased by a factor of 10-100 to a maximum of 10 <sup>5</sup> cells/ml medium (2. Fig. 3). The thiobacilli probably oxidize a small amount of the reduced sulphur compounds which are formed during the non-biological oxidation of pyrite. Experiments with sterilized potentially acid sulphate soil to which pure cultures of different thiobacilli or a suspension of unsterilized potentially acid sulphate soil were added aseptically corroborated the above-mentioned findings (2. Table 2). The initial acidification of sterile potentially acid sulphate soils was comparable with that of the inoculated soils. Only after the pH had decreased to values below 4.0 the oxidation of pyrite in the sterile soil was significantly slower than that in the soils inoculated with cultures of <em>Thiobacillus ferrooxidans.</em><br/>Although the addition of lime prevented acidification, the non- biological oxidation of pyrite was not abolished during the 68 days the process was followed (2. Table 2b).<br/>Attempts to isolate. other microorganisms that might be involved in the oxidation of pyrite such as <em>Leptospirillum ferrooxidans</em> or members of the genus <em>Metallogenium</em> were not successful. The only organism which is responsible for the oxidation of pyrite is <em>Thiobacillus ferrooxidans.</em><br/>Two mechanisms of the oxidation of pyrite by <em>Thiobacillus ferrooxidans</em> are known viz.: "the indirect contact mechanism" and the "direct contact mechanism" According to the former mechanism, the sulphur moiety of pyrite is oxidized to sulphate by ferric ions which in turn are reduced to ferrous iron. <em>Thiobacillus ferrooxidans</em> oxidizes the ferrous iron to ferric iron to complete a cyclic process. According to the second mechanism the bacterium is in direct contact with the mineral and it oxidizes the iron as well as the sulphur moiety of pyrite. When starting the experiments it was evident that pyrite can be oxidized by <em>Thiobacillus ferrooxidans</em> according to the indirect contact mechanism but there were some indications that the direct contact mechanism can also occur.<br/>Chapter 3 gives the results of a study in which it was clearly shown that <em>Thiobacillus ferrooxidans</em> is able to oxidize pyrite according to the direct contact mechanism. Inhibitors of the oxidation of Fe <sup>2+</sup> and S <sup>0</sup> , <em></em> NaN <sub>3</sub> and N-ethylmaleimide (NEM), respectively, partially abolished the oxidation of pyrite when added separately but when the inhibitors were added together the oxidation of the mineral completely stopped (3.Table 1).<br/>The electrons released during the oxidation of ferrous iron are transferred to cytochrome c, whilst the electrons derived from the oxidation of elemental sulphur probably reduce acceptors with a lower redox potential. Cytochromes of the b-type were clearly shown to be present in cells grown in media with S <sup>0</sup> or FeS <sub>2</sub> as energy source, but they were not observed in cells grown in media with Fe <sup>2+</sup> as electron donor (3. Fig. 3). The efficiency of the utilization of released electrons at pH 4.0 was highest with S <sup>0</sup> less with FeS <sub>2</sub> and lowest with Fe <sup>2+</sup> as energy source. This was investigated by measuring the incorporation of radio-active carbon dioxide ( <sup>14</sup> CO <sub>2</sub> ) into the bacteria in media with the three different electron donors (3. Table 3).<br/>Additional evidence for the direct oxidation of the sulphur moiety of pyrite by <em>Thiobacillus ferrooxidans</em> was obtained from the fact that the oxidation of Fe <sup>2+</sup> at pH 5.0 was negligible, whereas the oxidation of S <sup>0</sup> and FeS <sub>2</sub> clearly proceeded at this pH. Separation of bacteria and pyrite by means of a dialysis bag to prevent direct contact resulted in a decrease of the rate of oxidation of the mineral which was comparable with the inhibition of pyrite by NEM (3. Table 2). The oxidation of the sulphur moiety of pyrite by <em>Thiobacillus ferrooxidans</em> was relatively more important with increasing pH.<br/>During the study of the oxidation of pyrite by <em>Thiobacillus ferrooxidans</em> it appeared that cultures of this bacterium grown in media with Fe <sup>2+</sup> as energy source without exception contained <em>Thiobacillus acidophilus.</em> Nothing was known about factors which can explain the close association between the two organisms.<br/>Chapter 4 contains a survey of the results of an investigation into factors responsible for the presence of <em>Thiobacillus acidophilus</em> in <em>Thiobacillus ferrooxidans</em> cultures. The most obvious explanation would be that <em>Thiobacillus ferrooxidans</em> is able to utilize Fe <sup>2+</sup> as energy source. But attempts to adapt the mixotroph to the oxidation of Fe <sup>2+</sup> were not successful. <em>Thiobacillus acidophilus</em> grew in modest numbers on organic matter excreted by <em>Thiobacillus ferrooxidans</em> (4. Table 2). Oligocarbophilic growth also was observed (4. Table 6). Possible substrates for such a growth are: methanol and ethanol whilst hydrogen sulphide also can be utilized as energy source by the organism.<br/><em>Thiobacillus ferrooxidans</em> may profit from the presence of <em>Thiobacillus acidophilus</em> in mixed cultures containing organic compounds such as alcohols, amino acids or organic acids which are toxic to <em>Thiobacillus ferrooxidans.</em> Such compounds are utilized by the mixotroph after which the autotrophic organism can grow.<br/>Experiments with fluorescent labelled antibodies against <em>Thiobacillus acidophilus</em> showed that in mixed cultures of both organisms the cell numbers of the mixotroph were comparable with cell numbers of the autotroph (4. Table Also the fact that <em>Thiobacillus ferrooxidans</em> cultures are hard to purify indicates that both organism are present in almost equal numbers in heterogeneous cultures. These observations might be an indication that <em>Thiobacillus ferrooxidans</em> excretes more assimilable organic matter in the presence of <em>Thiobacillus acidophilus</em> than in pure cultures. More research is necessary to prove this assumption.<p/>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Mulder, E.G., Promotor, External person
Award date15 Feb 1980
Place of PublicationWageningen
Publication statusPublished - 1980


  • acid sulfate soils
  • soil
  • pyrites
  • clay soils


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