U.S. patent application number 10/301537 was filed with the patent office on 2003-06-26 for microbial enrichment using a container having a plurality of solid support particles.
This patent application is currently assigned to DIVERSA CORPORATION. Invention is credited to Keller, Martin, Mathur, Eric J., Rusterholtz, Karl, Stein, Jeffrey L..
Application Number | 20030119171 10/301537 |
Document ID | / |
Family ID | 25440980 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119171 |
Kind Code |
A1 |
Mathur, Eric J. ; et
al. |
June 26, 2003 |
Microbial enrichment using a container having a plurality of solid
support particles
Abstract
The present invention relates to a method for collecting and
concentrating microbes from complex or dilute microbial populations
in aqueous or terrestrial environments. The present invention also
relates to a method for collecting and concentrating microbes using
monomers of polymeric substances conjugated to surface
materials.
Inventors: |
Mathur, Eric J.; (Carlsbad,
CA) ; Stein, Jeffrey L.; (San Diego, CA) ;
Keller, Martin; (San Diego, CA) ; Rusterholtz,
Karl; (Clackamas, OR) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
DIVERSA CORPORATION
|
Family ID: |
25440980 |
Appl. No.: |
10/301537 |
Filed: |
November 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10301537 |
Nov 20, 2002 |
|
|
|
08918793 |
Aug 26, 1997 |
|
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Current U.S.
Class: |
435/243 ;
435/34 |
Current CPC
Class: |
C12Q 1/24 20130101; G01N
2001/2833 20130101 |
Class at
Publication: |
435/243 ;
435/34 |
International
Class: |
C12Q 001/04; C12N
001/00 |
Claims
What is claimed is:
1. A device for collecting a population of microorganisms from an
environmental sample comprising a solid support having a surface
for attaching a selectable microbial enrichment media.
2. The device of claim 1, wherein the selectable microbial
enrichment media comprises a microbial attractant.
3. The device of claim 2, wherein the microbial attractant is
selected from the group consisting of glucosamine, cellulose,
pentanoic or other acids, xylan, lignin, chitin, alkanes,
aromatics, chloroorganics, sulphonyls and heavy metals.
4. The device of claim 1, wherein the selectable microbial
enrichment media comprises a growth inhibitor for eukaryotic
organisms.
5. The device of claim 4, wherein a growth inhibitor specific for
eukaryotic organisms is selected from the group consisting of
nystatin, cycloheximide and pimaricin.
6. The device of claim 1, wherein the selectable microbial
enrichment media comprises a growth inhibitor for prokaryotic
organisms.
7. The device of claim 6, wherein a growth inhibitor specific for
prokaryotic organisms is selected from the group consisting of
polymyxin, penicillin and rifampin.
8. The device of claim 1, wherein the solid support is selected
from the group consisting of glass beads, silica aerogels, agarose,
Sepharose, Safeties, nitrocellulose, polyethylene, dextran, nylon,
natural and modified cellulose, polyacrylamide, polystyrene,
polypropylene, and microporous polyvinylidene difluoride
membrane.
9. The device of claim 1, wherein the population of microorganisms
is a population of uncultivated microorganisms.
10. The device of claim 9, wherein the uncultivated microorganisms
comprise a mixture of terrestrial microorganisms, a mixture of
marine microorganisms, or a mixture of terrestrial microorganisms
and marine microorganisms.
11. The device of claim 9, wherein the uncultivated microorganisms
are extremophiles.
12. The device of claim 11, wherein the extremophiles are selected
from the group consisting of thermophiles, hyperthermophiles,
psychrophiles, halophiles, acidophiles, barophiles and
psychrotrophs.
13. A method for isolating a population of microorganisms from an
environmental sample comprising: a) contacting the sample with a
device having a solid support and a surface for attaching a
selectable microbial enrichment media; and b) isolating the
population from the device.
14. The method of claim 13, wherein the selectable microbial
enrichment media comprises a microbial attractant.
15. The method of claim 14, wherein the microbial attractant is
selected from the group consisting of glucosamine, cellulose,
pentanoic or other acids, xylan, lignin, chitin, alkanes,
aromatics, chloroorganics, sulphonyls and heavy metals.
16. The method of claim 13, wherein the selectable microbial
enrichment media comprises a growth inhibitor for eukaryotic
organisms.
17. The method of claim 16, wherein a growth inhibitor specific for
eukaryotic organisms is selected from the group consisting of
nystatin, cycloheximide and pimaricin.
18. The method of claim 13, wherein the selectable microbial
enrichment media comprises a growth inhibitor for prokaryotic
organisms.
19. The method of claim 18, wherein a growth inhibitor specific for
prokaryotic organisms is selected from the group consisting of
polymyxin, penicillin and rifampin.
20. The method of claim 13, wherein the solid support is selected
from the group consisting of glass beads, silica aerogels, agarose,
sepharose, safeties, nitrocellulose, polyethylene, dextran, nylon,
natural and modified cellulose, polyacrylamide, polystyrene,
polypropylene, and microporous polyvinylidene difluoride
membrane.
21. The method of claim 13, wherein the population of
microorganisms is a population of uncultivated microorganisms.
22. The method of claim 21, wherein the uncultivated microorganisms
comprise a mixture of terrestrial microorganisms, a mixture of
marine microorganisms, or a mixture of terrestrial microorganisms
and marine microorganisms.
23. The method of claim 21, wherein the uncultivated microorganisms
are extremophiles.
24. The method of claim 23, wherein the extremophiles are selected
from the group consisting of thermophiles, hyperthermophiles,
psychrophiles, halophiles, acidophiles, barophiles and
psychrotrophs.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for capturing
samples for evaluation. More particularly, the present invention
relates to an approach which allows the collection and
concentration of microbes, possessing genes encoding specific
enzymes or small molecule pathways, from complex or dilute
microbial populations in aqueous or terrestrial environments.
BACKGROUND OF THE INVENTION
[0002] There is a critical need in the chemical industry for
efficient catalysts for the practical synthesis of optically pure
materials; enzymes can provide the optimal solution. All classes of
molecules and compounds that are utilized in both established and
emerging chemical, pharmaceutical, textile, food and feed,
detergent markets must meet stringent economical and environmental
standards. The synthesis of polymers, pharmaceuticals, natural
products and agrochemicals is often hampered by expensive processes
which produce harmful byproducts and which suffer from low
enantioselectivity. Enzymes have a number of remarkable advantages
which can overcome these problems in catalysis: they act on single
functional groups, they distinguish between similar functional
groups on a single molecule, and they distinguish between
enantiomers. Moreover, they are biodegradable and function at very
low mole fractions in reaction mixtures. Because of their chemo-,
regio- and stereospecificity, enzymes present a unique opportunity
to optimally achieve desired selective transformations. These are
often extremely difficult to duplicate chemically, especially in
single-step reactions. The elimination of the need for protection
groups, selectivity, the ability to carry out multi-step
transformations in a single reaction vessel, along with the
concomitant reduction in environmental burden, has led to the
increased demand for enzymes in chemical and pharmaceutical
industries. Enzyme-based processes have been gradually replacing
many conventional chemical-based methods. A current limitation to
more widespread industrial use is primarily due to the relatively
small number of commercially available enzymes. Only .about.300
enzymes (excluding DNA modifying enzymes) are at present
commercially available from the >3000 non DNA-modifying enzyme
activities thus far described.
[0003] The use of enzymes for technological applications also may
require performance under demanding industrial conditions. This
includes activities in environments or on substrates for which the
currently known arsenal of enzymes was not evolutionarily selected.
Enzymes have evolved by selective pressure to perform very specific
biological functions within the milieu of a living organism, under
conditions of mild temperature, pH and salt concentration. For the
most part, the non-DNA modifying enzyme activities thus far
described have been isolated from mesophilic organisms, which
represent a very small fraction of the available phylogenetic
diversity. The dynamic field of biocatalysis takes on a new
dimension with the help of enzymes isolated from microorganisms
that thrive in extreme environments. Such enzymes must function at
temperatures above 100.degree. C. in terrestrial hot springs and
deep sea thermal vents, at temperatures below 0.degree. C. in
arctic waters, in the saturated salt environment of the Dead Sea,
at pH values around 0 in coal deposits and geothermal sulfur-rich
springs, or at pH values greater than 11 in sewage sludge. Enzymes
obtained from these extremophilic organisms open a new field in
biocatalysis.
[0004] For example, several esterases and lipases cloned and
expressed from extremophilic organisms are remarkably robust,
showing high activity throughout a wide range of temperatures and
pHs. The fingerprints of five of these esterases show a diverse
substrate spectrum, in addition to differences in the optimum
reaction temperature. As seen in FIG. 1, esterase 5 (EST5)
recognizes only short chain substrates while esterase 2 (EST2) only
acts on long chain substrates in addition to a significant
difference in the optimal reaction temperature. These results
suggest that more diverse enzymes fulfilling the need for new
biocatalysts can be found by screening biodiversity.
[0005] Furthermore, virtually all of the enzymes known so far have
come from cultured organisms, mostly bacteria and more recently
archaea. Traditional enzyme discovery programs rely solely on
cultured microorganisms for their screening programs and are thus
only accessing a small fraction of natural diversity. Several
recent studies have estimated that only a small percentage,
conservatively less than 1%, of organisms present in the natural
environment have been cultured (see Table I). Amann et al.,
Microbiol. Rev. 59:143 (1995); Barnes et al., Proc. Natl. Acad.
Sci. 91:1609 (1994); Torvisk et al., Appl. Environm. Microbiol.
56:782 (1990). Hence, this vast majority of microorganisms
represents an untapped resource for the discovery of novel
biocatalysts.
[0006] Within the last decade there has also been a dramatic
increase in the need for bioactive compounds with novel activities.
This demand has arisen largely from changes in worldwide
demographics coupled with the clear and increasing trend in the
number of pathogenic organisms that are resistant to currently
available antibiotics. For example, while there has been a surge in
demand for antibacterial drugs in emerging nations with young
populations, countries with aging populations, such as the US,
require a growing repertoire of drugs against cancer, diabetes,
arthritis and other debilitating conditions. The death rate from
infectious diseases has increased 58% between 1980 and 1992 and it
has been estimated that the emergence of antibiotic resistant
microbes has added in excess of $30 billion annually to the cost of
health care in the US alone. As a response to this trend
pharmaceutical companies have significantly increased their
screening of microbial diversity for compounds with unique
activities or specificities.
[0007] There are several common sources of lead compounds (drug
candidates), including natural product collections, synthetic
chemical collections, and synthetic combinatorial chemical
libraries, such as nucleotides, peptides, or other polymeric
molecules. Each of these sources has advantages and disadvantages.
The success of programs to screen these candidates depends largely
on the number of compounds entering the programs, and
pharmaceutical companies have to date screened hundred of thousands
of synthetic and natural compounds in search of lead compounds.
Unfortunately, the ratio of novel to previously-discovered
compounds has diminished with time. The discovery rate of novel
lead compounds has not kept pace with demand despite the best
efforts of pharmaceutical companies. There exists a strong need for
accessing new sources of potential drug candidates.
[0008] The majority of bioactive compounds currently in use are
derived from soil microorganisms. Many microbes inhabiting soils
and other complex ecological communities produce a variety of
compounds that increase their ability to survive and proliferate.
These compounds are generally thought to be nonessential for growth
of the organism and are synthesized with the aid of genes involved
in intermediary metabolism hence their name--"secondary
metabolites". Secondary metabolites that influence the growth or
survival of other organisms are known as "bioactive" compounds and
serve as key components of the chemical defense arsenal of both
micro- and macroorganisms. Humans have exploited these compounds
for use as antibiotics, antiinfectives and other bioactive
compounds with activity against a broad range of prokaryotic and
eukaryotic pathogens. Approximately 6,000 bioactive compounds of
microbial origin have been characterized, with more than 60%
produced by the gram positive soil bacteria of the genus
Streptomyces. Of these, at least 70 are currently used for
biomedical and agricultural applications. The largest class of
bioactive compounds, the polyketides, include a broad range of
antibiotics, immunosuppressants and anticancer agents which
together account for sales of over $5 billion per year.
[0009] Despite the seemingly large number of available bioactive
compounds, it is clear that one of the greatest challenges facing
modern biomedical science is the proliferation of antibiotic
resistant pathogens. Because of their short generation time and
ability to readily exchange genetic information, pathogenic
microbes have rapidly evolved and disseminated resistance
mechanisms against virtually all classes of antibiotic compounds.
For example, there are virulent strains of the human pathogens
Staphylococcus and Streptococcus that can now be treated with but a
single antibiotic, vancomycin, and resistance to this compound will
require only the transfer of a single gene, vanA, from resistant
Enterococcus species for this to occur. When this crucial need for
novel antibacterial compounds is superimposed on the growing demand
for enzyme inhibitors, immunosuppressants and anti-cancer agents it
becomes readily apparent why pharmaceutical companies have stepped
up their screening of microbial diversity for bioactive compounds
with novel properties. There is still tremendous biodiversity that
remains untapped as the source of lead compounds.
[0010] The present invention provides a path to access biodiversity
for a variety of purposes, including the use in the eventual
discovery of novel bioactivities.
SUMMARY OF THE INVENTION
[0011] The present invention provides a means for selectively
attracting microbes to specific substrates chemically conjugated to
a solid surface. The invention further provides for the enrichment
of these microbes. This approach allows for the concentration and
collection of microbes, possessing genes encoding specific enzymes
or small molecule pathways, from complex or dilute microbial
populations in aqueous or terrestrial environments. The basis for
the attraction and subsequent enrichment is that microbes possess
specific receptors that signal chemotactic attraction towards
specific substrates. By binding the substrate to a surface and
subsequently incubating the substrate-surface conjugate in the
presence of a mixed microbial population, specific members of that
population can be collected.
[0012] It is an object of the present invention to provide a means
for selectively enriching for specific microorganisms from the
surrounding environmental matrix. In accomplishing these and other
objects, there has been provided, in accordance with one aspect of
the present invention, a device for collecting a population of
microorganisms from an environmental sample comprising a solid
support having a surface for attaching a selectable microbial
enrichment media.
[0013] In one aspect of the invention, microbial enrichment media
containing a microbial attractant is used to selectively lure
members of the environmental community to the device. In another
aspect of the invention, bioactive compounds which inhibit the
growth of unwanted organisms is included in the microbial
enrichment media to further enhance selection of desirable
microorganisms.
[0014] In yet another aspect of the invention, a method for
isolating microorganisms from an environmental sample comprising
contacting the sample with a device having a solid support and a
surface for attaching a selectable microbial enrichment media and
isolating the population from the device is provided.
[0015] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates the substrate spectrum fingerprints and
optimum reaction temperatures of five of novel esterases showing
the diversity in these enzymes. EST# indicates the different
enzyme; the temperatures indicate the optimal growth temperatures
for the organisms from which the esterases were isolated; "E"
indicates the relative activity of each esterase enzyme on each of
the given substrates indicated (Hepanoate being the reference).
[0017] FIG. 2 illustrates the capture of microbes from marine and
soil habitats as detailed in the present invention. These photos
demonstrate the difference in the types of microbes collected from
a soil environment when utilizing two different types of substrates
(cellulose and xylan). These photos also demonstrate the difference
in employing beads alone versus beads with substrate attached
(chitin).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] The present invention provides a device for the isolation
and containment of microorganisms and a method for acquiring in
situ enrichments of uncultivated microorganisms. The enrichment
process can increase the likelihood of recovering rare species and
previously uncultivated members of a microbial population.
[0019] In situ enrichment can be achieved in the present invention
by using a microbial containment device consisting of growth
substrates and nutritional amendments with the intent to
selectively lure members of the surrounding environmental matrix.
Choice of substrates (carbon sources) and nutritional amendments
(i.e., nitrogen, phosphorous, etc.) is dependent upon the members
of the community for which one desires to enrich. The exact
composition depends upon which members of the community one desires
to enrich and which members of the community one desires to
inhibit. These containment devices are then deployed in desired
biotopes for a period of time to allow attraction and growth of
desirable microbes.
[0020] Substrates of the invention can include monomers and
polymers. Monomers of substrates, such as glucosamine, cellulose,
pentanoic or other acids, xylan, chitin, etc., can be utilized for
attraction of certain types of microbes. Using monomers allows one
to depend on attraction for the collecting, versus the presence of
substrate receptors on cells. This could provide the added benefit
of allowing one to acquire more biodiversity. Polymers can also be
used to attract microbes that can degrade them.
[0021] Specific microbes of interest can be captured and
concentrated from dilute populations in aqueous environments
thereby obviating the need to concentrate microorganisms from large
volumes of water. These devices can also be implanted in soil
environments to enrich microbes from terrestrial habitats.
Substrates such as cellulose or chitin can be attached to the
surface material to attract specific classes of microbes, such as
the actinomyces, which are a rich source of secondary
metabolites.
[0022] Utilizing the present invention, in situ enrichment can be
readily achieved. FIG. 2 demonstrates the capture of microbes from
different habitats, as detailed in the present invention. These
photos demonstrate the difference in the types of microbes
collected from a soil environment when utilizing two different
types of substrates (cellulose and xylan). These photos also
demonstrate the difference in employing beads alone versus beads
with substrate attached (chitin).
[0023] In a preferred embodiment, the invention relates to a
microbial containment device for collecting a population of
microorganisms from an environmental sample comprising a solid
support having a surface for attaching a selectable microbial
enrichment media.
[0024] In another preferred embodiment of the invention, a method
for isolating microorganisms from an environmental sample
comprising contacting the sample with a device having a solid
support and a surface for attaching a selectable microbial
enrichment media and isolating the population from the device is
provided.
[0025] "Selective microbial enrichment media", as used herein, is
any medium containing elements which enhance the growth of certain
organisms and/or inhibit the growth of other organisms present in
the surrounding environment. The media of the present invention is
useful when the organism targeted for enrichment is present in
relatively small numbers compared to other organisms growing in the
surrounding matrix. For example, a selective microbial enrichment
media containing the antibiotics colistin and nalidixic acid will
inhibit the growth of gram-negative bacteria but not the growth of
gram-positives. The selectivity of the microbial enrichment media
can be further enhanced by the addition of a specific substrate
such as, for example, cellulose, to the colistin and nalidixic acid
containing media. Therefore, a microbial containment device
incorporating the aforementioned microbial enrichment media will be
selective for gram-positive organisms which are capable of
utilizing cellulose as an energy source.
[0026] The term "solid support", as used herein, is any structure
which provides a supporting surface for the attachment of a
selectable microbial enrichment media. Well known solid supports
that may be used for screening assays of the invention include, but
are not restricted to, glass beads, silica aerogels, agarose,
Sepharose, Sephadex, nitrocellulose, polyethylene, dextran, nylon,
natural and modified cellulose, polyacrylamide, polystyrene,
polypropylene, and microporous polyvinylidene difluoride membrane.
It is understood that any material which allows for the attachment
and support of a selectable media is included in the present
invention. By using large surface area materials, such as, for
example, glass beads or silica aerogels, a high concentration of
microbes can be collected in a relatively small device holding
multiple collections of substrate-surface conjugates.
[0027] In one aspect of the invention, substrates are conjugated to
solid surfaces prior to deployment into the environment of choice.
Such conjugation is preferably a chemical conjugation. Large
surface area materials, such as glass beads or silica aerogels are
preferably utilized as surfaces in the present invention. It is
anticipated that there are a variety of surface area materials that
could be utilized effectively in the present invention. Conjugation
or immobilization of substrates to the surface material may occur
via a variety of methods apparent to the skilled artisan. One
example of derivitization of glass beads is described in an Example
provided below. It is anticipated that any of a variety of
conjugation or immobilization strategies can be employed to
immobilize substrates to surfaces in the present invention.
[0028] Derivitized surface area materials, such as glass beads or
silica aerogels, of the present invention are contained in separate
device(s) before placement into the environment of interest.
Preferably, such containment devices are of the type which allow
migration of microbes in while simultaneously containing the
derivitized materials. For example, particularly preferred
containers are mesh filters, such as those available from Spectrum
in Houston, Tex., which have been manipulated to contain the
derivitized materials. For example, filters can be cut into
squares, derivitized materials can be placed in the center, the
filter can be folded in half and the three sides can be glued shut
to create a containment device. Mesh filters, or the like, can then
be placed in any device to be used as a solid support which will
contain the mesh filter for deployment into the environment.
Particularly preferred devices are made of inert materials, such as
plexiglass.
[0029] Alternatively, any device which allows migration of microbes
while simultaneously containing the materials can be employed with
the present invention. For example, Falcon tubes (VWR, Fisher
Scientific) or the like may be employed to contain the derivitized
materials directly. Said tubes can be punctured utilizing a sharp
instrument to yield a device which allows microbe migration into or
out of the device.
[0030] The anchored component of the selectable enrichment medium
can be immobilized by non-covalent or covalent attachments.
Non-covalent attachment can be accomplished by coating a solid
surface with a solution of, for example, a protein which is
specifically recognized by a receptor displayed on the cell
membrane of a target organism. Alternatively, an immobilized
antibody, preferably a monoclonal antibody, specific for the
protein to be immobilized can be used to anchor the protein to the
solid surface. The surfaces can be prepared in advance and
stored.
[0031] In another aspect, the present invention relates to a method
of selective in situ enrichment of bacterial and archaeal
microorganisms utilizing a microbial attractant attached to a solid
surface. A "microbial attractant", as used herein, is defined as
any composition which selectively precipitates or induces the
migration of microorganisms to a device containing a microbial
enrichment media. A microbial attractant is further defined as any
composition which selectively augments the survival of a
microorganism which contacts a microbial enrichment media contained
in a device of the present invention. For example, microorganisms
routinely display chemotactic responses to environmental stimuli
perceived as energy sources, such as a carbon source. Any
particular carbon source can be utilized by some members of the
community and not others. Carbon source selection thus depends upon
the members of the community one desires to enrich. For example,
members of the Streptomycetales tend to utilize complex, polymeric
substrates such as cellulose, chitin, and lignin. These complex
subtrates, while utilized by other genera, are recalcitrant to most
bacteria.
[0032] In another aspect, the use of additional nitrogen sources
may be called for depending upon the choice for carbon source. For
example, while chitin is balanced in its C:N ratio, cellulose is
not. To enhance utilization of cellulose (or other carbon-rich
substrates), it is often useful to add nitrogen sources such as
nitrate or ammonia. Further, the addition of trace elements may
enhance growth of some members of a community while inhibiting
others.
[0033] In another aspect of the invention, compounds useful as
growth inhibitors of eukaryotic organisms can be included in the
device of the present invention. Growth inhibitors of eukaryotic
organisms include any compound which selectively prevents the
growth of eukaryotic organisms. Such inhibitors can include, for
example, one or more commercially available compounds such as
nystatin, cycloheximide, and/or pimaricin or other antifungal
compounds. These compounds may be sprinkled as a powder or
incorporated as a liquid in the selectable microbial enrichment
medium. It is anticipated that other selective agents can be
employed to inhibit the growth of undesired species or promote the
growth of desired species. For example, obtaining bacterial and
archaeal species can be complicated by the presence of eukaryotic
organisms which can out-compete desired bacterial species for the
available substrate. Therefore, including selective agents, such as
antifungal agents or other eukaryotic growth inhibitors, in the
device of the present invention promotes the growth of target
microorganisms.
[0034] In yet another aspect, compounds which inhibit the growth of
some bacterial species, but not others, may be incorporated into
the enrichment medium. Growth inhibitors for prokaryotic organisms
include any compound which prevents the proliferation of
prokaryotic cells. Such compounds include, but are not limited to,
polymyxin, penicillin, and rifampin. Use of the compounds is
dependent upon which members of the bacterial community one desires
to enrich. For example, while a majority of the Streptomyces are
sensitive to polymyxin, penicillin, and rifampin, these may be used
to enrich for "rare" members of the family which are resistant.
Selective agents may also be used in enrichments for archaeal
members of the community.
[0035] In the context of the present invention, a containment
device containing a microbial enrichment medium can incorporate,
for example, a complex carbon source as an attractant, nystatin as
an inhibitor of eukaryotic organisms and rifampin as an inhibitor
of selected prokaryotic organisms. It is understood that
attractants, eukaryotic inhibitors and prokaryotic inhibitors can
be used individually, or in any combination, as a component of a
selectable microbial enrichment medium of the present invention. It
is further understood that a device of the present invention can
include any appropriate solid support in combination with any
microbial enrichment medium suitable for an environmental matrix or
for the isolation of microorganisms of interest. An environmental
matrix can include a marine environment, a terrestrial environment
or a combination of marine and terrestrial environment. Moreover,
an environmental matrix can include those organisms which exist in
surroundings which are neither solid nor liquid, such as those
organisms which remain airborne. The device of the present
invention can be used to filter such organisms from the atmosphere
or any other gaseous environment. It is further envisioned that a
containment device of the present invention can be used for the
isolation of microorganisms from non-terrestrial environments, such
as those existing on planets other than earth. For example, a
containment device containing a microbial enrichment medium
designed to attract microorganisms which can exist on the planet
Mars is included in the present invention. Such a device would
incorporate features designed to attract microorganisms capable of
existing in an environmental matrix not substantially different
from those which are currently encountered on earth. Further, a
sufficient amount of data concerning environmental conditions on
planets other than earth is available such that a containment
device of the present invention can be designed to incorporate
elements specific to those environments.
[0036] In another aspect, the present invention can be employed to
isolate and identify microorganisms useful in bioremediation.
Bioremediation is a process which utilizes microorganisms to remove
or detoxify toxic or unwanted chemicals from an environment. The
device of the present invention can be modified to contain a medium
which selectively enriches for those organisms capable of attaching
to, or detoxifying, toxic or unwanted chemicals. For example,
halogenated organic compounds have had widespread use as
fungicides, herbicides, insecticides, algaecides, plasticizers,
solvents, hydraulic fluids, refrigerants and intermediates for
chemical syntheses. As a result, they constitute one of the largest
groups of environmental pollutants. Chloroorganic compounds
comprise the largest fraction of these materials, having been
synthesized by large scale processes over the past few decades.
Their ubiquitous use and distribution in our ecosystem has raised
concern over their possible effects on public health and the
environment. Therefore, a need exists for the identification of
microorganisms which are capable of removing these, and other,
chemicals from the environment. The inclusion, for example, of
chlorinated organic compounds in a selectable enrichment medium of
the present invention can aid the isolation of organisms attracted
to such a compound. Once identified, the organism can be used as a
natural and inexpensive means of detoxifying environments known to
contain such pollutants.
[0037] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLE 1
Surface Derivitization--N-Acetyl
B-d-glucosamine-phenylisothiocyanate (activated substrate) onto
Glass Beads
[0038] Glass beads can be derivitized with N-Acetyl
B-d-glucosamine-phenylisothiocyanate as follows:
[0039] Bead Preparation:
[0040] 1. Mix:
[0041] 30 ml glass beads (Biospec Products, Bartlesville,
Okla.)
[0042] 50 ml APS/Toluene (10%) (APS--Aminopropyltriethoxysilane)
(Aldrich)
[0043] 2. Reflux overnight
[0044] 3. Decant and wash 3 times with Toluene
[0045] 4. Wash 3 times with ethanol and dry in oven
[0046] Derivitize with N-Acetyl
B-d-glucosamine-phenylisothiocyanate as follows:
[0047] 5. Combine in Falcon Tube:
[0048] 25 ml prepared glass beads from above
[0049] 15 ml 0.1M NaHCO.sub.3+25 mg N-Acetyl-B-d-glucosamine-PITC
(Sigma Chemical Co.)+1 ml DMSO
[0050] 6. Add 10 ml NaHCO.sub.3+1 ml DMSO.
[0051] 7. Pour over glass beads.
[0052] 8. Let shake in Falcon Tube overnight.
[0053] 9. Wash with 20 ml 0.1M NaHCO.sub.3.
[0054] 10. Wash with 50 ml ddH.sub.2O.
[0055] 11. Dry at 55.degree. C. for 1 hour.
EXAMPLE 2
Surface Derivitization--Birchwood Xylane (polymer) onto Sol Gel
[0056] Glass Mixture (slurry):
[0057] 1. Combine on ice:
[0058] 75 ml TMOS (Tetramethylorthosilicate--"Sol Gel")
(Aldrich)
[0059] 30 ml H2O
[0060] Slowly add 2 ml of 0.05M HCl
[0061] Slowly add while stiring 2 ml of 0.05M NaOH to increase
pH
[0062] 2. On stir block in container add:
[0063] 10 g of birchwood xylane (Fluka)
[0064] 200 ml of 10 mM phosphate buffer, pH 6.0
[0065] 3. Mix in 105 ml of glass mixture from above. Stir slowly to
solid (4-5 minutes).
[0066] 4. Incubate @ 55.degree. C. overnight.
[0067] The same protocol can be used to derivitize beads with
commercially available chitin or cellulose.
EXAMPLE 3
Surface Derivitization--Xylane Monomer onto Glass Beads
[0068] 1. Mix:
[0069] 15 ml 0.1M NaHCO.sub.3
[0070] 25 mg B-d-xylopyranosylphenylisothiocyanate (Sigma
Chemical)
[0071] 2. Pour over 25 mls prepared beads from Example 1 above.
[0072] 3. Add 10 ml 0.1M NaHCO.sub.3
[0073] 4. Let sit overnight.
EXAMPLE 4
Microbial Containment Devices with Antibiotics
[0074] 1. Cut both ends off a 15 ml Falcon Tube (VWR).
[0075] 2. Cut Spectra/Mesh Nylon Filters (Spectrum, Houston, Tex.)
(mesh opening: 70 .mu.m; % open area: 43; thickness: 70 .mu.m) into
2 circles of approximately 2.5 cm in diameter.
[0076] 3. Cover one end of the Falcon Tube with the mesh and attach
using Goop, Household Adhesive & Sealant.
[0077] 4. Make up and filter sterilize the following Dilute Basal
Medium:
1 K.sub.2HPO.sub.4 1.74 g NaH.sub.2PO.sub.4 1.38 g
(NH.sub.4).sub.2SO.sub.4 0.5 g NgSO.sub.4.7H.sub.2O 0.2 g
CaCl.sub.2.2H.sub.2O 0.025 g KNO.sub.3 0.5 g water 1 liter
cellobiose 0.001%
[0078] 5. Moisten 1 sterile gauze pad (North 3".times.3", Fisher)
with 10 ml of Dilute Basal Medium (make moist, not dripping wet).
This is best handled by laying gauze on a sheet of parafilm, adding
medium drop-wise.
[0079] 6. Allow medium to soak through gauze.
[0080] 7. Sprinkle approximately 0.5 g of cycloheximide powder on
pad and spread around using a spatula.
[0081] 8. Sprinkle approximately 0.5 g Nystatin (yellow) on the
same pad in the same manner.
[0082] 9. Using tweezers, jam treated gauze pad into Falcon
tube.
[0083] 10. Seal other end of tube with other circle as done
previously.
[0084] 11. Allow to dry (approximately 2 hours).
[0085] 12. Deploy as desired.
EXAMPLE 5
Sample Collection Using a Microbial Containment Device
[0086] The following example describes a type of microbial
containment device which can be generated according to the present
invention to attract organisms. The following protocol details one
method for generating a simple microbial containment device:
[0087] 1. Puncture small holes using a heated needle or other
pointed device into a 15 ml Falcon Tube (VWR, Fisher
Scientific).
[0088] 2. Place approximately 1-5 mls of the derivitized beads from
Examples 1, 2 or 3 into a Spectra/mesh nylon filter, such as those
available from Spectrum (Houston, Tex.) with a mesh opening of 70
.mu.m, an open area of 43%, and a thickness of 70 .mu.m. Seal the
nylon filter to create a "bag" containing the beads, using, for
instance, Goop, Household Adhesive & Sealant.
[0089] 3. Place the filter containing the beads into the ventilated
Falcon Tube and deploy the tube into the desired biotope for a
period of time (typically days).
[0090] 4. Recover the microbial containment device and associated
organisms.
[0091] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
2 TABLE 1 Habitat Cultured (%) Seawater 0.001-0.1 Freshwater 0.25
Mesotrophic lake 0.01-1.0 Unpolluted esturine waters 0.1-3.0
Activated sludge 1.0-15.0 Sediments 0.25 Soil 0.3
* * * * *