Götz Haferburg

Metallomics: lessons for metalliferous soil remediation

The term metallomics has been established for the investigation of transcriptome, proteome, and metabolome changes induced by metals. The mechanisms allowing the organisms to cope with metals in the environment, metal resistance factors, will in turn change biogeochemical cycles of metals in soil, coupling the metal pool with the root system of plants. This makes microorganisms key players in introducing metals into food webs, as well as for bioremediation strategies. Research on physiological and metabolic responses of microorganisms on metal stress in soil is thus essential for the selection of optimized consortia applicable in bioremediation strategies such as bioaugmentation or microbially enhanced phytoextraction. The results of metallomics studies will help to develop applications including identification of biomarkers for ecotoxicological studies, bioleaching, in situ soil regeneration, and microbially assisted phytoremediation of contaminated land. This review will therefore focus on the molecular understanding of metal resistance in bacteria and fungi, as can be derived from metallomics studies.

Arousing sleeping genes: shifts in secondary metabolism of metal tolerant actinobacteria under conditions of heavy metal stress

Numerous microbial habitats are strongly influenced by elevated levels of heavy metals. This type of habitat has developed either due to ore mining and metal processing or by pedogenesis above metal-rich base rocks. Most actinobacteria are soil-borne microbes with a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. One major obstacle in identifying secondary metabolites, however, is the known phenomenon of sleeping gene clusters which are present, but silent under standard screening conditions. Here, we proceed to show that sleeping gene clusters can be awakened by the induction in heavy metal stress. Both, a chemical and a biological screening with extracts of supernatant and biomass of 10 strains derived from metal contaminated and non-contaminated environments was carried out to assay the influence of heavy metals on secondary metabolite patterns of metal tolerant actinobacteria. Metabolite patterns of cultures grown in complex and minimal media were compared to nickel (or cadmium) spiked parallels. Extracts of some strains grown in the presence of a metal salt displayed intense antibiosis against Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus and Candida albicans. Contrarily to the widely held opinion of metals as hindrance in secondary metabolism, metals thus can induce or enhance synthesis of possibly potent and medically relevant metabolites in metal tolerant strains. Hence, re-screening of existing strain libraries as well as identification of new strains from contaminated areas are valid strategies for the detection of new antibiotics in the future.

Ni-struvite - a new biomineral formed by a nickel resistant Streptomyces acidiscabies.

Biomineralization dependent on bacterial activity has been described for struvite which is formed in soils, guano, putrescent matter and sediments. A new biomineral containing nickel instead of magnesium, Ni(NH4)(PO4) . 6H2O, has been identified. It was formed by nickel resistant Streptomyces acidiscabies E13, and putatively named nickel struvite. The mineral formation is dependent on biological activity since non-viable bacterial cells are not capable to induce formation of Ni-struvite under identical conditions. Formation of Ni-struvite was observed on colony surfaces upon prolonged incubation of solid minimal or complex media containing elevated concentrations of 8-15mM NiCl2. The formation of magnesium containing crystals was not observed although Mg2+ is present in the medium. However, the process was not depending on desiccation since small crystals attached to the mycelial biomass of the bacteria were observed microscopically also in liquid cultures of nickel supplemented minimal and complex media after two weeks of incubation. The capacity to induce biomineralization of a nickel containing mineral is postulated to constitute a resistance factor, allowing the soil bacterium to withstand high nickel concentrations. The strain shows nickel resistance as an adaption to its habitat, since this bacterium was isolated from a former uranium mining site in Eastern Thuringia, Germany, where nickel concentrations of up to 2000ppm (translating to appr. 30mM) occur as a result of former mining activities.

Bioprospecting at former mining sites across Europe: microbial and functional diversity in soils

The planetary importance of microbial function requires urgently that our knowledge and our exploitation ability is extended, therefore every occasion of bioprospecting is welcome. In this work, bioprospecting is presented from the perspective of the UMBRELLA project, whose main goal was to develop an integral approach for remediation of soil influenced by mining activity, by using microorganisms in association with plants. Accordingly, this work relies on the cultivable fraction of microbial biodiversity, native to six mining sites across Europe, different for geographical, climatic and geochemical characteristics but similar for suffering from chronic stress. The comparative analysis of the soil functional diversity, resulting from the metabolic profiling at community level (BIOLOG ECOPlates) and confirmed by the multivariate analysis, separates the six soils in two clusters, identifying soils characterised by low functional diversity and low metabolic activity. The microbial biodiversity falls into four major bacterial phyla: Actinobacteria, Proteobacteria, Firmicutes and Bacteroidetes, including a total of 47 genera and 99 species. In each soil, despite harsh conditions, metabolic capacity of nitrogen fixation and plant growth promotion were quite widespread, and most of the strains showed multiple resistances to heavy metals. At species-level, Shannon’s index (alpha diversity) and Sørensens Similarity (beta diversity) indicates the sites are indeed diverse. Multivariate analysis of soil chemical factors and biodiversity identifies for each soil well-discriminating chemical factors and species, supporting the assumption that cultured biodiversity from the six mining sites presents, at phylum level, a convergence correlated to soil factors rather than to geographical factors while, at species level, reflects a remarkable local characterisation.

Streptomycete heavy metal resistance: Extracellular and intracellular mechanisms

The responses of microorganisms to heavy metal stress involve both extracellular and intracellular mechanisms. In contaminated soils, streptomycetes are an important group with a particularly versatile secondary metabolism. The roles of extracellular and intracellular mechanisms for heavy metal retention in growth at contaminated sites were investigated, with emphasis placed on chelator and siderophore excretion, biomineralization, cell wall adsorption, and intracellular storage. The combined result of all of these processes is heavy metal resistance, which was specifically addressed for nickel. Strains withstanding up to 130 mM nickel in minimal media were isolated from a former uranium mining site near Ronneburg in Eastern Thuringia, Germany.

Plant–Microbe Interaction in Heavy-Metal-Contaminated Soils

Heavy metals are the most important inorganic pollutants, which are not degraded and progressively accumulate in the environment. The use of plants for rehabilitation of heavy-metal-contaminated soils is an emerging area of interest, because it provides an ecologically and environmentally sound and safe method for restoration and remediation. Although a number of plant species are capable of hyperaccumulation of heavy metals, however, this approach is not applicable for remediating sites with multiple contaminants. The biogeochemical capacities of microorganisms seem almost limitless and they can adsorb and accumulate metals in their cells and are being used in microbial leaching and also as agents of cleaning the environment. To overcome the metal stress, numbers of mechanisms have been evolved by microorganisms of agronomic importance by which they tolerate and promote the uptake of heavy metal ions. Such mechanisms include: the pumping of metal ions exterior to the cell, accumulation and sequestration of the metal ions inside the cell, transformation of toxic metal to less toxic forms, and adsorption/desorption of metals. The best approach would be to combine the advantages of plant–microbe interactions within the plant rhizosphere into an effective cleanup technology. The activities of plants and plant/microbial associations may offer viable means of accomplishing the in situ remediation of contaminated soils. This chapter examines the potential role of plant–microbe interactions in heavy-metal-contaminated soils toward phyto-bioremediation.

Biogeosciences in Heavy Metal-Contaminated Soils

Biogeosciences cover research from the micro to the macro scale. Element fluxes in microbial habitats have large impacts, e.g., in climate change phenomena. The strong focus on application of research and a high interdisciplinary, together with the understanding of the necessity for environmental protection, are the reasons for the fast growth of biogeosciences. This comparably young field of research integrates disciplines including hydro(geo)chemistry, plant physiology or microbiology and bacterial genetics, generally aiming at integration of effects of life (βίος) on Earth (γeος). The interference of humans with biogeochemical cycles leads to a dangerous imbalance in the overall mass balance of nature. Biogeosciences deliver tools and methods for an understanding of such anthropogenic imbalances. At the same time, the field develops means to counter-act adverse effects of disturbed matter cycling on the environment. The research field of biogeosciences arose as an answer to environmental degradation and delivers the scientific approach for measures to be taken to alleviate these deleterious effects of disequilibrium in bioremediation and phytoremediation approaches.

Geomicrobial Manganese Redox Reactions in Metal-Contaminated Soil Substrates

Geomicrobial cycles influence metal mobilities in soil. The formation of Fe and Mn(hydr)oxides in biogeochemical horizons and the subsequent mobilization of Mn from the substrate can lead to high mobility of Mn. This process of Mn mobilization was studied in substrates derived from a former uranium mining area in column experiments. Microbially influenced manganese release was investigated in columns with an autochthonous microbial community and columns additionally inoculated with Streptomyces. Additionally, azide-poisoned columns were analyzed. Levels of 1,060 μg l−1 Mn(II) were released from inoculated columns while batch experiments led to elution of up to 28 μg l−1 Mn(II). The microbial influence on element cycling correlated with decreasing redox potentials. To study the potential of microbial reduction processes, strains isolated from these columns were investigated. One prominent bacterium, identified as Cupriavidus metallidurans, tolerated up to 30 mM Mn(II). However, no aerobic microbial Mn reduction was found indicating that Mn release was either dependent on anaerobic conditions, or microbial respiration initiated abiotic Mn reduction by decreasing the redox potential necessary for these processes.

Laboratory Investigations on the Interactions of Soil, Water and Microorganisms with Manganese

Column experiments were carried out using contaminated geosubstrates and previously isolated Streptomyces strains from the former uranium mining area Ronneburg (Germany) to study transfer processes of heavy metals including radionuclides. Preliminary tests with comparatively low heavy metal and radionuclide contaminated surface material showed strongly elevated Mn concentrations up to 1060 #g/l after passage through inoculated columns. In contrast, the eluates of non inoculated columns showed, after a “first flush”, low Mn concentrations around 30 #g/l. Poisoned control columns showed decreasing concentrations after the “first flush” (maximum Mn release of 540 #g/l). Highest manganese release from the inoculated, non poisoned columns corresponded with strongly decreasing redox potentials (+200 to -270 mV), which probably indicates microbially catalysed manganese release through reductive processes. One of the strains isolated from the column material was identified as a potentially heavy metal resistant strain of Cupriavidus metallidurans. It showed tolerance of up to 30 mM Mn (II), however no aerobic Mn (IV) reduction processes were indicated.