Dunn Colin E. Biogeochemistry in Mineral Exploration

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Chapter 12

EXPLORATION GEOMICROBIOLOGY – THE NEW FRONTIER

FRANK REITH

CSIRO Exploration and Mining and Cooperative Research Centre for Landscape

Environment and Mineral Exploration

STEPHEN L. ROGERS

CEO-Cooperative Research Centre for Landscape Environment and Mineral

Exploration

INTRODUCTION

Mineral deposits characterized by outcropping hard-rock mineralization are

well explored and currently being depleted. Consequently, mineral exploration is

increasingly focussing on areas where thick layers of overburden conceal potential

mineralization (Smith, 1996). In Australia, significant parts of the landscape are

covered by deeply weathered residual regolith as well as transported cover of up to

100 m, which may mask underlying mineralization (Taylor and Eggleton, 2001).

Critical to the development of successful predictive exploration techniques in these

areas is an understanding of processes that lead to trace element dispersion and

re-concentration, and thus mediate the formation of surface and near surface

expression of buried mineralization.

Microbial processes are known to drive processes involved in the mobil ization,

distribution and speciation of many trace metals under a wide range of environ-

mental conditions (Ehrlich, 1996a,b). Understanding the relationship between

microbial mineral solubilization and precipitation and trace element bioavailability

in the rhizosphere (i.e., zone immediately surrounding the plant root) is critical in the

application of biogeochemical exploration using plant materials, because the biogeo-

chemical activity of micro-organisms and plants is closely interlinked. Bacteria and

fungi living in the rhizosphere of plants have been shown to increase the mobility and

solubility of commodity metals and pathﬁn der elements, such as As, Cd, Cu, Hg, Ni,

Pb, Se and Zn, which in turn leads to a increased uptake of these elements by the

plant (de Souza et al., 1999a,b; Whiting et al., 2001; Khan, 2005). However, other

studies indicate that rhizosphere microbiota may under different conditions reduce

plant metal uptake via surface adsorption and related processes such as ion -ex-

change, complexation, precipitation and crystallization on and within the cell wall

(Mattuschka and Straube, 1993; Cotoras et al., 1992; Morley and Gadd, 1995).

The structure and activity of the microbiota resident in the soil are strongly

inﬂuenced by the prevailing geochemical conditions such as pH, redox conditions

and element content (Brock et al., 1996; Ehrlich, 1996a,b; Kizilkaya et al., 2004).

Previous studies have shown that some micro-organisms indicate the presence of bur-

ied mineralization (Parduhn et al., 1985; Parduhn, 1991; Parduhn, 1995). In particular,

the use of Bacillus cereus as an indicator organism for Au and other metals has been

successfully applied in studies of different terrains in Belgium, China, Argentina

and Mexico (Neybergh et al., 1991; Melchior et al., 1994, 1996; Wang et al., 1999).

Cell numbers of Bacillus cereus in polymetallic soils, especially those with high Au

concentrations, were up to several orders of magnitude higher compared to soils

containing background concentrations, indicating an unambiguous association of

bacterial population with polymetallic soils (Watterson, 1985; Neybergh et al., 1991;

Melchior et al., 1994, 1996; Wang et al., 1999).

To generate an understanding of the relationship between micro-organisms, their

geochemical environment and processes relevant to mineral exploration, ‘exploration

geomicrobiology’ has emerged as a new area of research. Studies in this developing

ﬁeld provide the understanding of microbial populations and processes that is

required to successfully develop bio-prospecting tools and predictive biogeochemical

(sensu stricto) models. Figure 12-1 is a schematic diagram that demonstrates the links

between geomicrobial and biogeochemical processes (in grey) and mineral exploration

and bioprocessing of ores.

The aim of this summary is to introduce the ﬁeld of exploration geomicrobiology

to readers with a background in explora tion geosciences by (i) providing an overview

of microbial processes relevant to trace element dispersion and re-concentration

in the soil; (ii) introducing methods to study activities and structures of complex

microbial communities; and (iii) highlighting how these methods are ap plied in three

case studies relevant to mineral exploration.

SIGNIFICANCE OF MICRO-ORGANISMS AS BIOGEOCHEMICAL AGENTS

Microbes are the oldest and most diverse forms of life on the planet (Ehrlich,

1996b). Fossil records demonstrate that the oldest group of micro-organisms,

the single-celled prokaryotes (bacteria and archaea) inhabited the planet as early as

3.8 billion years ago, and that these early ancestors of today’ s prokaryotes display

cellular structures similar to their modern descendants (Ehrlich, 1998). Other groups

of micro-organisms that are impor tant in environmental systems are fungi and algae,

which belong to the eukaryotes and whose earliest ancestors have been around for

approximately 2.1 billion years (Han and Runnegar, 1992).

Micro-organisms are highly diverse and abundant in soils with population sizes of

up to 10

cells per gram of material (Brock et al., 1996; Paul and Clark, 1996).

They occur in ‘extreme’ environments such as deep marine sediments (Parkes et al.,

1994), deep-sea hydrothermal vents (Juniper and Tebo, 1995), deep rock fractures

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Exploration Geomicrobiology – the New Frontier

(Pedersen, 1993), deserts (Adams et al., 1992), polar-regions (Vincent and James,

1996) and acidic (pHo1) heavy metal-polluted mine wastes (Ledin and Pedersen,

1996; Baker et al., 2004). Besides being genetically and ecologically extremely

versatile, micro-organisms and in particular bacteria have developed a wide spectrum

of metabolic capabilities including the ability to utilize inorganic elements in the

production of energy (Brock et al., 1996; Nealson an d Stahl, 1997). Elements that

bacteria are known to oxidize or reduce in order to gain metabolic energy include H,

C, P, S, V, Mn, Fe, Co, Cu, As, Se, Br, Mo , Sn, Sb, Te, Hg, W and U (Woolfolk and

Whiteley, 19 62; Silverman and Ehrlich, 1964; Ehrlich, 1996b; Nealson and Stahl,

1997; Ehrlich, 1998). Furthermore, bacteria are known to concentrate extraordinarily

high amounts of many metals (see Table 1-I).

In the 19th century, Louis Pasteur demonstrated the existence of bacteria, and

their importance in disease and in agricultural fertility has been recognized for over

100 years. However, only in recent years has their significance for geological proc-

esses, such as rock weathering and secondary mineral formation, been recognized

(Ehrlich, 1996b). The particular importance of micro-organisms in processes relevant

to mineral exploration lies in their ability to promote mineral dissolution and

Fig. 12-1. Schematic diagram showing the integrated approach used in exploration geo-

microbiology to link geomicrobial and biogeochemical processes (in grey) with areas of re-

search and the requirements of the minerals industry.

395Biogeochemistry in Mineral Exploration

diagenesis as well as control major and trace metal mobilization, transport and

precipitation that may lead to the formation of secondary mineralization and

anomalies in and around mineralized zones (Ehrlich, 1998). Thus, it is increasingly

recognized that many mineral transport and transformation processes, previously

considered to be purely chemically driven, are in fact controlled by microbes

(Ehrlich, 1998).

Micro-organisms are able to alter the composition and structure of many

minerals, among them metal sulphides, silicates and carbonates (Garcia-Valle

s et al.,

2000). Iron- and sulphur-oxidizing bacteria and archaea mediate the direct oxidative

breakdown of sulphides, and thus contribute directly to the dispersion of metals

associated with these minerals. Several hundred different species of iron- and

sulphur-oxidizing bacteria and archaea are known and have been shown to promote

the breakdown of numerous economically important metal sulphides such as pyrite,

bornite, covellite, arsenopyrite, gallium sulphide, stibnite, cinnabar, cobalt sulphide,

galena, millerite and sphalerite. Thus, the mineral processing industry uses iron- and

sulphur-oxidizing bacteria in industrial bio-leaching processes to assist in the

extraction of metals from sulphide ores (Krebs et al., 1997).

Other bacteria, archaea and fungi promote rock weathering and trace metal

mobilization by excreting metabolites that corrode minerals through chemical inter-

action (Ehrlich, 1998; Sterflinger, 2000). The compounds that these micro-organisms

excrete include inorganic acids (such as HNO

or H

), and organic acids such as

acetic-, fumaric-, gluconic-, formic-, oxalic-, citric-, succinic-, malaic-, pyruvic- or

amino acids and complex molecules such as siderophores (Ehrlich, 1996a; Sterflinger,

2000). Many of these extracellular organic molecules readily form complexes with

free metal ions, and thus control their speciation and mobility in soils. These

microbial processes are particularly important in the rhizosphere, where they are stim-

ulated by plants excreting organic compounds as root exudates (Curl and Truelove,

1986; Khan, 2005). Root exudates directly increase metal mobilization, and provide

nutrition for rhizosphere micro-organisms which, due to their then higher metabolic

activity, further increase the turnover of metals. These microbe-plant interactions may

result in increased mobilization of trace metals in the rhizosphere, which may lead to

increased uptake and accumulation of trace metals by plants (Khan, 2005).

Micro-organisms have also been shown to promote the formation of minerals

at moderate temperatures (o501C) and atmospheric pressure that previously wer e

thought to form only at high temperatures and pressures (Ehrlich, 1998). In par-

ticular, carbonate- and silica minerals are precipitated by bacteria, archaea, fungi and

algae under a wide range of biogeochemical conditions (Castanier et al., 1999a,b).

Biogenic sulphides, which react with metal cations to form sulphide minerals, are

formed anaerobically by sulphate-reducing bacteria (Brock et al., 1996). Bacteria and

fungi also promote the formation of metallic minerals at their cell surface by binding

metal cations to negatively charged groups of the cell wall or cell envelope (Ehrlich,

1998); through this process the authigenic formation of secondary Au grains in soil has

been attributed to microbial processes, as shown in Case Study 1 (Reith et al., 2006).

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Exploration Geomicrobiology – the New Frontier

METHODS FOR IDENTIFYING MICRO-ORGANISMS AND MICROBIAL

PROCESSES

The task of developing a mechanistic understanding of element dispersion and

re-concentration in soils, which display complex inorganic and organic matrices and

distinct abiotic and biotic phases, is challenging. Understanding the biotic phases

in these systems is particularly demanding, because one gram of soil may contain several

thousand different microbial species (Paul and Clark, 1996). To assess the diversity of

these complex microbial populations as well as their function in soil, numerous methods

have been developed (Fig. 12-2). A synopsis with respect to their usefulness for explo-

ration geomicrobiology is given in the following section, and for an in depth look into

techniques introduced in this section the reader is referred to references given in the text.

Generally, methods to study microbial diversity are grouped into culture-

dependent and culture-independent techniques (Barns and Nierzwicki-Bauer, 1997).

Culture-dependent techniques

Referred to as classical methods, these techniques use artiﬁcial nutrient-rich growth

media to enrich and isolate micro-organisms from soil samples (Barns and Nierzwicki-

Bauer, 1997). They require the inoculation of a solid or liquid growth medium with a

small quantity (0.1–10 g) of the soil sample, and incubation under a variety of con-

ditions such as different pH, temperature, composition of gases, nutrient contents or

metabolic inhibitors (Brock et al., 1996). Once micro-organisms are growing in the

enrichment culture, the aim is obtain pure cultures of individual species for a com-

prehensive analysis of their biochemical, physiological and genetic characteristics.

Using selective media and conditions, a number of cell-counting methods (e.g., most-

probable-number counts and counting of colony-forming-units) have been developed

to assess the cell numbers of particular micro-organisms in soil samples (Atlas, 1984).

Community-level physiological proﬁling (CLPP) is a culture-dependent technique

commonly used to assess the diversity and function of complex microbial commu-

nities. This technique is based on the fact that differing bacterial and fungal pop-

ulations have differing abilities to utilize particular carbon sources such as sugars,

amines, organic acids, amino acids and complex organic molecules. CLPP can thus be

used to link bacterial and fungal diversity with their function in soils (Garland and

Mills, 1991; Garland, 1996a,b; Garland, 1997).

All culture-dependent methods share one main bias: they rely on having to culture

the organisms in a growth medium in vitro. Depending on the type of soil sample only

0.001 to 10% of the total microbial species contained therein can be successfully cul-

tured using existing culturing techniques (Alexander, 1977). This is a reﬂection of the

difﬁculty in accurately reproducing conditions of the natural micro-environments that

the organisms require, with the result that less than one percent of the total number of

bacterial species estimated to exist in the environment have been cultured and described

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Biogeochemistry in Mineral Exploration

to date (Amann, 1995; Macalady and Banﬁeld, 2003). In spite of this bias, the use of the

classical culture-dependent methods has led to the isolation and characterization of

thousands of micro-organisms from natural environments, and classical methods,

especially in combination with molecular techniques, will continue to be crucial for

Fig. 12-2. Molecular approaches for detection and identiﬁcation micro-organisms involved in

cycling of heavy metals and their catabolic genes from environmental samples (adapted from

Widada et al., 2002).

398 Exploration Geomicrobiology – the New Frontier

exploration geomicrobiology, because they allow comprehensive analyses of bio-

chemical processes and pathways in species important for geomicrobial processes.

Culture-independent methods

These methods for characterizing microbial populations in soil samples have been

developed in recent years. They are based on the analyses of cellular components such

as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or phospholipid fatty acid

(PLFA) (Olsen et al., 1986; Pace et al., 1986; Barns and Nierzwicki-Bauer, 1997). These

cellular components are extracted directly from the cells in the soils without prior

cultivation. The main advantage of molecular methods is that they do not rely on

culturing the organisms, and therefore provide a more accurate insight into the in situ

composition and activity of microbial communities in soil samples. Figure 12-2 shows

how they are used in combination to obtain a detailed representation of the phylo-

genetic (i.e., evolutionary) and functional relationships in microbial communities.

Phylogenetic methods aim to identify key-organisms, community structures and

genetic relationships (similar to DNA ﬁngerprinting used in criminal forensics) and are

based on the ampliﬁcation, ﬁngerprinting, sequencing and analyses of ribosomal DNA

and RNA (Barns and Nierzwicki-Bauer, 1997). Methods assessing the functional

aspects of microbial communities target genes that encode proteins/enzymes respon-

sible for key biogeochemical transformations. This can involve measuring the presence

(DNA-based) and expression (RNA-based) of the particular functional gene, e.g., the

genes responsible for Fe and S oxidation or reduction or metal resistance (Torsvik and

Øvrea

s, 2002; Widada et al., 2002). These methods can be summarized as follows:



DNA-based molecular methods allow detection of the presence or absence of a

particular gene in a soil sample, and thus establish if a microbial community is

capable of catalyzing a certain reaction (Barns and Nierzwicki-Bauer, 1997).



RNA-based methods are used to study the expression of a gene by the microbiota

in the sample, and establish if the function is utilized by the microbial community

at the time of sampling (Barns and Nierzwicki-Bauer, 1997).

MOLECULAR PROCEDURES

The steps required for the molecular procedures are as follows.

Extraction and puriﬁcation of nucleic acids from soils

The ﬁrst and most important step for any DNA- or RNA-based techn ique is the

isolation and clean-up of the nucleic acids (DNA or RNA) from the soil samples,

because all methods used in the following steps rely on the quality and the qua ntity

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Biogeochemistry in Mineral Exploration

of extracted nucleic acids. The ideal procedure for recovering nucleic acids from

soil samples was summarized by Hurt et al. (2001) and should meet the following

criteria:



Nucleic acid recovery efﬁciency should be high and not biased so that the nucleic

acids extracted are repres entative of the nucleic acids of the naturally occurring

microbial community.



Extracted RNA and DNA fragments should be as large as possible so that

molecular studies such as community gene libraries or gene expression analyses can

be carried out.



Extracted RNA and DNA should be sufﬁciently pure for reliable enzyme diges-

tion, hybridization, reverse transcription and polymerase chain reaction (PCR)

ampliﬁcation.



RNA and DNA should be extracted simultaneously from the same sample so that

direct comparative analyses can be performed.



Extraction and puriﬁcation protocols should be as simple as possible so that the

extractions are rapid and inexpensive.



Extraction protocols should be robust, reliable and generate reproducible results

from a variety of soil samples.

Most of the commercially available DNA or RNA extraction kits have been

designed to fulﬁll these criteria. However, preliminary experiments to test extraction

efﬁciency and reproducibility are recommen ded when applying commercial kits

to new groups of soil samples, because organic matter and clay have been shown to

significantly inﬂuence their pe rformance.

AMPLIFICATION OF TARGET GENES FROM EXTRACTED DNA OR RNA

Target genes have to be ampliﬁed, because their concentrations in soil samples are

too low to visualize on an electrophoresis gel without prior ampliﬁcation (Giraffa

and Neviani, 2001; Widada et al., 2002). For DNA-based methods the target gene

is ampliﬁed using the PCR. In the process of PCR-ampliﬁcation, the target DNA is

doubled during each of the 20–60 cycles, so that by the end of a PCR run several

millions of copies of a particular area of DNA have been produced (Fig. 12-3).

To assess if a PCR-ampliﬁcation was successful the ﬁnal product is visualized on

an agarose electrophoresis gel (Fig. 12-4). By using agarose electrophoresis gels it is

possible to separate DNA fragments based on molecular sizes (i.e., number of bases).

A different approach is used for RNA-based method s, because RNA has a different

molecular structure (Brock et al., 1998). Thus, the ﬁrst step of most RNA-based

techniques is the conversion of the RNA molecule to its DNA analogue using reverse

transcription polymerase chain reaction (RT-PCR) (Widada et al., 2002). To specif-

ically amplify only the target DNA or RNA of interest, primers speciﬁc to the genes

are used in the PCR reactions (Figs. 12-2 and 12-3). Primers are short sequences of

400

Exploration Geomicrobiology – the New Frontier

DNA that bind to the ends of extracted DNA and are the starting point from which

PCR-ampliﬁcation of a gene commences (Amann et al., 1995). Primers have been

developed to assess ribosomal or functional genes of individual species, phylogenetic

groups and whole domains; thus it is possible to assess the presence and activity of

Fig. 12-3. Scheme showing the selective ampliﬁcation of target DNA using polymerase chain

reaction (PCR).

Fig. 12-4. Agarose electrophoresis gel of the 510 bp (base pair) PCR-product from the

primer pair 27-GC and 534R amplifying 16S rDNA from Au grains obtained from the Hit or

Miss Gold Mine. Lanes marked with M contain a 100 bp ladder, fragment sizes (1500, 1000,

900, 800, 700, 600, 500, 400, 300, 200, 100). Lanes 1–19 contain PCR-product of the am-

pliﬁcation of bacterial DNA associated with the Au grains. E. coli DNA was used as positive

(+) control during ampliﬁcation, sterile MilliQ water was used as negative (–) control.

401Biogeochemistry in Mineral Exploration