Opinion
Exploring microbial diversity for
biotechnology: the way forward
Brajesh Kumar Singh1,2
1
Macaulay Land Use Research Institute, Aberdeen, AB15 8QH, UK
2
Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 3UU, UK
Environmental microbes are immensely diverse and
have numerous metabolic activities and products that
could have industrial applications. However, >99% of
environmental microbes cannot be cultured under cur-
rent laboratory conditions, leaving their potential largely
untapped. Metagenomic approaches have been used
successfully in recent years to obtain novel microbial
products from uncultured microorganisms. The activity,
efficiency and stability of these novel enzymes can be
further improved by the application of nanotechnology.
Here, I highlight the approaches that can be used to
obtain efficient microbial products from the uncultivable
majority. I propose that a multidisciplinary approach
combining different technologies including metage-
nomics and nanotechnology is the way forward for
tapping the real potential of microbial metabolism for
applications in biotechnology.
Introduction
Microbes are the most diverse and abundant group of
organisms on Earth, constituting 60% of the total biomass.
A current estimate suggests that, globally, the soil and
oceans consist of 4–5  1030 and 3.6  1029 microbial cells,
respectively [1–6]. Microbes are responsible for vital bio-
geochemical cycling without which life would not be
possible. Therefore, understanding the microbial com-
munity structure, diversity and function is essential to
understand fully the evolution and sustainability of life
on Earth [4,5]. As well as having a vital role in sustain-
ability, microbes are also a source of various industrial
products that have applications across all major indus-
tries. For example, microbial products are used as anti-
biotics, anti-tumour agents and immunosuppressants in
the pharmaceutical industries, and as biopesticides, anti-
parasite agents and food-processing agents in the agricul-
tural sector [7]. Similarly, microbial products are widely
used by the chemical industry for the production of amino
acids, vitamins, organic acids, detergents, bio-catalysts
and bioconversion agents, and by environmental indus-
tries for bio-remediation and the production of bioenergy
[3]. In fact, microbial products generate US$100 billions
within the industrial sector [7]. Table 1 lists some of the
most important industrial products of microbial origin.
Microbial enzymes present another field of application.
Biocatalysts of microbial origin have been used for several
decades and it is currently estimated that >500 commer-
cial products are being made using microbial enzymes.
Corresponding author: Singh, B.K. (b.singh@macaulay.ac.uk).
0167-7799/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2009.11.006 Available online 11 December 2009
The global market value for enzymes used in bio-catalysis
is estimated to be US$ 2.3 billion yr 1 [7]. The usage of
enzymes is distributed over various industries, including
food (45%), detergents (34%), textiles (11%), leather (3%)
and pulp and paper (1.2%). Several enzymes are also used
to prepare enantiomer-pure drugs from their racemic mix-
ture. Similarly, bio-conversion is becoming an essential
process in fine chemical industries, with a world-wide
market of U$55 billion, of which most lies within the
pharmaceutical (US$25 billion) and agro-chemical indus-
tries (US$10 billion). In the pharmaceutical industry,
microbial enzymes are not only used for production of
new drugs, but also as therapeutic agents. It is estimated
that, by 2010, 5% of all chemicals sold (US$160 billion) and
up to 60% of all fine chemicals will be produced using
methods that utilise microbes [7–9].
Current limitations of biotechnology
At present, there are increasing demands for new medicines
and food products that make use of environmentally friendly
technologies and that adopt sustainable approaches. This,
along with the economic and political pressure on industries
to be less dependent on politically unstable regimes for their
supplies, are driving the search for new biocatalysts for
maintaining a sustainable production. There is currently
a political initiative in the developed world to promote white
(industrial) biotechnology as a focal point for sustainable
economic development. Public support is gaining momen-
tum and it is believed by policy makers, analysts and
industrialists that white biotechnology will have a real
impact on industrial outputs on a global scale [3]. Microbial
enzymes could further revolutionise the sectors of white and
also red (pharmaceutical) biotechnologies, but it will require
considerable effort to overcome some of the practical pro-
blems associated with large-scale use of enzymes in bioca-
talysis and as therapeutic agents [10,11].
Three major challenges need to be addressed. First,
there is huge demand from both the white and red bio-
technological sectors for new enzymes and metabolites.
The diversity of desired products provides the basis from
which to select the most suitable enzymes for a particular
purpose [12]. At present, any industrial process has to be
designed within the constraints of available enzymatic
activities, leading to expensive systems with suboptimal
productivities [8]. It is commonly believed that the discov-
ery of more efficient enzymes will enhance productivity
and, thus, reduce the cost. Environmental microbes
are considered to be the main source of new enzymatic
111
 Opinion
Table 1. Selected products of industrial importance obtained
from environmental microbesa
Industrial products Examples
Industrial enzymes Amylase, lipase, protease
Organic acids Citric acid, lactic acid
Fine chemical Active ingredients of medicine
Antibiotics Streptomycin, ampicilin
Microbial insecticide Bacillus thuringiensis protein
Anti-parasite agents Avermectins
Vitamins Cyanocobalamin, riboflavin
Amino acids Glutamate, lysine
a
From Refs [3,7–9,11,14,19].
activities owing to their enormous metabolic capability and
diversity, much of which currently remains untapped.
Second, the large-scale availability of existing enzymes
is not always reliable and is often costly. In addition, their
low stability in fermenters is another limiting issue [11].
Enzyme engineering, for example with the aim to enable
activity in non-aqueous media, and protein engineering to
provide enzyme with altered structures, functions and
activities, would improve the efficiency in terms of cata-
lysis. This would improve stability, reduce the cost of the
processes and help to enhance production from non-pre-
ferential substrates.
Finally, the lack of mechanisms to protect enzymes
against protease attack occurring in biological systems is
another major hurdle to overcome to achieve optimal
activity. Proteases present in the native or added micro-
flora within a fermenter or, in a bioremediation context,
the enzymes present in environmental microbes, could
degrade exogenous enzymes and thus reduce their effi-
ciency. This is also true in the therapeutic use of microbial
proteins, which, as foreign proteins, might be destroyed by
blood proteases. Additionally, microbial proteins can also
illicit immunological responses when used for treatment
[10].
New source of microbial products
Soil, water and other environmental samples present a
vast reservoir of microbes and microbial products that
could be harnessed to revolutionise the productivity of
white and red biotechnologies. It has been estimated that
one gram of a pristine soil might contain up to 104 different
species, which potentially represent over one million open
reading frames encoding putative enzymes [8]. These
enzymes are potentially of enormous importance for indus-
trial processes. To date, most microbial products have been
obtained from microbes that have been isolated and
exploited in the laboratory. However, cultured microorgan-
isms only account for <1% of the total environmental
microbes and, thus, >99% of microbes are currently uncul-
tivable under laboratory conditions [2,13] and their poten-
tial for applications in industries remains untapped.
Recent advancements in molecular technologies offer
not only an opportunity to understand the fundamental
aspects of evolution and community formation, but also
provide an excellent opportunity to exploit the uncultiva-
ble microbes for biotechnological processes. A metage-
nomic approach is currently considered the most viable
method to search for these elusive enzymes and there has
already been some success with the use of several enzymes
112
Trends in Biotechnology Vol.28 No.3
discovered using metagenomics in the fine chemical and
pharmaceutical industries. For example, several nitrile
Microbial source hydratases, cellulases and lipases have been isolated using
Bacteria/fungi
metagenomics and are currently being marketed to the
Fungi
Bacteria fine chemical, pharmaceutical and detergent industries
Bacteria/ fungi [8,11,14].
Bacteria
Bacteria Metagenomics for novel products
Bacteria Metagenomics provides a novel way to extract valuable
Bacteria
products from environmental microbes without the need to
culture them in the laboratory (Box 1) [14–17]. The total
genetic material from all organisms present in an environ-
mental sample is obtained directly and transferred into
surrogate organisms to generate a metagenome clone
library [18]. Metagenomics provides two complimentary
approaches in the search for biological products; (i) mining
of the genetic information by sequencing and PCR; and (ii)
functional screening of clones (Figure 1). In the first
approach, when the metagenomic composition (i.e. the
genetic information of an environment) is known, the
search for a particular function or protein can be performed
by mining the metagenomic sequence data. Once putative
homologues are found, the exact sequence information can
Box 1. Metagenomics
Metagenomics involves the extraction of the total genetic material
from all organisms present in an environmental sample without the
need to culture them. The genetic material is then transferred into
surrogate organisms to generate a metagenome clone library
[14,15]. To obtain information about the diversity and community
structure of microbes, the metagenome library is then sequenced
[16,17]. To search for specific activities within the metagenome, the
surrogate organisms can be screened for particular enzymes, either
via DNA sequences or enzymatic functions, such as lipase, esterase,
anti-tumor agents and antibiotic production [7,8]. Based on current
estimates of the diversity of soil microbial communities, it has been
suggested that at least two million metagenomic clones will need to
be screened to ensure statistically that all of the initial genomes are
included in the metagenome library [18].
However, for functional screening, the number of clones can be
reduced by an initial enrichment step before metagenomic DNA
extraction that corresponds with a particular function. For example,
glycerol dehydrogenase could be isolated from a soil sample by
incubating the soil with glycerol before DNA extraction. This
increases the relative number of bacteria in the soil that exhibit
glycerol dehydrogenase activity, resulting in a higher ‘hit’ rate
during the later stages of the functional screening of clones [36]. The
number of clones in a functional screen can be further reduced by
applying the so-called ‘stable isotope probing’ (SIP) method. This
approach selectively enriches microbes utilising a specific substrate.
The DNA of enriched microbes can then be separated from other
microbes by using density centrifugation before generating a
metagenomic clone library [37].
The combined SIP–metagenomics approach has been used
successfully in isolating new genes and gene products [38–40]. For
example, a novel biphenyl-degrading gene was obtained and
expressed using the SIP–metagenomic approach [41]. However,
this approach can lead to the loss of some novel enzymes that are
only present in a few microbes, which can be overgrown by fast-
growing and more dominant bacteria. The success rate of finding
novel and desired enzymes and/or proteins can also be increased by
careful selection of environmental samples from which a metage-
nomic library is to be cloned [19,42]. For example, if the goal is to
find new enzymes for particular chemical pathways, such as
pesticides or specific pharmaceutical products, it is important to
choose collection sites that had been exposed to these compounds.
 Opinion Trends in Biotechnology Vol.28 No.3

Figure 1. Metagenomic library construction and following screening procedures. In situ enrichment technique, such as stable isotope probing (SIP), can be applied before
the cloning step to increase the hit rate for novel products. The total genetic material (i.e. DNA) obtained from environmental samples is then cloned into a vector and
transformed to a host. Screening for a particular function can be carried out by targeting a particular gene using PCR and/or by large-scale direct sequencing of clones and
any novel function found can be explored based on predicted protein structures. For functional screening, clones can directly be screened for particular activities.
Metagenomic libraries can also be used to examine the structure and functional diversity of the environmental community by sequencing DNA from clone libraries.

be obtained by PCR amplification and expression in sur-
rogate organisms. This approach has been successfully
used in the discovery of several new enzymes, including
chitinases, carboxypeptidases and lipases [3,11]. Nonethe-
less, this approach has a major drawback in that it depends
on the availability of homologous sequence data. There-
fore, it would fail to identify novel enzymes that have the
same function, but a different structure than known
enzymes.
The second approach, a functional screening of clones,
constitutes a function-based assay, in which surrogate
organisms are tested for a particular activity, such as
reactions catalysed by particular enzymes, or properties
attributed to a particular metabolite, for example anti-
biotics and anti-tumor agents. The major problem of this
approach is the logistics and the facilities required to
screen tens of thousands and up to millions of clones for
the desired functions. The simplest approach for functional
screening is a colour reaction, in which the enzyme of
choice converts a colour-less compound into a coloured
one, or vice versa, during host growth [10]. However, for
most enzymatic activities, colour assays are not available.
Despite these limitations, there have been some promising
advances made in function-based screening in metage-
nomics in past few years [19]. These include the so-called
‘substrate-induced gene expression’ (SIGEX) technology,
which selects clones with particular catabolic genes
induced by various substrates in concert with fluorescence
activated cell sorting (FACS) [20]. A further improvement
is the development of a laser-based high throughput
screening method, which claims to be able to screen 1
billion clones per day [3,21]. However, this approach needs
to be further tested and verified in various laboratories.
Thus, functional screening remains the main hurdle in
finding novel activities from metagenomics and, therefore,
more research and development is needed.
Moreover, creating, maintaining and sequencing meta-
genomic libraries is expensive and labour intensive and it
is often beyond the capabilities of one research group or
even one country to carry out metagenomic analysis from
samples taken from several environmental conditions.
Therefore, cross-country initiatives consisting of several
multi-disciplinary groups might be able to encompass the
necessary complimentary expertise in microbial genetics,
eco-physiology, bioinformatics, enzymology, chemistry,
structural biology and bioengineering, together with repre-
sentative end-users, such as industries and regulatory
agencies. Progress in this direction has recently been made
with the inauguration of a consortium of 52 international
teams, the so-called ‘TerraGenome’, which joined forces to
propose the screening and sequencing of two million clones
from one soil sample [22]. For continuing progress in
harnessing metagenomics for better biological products,
more such consortia will be needed to tackle samples from
various environmental systems and conditions.
Application of nanotechnology for improved
functionality
As well as the exploitation of environmental metagenomes,
the emerging field of nanotechnology also offers the poten-
tial for further advances in the use of microbial enzymes in
biotechnology (Box 2). Obtaining enzymes from uncultiva-
ble microbes is only a first step. Changes in enzyme proper-
ties and catalytic activities, probably via protein and
enzyme engineering, will be needed to achieve increased
efficiency and economic viability. However, as mentioned

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Opinion Trends in Biotechnology Vol.28 No.3

Box 2. Nanotechnology the enzyme, but can also increase the active enzyme con-
centration, protect against protease attack and minimise
Nanotechnology is the interdisciplinary study of the functional
system at atomic or molecular (nanometre) scales. Biotechnological solubility-related issues [25]. Furthermore, enzymes can
applications of nanotechnology include the entrapping and/or be immobilised on large nanostructured surface areas,
immobilising of microbial products (cells, proteins and enzymes) which enable them to be reused. This approach has been
in nanostructures with the aim of enhancing their activity and further improved with the development of the so-called
function [24–27]. Several nanostructures, such as nanoparticles,
nanofibres, mesoporous silicasol-gel and single enzyme nanoparti-
‘single enzyme nanoparticles’ (SENs), in which a porous
cles, have been tested successfully for enzyme immobilisation via organic/inorganic hybrid polymer network of <10 nm in
adsorption, entrapment and covalent bonding and further evaluated size surrounds each enzyme molecule [30,31]. The porosity
for their impacts on the catalytic activities of immobilised enzymes of the network enables easy transfer of a substrate while
[24,26,30]. Several review articles have covered these aspects covalent bonds between polymer and enzyme provide
[25–27] and, therefore, these are not discussed further here.
Nanomaterials range in size from 1 to 100 nm and exhibit unique
stability [25]. The so-called ‘ship-in-a-bottle’ approach is
magnetic, thermodynamic and catalytic properties [25,28]. These another means by which to improve the stability of
properties, along with their uniform size, enable nanostructures to enzymes in fermenters [25,32]. Here, the enzyme is first
improve enzyme properties, particularly in terms of enzyme stability stabilised onto nanoparticles and transferred into the
and activity [26]. Enzyme activity is increased because nanostruc-
‘bottleneck’ nanoporous media, where it is then cross-
tures provide a large surface area for improved enzyme loading.
This results in increased enzyme activity per unit mass or volume linked (Figure 2). The size of the resulting cross-linked
in comparison to conventional immobilisation approaches [25]. aggregated enzyme is greater than the ‘bottleneck’ pore
Covalent bonding or entrapment within the nanostructure provides size of the media and therefore, enzymes cannot leach out
enzyme stability under harsh bioreactor conditions. Thus, the from the nanoporous media. Moreover, it has been demon-
amount of enzyme required is reduced and the lifetime of enzymes
strated that this system is also resistant to protease
is prolonged, presenting the possibility of enzyme re-use [30].
The usefulness of nanotechnology has been successfully demon- activity as the nanoporous media prevents access to the
strated in several recent studies. For example, it was demonstrated enzyme aggregate by proteases (Figure 2).
in a 100-day experiment that an esterase enzyme–nanofibre Currently, however, there are only a few products in use
composite could be stable and functional in repeated batches and that have been manufactured using nanotechnology owing
continuous long-term operation modes [43]. Another advantage of
combining metagenomics and nanotechnology is based on the fact
to the limited understanding of the effects that nanopar-
that several novel metabolites of important industrial and medicinal ticles can have on biological systems. Despite a few large
values are produced by microbes, which live in a consortium. on-going initiatives, such as observatoryNano (http://
Reconstructing their environmental conditions in the laboratory is www.observatory-nano.eu/project/) and Engineered Nano-
currently impossible owing to a lack of understanding of the
particles: Review of Health and Environmental Safety
nutritional and behavioural aspects of these complex natural
systems. Similarly to cell encapsulation in gel by the micro-droplet (ENRHES; http://www.nano.org.uk), understanding of
technique [44], it might be possible to encapsulate single cells (i.e. the impacts that nanoparticles have on the environment
metagenomic clones) into nanostructures, thus enabling interspe- in general, and on human health in particular, remains to
cies chemical communication, which is required for the expression be advanced significantly before the full potential of nano-
of desired functions.
technology can be realised. In this context, it is important
that toxicological studies should be run in parallel with the
development of innovative nanotechnological products.
above, one of the main obstacles of using enzymes for
industrial or therapeutic purposes stems from their
reduced stability relating to solubility issues, mechanical
stress and protease attack in fermenters and also in bio-
logical systems [10,23]. Nanotechnology has emerged as a
promising tool with which to overcome these problems, as
it can provide enzyme stability and protection (Box 2;
[24–27]). It has been suggested that nanotechnology will
revolutionise diagnosis, drug delivery and tissue regener-
ation in health-care industries, as well as the field of
biocatalysis in the industrial sector [24,28]. Several drugs
with nanotechnology formulation are already in use,
such as Rapamune1, an immunosupressent (marketed
by Wyeth Pharmaceuticals; http://www.wyeth.com) and
Emend1, a medicine for emesis (introduced by Merck;
http://www.merck.com). Other drugs, such as Semapi-
mod1, an anti-cancer agent (produced by Cytokine Phar-
maSciences; http://www.cytokinepharmasciences.com/
index.shtml) and the antimicrobial Nucrystal1 (developed
Figure 2. The ‘ship-in-a-bottle’ approach for increasing enzyme stability. The
by Nucrystal Pharmaceuticals; http://www.nucryst.com) enzyme is first stabilised on nanoparticles (NP) by adsorption (grey circles) and
have been approved for clinical trials [28,29]. then transferred into a ‘bottleneck’ nanoporous media, where it is cross-linked
One major obstacle in any biocatalytic process is the using glutaraldehyde (GA). As the size of the resulting cross-linked enzyme
aggregate is larger than the pore size of the bottleneck (13 nm), the enzyme is
short catalytic life span of the enzyme. Nanotechnology can prevented from leaching out and is also protected against attack from proteases in
not only provide improved stability and a higher activity of the fermenter. Adapted with permission from Ref. [25].

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Opinion
The way forward
Recent progress in metagenomics and nanotechnology has
provided exciting new products for white and red biotech-
nology. However, the true potential of these approaches
can only be realized if they are integrated along with
protein and enzyme engineering in future research efforts.
Novel and potentially more efficient enzymes obtained
from metagenomics are likely to require further modifi-
cation by protein or enzyme engineering to obtain
increased efficiency and stability in large-scale fermenters.
More importantly, formulations are needed that can
stabilise the enzyme and protect them from adverse con-
ditions, including those arising from washing, leaching,
solvents, water and protease attack. Emerging nanotech-
nological tools could provide such protection to novel
enzymes. It is therefore important that these approaches
are combined from the conceptual stage onwards. Such an
interdisciplinary approach, combining genomics (mainly
functional metagenomics), protein engineering and nano-
technology, might expedite the exploitation of metabolic
capabilities of environmental microbes, which in turn will
bring sustainable wealth creation, and efficient resource
management and other socio-economic benefits.
Further exploitation of environmental microbes is
anticipated to increase the production of renewable and
efficient fuels and of high-value bioproducts used in indus-
try and medical treatments [7,9,11]. There is a need for a
sustained metagenomic data archive comprising data col-
lected by different research groups. The CAMERA inven-
tory, which archives data from marine metagenomes, is a
good example [33]. However, it is desirable to create a
global inventory database that contains exclusively meta-
genomic data obtained from various environments and
that should be freely available to researchers and other
interested groups [6]. The best model of such an archive is
the Human Genome Project, where generated sequence
data are freely accessible.
The promise of metagenomics and nanotechnology can
not be realised without co-ordinated and well-funded
research and development. For this to happen, a strong
and sustained political support is essential. Policy guidance
on public funding with an emphasis on the social impacts
along with minimal disturbance to the environment will go a
long way to help on this count. To accomplish these goals
successfully, a strong legal framework is needed that would
clearly define the freedom and limitation under which
experiments can be carried out for creating new biological
and/ or nanotechnology based products. Currently, several
misconceptions regarding nanotechnology prevail among
the general public and these need to be tackled urgently.
A legal framework, along with the recognition that infor-
mation generated by these emerging technologies is for
common societal benefit, would enhance the public confi-
dence in this technology. Thus, making research data freely
available will go a long way in gaining approval and accept-
ance of stakeholders so that mistakes, such as those made by
GM industries, are not repeated. The public opposition to
GM crops was not driven by lack of scientific data or risk of
hazard but was due to the perception of lack of institutional
and cultural responsibilities associated with new inno-
vations; for details see reviews [34,35]. Therefore, public
Trends in Biotechnology Vol.28 No.3
involvement from the onset, together with openness and
availability of research data with regard to environmental,
social and health impacts of these emerging technologies
and enforceable regulations, would be essential for public
support and ultimately their successful application. If the
global research and industrial communities are able to
overcome the above challenges, then they are poised to
harness the enormous potential of uncultivable environ-
mental microbes for industrial applications.
Acknowledgements
I thank Catriona Macdonald, Emma Sproston and Philippa Booth for
useful comments on the article. My laboratory is funded by the Scottish
Government.
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