Dunn Colin E. Biogeochemistry in Mineral Exploration

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individual species, groups or domains such as bacteria, archaea or eukaryotes in a soil

sample (Amann et al., 1995; Barns and Nierzwicki-Bauer, 1997).

Recently, an accurate quantitative analysis has been developed of the target DNA

from community DNA and RNA (so-called real-time PCR). This technique allows

quantifying how many copies of a particular gene are present in the soil sample

(Widada et al., 2002). These results can be correlated to the total amount of organ-

isms that express this gene in a sample and the rates of biogeochemical processes in

the soil samples. This makes real-time PCR a very powerful method to establish a

mechanistic link between the genetic composition of a microbial community and the

microbially mediated geochemical processes in soil materials that are relevant to

exploration geochemistry (Widada et al., 2002).

Assessing genetic diversity

To assess genetic diversity and identify key-species of microbial communities in

environmental samples, DNA libraries of unknown specie s are created (Fig. 12-2;

Barns and Nierzwicki-Bauer, 1997; Giraffa and Neviani, 2001). The DNA sequences

in these libraries can then be compared to other sequences deposited in global

sequence databases, which are an increa singly accurate and fast way of identiﬁcation,

similar to the way that human DNA samples can be used to identify individuals in

criminal forensics.

Genetic ﬁngerprinting

A number of techniques have been developed to obtain genetic ﬁngerprints of

microbial communities in soil samples, and to relate these structures to environmental

conditions of different samples or treatments. The most commonly used techniques,

shown in Fig. 12-2, are as follows:



DGGE – denaturing gradient gel electrophoresis



TGGE – thermal gradient gel electrophoresis



SSCP – single strand conformation polymorphism



T-RFLP – terminal restriction fragment length polymorphism.

Initially, these methods were used to assess the phylogenetic relationships and

genetic diversity of microbial communities based on 16S rDNA or 18S rDNA, but

now they are also used to assess microbial communities based on functional genes

(Giraffa and Neviani, 2001; Widada et al., 2002).

DGGE and TGGE are methods by which similar sized fragments of PCR-

ampliﬁed DNA can be resolved electrophoretically by their nucleotide composition

(Giraffa and Neviani, 2001; Widada et al., 2002). The double-stranded DNA

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Exploration Geomicrobiology – the New Frontier

molecules have different melting behaviour and will stop at different pos itions

along the gel, as shown in Fig. 12-5. The emerging banding pattern is stained

to allow visualization and then analysed for similarity using cluster analysis

(Fig. 12-6) or non-metric multidimensional scaling (nMDS, Powell et al., 2003).

Thus, PCR-DGGE and -TGGE methods provide an ecological insight into the

structure of microbial communities in environmental samples, and may also be

used to identify key organisms (see Case Study 1) whi ch can be cloned, sequenced

and subsequently identiﬁed.

SSCP also detects sequ ence variations between different ampliﬁed target DNA

fragments.

T-RLFP is a method to study mixed populations in soil samples that is based on

restriction enzyme digestion of ﬂuorescently marked PCR products.

Phospholipid fatty acid (PLFA) analysis is an additional ﬁngerprinting technique,

based on using cell constituents other than DNA or RNA ( Barns and Nierzwicki-

Bauer, 1997). PLFA are fatty acids present in the lipid bi-layer membranes of

living micro-organisms that are unique for speciﬁc groups of micro-organi sms.

Thus, PLFA analyses can be used to assess the diversity of a microbial community

and to evaluate changes in microbial community structures as a result of changing

biogeochemical conditions.

CASE STUDIES

Three case studies are presented to demo nstrate how classical and molecular tools

enhance the understanding of micro-organisms and microbially mediated processes

associated with the turnover of trace metals in soils and deeper soil materials, and

how this increased understanding may lead to the following:

(1)

The prediction of mineral transport and transformation pathways and the

location of secondary mineralized zones in the soil.

(2)

The development of biosensors for masked mineralization.

Case Study 1 – The geomicrobiological cycling of gold

This study assessed the ability of natural soil microbiota to mediate the

solubilization, transport and precipitation of Au in the Australian regolith, and is

taken from Reith et al. (2005), Reith and McPhail (2006), Reith et al. (2006) and

Reith and McPhail (2007).

Whereas it is now well established that micro-organisms play a key in role in the

cycling of major and trace elements in the environment, current evidence for the role

of micro-organisms in the biogeochemical cycling of Au is at best equivocal (Mossman

et al., 1999). Laboratory experiments using pure cultures of common bacteria such as

Bacillus subtilis or Bacillus megaterium have shown that these organisms can solubilize

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Biogeochemistry in Mineral Exploration

Fig. 12-5. DGGE (denaturing gradient gel electrophoresis) patterns obtained after amplifying the V3 region of the 16S ribosomal DNA

extracted from Au grains and surrounding soils collected at the Tomakin Park- (T) and the Hit or Miss (P) gold mines. Bands designated

with acronyms were excised from the gels, re-ampliﬁed and sequenced (Reith et al., 2006).

404 Exploration Geomicrobiology – the New Frontier

Au (Korobushkina et al., 1983). The ability of some bacteria (Escherichia coli,

Pseudomonas aeruginosa)andfungi(Aspergillus niger, Penicillium chrysogenum)to

precipitate mobile Au complexes and colloids in vitro has also been demonstrated

(Karthikeyan and Beveridge, 2002; Nakajima, 2003). Furthermore, morphological

evidence for a microbially mediated formation of Au grains and nuggets in soils and

deeper soil materials has been presented (Mann, 1992; Keeling, 1993; Bischoff, 1994,

1997). Using biochemical tools, Levchenko et al. (2002) demonstrated that a Au (I/III)-

redox couple acts as a metal centre in the membrane-bound enzyme NADH-oxidase

utilized by the common bacterium Micrococcus luteus during the oxidation of methane.

Whereas this limited evidence suggested that microbial biogeochemical mechanisms

may exist for the solubilization and precipitation of Au, the relative importance of

these mechanisms in the regolith compared to abiotic processes and the organisms

Fig. 12-6. Molecular ﬁngerprinting analyses showing DGGE-patterns of 16S rDNA and

dendrogram of subsequent cluster analysis from auriferous (A, B) and non-auriferous (A100,

B100) soils from the Tomakin Park Gold Mine.

405Biogeochemistry in Mineral Exploration

driving these processes are not known. Thus, the aim of the research was to obtain

evidence for the presence and significance of a biologically mediated cycle of Au in the

regolith, and subsequently identify and quantify microbial processes that are important

in mediating the dispersion and concentration of Au.

Figure 12-7 shows, from the sequence of observations, that microbiota resident in

auriferous Australian soils and deeper regolith materials are capable of mediating a

geomicrobiological cycle of Au. The indigenous microbiota in biologically active soil

microcosms were able to solubilize up to 80 wt.% of the Au contained in these

materials during the ﬁrst 50 days of incubation, after which the solubilized Au was

re-adsorbed by mineral- and organic soil fractions. In contrast, no Au was solubilized

in sterilized microcosms incubated under otherwise identical conditions. Molecular

(PCR-DGGE, cloning and sequencing of 16s rDNA) and physiological proﬁling

(CLPP) of bacterial communities during the incubation of the microcosms combined

with amino acids analyses indicated that changes in the structure of the bacterial

community from carbohydrate- to amino acid-utilizing populations occurred concur-

rently with, and appear to be linked to, the observed solubilization and re-precipitation

of Au.

Fig. 12-7. Conceptual model linking the processes of Au solubilization, transport, precip-

itation and authigenic Au nugget formation to form a geo(micro)biological cycle for the

behaviour of Au in the regolith.

406 Exploration Geomicrobiology – the New Frontier

These results suggest the following model of Au solubilization and re-precipitation

in the soil microcosms (Fig. 12-8).



The bacterial community in the early stages of incubation apparently produced

surplus amino acids, which are known to solubilize native Au a nd stabilize it in

solution.



The bacterial community in the later stages of incubation utilized these ligands,

and as a result the Au complexes were destabilized and Au was re-precipitated to

the solid soil fractions.

Molecular proﬁling also allowed the differentiation of bacterial communities

from auriferous and adjacent non-auriferous soils (Fig. 12-6). These results in com-

bination with results of Bacillus cereus spore counts (which were up to 1000 times

higher in soils that displayed Au concentrations of 150–1000 ppb compared to soils

with background Au concentrations), and microcosms amended with dissolved

AuCl

–

, suggest that the presence of highly mobile Au in the regolith as observed

at many Australian sites may inﬂuence the composi tion of the resident microbiota.

The results indicate that a geomicrobial exploration technique may be developed,

where B. cereus spore counts are measured and used as a pre-screening method to

target areas useful for further sampling and complete geochemical analysis (Reith

et al., 2005).

Fig. 12-8. Conceptual model linking Au solubilization and re-precipitation in microcosm

experiments with auriferous soils to the results of microbial community structure analyses.

407Biogeochemistry in Mineral Exploration

Scanning electron microscopy (SEM) revealed bacterial pseudomorphs on

untreated secondary Au grains from two ﬁeld sites used in this study (Fig. 12-9).

The presence of active bacterial bioﬁlms on the surface of Au grains was conﬁrmed

using confocal stereo laser microscopy (CSLM) combined with nucleic acid staining.

Molecular proﬁling showed that unique, site-speciﬁc bacterial communities are

associated with these Au grains, which differed from those dominating the surrounding

soils (Figs. 12.4 and 12.5). 16S ribosomal DNA clones belonging to the genus Ralstonia

and bearing 99% similarity to Ralstonia metallidurans were present on all 16S rDNA

positive Au grains from both locations, but were not detected in the surrounding soils

(Fig. 12-5). The ability of R. metallidurans to actively accumulate Au from solution

was then successfully tested suggesting that R. metallidurans may contribute to the

formation of secondary Au grains and nuggets in the regolith.

These studi es have shown for the ﬁrst time that microbiota resident in Australian

regolith are capable of actively mediating a biogeochemical cycle of Au in the

environment, and that the micro bially induced turnover of Au in the regolith may be

rapid. Furthermore, there is evidence for a number of processes and organisms that

may be associated with this cycle. Future experiments will use molecular microarrays

to assess Au solubilizing and precipitating organisms, such as R. metallidurans,in

order to understand the speciﬁc biochemical mechanisms regulating Au solubilizat-

ion and precipitation. This may enable the development of speciﬁc gene probes for

genes associated with the biogeochemical cycle of Au. Novel biosen sor technology

(as shown in Case Study 3) in combination with molecular characterization of

Au-transforming organisms and associated biochemical processes will then have

the potential to facilitate the development of molecular biosensors speciﬁc to Au for

use in the ﬁeld.

Fig. 12-9. Secondary electron micrographs of surface features of secondary Au grains from

the Hit or Miss Gold Mine in the Palmer River Goldﬁelds in north eastern Queensland,

Australia. (a) ‘Bacterioform’ Au consisting of bacterial cell-like structures on the surface of the

grain (scale bar ¼ 5 mm) and (b) Initial stages of ‘bacterioform’ Au formation in a surface

depression of a Au grain (scale bar ¼ 20 mm).

408 Exploration Geomicrobiology – the New Frontier

Case Study 2 – Microbial diversity in soils and sediments contaminated with Zn

and Cu

Brim et al. (1999) examined bacterial communities of Zn-contaminated soils

from Belgium using a combination of molecular and classical methods. From an

exploration geomicrobiology perspective these results are interesting, because they

show that the composition of the resident bacterial community was dominantly

inﬂuenced by the concentration and speciation of Zn in these soils, as may be the

case in soils overlying buried mineralization. The site had been monitored for two

decades, and previous studies using cultivation-based techniques had shown that

Ralstonia eutropha-like strains had dominated in the microbial community in the

soil, i.e., in previous studies up to 40% of cultured cells belonged to the Ralstonia

group.

In this study using a colony-forming-units technique, 10

–10

–1

culturable soil

bacteria were counted in a number similar to results of previous studies. However, 23

of the isolates belonged to the Arthrobacter group of bacteria, but no R. eutropha-like

isolates were detected. Most of the isolates were Zn tolerant but only seven were

considered Zn resistant. Sequences obtained from 16S rDNA clones from a clone

library, established from the soil community DNA, were afﬁliated with a number of

different groups such as a- and b-proteobacteria and the Cytophaga- Flexibacter-

Bacteroides group. Molecular proﬁling, using TGGE, of ampliﬁed 16S rDN A from

isolates, soil clones and the extracted soil community DNA showed that isolates and

clones only represented a pa rt of the microbial population of these soils. Arthrobacter

was the dominant band when 16S rDNA had been ampliﬁed from DNA extracted

from the soil in a procedure with a bead -beating step, but was absent or faint when

soil DNA was extracted without bead beating. From these results, Brim et al. (1999)

concluded that the microbial community in previous studies was highly resistant to

Zn toxicity concentration but was replaced with a less resistant bacterial microﬂora.

They suggest ed that Zn toxicity levels had been reduced because of the activity of

the Zn resistant R. eutropha-like bacteria that had led to the formation of insoluble

Zn carbonates. The results also showed that molecular techniques such as TGGE

in combination with classical techniques are powerful tools to assess microbial

community structures, as they allow monitoring of the temporal and spatial changes

of microbial communities and may facilitate the identiﬁcation of key organisms, i.e.,

R. eutropha- and Arthrobacter-like strains.

In another study published by Konstantinidis et al. (2003) Ralstonia-and

Arthrobacter-like strains were the only Cu resistant isolates cultivated from lake

sediments contaminated with 200–5500 ppm Cu. T-RFLP showed that a numb er of

OTUs (operational taxonomic units), among them Ralstonia sp., were present uni-

versally the entire width of the sedim ent. A number of other studi es also demon-

strated the presence Ralstonia sp. and Arthrobacter sp. in samples with elevated

concentrations of heavy metals and may as such be tried as bio-indicator organisms

in geomicrobial mineral exploration.

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Biogeochemistry in Mineral Exploration

Case Study 3 – Bacterial biosensors as alternatives for measuring heavy metals

in soil extracts

This study tested the applicability of metal-speciﬁc whol e-cell bacterial sensors for

the analysis of arsenite and Hg in soil extracts from polluted soils by comparing them

to resul ts from ICP-AES analyses. The luminescence-based bacterial sensor strain

Pseudomonas ﬂuorescens OS8 (pTPT11) was used for Hg detection and Pseudomonas

ﬂuorescens OS8 (pTPT31) for arsenite detection. Petanen and Romantschuk (2002)

spiked three different soil types (humus, mineral and clay) with 1100 or 500 mgg

–1

(ppm) of dissolved Hg(II) or As(III). They took samples after 1, 14 and 30 days

of incubation and extracted with water, ammonium acetate, hydrogen peroxide

and nitric acid to represent water soluble, bioavailable, organic matter-bound and

residual fractions, respectively.

Concentration results with chemical and biosensor analysis were similar in the

case of Hg-spiked samples. However, the lowest Hg concentration measured in the

soil extracts using the bacterial sensor was 0.003 mgkg

–1

(ppb) which was consid-

erably lower than by the chemical method 0.05 mgkg

–1

(ppb). The sensor strain with

pTPT31 for arsenite had a useful detection range similar to that of chemical methods.

Thus, they demonstrated that the bacterial sensors were sufﬁciently sensitive to

measure concentrations of As and Hg that are relevant to mineral exploration

in their soil extracts. Recently, other authors found similar low-detection limits in

biosensors for Cu, Co, Zn, Pb and Cd (Tibazarwa et al., 2001; Rensing and Maier,

2003).

FUTURE TECHNOLOGIES FOR BIO-PROSPECTING

There are several emerging molecular techniques that have the potential to

become the basis of future bio-prospecting technologies – these are nucleic acid

probes, molecular micro-arrays, biosensors and immuno-assays. These techniques

will provide the platform on which bio-prospecting test kits will be developed for the

analyses of soil samples in the ﬁeld. It is expected that within the next ten years ﬁeld

test kits will be developed that permit analyses of up to several hundred samples

per day. Use of these methods to supplement traditional chemi cal methods will have

a number of advantages for mineral exploration in that, apart from providing

an additional layer of information, they are rapid, inexpensive, simple to perform,

portable, highly sensitive and selective and thereby allow rapid assessment of

potential mineralization in the ﬁeld. These techniques will not replace traditional

chemical analysis techniques but will provide an additional level of information and

they represent an additional tool in the mineral explorationist’s tool box. Molecular

techniques will add value to existing techniques, most importantly ‘green ﬁeld’

exploration where they can narrow down the ﬁnal drilling target in relatively cheap

and rapid ways.

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Exploration Geomicrobiology – the New Frontier

The iden tiﬁcation of bio-indicator organisms or genes present in elevated

numbers in soil samples with anomalous trace metals concentrations may lead to the

development of speciﬁc nuclei c acid probes for these bio-indicators that can be used

directly for bio-prospecting. Nucleic acid probes are complementary to signature

sequences of functional or ribosomal DNA or RNA of particular species or groups

of organisms and are ﬂuorescently- or isotopically labelled allowing for their detec-

tion at very low concentration (Barns and Nierzwicki-Bauer, 1997). Nucleic acid

probes can bind to bulk community DNA or RNA bound in ﬁlter membranes

making it possibl e to quantify the amoun t and activity of particular bio-indicator

organisms (Barns and Nierzwi cki-Bauer, 1997).

Up to several hundred thousan d nucleic acid probe s can be used together as a

molecular micro-array, as shown in Fig. 12-10 (Widada et a l., 2002; Zhou and

Thomps on, 2002;, Zhou (2003) ; Bae and Park, in press). Molecular micro-arrays,

which all ow researchers t o study comple x microbial communities, comprise one

of the new tools in m olecular m icrobiology. Micro-arrays are powerful tools

for detection of multiple genes from soil samples, g iving a vast amount of i n-

formation relating to the phylogenetic and/or functional structure of microbial

ecosystems.

Expression micro-arrays are used to assess gene expression from microbial

cultures or environm ental samples. This type of micro-array provides information

relating to the activity of the microbe(s), i.e., ‘listening-in’ to the message provided by

the microbes. Furthermore, key genes associated with environmental attributes can

be identiﬁed. For example, genes can be identiﬁed that are important when a species

is subjected to elevated concentrations of heavy meta ls. Identiﬁcation of genes

that facilitate heavy metal transformations may then lead to the development of

micro-arrays with speciﬁc probes for these genes. These can then be applied as

exploration tools after RNA extraction from soil samples.

Heavy metal speciﬁc bacterial sensors provide another promising bio-prospecting

tool, allowing the measurement of mobile heavy metals in soil samples to ppb levels

(Tibazarwa et al., 2001; Rensing and Maier, 2003). This technique, which is high-

lighted in Case Study 3, was initially developed for ecotoxicological testing of heavy

metal polluted sites in Europe and the US to distinguish between mobile and

bioavailable, and non-mobile and non-bioavailable fractions of heavy metals in soils

and sediments (Rensi ng and Maier, 2003). Metal speciﬁc bacterial sensors have been

developed for a number of metals and metalloids such as As, Cu, Ni, Co, Zn, Pb, Hg

and Cd, and could be tested as exploration tools in bio-prospecting (Rensing and

Maier, 2003). Such biosensors are able to distinguish between mobile transported

heavy metals and in-situ bac kground concentrations much better than by wet chem-

ical methods (Rensing and Maier, 2003). This is likely to be of speciﬁc interest to

geochemical exploration.

Antibody-based or immuno-assays offer an alternative approach for metal ion

detection in natural samples. An immuno-assay makes use of the binding between an

antigen and its homologous antibody in order to quantify the speciﬁc antigen or

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Biogeochemistry in Mineral Exploration