Pilot drain system for rapid biofilm formation

Details Pilot drain system for rapid biofilm formation Pilot drain system for rapid biofilm formation

1998-06-29

2001-03-27

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S283100, C435S289100

Reexamination Certificate

active

06207405

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention generally relates to methodology for production of experimental biofilm matrices on selected surfaces. More specifically this invention relates to the production of simulated natural biofilms for testing the activities of formulated products for inhibition or removal of the simulated biofilms from a drainage system.
Since 1943 a vast technical literature has developed in connection with advances in biofilm research. The understanding of biofilm processes has progressed rapidly in the last decade. One of the ultimate goals in studying biofilms is to evolve means for manipulating these processes for technological and ecological advantage. Biofilm science is reviewed in publications such as Food Reviews International, 8 (4), 573 (1992), Microbial Ecology in Health and Disease, 8, 305 (1995); Annu. Rev. Microbial, 49, 711 (1995); Applied and Environmental Microbiology, 4014 (November 1996); and “Biofilms” by W. G. Characklis and K. C. Marshall (John Wiley & Sons, Inc., New York, 1989).
As elaborated in the technical literature, a biofilm consists of cells immobilized on a substratum and embedded in an organic polymer matrix of microbial origin. A biofilm is a surface accumulation, which is not necessarily uniform in time or space. A biofilm may be composed of a significant fraction of inorganic or abiotic substances held cohesively by the biotic matrix. A biofilm is a protective matrix for bacteria, with the essential purpose of survival in an environment of limited nutrient supply.
Biofilms consist of both host microbes and their extracellular products, usually exopolysaccharides. Microbes have a tendency to form these protective exopolysaccharide matrices after they have adhered to a surface. The formation of biofilm complexes requires only humid conditions and/or water systems and contact with a support surface and/or interface. With respect to nutrients, a nutrient deficiency in fact may increase the biofilm formation capacity of microbes, as reported in Adv. Appl. Microbiol., 29, 93 (1983).
Biofilms generally can be produced by almost all microbes under suitable conditions. The most common biofilm producers belong to the genera Pseudomonas, Enterobacter, Flavobacterium, Alcaligenes, Staphylococcus, and Bacillus. There also are anaerobes that can construct corrosive biofilms.
Besides causing problems in cleaning and hygiene, biofilms can cause energy losses and blockages in condenser and heat exchange tubes, interfere with water and waste water systems, and form drag-inducing encrustations on ship hulls. In the medical disciplines, a biofilm (referred to as “glycocalyx”) formed by bacteria such as a Pseudomonas species can be the systemic causation of diseases of the lungs or the gastrointestinal and urinary tracts. Additionally, a biofilm formed by bacteria such as Staphylococcus species can be a serious contamination problem in foreign-body instruments such as cardiac pacemakers, catheters, prostheses, artificial valves, and the like. Dental plaque is also a typical form of biofilm.
One of the main purposes of natural biofilm formation is for the protection of the host microbes from a hostile environment. As a consequence, there is a combative interaction between microbes in biofilms and biocidal vehicles such as preservatives, disinfectants and antibiotics. Further, the sessile mode of bacterial growth in biofilms differs from that of the same bacteria species that are present as planktonic cells in a circulating aqueous medium which interfaces with the biofilm. Biofilms also act as a trap for nutrient acquisition, which is an important factor when bacteria grow on surfaces and the nutrient supply is oligotrophic.
Because of the manifold ramifications of biofilm formation, there is a serious commitment to biofilm research in a broad range of scientific investigations. Methods of studying biofilm formation include microbiological, physical, and chemical methods. When microbes from extreme natural environments are cultured, standard plate counts usually do not provide accurate estimates. Thus, the classical evaluation methods relying on microbiological plating have questionable value with respect to the laboratory study of biofilms which are intended to achieve authentic correspondence with natural biofilms which exist in the biosphere. In addition, formation of a natural type biofilm in the laboratory environment is difficult, mainly because there are no standardized methodologies currently available.
There is increasing interest in the research and development of methodologies for the production and study of biofilms in a laboratory environment. Accordingly, it is an object of this invention to provide an improved method for the laboratory production of biofilms which simulate natural biofilms that grow under biospheric conditions.
It is another object of this invention to provide a laboratory protocol for simulated natural biofilm production, in combination with a further protocol for testing the activities of formulated products for inhibition or removal of the simulated biofilms as a reliable indicator of the same activities under natural environmental conditions.
Other objects and advantages of the present invention shall become apparent from the accompanying description and examples.
Publications of background interest with respect to the present invention include Water Research Pergamon Press, 2, 597 (1968); Corrosion 97, Paper No. 405 by M. L. Ludyanskiy and F. J. Himpler (NACE Int. Conf. Div., Houston, Tex.); and references cited therein; incorporated herein by reference.
SUMMARY OF THE INVENTION
One or more objects of the present invention are accomplished by the provision of a method for rapid biofilm formation in accordance with a monitored protocol which comprises (1) providing a pilot drain system for liquid flow through a conduit having an average internal diameter between about 0.9-5 centimeters (preferably 0.9-2 centimeters); (2) introducing aqueous nutrient medium and bacterial inoculum comprising sheathed bacteria into the influent end of the conduit to full volume capacity without drainage; (3) maintaining the aqueous suspension medium in the drain conduit for an incubation period sufficient to initiate bacterial attachment to the conduit inner surface; (4) draining the suspension from the effluent end of the drain conduit; (5) recharging and draining the conduit with aqueous nutrient medium at least once every 24 hours of elapsed time period under simulated drain usage conditions to promote biofilm formation on the conduit inner surface; and (6) repeating the aqueous medium recharging and draining cycle of step (5) for an elapsed time period up to about 40 days (preferably up to about 15 days) that is sufficient to yield a biofilm mass which occupies at least about 15 percent of the conduit volume.
Sheathed bacteria genera and species are described in “Bergey's Manual Of Systemic Bacteriology” (Volume 3; Williams & Wilkins; Baltimore); “Biology Of Microorganisms” (Seventh Edition; Prentice Hall, Englewood Cliffs, N.J.); and Microbiological Reviews, p. 329-356 (June 1978); incorporated by reference.
The Sphaerotilus-Leptothrix group of sheathed bacteria are of particular advantage for purposes of the present invention. Genera species include
Sphaerotilus natans, Leptothrix lopholea, Leplothrix ochracea, Leptothrix cholodnii
and
Leptothrix discophora.


REFERENCES:
patent: 5928889 (1999-07-01), Bakich et al.
Effect of Growth Conditions and Substratum Composition on the Persistence of Coliforms in Mixed-Population Biofilms,Applied&Environmental Microbiology, Nov. 1996, p. 4014-4018, A. K. Camper, W. L. Jones & J. T. Hayes, Month Not Available.
The Biology of Sphaerotilus Species,Water ResearchPergamon Press 1968. vol. 2, pp. 597-614, John D. Phaup, Month Not Available.
The Effect of Halogenated Hydantoins on Biofilms,Corrosion 97, Paper No. 405 by M. L. Ludyanskiy and F. J. Himpler (NACE International Conference Division, Houston , Texas) (1997), Month Not Available.
Bergey's Manual ® of Systematic Ba

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