CA2098331C - Method for the isolation of degradative microbial consortia and apparatus therefore - Google Patents

Abstract

There is disclosed a method for isolating microorganisms which are capable of metabolizing a selected contaminant at a variety of concentrations. An apparatus in the form of an extended concentration gradient gel cell is disclosed for the purpose of sustaining the contaminant in various concentration levels on the gel. The microorganisms self colonize on the gel cell at a concentration suitable for metabolic activity of the microorganisms. This permits easy identification and isolation of microorganisms capable of metabolizing various concentrations of a contaminant.

Description

METHOD FOR THE ISOLATION OF DEGRADATIVE MICROBIAL
CONSORTIA AND APPARATUS THEREFOR
FIELD OF THE INVENTIO~1 The present invention relates to a method and apparatus for isolating degradative microorganisms on an extended gradient of contaminant concentrations.
BACKGROUND OF THE INVENTION
Increase in the amounts and variety of xenobiotic compounds introduced to the environment, together with recognition of the importance of microorganisms in the attenuation of these compounds, has stimulated microbiological research in the subsurface environment. Involvement of mixed microbial consortia in degradation processes has led to a change in the emphasis of biodegradation related research from the traditional approach of studying pure isolates to the study of naturally occurring, mixed microbial populations. In many cases, traditional techniques used to isolate and study degradative microorganisms also lack effective means to reproduce the natural micro-environment, especially gradients, such as nutrients and contaminants.
Current practice utilizes a series of batch reactors, or continuous culture systems each one representing a specific set of conditions. Accordingly, many tests would be required to detect degradative organisms and assess the effect of contaminant concentration and coexisting compounds on the growth and development of the degradative organisms.
In view of the presently employed protocols for achieving the isolation of degradative organisms there exists a need for an inexpensive diffusion gradient system whereby an infinite range of concentrations of a test compound is produced.
The gradient may be inoculated with microorganisms in environ mental samples.

SUMMARY OF THE INVENTION
In accordance with one aspect of the present invention there is provided an improved method for isolating degradative microorganism consortia. Such microbial consortia are useful, for example, in ground water clean-up, remediation schemes and wastewater treatment. The gel is also useful for bioreactor monitoring and laboratory screening procedures, an example of which is antimicrobial agent testing.
The method may employ a substrate such as a poly-acrylamide, agar gel, porous glass materials, porous plastic materials, etc., for support of the contaminant and more particularly the concentration gradient thereof.
Creation of an extended gradient whereby the placement of sources under the gel at intervals results in a linear gradient 3-5X as long as that achieved by conventional means using a single source. Unlike other gel stabilized systems, the gradient is sufficiently long for analyses, the time to steady state is known and the fluxes from the gels are determined. Thus the gel cell system, according to the present invention, is useful in isolating degradative consortia. In addition, the system can be modified to allow testing of multiple one dimensional gradients to examine to efficacy of nutrient additions to stimulate microbial growth/degradation in the same system.
In accordance with a further aspect of the present invention, there is provided a method of isolating microorganisms capable of degrading a selected contaminant from a sample containing the contaminant comprising the steps of: providing a substrate onto which the microorganisms may be isolated;
contacting the substrate with the contaminant such that a variety of concentrations of the contaminants are provided on the substrate: exposing the microorganisms to the substrate; and isolating microorganisms at the concentrations.

In another aspect of the present invention there is provided a method for selectively isolating microorganisms capable of metabolizing contaminants from a sample containing the contaminants, comprising the steps of: providing a gel substrate onto which the microorganisms may be isolated; contacting the gel substrate with the sample such that the contaminants diffuse in varying concentrations in the gel; inoculating the gel substrate with the microorganisms: and effecting isolation of the microorganisms at different concentrations of the varying concentrations on the gel substrate.
A further aspect of the present invention relates to a method of identifying specific microorganisms, from a sample containing microorganisms, for degrading a selected contaminant at a variety of concentrations comprising the steps of : providing a sample containing microorganisms to be identified; providing a substrate on which the microorganisms may be supported; varying the concentration level of the contaminant on the substrate;
exposing the microorganisms to the contaminant on the substrate:
and identifying microorganisms at each concentration level.
Apparatus to effect the method of the present invention includes a gel plate which effectively functions as substrate onto which the microorganisms may colonize at optimum concentrations so that metabolic activity of the microorganisms is maintained while the contaminant is metabolized.
According to a further aspect of one embodiment of the present invention, there is provided a cell for the isolation of degradative microorganisms from a sample containing the microorganisms, comprising: a body having a substrate therein adapted for supporting microorganisms; at least one first inlet means connected to the body for permitting exposure of the _ substrate to a source of sample fluid containing a contaminant;
second inlet means for admitting a purging fluid for purging excess sample fluid from the substrate; and outlet means for facilitating removal of the excess sample fluid.

According to yet another aspect of the present invention there is provided a cell for isolation of degradative microorganisms from a sample containing the microorganisms, the cell comprising: a body: well means for receiving and retaining a sample of a contaminant: the well means having inlet means for receiving the sample and outlet means; a substrate receiving zone above the well means and in communication therewith, the zone adapted to receive a microorganism supporting substrate: and means for passing a fluid over the substrate when the substrate is in the zone.
Advantageously, the cell is relatively small, lightweight and portable thus affording field use.
The cell conveniently provides a plurality of wells, in one embodiment, for the isolation of degradative microorganisms or testing of the response of microorganism communities to antimicrobial agents used in medicine, industry and agriculture.
Generally, many industries add biocides to the water supply employed for various processes, etc. to prevent microbial fouling of the water supply lines, cooling systems and other related equipment. Presently, it is difficult to determine an effective biocidal dose to alleviate these problems; the method and apparatus according to the present invention effectively satiate the above-mentioned existing limitations.
In addition to the aforementioned, the apparatus and method are effective for determining antimicrobial sensitivity.
In such an application, a gradient of the antimicrobial agent is formed in the gel, the fluid supply passed over the gel or microorganisms of interest and the inhibitory concentration, i.e., the zone at which no microbial growth occurs, is detected.
According to another attractive feature of the present invention, there is provided a method of testing the sensitivity of a microorganism to a selected test compound comprising the steps of: providing a substrate capable of sustaining the microorganism: contacting the substrate with the test compound to provide a range of concentrations of the test compound;

5 exposing the microorganism to the substrate containing the test compound: and detecting a concentration at which microorganism growth is inhibited.
It will be readily appreciated that auxiliary analysis means l0 may be employed with the apparatus. As an example, optical scanning means may be employed to scan the gel surface. The readings obtained could be used to determine the response pattern of the microorganisms to the gradient of the chemical compound.
Further, direct microscopic observation of microbial growth or lack thereof may be possible by placing a coverslip on the cell.
Numerous other means of auxiliary analysis means may be employed and will be readily apparent to those skilled in the art.
Having thus generally described the invention, refer-ence will now be made to the accompanying drawings illustrating preferred embodiments.
ERIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a perspective view of one embodiment of the present invention;
Figure 2 is a longitudinal sectional view of the cell illustrated in Figure l;
Figure 3 is a sectional view along line 3-3 of Figure 1:
Figure 4 is a top plan view of the cell;
Figure 5 is an end elevational view of the cell of Figure 1;

2098~~1 Figure 6 is a view similar to Figure 2 with parts removed and illustrating schematically the disposition of the elements in the cell:
Figure 7 is a graphical representation of the concen-tration gradients of diclofop and fluorescein as measured in a single well diffusion gradient plate. The reservoirs contained 14 mg/1 diclofop and 50 mg/1 fluorescein, respectively;
Figure 8 is a graphical representation of the diffusion of fluorescein through a.1.5% agarose gel. Concentrations were measured and compared with calculated values:
Figure 9 is a representation of the concentration gradient developed in 1.5% agarose gel, determined with the numerical model. The profile shown here developed over 30 hours and was exactly the same as profiles for 60 and 90 hours, respectively, indicating that steady-state conditions were reached within 5 hours. Concentration values are in log format.
The water-gel interface is at a height of 0.2 cm:
Figure 10 is a 3 dimensional representation of microbial responses to a diclofop gradient after two days.
Enumeration was done with an epifluorescent microscope, following a grid pattern with 2 mm intervals across the length and width of the gel surface. X-axis represents the distance from the reservoir: Y-axis represents numbers of cells per microscope f field:
Figure 11 is a schematic representation to illustrate the thickness of a biofilm developed on the surface of the gel, determined with the scanning confocal laser microscope:
Figure 12 is a graphical representation of a calculated concentration gradient (log values) developed in the multi well gradient plate, determined with the numerical model. The water-gel interface is at a height 0.2 cm:

Figure 13 is a graphical representation of the development of a fluorescein gradient in a multi well gradient plate with similar dimensions as the numerical model illustrated in Figure 12, showing resemblance to the calculated gradient; and Figure 14 is a graphical representation of microbial growth in response to a diclofop concentration gradient in the multi well plate, determined with the scanning confocal laser microscope. Biomass is expressed as percentage of total surface area covered by cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTB
Referring initially to Figure 1, shown is a perspective view of one embodiment of the gel cell, generally denoted by numeral 10 . The cell 10 provides a bottom wall 12 , a pair of spaced apart side walls 14 and 16, end walls 18 and 20 and top wall 22 having a opening 24.
A coverslip 25, is shown exploded from the top 22 of cell 10, but, when in position, the same is sealed in place to enclose the gel and thus permit observation.
A fluid pathway is established. in the cell 10 by an inlet 26 in end wall 18, shown more clearly in Figure 2, an outlet 28 in end wall 20 and opening 24 in top wall 22, the latter extending between inlet 26 and outlet 28. The fluid is preferably sterile water and the purpose will be described hereinafter.
Opening 24 in top wall 22 includes a recessed area 30.
This is illustrated in Figure 2. The recessed area 30 includes a plurality of individual sections 32 recessed from, but in communication with area 30. In the example, four individual sections are illustrated, although it will be appreciated that more or less may be included depending on the specific application.

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Each section 32 includes an inlet 34 and outlet 36 in fluid communication with the respective section 32. Each inlet 34 and outlet 36 extend through side wall 14 and 16, respec-tively, as illustrated in phantom in Figures 4 and 5.
In the preparation of the cell 10, the sections 32, which receive the compound to be tested, are initially filled with cold water. Subsequently, liquid or molten gel is poured into recessed area 30. Figure 6 illustrates a sectional view of the cell 10 with the materials therein. The coverslip 25 is positioned on opening 24 (Figure 1). The individual sections 32 are then refilled with the test compound 42 with the concentra-tion gradually decreasing in the direction from end wall 20 to end wall 18. Within each section 32, the concentration is maintained by constant introduction of fresh solution using inlets 34 and outlets 36 (not shown in Figure 6).
The sterile water, indicated generally by numeral 44, referred to hereinabove, is passed over the surface of the gel 40, occupying recess 30, at a given rate. The rate will be determined based on the number of section 32 and the minimum rate required to maintain a nutrient free environment above the gel surface. The flow is continued until steady state conditions prevail (discussed hereinafter).
The microorganisms (not shown) are introduced into the system and specifically on the gel through the fluid inlet 26.
The flow of the water 44 is in opposition to the direction of the concentration gradient between ends 20 and 18.
This additionally assists in maintaining a nutrient free environment on gel 40.
The sterile water may be pumped using suitable pumps of the continuous or peristaltic variety. In addition, the test compound may be introduced into inlet 34 by suitable hosing or other fluid transportation means.

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Turning now to greater detail of the method aspect of the present invention, a gel cell l0.consisting of 1.5% agarose gel was used. A constant flow of sterile water over the surface of the gel, against the direction of the gradient, removed the test compound diffusing from the gel. Concentration gradients were determined for diclofop and fluorescein. Gradients were allowed to develop over a period of five days. Plugs (1 mm diameter) were then removed from the diclofop gel, weighed, and left in acidified, sterile water for 24 hours to allow diffusion of diclofop into the water for analyses. The acidified samples were extracted with diethyl ether, concentrated, methylated, and solvent switched to hexane before injection into a VARIANTM 3500 capillary gas chromatograph (GC-ECD). The concentration gradient of fluorescein was measured directly with a BIORADTM MRC - 600 scanning confocal laser microscope, 10 ~m below the surface, at various points along the length of the gel.
Diclofop and fluorescein gradients developed as illustrated in Figure 7. The slopes of both gradients were steep resulting in a rapid decrease in concentration with distance from the source. These results suggested that microbial responses to the test compounds would be restricted to a relatively thin band close to the source.
A numerical transport model, *FracTran, (Ground Water Simulations Group - University of Waterloo) was used to assess the accuracy of the diffusion gradients measured in the physical model shown in Figure 7. Fluorescein, which can be analyzed with greater ease and accuracy, was used as a biologically conservative tracer for predicting diclofop.gradients. This decision was based on the small difference in molecular weights of diclofop and fluorescein (341 and 376,.respectively), as well as the similarity in the concentration gradients, developed by these two compounds (Figure 7). The,diffusion coefficient for fluorescein was determined by using glass plates, 250 mm long and 120 mm wide and containing a 5 mm thick agarose gel (1.5%) , which was placed with one end in a 0.5 g/1 fluorescein solution. The *Trade-mark '~Q9833i ,,~.~..
gel was removed from the solution after 47 days and one glass plate carefully removed. Plugs were then removed at 10 mm intervals along the length of the gel. Each plug was weighed and placed in a dibasic sodium phosphate solution (NaZHP047H20) for 5 24 hours (in the dark), followed by measurement of fluorescence, using a TURNER DESIGNT" model 10-005 fluorometer. The diffusion coefficient was then determined by fitting calculated concentra-tion values (determined using an analytical solution for dif-fusive transport) with measured values (Figure 8).
Hydraulic conductivity for agarose was determined using the constant head method. The measured K-value was 8.5 x 10-9 m/s, which indicated that solute transport through the agarose was dominated by diffusion. The calculated diffusion coefficient and hydraulic conductivity values were then applied in a finite element mass-transport mathematical model to define and evaluate the development of concentration gradients in diffusion gradient plates. Parameters defined included prediction of the length and steepness of gradients, as well as fluxes out of the gel for specific Co values. The optimum flow velocity across the gel was also determined.
The model thus not only provided information on the gradient within the gel, but also the actual concentrations of the compound available to the microorganisms on the surface gel.
The results obtained with the mathematical model (Figure 9) indicated a rapid decrease in concentration with distance from the reservoir, with a C/Co value of only 0.01 at a distance 0.75 cm from the reservoir, which is in good agreement with the observations made with the physical model (Figure 7).
ASSESSMENT OF MICROBIAL RESPONSES TO DICLOFOP GRADIENTS
(a) Epifluorescent microscopy. Ground water containing a natural microbial community was used to inoculate a gradient plate to assess microbial responses to a diclofop gradient. The surface of the gel was stained with 4'6-diamidino-2-phenylindole (DAPI) after a 2 day incubation period, and numbers of micro-~~98331 organisms attached to the surface determined using an epi-fluorescent microscope. (b) Laser Microscopv. The scanning confocal laser microscope was used to image the biofilm developed on a diclofop - gradient plate after an incubation period of 5 days. Fluorescein isothiocyanate (FITC) was used to stain the cells. A minimum salts solution, inoculated with a natural microbial community, was used to irrigate the gel. Diclofop diffusing from the gel was thus the only carbon and nutrient source provided to the microbes.
Microorganisms responded to the diclofop gradients, accumulating at the higher end of the concentration range.
Development of biofilms with more than one layer of cells prevented the use of DAPI for counting biofilms older than two days. However, XZ sagittal scans, which were used to determined biofilm depth utilizing a scanning confocal laser microscope, provided a reliable and accurate alternative to epifluorescence microscopy for analyzing biofilms. As shown in Figures l0 and 11, microbial colonization of the gel surface correlated with the gradients measured in the physical model (Figure 7) and those calculated with the numerical model (Figure 9).
Results from both the physical and numerical models indicated that the concentration gradient developing as a result of diffusion did not extent over the length of the gel, regard-less, of the Co value of the contaminant in the reservoir.
Optimum utilization of the contaminant by degradative microor-ganisms may be possible. However, selective isolation and quantitative analyses of microbial behaviour to the concentration gradient, would be difficult, if not impossible in this narrow band over which the concentration was spread. It was therefore concluded that certain modifications to existing plates were necessary to make it applicable for in situ use.
The numerical model was utilized for the design of a modified gradient plate. Data obtained with the basic gradient plate was applied in the numerical model to develop a system in which the concentration gradient was extended over the length of the gel by using a series of parallel wells. Manipulation of the gradient was possible by altering either the concentrations in the wells, or the distance between the wells. Using the numerical model to manipulate and select gradients prior to the construction of experimental physical models, allowed for mathematical evaluation of the modified plates, thereby avoiding numerous experimental trial and error. This approach made it possible to modify the steepness (and thus length) of the gradient to suit any specific requirement.
Figure 12 illustrates a calculated concentration gradient developed in the multi well gradient cell, determined with the~numerical model.
Figure 13 illustrates the development of a fluorescein gradient in a multi-well cell similar to the model in Figure 12.
Figure 14 illustrates the extent of microbial growth in response to a diclofop concentration gradient determined with the scanning confocal laser microscope.
As those skilled in the art would realize these preferred illustrated details can be subjected to substantial variation, without affecting the function of the illustrated embodiments.
Although embodiments of the invention have been described above, it is not limited thereto and it will be apparent to those skilled in the art that numerous modifica-tions form part of the present invention insofar as they do not depart from the spirit nature and scope of the claimed and described invention.

Claims (21)

1. A method of isolating microorganisms capable of degrading a selected contaminant from a sample containing said contaminant comprising the steps of:
providing a substrate onto which said microorganisms may be isolated and through which said contaminant may diffuse;
predetermining substrate interval spacing to provide an extended concentration linear gradient;
contacting said substrate at a plurality of separate intervals with said contaminant, each interval having a different concentration of said contaminant:
exposing said microorganisms to said substrate; and isolating microorganisms at said concentrations.

2. The method as set forth in claim 1, wherein the step of contacting said contaminant with said substrate includes the diffusion of said contaminant in said substrate to form a concentration gradient.

3. The method as set forth in claim 2, wherein said concentration gradient comprises a high concentration of said contaminant and intervening concentrations in a gradient of decreasing concentrations of said contaminant.

4. The method as set forth in claim 1, wherein said substrate comprises a gel substrate.

5. The method as set forth in claim 1, wherein the step of isolating said microorganisms includes colonization of said microorganisms at concentrations optimal for metabolism of said microorganisms.

6. A method for selectively isolating microorganisms capable of metabolizing contaminants from a sample containing said contaminants, comprising the steps of:
providing a gel substrate onto which said microorganisms may be isolated and through which said contaminant may diffuse:
predetermining gel substrate interval spacing to provide an extended concentration linear gradient:
contacting said gel substrate at a plurality of separate intervals with said sample, each interval having a different concentration of said contaminants:
inoculating said gel substrate with said microorganisms:
and effecting isolation of said microorganisms at different concentrations of said varying concentrations on said gel substrate.

7. The method as set forth in claim 6, wherein said contaminants diffuse on said substrate such that a concentration gradient is formed on said substrate.

8. The method as set forth in claim 7, wherein said microorganisms migrate to a concentration on said substrate where said microorganisms metabolize said contaminant.

9. The method for identifying specific microorganisms capable of degrading a selected contaminant at selected concentrations of said contaminant from a sample containing microorganisms, comprising:
providing a sample containing microorganisms to be identified:
providing a substrate on which said microorganisms may be supported:
predetermining substrate interval spacing to provide an extended concentration linear gradient;
varying the concentration level of said contaminant on said substrate:

exposing said microorganisms to said contaminant on said substrate; and identifying microorganisms at each concentration level.

10. A cell for the isolation of degradative microorganisms from a sample containing said microorganisms, comprising:
a body having a plurality of wells each having a substrate therein adapted for supporting microorganisms;
each of said wells having first inlet means for permitting exposure of said substrate to a source of sample fluid containing a contaminant;
second inlet means for admitting a purging fluid for purging excess sample fluid from said substrate; and outlet means for facilitating removal of said excess sample fluid.

11. A cell for isolation of degradative microorganisms from a sample containing said microorganisms, said cell comprising:
a body;
a plurality of well means for receiving and retaining a sample of a contaminant;
each of said well means having inlet means for receiving said sample and outlet means;
a substrate receiving zone above said plurality of well means and in communication therewith, said zone adapted to receive a microorganism supporting substrate; and means for passing a fluid over said substrate when said substrate is in said zone.

12. The cell as set forth in claim 11, wherein said means for passing a fluid over said substrate includes an inlet and an outlet, said inlet being positioned within an end wall of said cell, said outlet being positioned in an opposed end wall.

13. The cell as set forth in claim 11, wherein said inlet means and said outlet means of said plurality of well means extend between side walls of said cell.

14. The cell as set forth in claim 11, wherein said cell includes an open top, said plurality of well means being spaced vertically downwardly therefrom.

15. A cell for the isolation of degradative microorganisms from a sample containing said microorganisms, comprising:
a body;
a plurality of wells for receiving and retaining a sample of a contaminant, said plurality of wells each including a first inlet for receiving said sample and a first outlet:
a recessed area in said body above said plurality of wells and in communication therewith, said area for receiving a substrate capable of sustaining microorganisms; and a second inlet and a second outlet in fluid communication adjacent said recessed area, said second inlet and outlet for receiving and removing, respectively, fluid to be passed over said substrate.

16. The cell as set forth in claim 15, wherein said first inlet and said first outlet are angularly arranged relative to said second inlet and said second outlet.

17. The cell as set forth in claim 16, wherein said first inlet and said first outlet are substantially orthogonal to said second inlet and said second outlet.

18. A method of testing the sensitivity of a microorganism to a selected test compound comprising the steps of:
providing a substrate on which a microorganism may colonize predetermining substrate interval spacing to provide an extended concentration linear gradient;

contacting said substrate with said test compound at a plurality of separate intervals, each interval having a different concentration of said contaminant:
exposing said microorganism to said substrate containing said test compounds and detecting a concentration at which microorganism growth is inhibited.

19. The method as defined in claim 18, wherein said test compound comprises a biocide.

20. The method as defined in claim 18, wherein said test compound is an antimicrobial agent.

21. The method as defined in claim 18, wherein said test compound is a chemical compound.