Microbial (per)chlorate reduction in hot subsurface environments

M. Liebensteiner

Research output: Thesisinternal PhD, WU



The microbial reduction of chlorate and perchlorate has been known for long as a respiratory process of mesophilic bacteria that thrive in diverse environments such as soils, marine and freshwater sediments. Chlorate and perchlorate are found in nature deriving from anthropogenic and natural sources and can, in the absence of oxygen, be reduced by respective microorganisms to chloride coupled to energy conservation and growth. These classical chlorate- and perchlorate-reducing microorganisms employ enzymes that reduce perchlorate (or chlorate) to the intermediate chlorite, followed by the disproportionation of chlorite to chloride and dioxygen. The latter has been regarded as key reaction for complete (per)chlorate reduction, catalyzed by the enzyme chlorite dismutase, which forms oxygen under anaerobic conditions. This de novo produced oxygen is reduced by terminal oxidases in the metabolism of facultative anaerobic (per)chlorate-reducing microorganisms and can be used by oxygenases for the activation of recalcitrant substrates, as was shown earlier for hydrocarbons.

The potentially stimulating effect of chlorate and perchlorate on microorganisms indigenous to petroleum reservoirs was discussed, seeking new strategies for microbial enhanced oil recovery (based on subsurface growth stimulation and partial hydrocarbon degradation) and reservoir souring control (by inhibiting sulfate-reducing prokaryotes and diminishing sulfide formation).

This thesis reports the capability of hyperthermophilic and thermophilic prokaryotes that originate from subsurface environments to grow by the reduction of chlorate and/or perchlorate. In contrast to the classical metabolism of mesophilic (per)chlorate-reducing microorganisms this study demonstrated that a chlorite-disproportionating enzyme is commonly absent in (hyper)thermophilic (per)chlorate reducers. The absence of this enzyme that was previously defined as prerequisite for (per)chlorate reduction is overcome by the chemical reactivity of reduced sulfur compounds with chlorite generated. In the here more closely investigated hyperthermophilic archaea (Archaeoglobus fulgidus and Aeropyrum pernix) and thermophilic Firmicutes (Carboxydothermus hydrogenoformans and Moorella glycerini strain NMP) chlorite is formed by the activity of molybdopterin oxidoreductases. The respective enzymes are remotely related to perchlorate reductases of mesophilic bacteria and nitrate reductases of the bacterial Nar-type. In contrast to classical bacterial Nar-type enzymes, above-mentioned enzymes seem to have their catalytic subunits outside of the cell. As a consequence the reduction of (per)chlorate forms chlorite extracellularly where it reacts with reduced sulfur species present in the medium/environment (e.g. sulfide), forming chloride anions and oxidized sulfur species (SxOyz-).

The hyperthermophilic archaeon Archaeoglobus fulgidus reduces these chemically formed sulfur species concomitantly to (per)chlorate reduction, which regenerates sulfide for the continuous reduction of (per)chlorate. This interaction of biotic and abiotic reactions during (per)chlorate reduction establishes an intraspecies “sulfur loop” that enables complete reduction of perchlorate to chloride.

Whereas A. pernix also relies on the chemical reactivity of chlorite with thiosulfate, this archaeon does not employ systems for regenerating the reducing agents biologically; which is reflected by the accumulation of sulfate during perchlorate reduction. The Crenarchaeon A. pernix, formerly known as a strictly aerobic microorganism, expands the trait of microbial (per)chlorate reduction up to 100°C.

In addition to the intraspecies “sulfur loop” of A. fulgidus, there were indications that the reduction of perchlorate may also proceed syntrophically, as indicated by a thermophilic bacterial consortium. In the respective culture, it seems that one microorganism reduces perchlorate, forming chlorite, which is chemically reduced by sulfide to chloride anions and oxidized sulfur compounds. Another group of microorganisms uses the respective sulfur compounds as electron acceptors and  regenerates sulfide. Sulfur (of different redox states) depicts the mediating agent in this interspecies “sulfur loop”, but may possibly be substituted in nature by other compounds such as ferrous/ferric iron.

Here presented (per)chlorate reduction sensu lato, which lacks the action of a chlorite-disproportionating enzyme may be widely spread among prokaryotes. For example enzymes closely resembling the designated (per)chlorate-reducing enzyme in Archaeoglobus fulgidus are also found in other strictly anaerobic thermophiles, of which C. hydrogenoformans and M. glycerini NMP were already confirmed to grow by the reduction of (per)chlorate as well.

The substrate ambiguity of particular periplasmic DMSO enzymes may enable a broader group of microorganisms of (per)chlorate reduction sensu lato, in case sulfide is present in the environment. A broadened substrate spectrum of respective enzymes (beyond their canonical function) may possibly have had evolutionary advantages. Chlorine oxyanions are naturally formed and have been introduced on Earth for ages already. The reduction of (per)chlorate and formation of chlorite in ancient anaerobic microorganisms may even have contributed to the evolution of proteins adapted to oxidizing conditions on early Earth and preceded the evolution of oxygenic photosynthesis.

It is shown that subsurface-inhabiting (hyper)thermophiles are able to grow by the reduction of (per)chlorate, which is also of interest for applications in the field of oil recovery. The finding that (per)chlorate reduction is interfering with the sulfur metabolism of a major contributor to reservoir souring in hot oil fields, A. fulgidus, draws promising scenarios for future attempts in developing novel souring control strategies.

(Per)chlorate reduction by A. fulgidus was also coupled to the oxidation of butyrate, a volatile fatty acid commonly present in petroleum reservoirs. For sustainable applications in the oil recovery business, it is desirable to rely, as little as possible, on external substrates. In this respect the fact that A. fulgidus couples (per)chlorate reduction to the oxidation of butyrate is advantageous. Possibly the microorganism can also degrade long-chain alkanes and alkenes coupled to (per)chlorate reduction, a feature that was shown earlier coupled to sulfate reduction.

All together a shift of A. fulgidus from sulfate reduction to (per)chlorate reduction in oil fields would not only diminish souring, but maintain/stimulate in-situ growth of the microorganism (based on intrinsic carbon sources) which has additionally advantageous effects for improved sweeping efficiencies during water flooding.


Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Stams, Fons, Promotor
  • Lomans, Bart, Co-promotor
Award date17 Oct 2014
Place of PublicationWageningen
Print ISBNs9789462571259
Publication statusPublished - 2014


  • perchlorates
  • chlorates
  • reduction
  • microbiology
  • microorganisms
  • subsurface layers

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