Thore Rohwerder

Bioleaching review part A

Bioleaching of metal sulfides is caused by astonishingly diverse groups of bacteria. Today, at least 11 putative prokaryotic divisions can be related to this phenomenon. In contrast, the dissolution (bio)chemistry of metal sulfides follows only two pathways, which are determined by the acid-solubility of the sulfides: the thiosulfate and the polysulfide pathway. The bacterial cell can effect this sulfide dissolution by “contact” and “non-contact” mechanisms. The non-contact mechanism assumes that the bacteria oxidize only dissolved iron(II) ions to iron(III) ions. The latter can then attack metal sulfides and be reduced to iron(II) ions. The contact mechanism requires attachment of bacteria to the sulfide surface. The primary mechanism for attachment to pyrite is electrostatic in nature. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions in the contact mechanism is mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in the non-contact mechanism. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a complex redox chain located below the outer membrane, the periplasmic space, and the cytoplasmic membrane of leaching bacteria. The dominance of either At. ferrooxidans or Leptospirillum ferrooxidans in mesophilic leaching habitats is highly likely to result from differences in their biochemical iron(II) oxidation pathways, especially the involvement of rusticyanin.

The Alkyl tert-Butyl Ether Intermediate 2-Hydroxyisobutyrate Is Degraded via a Novel Cobalamin-Dependent Mutase Pathway

ABSTRACT Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated β-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

Intermediary sulfur compounds in pyrite oxidation: implications for bioleaching and biodepyritization of coal

Abstract Accumulation of elemental sulfur during pyrite oxidation lowers the efficiency of coal desulfurization and bioleaching. In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an iron(II)-ion-oxidizing organism without sulfur-oxidizing capacity, from the pyritic sulfur moiety about 10% elemental sulfur, 2% pentathionate, and 1% tetrathionate accumulated by a recently described cyclic pyrite oxidation mechanism. In the case of pure cultures of Thiobacillus ferrooxidans and mixed cultures of L. ferrooxidans and T. thiooxidans, pyrite was nearly completely oxidized to sulfate because of the capacity of these cultures to oxidize both iron(II) ions and sulfur compounds. Pyrite oxidation in acidic solutions, mediated chemically by iron(III) ion, resulted in an accumulation of similar amounts of sulfur compounds as obtained with L. ferrooxidans. Changes of pH to values below 2 or in the iron ion concentration are not decisive for diverting the flux of sulfur compounds. The literature on pyrite bioleaching is in agreement with the findings indicating that the chemistry of direct and indirect pyrite leaching is identical.

Novel combination of atomic force microscopy and epifluorescence microscopy for visualization of leaching bacteria on pyrite.

ABSTRACT Bioleaching of metal sulfides is an interfacial process comprising the interactions of attached bacterial cells and bacterial extracellular polymeric substances with the surface of a mineral sulfide. Such processes and the associated biofilms can be investigated at high spatial resolution using atomic force microscopy (AFM). Therefore, we visualized biofilms of the meso-acidophilic leaching bacterium Acidithiobacillus ferrooxidans strain A2 on the metal sulfide pyrite with a newly developed combination of AFM with epifluorescence microscopy (EFM). This novel system allowed the imaging of the same sample location with both instruments. The pyrite sample, as fixed on a shuttle stage, was transferred between AFM and EFM devices. By staining the bacterial DNA with a specific fluorescence dye, bacterial cells were labeled and could easily be distinguished from other topographic features occurring in the AFM image. AFM scanning in liquid caused deformation and detachment of cells, but scanning in air had no effect on cell integrity. In summary, we successfully demonstrate that the new microscopic system was applicable for visualizing bioleaching samples. Moreover, the combination of AFM and EFM in general seems to be a powerful tool for investigations of biofilms on opaque materials and will help to advance our knowledge of biological interfacial processes. In principle, the shuttle stage can be transferred to additional instruments, and combinations of AFM and EFM with other surface-analyzing devices can be proposed.

Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108.

Growth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 degrees C were 0.045 h(-1), 0.06 h(-1) and 0.055 h(-1), respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h(-1), 0.1 h(-1) and 0.17 h(-1), respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE)(-1), 0.53 g dm (g ETBE)(-1), 0.81 g dm (g TAME)(-1), 0.48 g dm (g TBA)(-1), 0.76 g dm (g TAA)(-1) and 0.54 g dm (g 2-HIBA)(-1) were obtained. Maximum specific degradation rates were 0.92 mmol MTBE h(-1) (g dm)(-1), 1.11 mmol ETBE h(-1) g(-1), 0.66 mmol TAME h(-1) g(-1), 1.19 mmol TAA h(-1) g(-1), 2.82 mmol TBA h(-1) g(-1), and 3.27 mmol 2-HIBA h(-1) g(-1). The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 degrees C, but at 5 degrees C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 degrees C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups.

Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.

MECHANISMS AND BIOCHEMICAL FUNDAMENTALS OF BACTERIAL METAL SULFIDE OXIDATION

The interest in the fundamentals of metal sulfide bioleaching has significantly increased in the course of the last decade. One driving force is the metal winning industry, as the recovery of heavy metals from sulfidic ores employing microorganisms is now an established biotechnology (Bosecker, 1997; Rawlings, 1997, 2002; Rohwerder et al., 2002; Olson et al., 2003; Rawlings et al., 2003). On the other hand, the problem of acid rock/mine drainage (ARD/AMD) increasingly attracts attention, because environmental awareness and legislation force mining companies and state agencies into alliances for fighting effectively these problems or at least reducing their impact on the environment. Consequently, the underlying reaction mechanisms have been the subject of intensive research. In combination with the emerging possibilities resulting from exciting developments in the fields of chemical and surface analysis as well as biochemistry and molecular biology, our understanding of the responsible bioprocesses have been dramatically improved. The first and decisive step for the renewed interest was the clarification of the sulfur chemistry behind metal sulfide oxidation (Schippers et al., 1996; Schippers & Sand, 1999; Schippers, 2004), which brought a decades-long, often contradictory discussion to an end and, as a consequence, changed the scientific approach for studying these processes. It has been shown that by only two different pathways all relevant redox reactions and sulfur compound speciation may be explained. Most important was that the original concept of a “direct leaching mechanism” i.e. the direct enzymatic oxidation of the sulfur moiety of heavy metal sulfides (reviewed by Ehrlich, 2002 and Sand et al., 1995), lost any justification. The “indirect mechanism”, i.e. the non-enzymatic metal sulfide oxidation by iron(III) ions combined with the enzymatic (re)oxidation of the resulting iron(II) ions, is to be considered as the only relevant mechanism. It is now divided into

Carbon conversion efficiency and limits of productive bacterial degradation of methyl tert-butyl ether and related compounds.

ABSTRACT The utilization of the fuel oxygenate methyl tert-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the YATP concept. Experiments were conducted to derive realistic maintenance coefficients and Ks values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g−1, which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient ms and its associated biomass decay term b, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations, Smin, below which growth would not further be supported. Smin strongly depended on the maximum growth rate μmax, and b and was directly correlated with the half maximum rate-associated substrate concentration Ks, meaning that any effect impacting this parameter would also change Smin. The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase Ks and Smin for MTBE.

Determination of reaction energy values for biological pyrite oxidation by calorimetry

Abstract The reaction energy values for pyrite oxidation by chemolithotrophic leaching bacteria (pure cultures of Thiobacillus ferrooxidans, Leptospirillum ferrooxidans , and mixed cultures of L. ferrooxidans and T. thiooxidans ) were calorimetrically measured in batch experiments. The obtained data were compared with the nonconservative values for the corresponding abiotic processes that were calculated from standard enthalpies and chemical analyses of the leaching products. Due to the different abilities of the investigated species to oxidize the sulphur moiety of pyrite, the measured reaction energy values ranged from −1100 to −1600 kJ/mol with an accuracy of 4–16%. In samples with T. ferrooxidans and with the mixed cultures, no significant difference between the calorimetric and theoretical reaction energy values occurred. In contrast, pure cultures of L. ferrooxidans exhibited measured values which were up to 200 kJ/mol lower than the theoretical ones. It is highly unlikely that this difference may be explained by a higher energy conservation efficiency of L. ferrooxidans compared to the one of T. ferrooxidans or T. thiooxidans . Besides use in efficiency studies, the collected data can be used to determine the activity of leaching bacteria in natural biotopes by calorimetric measurements.

Critical evaluation of the 2D-CSIA scheme for distinguishing fuel oxygenate degradation reaction mechanisms.

Although the uniform initial hydroxylation of methyl tert-butyl ether (MTBE) and other oxygenates during aerobic biodegradation has already been proven by molecular tools, variations in carbon and hydrogen enrichment factors (ε(C) and ε(H)) have still been associated with different reaction mechanisms (McKelvie et al. Environ. Sci. Technol. 2009, 43, 2793-2799). Here, we present new laboratory-derived ε(C) and ε(H) data on the initial degradation mechanisms of MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by chemical oxidation (permanganate, Fenton reagents), acid hydrolysis, and aerobic bacteria cultures (species of Aquincola, Methylibium, Gordonia, Mycobacterium, Pseudomonas, and Rhodococcus). Plotting of Δδ(2)H/ Δδ(13)C data from chemical oxidation and hydrolysis of ethers resulted in slopes (Λ values) of 22 ± 4 and between 6 and 12, respectively. With A. tertiaricarbonis L108, R. zopfii IFP 2005, and Gordonia sp. IFP 2009, ε(C) was low (<|-1|‰) and ε(H) was insignificant. Fractionation obtained with P. putida GPo1 was similar to acid hydrolysis and M. austroafricanum JOB5 and R. ruber DSM 7511 displayed Λ values previously only ascribed to anaerobic attack. The fractionation patterns rather correlate with the employment of different P450, AlkB, and other monooxygenases, likely catalyzing ether hydroxylation via different transition states. Our data questions the value of 2D-CSIA for a simple distinguishing of oxygenate biotransformation mechanisms, therefore caution and complementary tools are needed for proper interpretation of groundwater plumes at field sites.