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Journal of Basic Microbiology 2007, 47, 453 – 467 453

Microbes and metals: interactions in the environment

Götz Haferburg, Erika Kothe

Institute of Microbiology, Friedrich-Schiller-University, Jena, Germany

Research on the behaviour of microorganisms in geogenic or anthropogenic metallomorphic

environments is an integral part of geomicrobiology. The investigation of microbial impact on
the fate of minerals and geologically significant compounds of mining areas can lead to an
understanding of biogeochemical cycles. Metabolic processes of microorganisms are the cause
for the dissolution of minerals, and especially pyrite oxidation results in the generation of acid
mine drainage which, in turn, leads to heavy metal contamination as a result of mining
activities. On the other hand, microbial metabolism can also contribute to the formation of
certain ore deposits over geological time.
The adaptation to heavy metal rich environments is resulting in microorgansims which
show activities for biosorption, bioprecipitation, extracellular sequestration, transport me-
chanisms, and/or chelation. Such resistance mechanisms are the basis for the use of
microorganisms in bioremediation approaches. As only a small part of the worldwide
occurring prokaryotes has been described yet, the understanding of the role bacteria play in a
geogenic and pedogenic context is very likely to change deeply as soon as more habitat relevant
microbial functions can be described. Examples for the identification of microbial processes
from case studies may help to advance this field. The strongly interdisciplinary field of bio-geo-
interactions spanning from the microorganism to the mineral holds much promise for future
developments in both basic research as well as applied sciences.

Keywords: Actinobacteria / Metal resistance / Bioremediation / Mining / Metallomorphic habitats

Received: August 30, 2007; accepted: October 15, 2007

DOI 10.1002/jobm.200700275

Metals in the environment* and destruction of biotopes. Biotopes are rarely pro-
tected by geo- or pedological barriers from the intru-
“When you create a mine there are two things you sion of pollutants; on the contrary, they maintain an
can’t avoid: a hole in the ground and a dump for waste intense interconnection with the mining site itself.
rock.” As simple as this comment of Charles Park in a The lack of spatial and temporal separation from the
novel by John McPhee [78] sounds, as severe is the con- site leads to ecological disturbances. Most important is
sequence. The surface of the Earth is affected by min- the transmission of pollutants like heavy metals from
ing operations with an area of 240,000 square kilome- waste piles and pits with the waterpath which can be
tres [34]. The inevitably injurious effects on the noxious to microbes, plants, animals and human be-
biosphere, not only within the mining sites but across ings. The unearthing of geological formations with its
stretched regions in the surrounding as well, are hard subsequent scarcely preventable weathering and
to foresee and to estimate. Long-term effects, the delay chemical alteration of minerals can cause the genera-
of effects, and the dimension of the affected areas are tion of acidic seepage waters, which trickle through soil
only some of the crucial factors determining alteration habitats and are distributed vertically and horizontally
into microbial habitats. Microbes, however, play the
Correspondence: Götz Haferburg, Friedrich-Schiller-University, Institute key role in mineralization of biological compounds,
of Microbiology, Neugasse 25, 07743 Jena, Germany especially biopolymers like, e.g., lignocellulose and
Tel.: +49(0)3641-949299
chitin by decomposing [27, 77]. Thus, they are essential
Fax: +49(0)3641-949292 for the global biogeochemical cycling of elements. Per-

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

454 G. Haferburg and E. Knothe Journal of Basic Microbiology 2007, 47, 453 – 467

turbations of this particular type of habitat by infiltra- saccharides [51], and (3) influence the biogeochemical
tion of metals can have enormous effects on the bio- cycling of elements, e.g., sulphur [106]. The impairment
sphere. of the biological activity of soils due to metal loading
According to Ross [102], the anthropogenic sources of leads basically to a reduction in decomposition and
metal contamination can be divided into five main turnover rates of organic matter [5]. Ultimately, this
groups: (1) metalliferous mining and smelting, (2) in- interference can cause a reduction in primary produc-
dustry, (3) atmospheric deposition, (4) agriculture, and tion [118].
(5) waste disposal. Worldwide, there is an increasing For the availability of nutrients in the microbial
market for raw materials causing intensified mining habitat the intimate contact between water and soil is
activities. Use and dispersion of metals has assumed of utmost importance. The distribution of nutrients as
enormous proportions during the last century, and the well as the availability of trace elements and toxic met-
behaviour of metals in the environment is therefore a als is determined by the waterpath. The bioavailability
matter of rising concern [89]. The society as profiteer of of metals in the habitat is influenced by the constitu-
mining products has to accept responsibility for mini- tion of the soil matrix, climatic conditions, microbial
mizing the impact of mining operations on the bio- activity, and especially the water flow. The metals con-
sphere, for the development of methods to protect bio- tained in soil minerals are released into the soil solu-
topes, and for the remediation of contaminated areas. tion as a result of weathering processes. Among the
many parameters that govern the behaviour of a metal
in the soil, the hard-soft character of a metal is not to
Metallomorphic microbial habitats underestimate as it determines the ligand preferences
of the metal [3]. The ligand preference, in turn, affects
The most characteristic feature of microbial habitats is the distribution and speciation of the metal, thereby
the great variability of environmental parameters like, influencing the organisms of the habitat [85]. Biologi-
e.g., temperature or nutrient availability over short cally essential metals, like nickel, prefer oxygen ligands
distances. Many basic requirements of heterogeneous and usually form ionic bonds with the ligands [55]. On
microorganisms are satisfied. In ecological terms, the the other hand, many toxic metals, e.g., cadmium, are
microbial habitat consists of a multiplicity of niches. often associated with environmental pollution, have a
The microbial community, then, can be composed of higher affinity for nitrogen and sulphur containing
diverse taxa with different nutritional demands within ligands and form bonds of covalent character.
small microenvironments. ‘Every microbe can be found
everywhere’ and ‘the environment selects’ are the two
seemingly contradictory hypotheses still discussed [75]. Acid mine drainage and acid rock drainage
For the habitats of mining areas it is a clear mutual
influence: microbes in soil are not only affected by The cause for the frequently widely dispersed metal
their environment directly and indirectly, but they also load of habitats in mining areas has been found in the
control particular soil parameters. Growth and metabo- formation of acid mine drainage (AMD). The run-off
lism can lead to changes in pH, redox potential , and from mining heaps of active and abandoned mines can
ionic strength of the soil. For example, oxidation of reach pH values as low as pH 2. The microbes mainly
pyrite by members of the genus Acidithiobacillus results responsible for the formation of AMD are metabolically
in a strong pH decline and thereupon in higher mobil- active even below pH 2 [99]. If the chemical and micro-
ity of heavy metals. This process of metal mobilization, bial processes in the exposed overburden are set into
in turn, determines the species composition within the motion once, AMD formation is hard to control again
habitat to a great extent. and can last for incalculably long times. Chemical and
The microflora, again, strongly participates in pro- biological oxidation of the abundant mineral pyrite
cesses like decomposing soil constituents as well as (FeS2) takes place after the unearthing of pyrite contain-
particle aggregation and influences soil texture and ing rock formations and results in an acidification of
availability of nutrients for plants [64]. This means that the dump material [24]. AMD has a typical orange or
the food web in the soil is constituted to a high degree ocherous appearance which is due to the iron hydrox-
by microbes, which (1) produce substances that change ide that is formed during the oxidation. The iron hy-
the microenvironment by, e.g., solubilization of miner- droxide precipitates as sludge, coating the bottoms of
als and subsequent rock breakdown [22], (2) modify the streams and canals (Fig. 1). Under acidic conditions,
soil structure by, e.g., production of extracellular poly- most heavy metals are leached from the dump waste

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of Basic Microbiology 2007, 47, 453 – 467 Microbes and metals: interactions in the environment 455

Formation of AMD is both a problem of active min-

A B C ing and of abandoned mines. The high number of aban-
doned mines worldwide poses a threat to the potable
water protection areas. Generation of AMD is hard to
avoid, because pyrite is the most common sulphide
mineral and pyrite containing excavated matter is the
result of worldwide operating metal and coal mining. It
is a global issue, affecting not only countries where
mining activities take place, but also neighbouring
countries, whose environments may be adversely af-
Figure 1. Acid mine drainage as metallomorphic habitats. (A) AMD fected by migrating pollution. There are several options
formation at a former uranium mining site in Thuringia. (B)
Accumulated AMD with typically precipitated iron hydroxide. (C) to reduce the rise of AMD. Access of oxygen to the
AMD collected in canals and pumped to a treatment plant. Photos: dump material can be prevented by water saturation of
S. Senitz. the sulphidic material. In some cases the mining opera-
tions can be performed in the absence of water. In
and are subsequently transported as AMD in streamwa- many remediation sites the dump material is sealed
ters, if they are not collected. Conditions required for with watertight substrates. Liming of the dump mate-
the generation of AMD are: (1) contact with the atmos- rial supports neutralization of acidic seepage waters.
pheric oxygen, (2) an aqueous environment, (3) and the However, there is no perfect barrier to separate the
occurrence of iron oxidizing, acidophilic bacteria. reaction components and therefore the resulting AMD
Iron oxidizing bacteria like members of the genera has to be collected in reservoirs. Precaution and per-
Thiobacillus, Leptospirillum and Ferroplasma use Fe2+ as manent monitoring are of utmost importance for the
electron donor to satisfy their energetic demands. But protection of nearby biotopes. In some cases the AMD
due to the high energy demand for autotrophic life – treatment can comprise the recovery and recycling of
supply of reducing power for CO2 fixation – the ener- precious metals by using biomass material as biosor-
getic yield of the Fe2+ to Fe3+ oxidation is relatively bent [122]. There are terrains known for generation of
scarce for the overall energy requirement of the cell. To acidic drainage with the same chemical and microbial
satisfy the energy demand and to maintain the vital processes, but not initiated by human intervention.
functions of the cell, the substrate turnover has to be This natural type of alteration of sulphidic rocks, for
high. The formation of one gram biomass (dry weight) example, in Rio Tinto, Spain, is considered as acid rock
requires the oxidation of an amount of about 55 gram drainage (ARD). The high acidity of both, AMD and
Fe2+. In turn, Fe3+ oxidizes pyrite in a fast autocatalytic ARD, and the high amounts of dissolved heavy metals
mechanism in the presence of water under generation generally lead to an extreme toxicity to most organisms
of protons which lead to a pH decrease. In the overall [95]. Nevertheless, there are microbes thriving even in
reaction, the part of the abiotic oxidation of iron is this type of environment. The phylogenetic diversity of
comparatively slow under acidic conditions. both, prokaryotes and eukaryotes dwelling in drainage
(a) Abiotic process, slow oxidation rate, initiator reac- influenced habitats can reach unexpected dimensions
tion, acidification of the site: as has been shown, e.g., for the extremely acidic envi-
FeS2 + 7/2 O2 + H2O → Fe2+ + 2 SO42– + 2 H+ ronments (pH 1.7 – 2.5) of the Rio Tinto [130].

(b) Biotic process, energy-yielding reaction of iron-

oxidizing bacteria, high turnover: Geogenic metallomorphic habitats
4 Fe2+ + O2 + 4 H+ → 4 Fe3+ + 2 H2O
Microbes have to cope with high concentrations of
(c) Abiotic process, fast, autocatalytic mechanism, re- different heavy metals in various kinds of habitats. For
generates electron donor for (b): life under extreme conditions, habitats in areas in
which mining is pursued are probably the most promi-
FeS2 + 14 Fe3+ + 8 H2O → 15 Fe2+ + 2 SO42– + 16 H+ .
nent, but in terms of evolutionary time those habitats
But due to the regeneration of the ferrous ion as within naturally metalliferous biotopes are more influ-
electron donor for the bacteria, the change of iron from ential on adaptation and expression of microbial resis-
the ferrous (Fe2+) to the ferric (Fe3+) state enters a tance determinants. It has been found that bacteria
propagation cycle, and acidification accelerates [114]. isolated from serpentine soils have developed strong

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

456 G. Haferburg and E. Knothe Journal of Basic Microbiology 2007, 47, 453 – 467

resistance mechanisms that seem to be fundamental metallomorphic microbial habitat and harbour bacteria
for survival in worldwide occurring, naturally nickel with remarkable resistance attributes.
enriched soils. Serpentine soils are depleted in nutri- If we continue to focus on time available for evolu-
ents causing a remarkably low number of microbes of tionary adaptation, then the extreme habitats of min-
any physiological group [68]. This soil type is character- ing areas can be positioned between long-lived metal
ized by deficiency in available phosphate, paucity in containing outcrops (e.g., 40 million years of influence
ammonia and lack of readily decomposable carbon on adaptation in Neocaledonia) and short-lived, man-
source. The magnesium to calcium ratio is high. Fur- created metal contaminations of industrial installa-
thermore, serpentine soils are not only enriched in tions. Microbial strains of various taxonomic categories
nickel, but due to the mineral composition of the base have been isolated from metal mining sites [105, 115].
rock they display elevated levels of chromium, cobalt Usually, studies on distribution and phylogenetic
and iron as well. Taking all the pedological facts in grouping involve the investigation of resistance
consideration, the occurrence of typical serpentino- mechanisms.
phytes [97] and a characteristic microbial community
structure become understandable. These soils can con-
tain enormous amounts of various metals, as it was Microbe-metal-interactions
shown on, e.g., soil samples from Andaman (India) with
up to 8 g nickel, 4 g chromium and 150 g iron per kg With 106 – 109 viable cells cm–3 bacteria are usually the
dry soil [92]. A multiple metal-resistance of resident most numerous organisms in soil [71]. Due to their
microbes is prerequisite for the occupation of this eco- small size, bacteria have a high surface to volume ratio
logical niche. Bacterial isolates of serpentine soils of and therefore provide a large contact area for interac-
Tuscany were investigated on their resistance pattern tions with the surrounding environment. Besides their
towards several heavy metals and the magnitude of occurrence in high numbers and their high surface to
resistance in relation to the distance from the typical volume ratio, it is the negative net charge of the cell
serpentinophyte Alyssum bertolonii. A simultaneous re- envelope that makes these organisms prone to accumu-
sistance to a set of metals and highest resistance from late metal cations from the environment [23]. Microbes
isolates of the rhizosphere were found to be character- can potentially accumulate metals either by a metabo-
istic [79]. It is known that nickel hyperaccumulating lism-independent, passive, or a metabolismdependent,
plants as, e.g., A. bertolonii provide a niche for nickel active process. Thus, overall accumulation is deter-
resistant bacteria [108]. An example for the adaptation mined by two characteristics of the cell: sorptivity of
of microbes to the soil substrate of metallomorphic the cell envelope and capacity for taking up metals into
habitats has been presented and shows the influence of the cytosol. Active uptake into the cytosol is usually
nickel on soil structure of neocaledonian soils [52]. The slower than passive adsorption and is dependent on
extreme environments in serpentinized neocaledonian element-specific transport systems [35]. Passive adsorp-
soils are microbiologically well investigated. Owing to tion is likely to be the dominant mechanism in metal
the particular mineral composition and to the distribu- accumulation, since scarcity of nutrients is the ground
tion in isolated patches serpentinized outcrops have state for many natural environments in soils, and ac-
been considered ecological islands [65]. tive uptake requires energy. Additionally, microbes
Anthropogenically created metalliferous habitats can probably lack highly specific uptake systems for most
be understood as ecological islands in a similar sense. metals. The surface characteristics of the bacteria de-
Isolation of highly resistant organisms from anthropo- termine their metal-adsorption properties. The differ-
genic metal rich habitats is not unusual. Publications ences in cell wall construction of Gram-positive and
on isolation of metal resistant bacteria from the sewage Gram-negative bacteria have minor influence on the
sludge of waste water treatment plants and metal- sorption behaviour of different metals [57]. The bulk
processing industry are numerous. Stoppel and col- functional group chemistry of both classes of bacterial
leagues isolated from a mineral oil emulsion tank a surfaces is similar, but particular single constituents of
Klebsiella oxytoca strain resistant to 10 mM nickel [116]. the cell envelope can have great importance for metal
From a decantation tank of a zinc factory a strongly binding. For example, phosphoryl groups of lipopoly-
metal resistant Alcaligenes xylosoxidans strain could be saccharides, carboxylic groups of teichoic and teichuro-
isolated [111]. This implies that ephemeral locations of nic acids, or capsule forming extracellular polymers
technical facilities with a partially extremely high en- influence the metal sorption of the cell envelope
richment of metal containing compounds can act as (Tab. 1). The interplay of metal mobilizing mechanisms

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of Basic Microbiology 2007, 47, 453 – 467 Microbes and metals: interactions in the environment 457

Table 1. Metal-binding functional groups in bacterial surface tolerated in higher concentrations. They accomplish
either metabolic functions as constituents of enzymes
Functional Location Organism Refe- or meet structural demands as, e.g., in supporting the
group/compound rence cell envelope. The concentration and the speciation of
class the metal determine whether it is useful or harmful to
Caroxyl Lipopolysaccharide Pseudomonas [67] the bacterial cell. Homoeostasis is therefore essential
aeruginosa and bacteria have developed a fine-tuned regulatory
Peptidogycan Escherichia coli [54]
Peptidogycan Bacillus subtilis [9]
system of incorporation and excretion. Adverse effects
Amine/imidazole Polypeptide B. subtilis [9] of metals on the microbial cell are decreased decompo-
Polypeptide Klebsiella [83] sition of soil organic matter, reduced soil respiration,
pneumoniae lower diversity, and decreased activity of several soil
Thiol Phytochelatins E. coli, GMO [6]
Metallothioneins Ralstonia [121] enzymes [103, 119]. Depending on the external condi-
eutropha, GMO tions microbial cells have developed mechanisms to
Phosphoryl Lipopolysaccharide E. coli [31] cope with high concentrations of metals [113].
Teichoic acid B. subtilis [10]
Phospholipids Bacteria [11]

Survival strategies
and metal fixation forces is highly complex and de- of heavy metal resistant bacteria
pendent on a number of soil characteristics (Fig. 2).
Metals without biological function are in general Heavy metals affect the microbial cell in various ways.
tolerated only in minute concentrations, whereas es- On the macro- and microscopic level general changes in
sential metals with biological functions are usually morphology, as e.g., the disruption of the life cycle and
the increase or decrease of pigmentation are easy to
observe and evaluate. It has been shown that the im-
Microbial Cell pact of metals on the metabolism depends on the
growth form. In consortia from mining sites the resis-
1 2 4 3 tance towards different metals seems to be higher than
for pure cultures [115]. In addition, the species compo-
sition of an AMD site fluctuates seasonally, as has been
Soluble Insoluble
Metal Compounds
5 6 Metal Compounds shown for Iron Mountain, Richmond, USA [28].
A great number of heavy metal resistant bacteria
8 7 7 8 such as, e.g., Cupriavidus metallidurans is known to pos-
sess efflux transporters that excrete toxic or over-
concentrated metals [86, 87]. This type of transporters
is characterized by a high substrate affinity and can
therefore keep low the cytosolic metal concentration.
Figure 2. Interaction of metal mobilization and fixation (modified From genomic data mining, actinobacteria presumably
after 39). Microbial metabolism and growth can lead to changes in can have cation efflux transporters, but they were not
metal solubility. The relative balance between the processes functionally identified yet. The ATP binding cassette
depends on various environmental factors, organisms, and anthro-
pogenic activities. (1) Metal solubilization by, e.g., heterotrophic (ABC) transporters of actinobacteria are, in contrast,
leaching, metabolite excretion including organic acids and H+, redox well investigated and are responsible for antibiotic
reactions. (2) Effects of soluble metal compounds on microbes and resistance. Some of the ABC transporters function as
metal immobilization by biosorption, transport, intracellular
metal efflux pumps as well [14]. Alternatively, the mi-
sequestration, and precipitations. (3) Effects of insoluble metal
species on microbes, particulate adsorption, and entrapment by crobial cell can prevent itself from being intoxicated by
polysaccharide and/or mycelial network. (4) Metal immobilization the release of metal binding compounds into the ex-
by, e.g., precipitation, or reduction. (5) Influence of environmental tracellular surrounding. The metals are chelated out-
factors, e.g., pH, O2, CO2, nutrients, salinity and metal toxicity on
microbial growth and metabolism. (6) Influence of microbial
side the cell and thus blocked from entering the cell
activities on the environment, e.g., alterations in pH, O2, CO2, and through the unspecific membrane transporters that
redox potential; depletion of nutrients; mineralization of polymers by otherwise would facilitate the influx. Membrane trans-
exoenzymes, and metabolite excretion. (7) and (8) Environmental port systems of the cell can not differentiate between
factors which direct the equilibrium between soluble and insoluble
metal species towards metal mobilization (step 7) or metal the trace elements needed for metabolic actions and
immobilization (step 8). toxic metals that would – once inside the cell – inter-

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

458 G. Haferburg and E. Knothe Journal of Basic Microbiology 2007, 47, 453 – 467

fere with, e.g., the phosphoryl groups of nucleic acids molecular level is the prerequisite necessary for the
or the thiol groups of proteins. Some fungal and bacte- biological treatment of solid mining waste and result-
rial organisms are able to keep metals outside the cell ing effluents. The combination of genomic approaches
by the extracellularly active melanin [7, 32]. This sec- with geochemical and hydrological models is the ulti-
ondary metabolite has powerful cation chelating prop- mate goal to accelerate bioremediation [69].
erties through the anionic function such as the car- Microbial life has conquered extremely hostile envi-
boxyl, and the deprotonated hydroxyl groups [101]. For ronments. It has been reported that habitats like AMD
the metal burden of soil habitats the interplay of bio- run-offs characterized by a pH of zero and a content of
solubilization and bioprecipitation is of great impor- more than 100 g/l iron as well as the metals copper,
tance. Numerous soil microbes, for example the wide- arsenic, cadmium, zinc up to a range of tenths of grams
spread fungus Aspergillus niger, solubilize metals by the per litre have been colonized by the archaeal iron-
release of organic acids, while others – or even the oxidizer Ferroplasma [29]. Colonization of this type of
same microorganisms – immobilize metals through the niche requires a high measure of adaptability. Never-
excretion of compounds as, e.g., oxalates [37, 128]. If theless, in a general view, the richness of bacterial di-
toxic metals have entered the cell and can not be ex- versity is yet unexplored to a very large extent. The
creted by efflux transporters several organisms have difficulties in imitating the natural conditions that
developed a cytosolic sequestration mechanism for occur in the investigated niches and the duplication of
protection. It has been shown for many metal resistant niche characteristics on a laboratory-scale are the most
organisms that internal inclusion bodies, like, e.g., challenging obstacles that have to be overcome for
polyphosphate granules (volutin) bind large amounts of obtaining new taxa. The physiology of metal-adapted
metal cations [46]. The cell envelope equips the cell microorganisms determines the methodology that has
with an additional metal resistance feature. The cell to be applied for isolation and cultivation. There is a
wall, in combination with the cell membrane, supports remarkable contradiction between the fact of more
the sorption of metals and facilitates bioreduction as than 600 completely sequenced bacterial genomes (as of
well. It has been shown that, for example, Penicillium January, 2007) and the numbers reported for yet undis-
chrysogenum has the capacity to reduce silver. After covered bacteria that range from 80% [124] to 99.9%
reduction the metallic silver precipitates at the cell wall [26]. In order to expand our knowledge on microbial
[36]. functions in an ecological context, it seems worth-while
In Fig. 3 the mechanisms of metal resistance of mi- to put more effort especially in the isolation of micro-
crobes are summarized schematically: (1) Metal resis- organisms from endangered and extreme environments
tance of microbes is accomplished by intra- and ex- like, e.g., mining sites and metallurgical plants. Well-
tracellular mechanisms; (2) Metals can be excreted via adapted microbes isolated from these types of habitats
efflux transport systems; (3) Sequestering compounds can probably support remediation and are of great in-
of the cytosol can bind and detoxify metals inside the terest for strategies on environmental conservation.
cell; (4) The release of chelators into the extracellular
milieu leads to bound and fixed metals; (5) The struc-
ture of the cell envelope is prone to bind large amounts Release of chelators
of metals by sorption thus preventing influx.
The investigation of microbial resistance mecha- The genus Streptomyces is known to be the largest anti-
nisms towards heavy metals is essential for the poten- biotic-producing group and is still of central impor-
tial applications of microorganisms in bioremediation. tance in the identification of medically relevant natural
The understanding of the resistance phenomena at the compounds. With the discovery of streptothricin and

M2+ M-X X X X-M
Export of chelating Intracellular
compounds sequestration
M2+ X X M-X M2+ M2+
M-X M2+ transporter

Figure 3. Overview of microbial resistance mechanisms. (X) – Cell constituents interacting with metal cations, (M) – Metal cation.

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of Basic Microbiology 2007, 47, 453 – 467 459

streptomycin in the 1940ies, a systematic screening of An additional feature relating to metal mobility and
antibiotics on isolates of this genus and related taxa availability in soil habitats is the microbial initiation of
was initiated [8, 123]. Later on, the screening efforts secondary mineral formation. The most basic processes
started to decrease due to the difficulties to obtain in biomineralization operate at the nanometer scale
novel compounds. This is in contrast to the continued and involve macromolecules, like proteins, directly
demand for new antibiotics against resistant pathogens controlling the nucleation and growth of the mineral
[18]. Interestingly, it was calculated that only a tiny phase [74]. The cell-mediated deposition of crystalline
fraction of the antimicrobial compounds the genus is materials can proceed both within and outside the cell,
capable to produce has been discovered so far. A total but always involves anionic matrix molecules which
number in the order of 100,000 antibiotics was calcu- function to sequester the relevant ions [15]. Such pro-
lated using a conservative estimate [125]. It can be con- cesses also would limit the bioavailable fraction of met-
cluded that the decline in the discovery of new metabo- als in the environment.
lites is due to limitations in the screening efforts rather
than an exhaustion of potentially produced compounds
raising the question of developing new screening strate-
Using rare earth elements to understand
microbial processes in metalliferous habitats
Could the metabolic response of actinobacteria on
metals be used to trace new secondary metabolites or
could metal supply even direct and support metabolite There is only limited literature available on methods
synthesis? Metals facilitate secondary metabolism not necessary for description of metallomorphic microbial
only in actinobacteria, but in some other prokaryotic habitats. In order to understand the role microbes play
taxa and fungal groups as well [17, 126]. The complex in mobilisation/immobilisation of metals in soils, it has
effect of metals on secondary metabolism can be seen to be differentiated between microbial activity and the
best with results of product research: A Streptomyces effects of the soil matrix itself on mobility and sorption
galbus strain producing an antifungal antibiotic is en- of metals.
hanced in production if the fermentation medium is Rare earth elements (REE), or lanthanides, comprise a
supplemented with copper, zinc or iron, whereas nickel group of 15 elements with fairly similar geochemical
and cadmium addition lead to a reduction of antibiotic properties and an approximately similar content in
concentration in the same strain [94, 100]. The addition different soils of various regions. However, the concen-
of as little as 0.01 μg/ml of cobalt to the medium results trations of REE in mining areas can reach high levels.
in a strong increase in the production of coumermycin During weathering processes, the REE are fractionated
A1 by Streptomyces rishiriensis [19]. Chromium is known [60]. The differences in the fractionation of REE are
to have a stimulatory effect on both actinorhodin pro- dependent of the sorption characteristics of the sub-
duction and growth yield of the model streptomycete strate.
S. coelicolor [1]. Thus, gene regulation by metals can af- Numerous studies refer to the sorption of REE by
fect morphological differentiation and control produc- microorganisms [58, 81, 82]. The potential role of bacte-
tion of antibiotics and pigments. ria on REE fractionation processes is not yet clearly
It could be shown that the rare earth element ytter- understood [91, 117]. However, if REE fractionation is
bium induces at a concentration of 25 mg/l the produc- applied as a monitoring tool for remediation, the rea-
tion of a reddish pigment with a naphthoquinone-like son of the different fractionation behaviour of geogenic
structure in a Streptomyces isolate [61]. Melanins, pig- and biogenic substrates has to be elucidated. In con-
ments produced by a wide variety of microorganims, trast to a geogenic matrix, the microbial REE sorption
are characterized by a strong metal affinity [88]. A comprises the two subprocesses of passive cation at-
number of secondary metabolites, among them antibi- tachment to the cell surface and metal uptake with
otics like isatin [47] and pigments like actinorhodin [21] intracellular accumulation. For a Pseudomonas aeruginosa
or melanin [7], are known for their metal chelating strain endowed with a high metal uptake capacity, it
behaviour and participate very likely in extracellular was shown that around 85% of the REE can be desorbed
metal resistance processes. The influence of such che- using citrate buffer [96]. The fractionation analysis of a
lating compounds on the metal mobility in soil is easily Bacillus subtilis strain and an Escherichia coli strain
underestimated and hence research is needed to eluci- showed an enrichment of especially heavy REE on the
date their role in metal tranport in the compartments cell surface with at least two binding sites, i.e. carboxy-
water and soil. late and phosphate groups [117].

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

460 G. Haferburg and E. Knothe Journal of Basic Microbiology 2007, 47, 453 – 467

Microbial biosorption of metals Examples from a case study:

the former uranium mining area
The capacity of microbial cells for biosorption is con- in Eastern Thuringia (Germany)
siderable. Bacteria, for instance, have a cell volume of
1.5 – 2.5 μm3 resulting in a high surface to volume ratio. The mine field covers an area of 74 km2 including
If a cube with the edge length of 1 cm is divided into 40 mine shafts and 3000 km mine drifts. Between 1952
1012 cubes with an edge of 1 μm, the entire surface of and 1990 113,000 metric tonnes of uranium extracted
the 1012 small cubes is 10,000 times larger than the from 154 million tonnes crude ore were produced in
surface of the big cube. The large surface area permits this district [41]. 14 mine dumps with a total volume of
not only the efficient uptake of nutrients and the re- 125 million m3 of waste rock – containing considerable
lease of metabolic waste products, but the interaction amounts of pyrite – are the major source for AMD gen-
with the mobile metal fraction of the environment as eration. The AMD, in turn, determines the character of
well. Although Gram-positive and Gram-negative bacte- the habitats and their colonization by microorganisms
ria differ markedly in the structure of the cell envelope, through parameters like pH, metal content, ionic
the potential for biosorption is comparable, due to the strength and redox potential. The microbial population
similar composition of functional groups appropriate to of the waste rock heaps in the district itself is domi-
bind (metal) cations [57]. Nevertheless, microbial iso- nated by thiobacilli, but species of the genera Sulfolobus
lates from contaminated environments behave re- and Acidianus, and Leptospirillum ferrooxidans occur as
markably different in biosorption of metals from AMD. well [105]. Members of the genera Pseudomonas and
Many parameters besides metal toxicity, metal compo- Aeromonas, among others, could be isolated from the
sition of the mix and total metal content of the solution uranium waste piles of the neighbouring mining field
influence the biosorption. The age of the culture de- in Saxony [112]. But also members of the actinobacteria
termines to a great extent the biosorption capacity of can be found in dump material, as it was shown for the
the cells as it was tested for uranium uptake of Strepto- metal leaching actinomycete Nocardiopsis metallicus
myces longwoodensis [33]. Older cells (> 14 h) accumulated [107].
about double the amount of uranium of younger cells The investigated substrate samples of the former
(< 14 h). Streptomyces albus in contrast to Pseudomonas uranium mining area were strongly influenced by AMD
stutzeri accumulates metals from a mixed metal solu- containing, e.g., approximately 50 mg/l aluminum,
tion preferentially at the cell wall. However, the elec- more than 10 mg/l nickel and around 650 μg/l uranium
tron-microscopic analysis revealed that the cell walls of [50]. As long as AMD generation occurs, the metals are
neighbouring cells contained metals to a strongly vary- enriched in the soil/substrate. Only a fraction of the
ing degree [76]. metals bound to the pedogenic matrix becomes easily
Metals are immobilized by either complexation with mobilized and therefore bioavailable as it has been
released chelating compounds or precipitation across described for cadmium [63]. The bioavailability of dif-
the cell envelope depending on structural features. ferent metals can be estimated by sequential extrac-
Particular structures of the cell envelope as, e.g., S-layer tion. It has been shown that the concentration of mo-
proteins [12], act as nucleation centre and facilitate bile metals in mining habitats has an influence on the
crystal growth resulting in the formation of biominer- sporulation of actinobacteria [109]. The high metal
als. However, precipitation and biosorption are some- burden in the habitat results in the loss of the capacity
times overlapping phenomena and it can be difficult to for sporulation. Nevertheless, strains obtained from
assign the contribution of each to metal immobilisation these habitats displayed different grades of resistance
[43]. towards nickel. Six actinobacteria isolates originating
The differing sorption by cells of one isolate hint at from an isolation campaign along an AMD contamina-
the dependence of the biosorption on the stage in the tion gradient were able to grow on minimal media
life cycle. Biosorption of uranium from a solution by containing up to 6000 mg/l nickel – comparable to
Streptomyces strains seems to be a fast process. Within strongly nickel enriched dump material. Since isolation
seconds or only few minutes, the tested dead and vital was achieved from the non-dormant state of the cells,
biomass is saturated [44, 45, 53]. In the context of bio- metabolically active populations of actinobacteria are
remediation the outstanding importance of research on therefore supposed to occur in the investigated habi-
both biosorption and resistance of microorganisms that tats. The occurrence of actinobacteria in AMD influ-
can thrive in metalliferous environments cannot be enced, extreme environments implies cellular resis-
overlooked. tance mechanisms. Metal resistance of a number of

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of Basic Microbiology 2007, 47, 453 – 467 Microbes and metals: interactions in the environment 461

bacteria is well investigated and partly used in biore- energy conservation by re-oxidising of the hydrogen
mediation [70, 80]. In contrast to many microorganisms liberated by nitrogenase.
that were isolated from metalliferous habitats, metal In order to survey the behaviour of actinobacteria –
resistance of actinobacteria is a rather recent research isolated from different habitats – in presence of nickel,
topic and not comprehensively outlined yet. an isolation campaign was conducted. The distribution
of nickel resistance among 100 Streptomyces strains
originating from contaminated and noncontaminated
Nickel resistance among actinobacteria sites and adaptation of the actinobacteria isolates to
examplifying metal-microbe interactions metalliferous environments was tested [49]. It could be
shown that the reservoir for potentially useful, metal
Nickel is both an essential trace nutrient in nanomolar resistant strains in such polluted environments is huge
concentrations and a toxicant to microbial metabolism and strains for different uses in bioremediation pro-
if over-concentrated, i.e. in the micro- or milimolar cesses can be easily identified from different, metal
range. Enzymes like, e.g., hydrogenases, ureases, CO poluted sites.
dehydrogenases and a particular type of superoxide
dismutase depend in their function (and partially struc-
tural composition) on sufficient nickel supply. A sur- Search of microbes applicable
plus of intracellular nickel results in protein damaging to bioremediation processes
especially by complexation of thiol groups, and can
furthermore interfere with nucleic acids. The uptake of It is the pragmatic goal of current bioprocess research
nickel is accomplished by the Mg2+ transport system or, on metal removal from treatable sources to identify
in case of nickel deficiency, by specific nickel uptake species of microorganisms that are capable of efficient
systems. Intoxication of the cell with nickel can only uptake of environmentally and economically important
marginally be prevented by regulation of the uptake metals [120]. As a result of metal toxicity, living cells
systems since magnesium is an essential macronutrient may be inactivated; therefore most living-cell systems
and required in larger quantities than nickel. The cellu- exploited to date have been used to decontaminate
lar counteractions are either performed by expression effluents containing metals at subtoxic concentrations
of active nickel efflux transport systems or by com- [40]. The average concentration of trace metals in soil
plexation with nickel chelating substances, limiting solution – iron and manganese excepted – is usually
free Ni2+ within the cell. Resistance of actinobacteria within the range of 1 – 100 mg/l, depending on the
towards cadmium, copper and mercury has been re- abundance of the element in the lithosphere [59]. Bac-
ported and is well investigated in the case of mercury teria isolated from non-polluted environments are
[4, 98]. For nickel resistance, knowledge has been ac- adapted to cope with concentrations within or below
cumulated regarding various isolates of, e.g., Cupria- the micromolar range. However, for soil remediation
vidus metallidurans [48], Achromobacter xylosoxidans [110], with biological treatments, microbes adapted to far
Hafnia alvei [93] and Pseudomonas aeruginosa [104], but higher concentrations are required. Therefore, screen-
only limited literature is available on nickel resistance ing for microbes with high accumulation capacities and
of members of the group of actinobacteria. Neverthe- stable resistance characteristics is an inevitable part of
less, as has been reported for the actinobacterial genus any remediation strategy. Bioremediation is the use of
Microbacterium, the accumulation of nickel by nickel living organisms to reduce, eliminate or immobilize
hyperaccumulator plants is influenced by the micro- environmental hazards resulting from accumulation of
flora of the rhizosphere [2]. Microbacterium arabinogalac- toxic chemicals and other hazardous wastes. The
tanolyticum survives in nickel rich soils and promotes treatment is performed ex-situ or in-situ with solid and
the nickel accumulation of the nickel accumulator liquid waste [38]. The technology is based on the utilisa-
Alyssum murale. Another example of nickel tolerating tion of naturally occurring (or genetically engineered)
actinobacteria of the rhizosphere is Frankia. Members of microorganisms or plants to transform organic and
the nitrogen-fixing genus Frankia, living in symbiosis inorganic compounds. Often, biostimulation and bio-
with alder (Alnus glutinosa), were found to tolerate more augmentation are components of a bioremediation
than 2.0 mM nickel and to increase yield when nickel is strategy. Biostimulation utilizes the site-specific in-
added. This has been correlated with an enhanced hy- digenous microorganisms. Nutrients or electron accep-
drogenase synthesis [127]. The increased synthesis of tors are added to the contaminated material. Bioaug-
nickel containing hydrogenases is thought to be due to mentation is the introduction of specific competent

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

462 G. Haferburg and E. Knothe Journal of Basic Microbiology 2007, 47, 453 – 467

microbes to the local microbial population in order to For this preventive measure the introduced microbes
increase the metabolic capacities needed for remedia- should: (1) be resistant towards the metals occurring in
tion [42]. the habitat, (2) possess a high capacity for metal uptake
The processes of bioremediation are based on two or sorption, (3) be capable to adapt to the conditions of
premises: (1) the removal or detoxification of the pol- the habitat, (4) fix metals stably, in order to prevent a
lutant and (2) the maintenance/improvement of soil flush due to, e.g., a dying off in the cold season, and (5)
fertility. The characteristics of the soil are not – or only allow biomonitoring. Literature on bioaugmentation
to a limited extent – altered by chemical and mechani- using biocurtains for immobilization of heavy metal
cal measures. Phytoextraction applies selected plants contaminants is, however, scarce.
which can take up a plantspecific set of metals as hy-
peraccumulators. These metals are subsequently en-
riched in the biomass and in comparison to excluder
plants concentrated up to 1000 fold [30]. By phytoex- Research on the behaviour of microorganisms in metal-
traction, large quantities of the bioavailable metal frac- liferous environments is an integral part of geomicro-
tion of the soil solution can be removed from the biology. The microbial impact on the fate of minerals
ground with the harvest, and are further processed for and geologically significant compounds of mining areas
metal recycling. In comparison to conventional meth- is of major interest since its investigation leads to an
ods like soil excavation (ex-situ remediation), phytoex- understanding of the cycling (or accumulation) of me-
traction is time consuming, but cost effective and less tallic elements, the formation and stability of biotopes
labour-intensive. Interestingly, the role microbes play and the formation of altered compounds.
in phytoextraction is still underestimated. Often, the How inseparable geological processes and microbial
application of plants for bioremediation is restricted by activity in the lithosphere are allied has been expressed
the immature and poor soils of typical metal contami- with the term geosymbiosis [16]. Such geo-bio-
nated mining sites and mining affected grounds. High interactions describe the reciprocal relationship be-
concentrations of heavy metals lead to retardation in tween restructuring/proliferation of a mineral and a
growth and subsequently low yield during harvest microorganism. In the microbe-mineral interaction,
whereas soils with lower metal concentrations do not both geosymbiotic partners affect and are affected in
warrant extraction. In both cases, the soil shows an return.
unvaried load of the pollutant. For soils only weakly Metabolic processes of microorganisms have not only
contaminated, another type of bioremediation can be been identified as cause for the dissolution of pyrite
applied. Biogenic barriers made of microbial biomass, and other rocks often resulting in the generation of
so called biocurtains, are introduced into soil areas of AMD. In contrast, reverse effects of the microbial me-
contamination. The microbial cells act directly on the tabolism can also contribute to the formation of certain
spot of contamination. Cost-effective biocurtains for ore deposits over geological time (Table 2). The micro-
the degradation and removal of halogenated hydrocar- bial formation of ore deposits is based on the capability
bons are already in use [56]. For metal pollution, con- of organisms to precipitate metals from solution. Thus,
taminants are immobilized at (cell surface) or inside microbes participate constitutively in shaping the
(cytosol) the cell. Thereby, heavy metals will be retained physical world by precipitating ore deposits [84].
in the soil and the water path can be protected from
metal pollution by blocking the contamination route.

Table 2. Formation and dissolution of ore deposits by microorganisms’ metabolic processes.

Formation of ore deposits Microbial activity Dissolution of ore deposits Reference

Banded iron formation Iron-oxidizing phototrophs [129]
Sulfide ores (e.g. ZnS) Sulfate reducing bacteria [66]
Magnetite (Fe3O4), uranitite (UO2) ores Hyperthermophilic microbes [62]
Siderophore producing microbes Iron leaching from minerals [20, 73]
Anaerobic resiration Reductive dissolution of Fe(III) oxides [25]
Generation of acid mine drainage Pyrite oxidation [13]
Biomineralization and Heavy metal resistance Chelator production [36]
bioaccumulation mechanisms

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Journal of Basic Microbiology 2007, 47, 453 – 467 463

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