U.S. patent application number 12/942610 was filed with the patent office on 2011-05-12 for methods, compositions and systems for controlling fouling of a membrane.
This patent application is currently assigned to NOVOZYMES BIOLOGICALS, INC.. Invention is credited to DAVID DRAHOS, SVEND PETERSEN.
Application Number | 20110110894 12/942610 |
Document ID | / |
Family ID | 43810579 |
Filed Date | 2011-05-12 |
United States Patent
Application |
20110110894 |
Kind Code |
A1 |
DRAHOS; DAVID ; et
al. |
May 12, 2011 |
METHODS, COMPOSITIONS AND SYSTEMS FOR CONTROLLING FOULING OF A
MEMBRANE
Abstract
The present invention provides methods and compositions for
improving permeability and flux in a membrane filtration system,
especially in water or wastewater treatment processes.
Inventors: |
DRAHOS; DAVID; (ROANOKE,
VA) ; PETERSEN; SVEND; (LYNGBY, DK) |
Assignee: |
NOVOZYMES BIOLOGICALS, INC.
SALEM
VA
|
Family ID: |
43810579 |
Appl. No.: |
12/942610 |
Filed: |
November 9, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61259936 |
Nov 10, 2009 |
|
|
|
61369801 |
Aug 2, 2010 |
|
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Current U.S.
Class: |
424/93.3 ;
424/93.1; 424/93.4; 424/93.46; 424/93.461; 424/93.462;
424/93.5 |
Current CPC
Class: |
C02F 3/34 20130101; C02F
3/1268 20130101; Y02W 10/15 20150501; C02F 3/2853 20130101; Y02W
10/10 20150501; C02F 2303/20 20130101 |
Class at
Publication: |
424/93.3 ;
424/93.1; 424/93.4; 424/93.5; 424/93.46; 424/93.461;
424/93.462 |
International
Class: |
A01N 63/00 20060101
A01N063/00; A01N 63/04 20060101 A01N063/04; A01P 1/00 20060101
A01P001/00 |
Claims
1. A method of improving the permeability of a membrane used in a
process or the flux through a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane.
2. The method of claim 1, wherein microorganisms includes one or
more bacterial strains capable of reducing or preventing
undesirable biofilm formation on the membrane.
3. The method of claim 1, wherein the one or more microorganisms
are spore forming microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane.
4. The method of claim 2, wherein the one or more bacterial strains
are spore forming bacterial strains capable of reducing or
preventing undesirable biofilm formation on the membrane.
5. The method of claim 1, wherein microorganisms includes one or
more bacterial strains, one or more fungal strains, or a mixture of
one or more bacterial and fungal strains capable of reducing or
preventing undesirable biofilm formation on the membrane.
6. The method of claim 1, wherein the membrane is subjected to a
strain of Bacillus spp., e.g., Bacillus amyloliquefaciens; Bacillus
atrophaeus; Bacillus azotoformans; Bacillus brevis; Bacillus
cereus; Bacillus circulans; Bacillus clausii; Bacillus coagulans;
Bacillus firmus; Bacillus flexus; Bacillus fusiformis; Bacillus
globisporus; Bacillus glucanolyticus; Bacillus infermus; Bacillus
laevolacticus; Bacillus licheniformis; Bacillus marinus; Bacillus
megaterium; Bacillus mojavensis; Bacillus mycoides; Bacillus
pallidus; Bacillus parabrevis; Bacillus pasteurii; Bacillus
polymyxa; Bacillus popiliae; Bacillus pumilus; Bacillus sphaericus;
Bacillus subtilis; Bacillus thermoamylovorans; or Bacillus
thuringiensis.
7. The method of claim 1, wherein the improved flux allows for the
use of a membrane having a smaller membrane surface area.
8. The method of claim 1, wherein the membrane is part of a
membrane bioreactor system.
9. The method of claim 1, wherein the process is a water treatment
process.
10. The method of claim 1, wherein the one or more microorganisms
are capable of preventing or reducing biofilm formation through
quorum sensing inhibition.
11. The method of claim 6, wherein the one or more strains of
Bacillus are selected from the group consisting of: the Bacillus
megaterium strain having the deposit accession number ATCC 14581;
the Bacillus pumilus strain having the deposit accession number
ATCC 700385; the Paenibacillus azotofixans strain having the
deposit accession number ATCC 35681; the Bacillus licheniformis
strain having the deposit accession number NRRL B-50014; the
Bacillus licheniformis strain having the deposit accession number
NRRL B-50015; the Bacillus pumilus strain having the deposit
accession number NRRL B-50016; the Bacillus subtilis strain having
the deposit accession number ATCC 6051A; the Bacillus
amyloliquefaciens strain having the deposit accession number NRRL
B-50017; the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50018; the Bacillus subtilis strain having
the deposit accession number NRRL B-50136; the Bacillus
amyloliquefaciens strain having the deposit accession number NRRL
B-50141; the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50304; the Bacillus amyloliquefaciens
strain having the deposit accession number NRRL B-50349; the
Bacillus megaterium strain having the deposit accession number
PTA-3142; the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7541; the Bacillus amyloliquefaciens strain
having the deposit accession number PTA-7542; the Bacillus
atrophaeus strain having the deposit accession number PTA-7543; the
Bacillus amyloliquefaciens strain having the deposit accession
number PTA-7544; the Bacillus amyloliquefaciens strain having the
deposit accession number PTA-7545; the Bacillus amyloliquefaciens
strain having the deposit accession number PTA-7546; the Bacillus
subtilis strain having the deposit accession number PTA-7547; the
Bacillus amyloliquefaciens strain having the deposit accession
number PTA-7549; the Bacillus amyloliquefaciens strain having the
deposit accession number PTA-7790; the Bacillus amyloliquefaciens
strain having the deposit accession number PTA-7791; the Bacillus
atrophaeus strain having the deposit accession number PTA-7792; and
the Bacillus amyloliquefaciens strain having the deposit accession
number PTA-7793; or a mixture of two or more of the strains.
12. A method of increasing critical flux of a membrane used in a
process, comprising subjecting the membrane to one or more
microorganisms capable of reducing or preventing undesirable
biofilm formation on the membrane.
13. The method of claim 12, wherein microorganisms includes one or
more bacterial strains, capable of reducing or preventing
undesirable biofilm formation on the membrane.
14. The method of claim 12 wherein the one or more microorganisms
are spore forming microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane.
15. The method of claim 13 wherein the one or more bacterial
strains are spore forming bacterial strains capable of reducing or
preventing undesirable biofilm formation on the membrane.
16. The method of claim 12, wherein microorganisms includes one or
more bacterial strains, one or more fungal strains, or a mixture of
one or more bacterial and fungal strains capable of reducing or
preventing undesirable biofilm formation on the membrane.
17. The method of claim 12, wherein the membrane is part of a
membrane bioreactor system.
18. The method of claim 12, wherein the process is a water
treatment process.
19. A method of reducing or preventing fouling of a membrane used
in a process, comprising subjecting the membrane to one or more
microorganisms capable of reducing or preventing undesirable
biofilm formation on the membrane.
20. The method of claim 19, wherein microorganisms includes one or
more bacterial strains capable of reducing or preventing
undesirable biofilm formation on the membrane.
21. The method of claim 19, wherein the one or more microorganisms
are spore forming microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane.
22. The method of claim 20, wherein the one or more bacterial
strains are spore forming bacterial strains capable of reducing or
preventing undesirable biofilm formation on the membrane.
23. The method of claim 19, wherein microorganisms includes one or
more bacterial strains, one or more fungal strains, or a mixture of
one or more bacterial and fungal strains capable of reducing or
preventing undesirable biofilm formation on the membrane.
24. The method of claim 19, wherein the membrane is part of a
membrane bioreactor system.
25. The method of claim 19, wherein the process is a water
treatment process.
26. The method of claim 19, wherein the one or more microorganisms
are capable of preventing or reducing biofilm formation through
quorum sensing inhibition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. 119 of
U.S. provisional application Nos. 61/259,936 and 61/369,801 filed
Nov. 10, 2009 and Aug. 2, 2010, respectively, the contents of which
are fully incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention provides methods and compositions for
improving permeability and flux in a membrane filtration system,
especially in water or wastewater treatment processes.
BACKGROUND OF THE INVENTION
[0004] Membrane bioreactor (MBR) systems are becoming an
increasingly popular solution for water and wastewater treatment.
Although membrane systems for water treatment and purification have
been in use for decades, the employment of MBR systems as a
widespread solution for water and wastewater treatment has
generally been disregarded in favor of more conventional
biotreatment plants. One significant reason for such disregard is
that MBR systems are often comparatively more expensive than
conventional treatment systems. However, the higher purity of the
product and the decreased footprint make the employment of MBR
systems desirable.
[0005] MBR systems typically include one or more biological
reactors, such as anaerobic, anoxic and aerobic reactors, followed
by one or more membrane tanks with each tank containing one or more
membrane modules. Water or wastewater is induced into the membrane
modules by gravity feed or suction created by a pump. During the
process, the membranes filter out contaminants and other solids and
a permeate is produced.
[0006] One major drawback to membrane filtration processes is
membranes tend to foul. As the membranes foul, the permeability of
the membranes decrease, and the effectiveness of the whole process
is reduced. It is generally understood that the rate of membrane
fouling is increased roughly exponentially with an increase in the
flux. Study of this phenomenon has lead to the theory of critical
flux. Although critical flux is described in a number of ways, the
general definition of critical flux is the flux below which
permeability decline is considered negligible. Therefore,
controlling the flux, preferably maintaining it at or below the
critical flux, reduces the rate of permeability decline and
provides sustainable operation of membrane systems.
[0007] Even if a membrane system is run at or below the critical
flux rate, membrane fouling still occurs and methods of cleaning
the membranes must be employed. In membrane systems such as MBRs,
air scouring is often utilized to continually clean the membranes
and help sustain permeation. Air scouring creates turbulence and
shear force at the surface of the membrane to help reduce fouling
and cake layer buildup. However, air scouring significantly
increases operating costs and is not completely effective at
maintaining adequate critical flux rates.
[0008] Other physico-mechanical and/or chemical membrane cleaning
or treatment methods are used to remove fouling material and
maintain membrane permeability. Most widely used physico-mechanical
methods include backwashing, vibration, and air-scouring. These
methods are energy-intensive and not applicable to all membrane
types.
[0009] Chemical cleaning or treatment methods include pretreatment
with coagulants and/or polymers, and treatment with antiscalants,
biocides, and/or cleaning products such as NaOCl or citric acid.
Mineral or organic acids, caustic soda, or sodium hypochlorite are
also often used in chemical cleaning methods. However, frequent
chemical cleaning is costly due to the loss in system operation
time, decreased life expectancy of the membranes, and large
consumption of cleaning chemicals.
[0010] Physical cleaning methods such as air scouring are most
effective at removing gross solids from the membranes, the
substances that cause fouling sometimes referred to as "temporary"
or "reversible" fouling. Chemical cleaning methods are effective at
removing more tenacious fouling substances, the substances that
cause fouling sometimes referred to as "irreversible" or
"permanent" fouling. However, chemical cleaning cannot remove all
permanent or irreversible fouling substances and residual
resistance of the membrane remains. This residual resistance or
"irrecoverable" fouling is the fouling that builds up on the
membrane over a number of years and ultimately limits the lifetime
of the membrane.
[0011] Combinations of the mentioned methods are also commonly
used, such as chemically enhanced backwashing, often as a daily
cleaning measure. Weekly cleaning measures may include cleaning
with higher chemical concentration, and less often regular cleaning
may include even more intensive chemical cleaning with a
significant negative effect on membrane lifespan.
[0012] The mechanisms of membrane fouling have been studied
extensively. Fouling occurs over time and often in various stages
depending upon flux rate and consistency, as well as the
composition of the substance being passed through the membrane. The
stages of fouling are sometimes described as initial fouling (or
conditioning fouling), steady fouling, and transmembrane pressure
(TMP) jump. Initial fouling is believed to be a result of colloid
adsorption, small particulates blocking the membrane pores, and
small flocs or extracellular polymeric substances (EPS) left from
temporary attachment of biological aggregates to the membrane. The
overall resistance change by this initial fouling often has only a
negligible effect on flux and TMP once active filtration occurs.
However, initial fouling is believed to play a bigger role in
providing a favorable matrix for further or steady fouling. The
steady fouling stage includes further pore blocking by particulate
matter, but is also disadvantageous due to increased cake formation
and biofilm growth on the membranes. This stage of fouling does not
always occur homogeneously across the membrane, but steady fouling
increases TMP and decreases permeability, resulting in a decrease
in flux. The final stage of fouling is referred to as TMP jump
where permeation lessens significantly in a relatively short period
of time. There are a number of theories postulating the mechanisms
that cause TMP jump. However, regardless of the mechanism, once TMP
jump occurs, the membrane is so significantly fouled that it often
is ineffective for use in the process.
[0013] Other process parameters can affect membrane flux. One
example is the temperature that the process is run at. Generally,
an increase in process temperature results in an increased flux
rate. This flux improvement with higher temperature may be due to a
decrease in permeate viscosity, and may decrease the rate of
fouling. However, controlling the temperature of the water or
wastewater treatment process is typically not feasible and would be
cost prohibitive.
[0014] Solutions to reduce or prevent membrane fouling have
targeted all types and stages of fouling. Particularly, targeting
biofilm formation has been of recent interest. For example, Yeon et
al., 2009, Environ. Sci. Technol. 43: 380-385 discuss targeting the
quorum sensing (QS)-based membrane fouling mechanism of organisms
that are involved in steady fouling.
[0015] U.S. Patent Application Publication No. 2008/0233093
discloses a small number of strains of the genus Bacillus that can
reduce and/or prevent biofilm formation and/or planktonic
proliferation when co-cultured with certain undesirable
microorganisms.
[0016] Due to the critical need for effective water and wastewater
treatment, solutions that decrease membrane fouling and/or increase
critical flux rates in membrane applications including MBR systems
are highly desirable.
SUMMARY OF THE INVENTION
[0017] In one aspect, the present invention provides a method of
improving permeability or flux of a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing the development of undesirable
biofilm on the membrane.
[0018] In another aspect, the present invention provides a method
of increasing the critical flux of a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing the development of undesirable
biofilm on the membrane.
[0019] In another aspect, the present invention provides a method
of reducing or preventing fouling of a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing the development of undesirable
biofilm on the membrane.
[0020] In another aspect, the present invention provides a
composition comprising one or more cultures of microorganisms
capable of reducing or preventing the development of undesirable
biofilm on the membrane.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic illustration of the layout of the MBR
pilot plant.
[0022] FIG. 2 shows the effect of NRRL B-50141 on membrane
permeability over time.
[0023] FIG. 3 shows the effect of NRRL B-50141 on membrane
permeability over time.
[0024] FIG. 4 shows the effect of relaxation events on membranes
with or without treatment with NRRL B-50141.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention relates to methods of and compositions
for improving permeability and flux in a membrane filtration system
as well as methods and compositions for reducing and/or preventing
fouling of membranes in water and wastewater treatment
processes.
[0026] Fouling of membranes occurs by many mechanisms and at
differing rates due to a number of process variables in the
filtration systems as well as the content of the water or
wastewater being treated. One commonality of membrane fouling,
however, is that microorganisms from the water or wastewater create
biofilms on the membranes during the fouling process, and as a
result, TMP increases and permeability or flux decreases. The
relationship between the rate of fouling of a membrane and the rate
of flux through a membrane are known to be inversely correlative.
Thus, the theory of "critical flux" or the maximum flux rate at
which fouling can be reduced or slowed has been developed. However,
maintaining the flux at or below the critical flux does not prevent
fouling from occurring. Such fouling eventually increases the TMP
and decreases the flux so much that the membranes must be cleaned
in order to maintain effectiveness of the filtration process. The
ability to raise the critical flux is advantageous in a membrane
system. Increased flux improves capacity of an existing system,
enables lower investment requirements for new systems due to
smaller dimensioning, and/or increases operational efficiency and
flexibility since a larger volume of water or wastewater can be
treated before the membranes have to be cleaned, and the overall
lifespan of the membrane may be increased. Plant investments costs,
as well as the cost of cleaning and replacing the membranes, are
high, and the loss of productivity during the cleaning process
results in lost operation time and revenue. Therefore, methods that
can allow more water or wastewater to be treated per area of
membrane and/or methods that allow more water or wastewater to be
treated between membrane cleanings and or increase the lifespan of
the membranes are financially advantageous.
[0027] Surprisingly, addition of certain microorganisms to a
membrane filtration system allows the system to maintain or even
increase flux rates. The microorganisms employed in the present
invention adhere to the membranes in the same or similar fashion as
the microorganisms that create the undesirable biofilm formation
do. However, the use of the microorganisms according to the method
of the present invention surprisingly does not have a negative
effect on permeability and may even improve flux (e.g., increase
the critical flux) as compared to permeability or flux through an
untreated membrane in the same process over the same period of
time.
[0028] Permeability of a membrane or flux through a membrane
generally declines over a period of time during a process that
employs the membrane. It is generally accepted that this decline is
due to membrane fouling. According to the present invention,
"improving permeability" or "improving flux" means that the
membrane permeability or flux is the same or declines less over a
certain period of time during a process as compared to a same or
similar membrane during the same process without applying a
bacterial strain over the same certain period of time at the same
conditions such as flow rate, temperature, and pressure.
[0029] In an embodiment, the method of reducing and/or preventing
of fouling of membranes in water treatment processes comprising
subjecting the membranes to one or more bacterial strains capable
of reducing or preventing undesirable biofilm formation on the
membrane, wherein the bacterial strain is of the genus
Bacillus.
[0030] In an embodiment a blend of bacteria may be used according
to the method of the invention. Examples of blends can be found
below in the section "Bacterial Strains and Blends of Bacterial
Strains" section below.
[0031] The term "biofilm" or "biofilm formation" as used herein
means the slime layer or film or the formation of a slime layer or
film by undesired microorganisms on a membrane. Biofilm formation
is a consequence of growth of undesired microorganisms which attach
singly or in colonies to a membrane.
[0032] The invention also relates to a method of improving
permeability or flux of a membrane used in a process, comprising
subjecting the membrane to one or more microorganisms capable of
reducing or preventing undesirable biofilm formation on the
membrane. These novel microbes may cause no direct impact on the
health or viability of these undesirable strains, but only compete
against them in developing a biofilm on the membrane surface. As
used herein, "subjecting" means applying the one or more bacterial
strains to the water and/or membrane, such as, e.g., by
introducing, inoculating dispensing, applying, treating the water
to be treated and/or directly the membrane to be treated with the
one or more microorganisms or bacterial strains recited herein for
use in the present invention in reducing or preventing undesirable
biofilm formation on the membrane. Subjecting also includes
intentionally biasing the microbial content of the water and/or
membrane to contain an effective amount of the desire
microorganism. Such biasing can be achieved by introducing,
inoculating dispensing, applying, treating the water to be treated
and/or directly the membrane to be treated with the one or more
microorganisms or bacterial strains recited herein or any other
method effective to obtain the desired microorganism population in
the water and the membrane to be treated.
[0033] In one embodiment, the present invention provides a method
of increasing critical flux of a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane.
[0034] In another embodiment, the present invention provides a
method of reducing or preventing fouling of a membrane used in a
process, comprising subjecting the membrane to one or more
microorganisms capable of reducing or preventing undesirable
biofilm formation on the membrane.
Microorganisms, Bacterial Strains, and Blends of Microorganisms and
Bacterial Strains
[0035] It is to be understood that the microorganism or bacterial
strain used in accordance with methods of the invention reduces or
prevents undesirable biofilm formation on membranes. In order to
determine if a microorganism or bacterial strain reduces or
prevents undesirable biofilm formation on membranes, a comparison
is made with Pseudomonas aeruginosa PAO1 (ATCC 47085). In
particular, a microorganism or bacterial strain is useful in the
compositions and methods of the present invention if the strain
reduces or prevents undesirable biofilm formation on membranes
compared with the biofilm formation caused by Pseudomonas
aeruginosa PAO1 (ATCC 47085), as measured by flux reduction, as
described in Example 1. The microorganism or bacterial strain may
be a culture of a strain. Preferred properties for the
microorganisms or bacterial strains include, for example, one or
more of the following properties: minimal output of extracellular
polymeric substances (EPS), low biocake formation tendencies, and
low mucoidal substance release, and preferably, the microorganisms
or bacterial strains include all of these properties.
[0036] In one embodiment, the microorganism is a spore forming
microorganism. In another embodiment, the microorganism is a spore
forming bacteria. In yet another embodiment, the microorganism is
in the form of a stable spore. In yet another embodiment, the
microorganism is the form of a stable bacterial spore. As used
herein, "stable" is a term that is known in the art and in a
preferred aspect stable is used in the present invention to mean
the ability of the microorganism to remain in a spore form until it
is applied in the present invention to reduce or prevent
undesirable biofilm formation on the membrane.
[0037] In an embodiment, the bacterial strain is a gram-positive
bacterial strain.
[0038] In an embodiment, a bacterial strain for use in the present
invention is a strain of Agrobacterium spp., e.g., Agrobacterium
atlanticum; Agrobacterium rubi; Agrobacterium tumefaciens; or
Agrobacterium vitis, and combinations thereof.
[0039] In another embodiment, a bacterial strain for use in the
present invention is a strain of Arthrobacter spp., e.g.,
Arthrobacter oxydans; Arthrobacter aurescens; Arthrobacter
globiformis; Arthrobacter ramosus; or Arthrobacter viscosus, and
combinations thereof.
[0040] In another embodiment, a bacterial strain for use in the
present invention is a strain of Bacillus spp., e.g., Bacillus
amyloliquefaciens; Bacillus atrophaeus; Bacillus azotoformans;
Bacillus brevis; Bacillus cereus; Bacillus circulans; Bacillus
clausii; Bacillus coagulans; Bacillus firmus; Bacillus flexus;
Bacillus fusiformis; Bacillus globisporus; Bacillus glucanolyticus;
Bacillus infermus; Bacillus laevolacticus; Bacillus licheniformis;
Bacillus marinus; Bacillus megaterium; Bacillus mojavensis;
Bacillus mycoides; Bacillus pallidus; Bacillus parabrevis; Bacillus
pasteurii; Bacillus polymyxa; Bacillus popiliae; Bacillus pumilus;
Bacillus sphaericus; Bacillus subtilis; Bacillus thermoamylovorans;
or Bacillus thuringiensis, and combinations thereof.
[0041] In another embodiment, a bacterial strain for use in the
present invention is a strain of Bacteriodes spp., e.g.,
Bacteriodes cellulosolvens; Bacteriodes galacturonicus; Bacteriodes
pectinophilus; or Bacteriodes vulgates, and combinations
thereof.
[0042] In another embodiment, a bacterial strain for use in the
present invention is a strain of Beggiatoa spp., e.g., Beggiatoa
alba, and combinations thereof.
[0043] In another embodiment, a bacterial strain for use in the
present invention is a strain of Beijerinckia spp., e.g.,
Beijerinckia derxia; Beijerinckia fluminensis; Beijerinckia indica;
or Beijerinckia mobilis, and combinations thereof.
[0044] In another embodiment, a bacterial strain for use in the
present invention is a strain of Bifidobacterium spp., e.g.,
Bifidobacterium animalis; Bifidobacterium inducum; Bifidobacterium
magnum; Bifidobacterium minimum; or Bifidobacterium subtile, and
combinations thereof.
[0045] In another embodiment, a bacterial strain for use in the
present invention is a strain of Brachybacterium spp., e.g.,
Brachybacterium alimentarium; Brachybacterium nesterenkovii; or
Brachybacterium rhamnosum, and combinations thereof.
[0046] In another embodiment, a bacterial strain for use in the
present invention is a strain of Bradyrhizobium spp., e.g.,
Bradyrhizobium elkanii; Bradyrhizobium japonicum; or Bradyrhizobium
liaoningense, and combinations thereof.
[0047] In another embodiment, a bacterial strain for use in the
present invention is a strain of Brevibacillus spp., e.g.,
Brevibacillus brevis; Brevibacillus formosus; Brevibacillus
laterosporus; or Brevibacillus parabrevis, and combinations
thereof.
[0048] In another embodiment, a bacterial strain for use in the
present invention is a strain of Burkholderia spp., e.g.,
Burkholderia andropogonis; Burkholderia sacchari; or Burkholderia
vandii, and combinations thereof.
[0049] In another embodiment, a bacterial strain for use in the
present invention is a strain of Carnobacterium spp., e.g.,
Carnobacterium divergens; Carnobacterium funditum; Carnobacterium
mobile; or Carnobacterium pleistocenium, and combinations
thereof.
[0050] In another embodiment, a bacterial strain for use in the
present invention is a strain of Caulobacter spp., e.g.,
Caulobacter bacteriodes; Caulobacter fusiformis; Caulobacter
variabilis; or Caulobacter viriodoes, and combinations thereof.
[0051] In another embodiment, a bacterial strain for use in the
present invention is a strain of Cellulomonas spp., e.g.,
Cellulomonas humilata or Cellulomonas xylanilitica, and
combinations thereof.
[0052] In another embodiment, a bacterial strain for use in the
present invention is a strain of Citrobacter spp., e.g.,
Citrobacter amalonaticus; Citrobacter koseri; or Citrobacter
freundii, and combinations thereof.
[0053] In another embodiment, a bacterial strain for use in the
present invention is a strain of Corynebacterium spp., e.g.,
Corynebacterium flavescens or Corynebacterium glutamicum, and
combinations thereof.
[0054] In another embodiment, a bacterial strain for use in the
present invention is a strain of Enterobacter spp., e.g.,
Enterobacter cloacae; Enterobacter dissolvens; Enterobacter
gergoviae; Enterobacter nimipressuralis; or Enterobacter pyrinus,
and combinations thereof.
[0055] In another embodiment, a bacterial strain for use in the
present invention is a strain of Escherichia spp., e.g.,
Escherichia albertii; Escherichia blattae; Escherichia coli;
Escherichia fergusonii; Escherichia hermannii; or Escherichia
vluneris, and combinations thereof.
[0056] In another embodiment, a bacterial strain for use in the
present invention is a strain of Erwinia spp., e.g., Erwinia
amylovora or Erwinia caratovora, and combinations thereof.
[0057] In another embodiment, a bacterial strain for use in the
present invention is a strain of Flavobacterium spp., e.g.,
Flavobacterium acidurans or Flavobacterium resinovorum, and
combinations thereof.
[0058] In another embodiment, a bacterial strain for use in the
present invention is a strain of Gluconobacter spp., e.g.,
Gluconobacter oxidans, and combinations thereof.
[0059] In another embodiment, a bacterial strain for use in the
present invention is a strain of Halomonas spp., e.g., Halomonas
elongate or Halomonas salinas, and combinations thereof.
[0060] In another embodiment, a bacterial strain for use in the
present invention is a strain of Hyphomicrobium spp., e.g.,
Hyphomicrobium facilis or Hyphomicrobium indicum, and combinations
thereof.
[0061] In another embodiment, a bacterial strain for use in the
present invention is a strain of Lactobacillus spp., e.g.,
Lactobacillus casei; Lactobacillus helveticus; Lactobacillus
johnsonii; or Lactobacillus paracasei, and combinations
thereof.
[0062] In another embodiment, a bacterial strain for use in the
present invention is a strain of Lactococcus spp., e.g.,
Lactococcus lacti, and combinations thereof.
[0063] In another embodiment, a bacterial strain for use in the
present invention is a strain of Leuconostoc spp., e.g.,
Leuconostoc citreum or Leuconostoc mesenteroides, and combinations
thereof.
[0064] In another embodiment, a bacterial strain for use in the
present invention is a strain of Lysobacter spp., e.g., Lysobacter
antibioticus; Lysobacter brunescens; or Lysobacter enzymogenes, and
combinations thereof.
[0065] In another embodiment, a bacterial strain for use in the
present invention is a strain of Methylobacterium spp., e.g.,
Methylobacterium organophilum or Methylobacterium rhodesianum, and
combinations thereof.
[0066] In another embodiment, a bacterial strain for use in the
present invention is a strain of Microbacterium spp., e.g.,
Microbacterium laevaniformans and combinations thereof.
[0067] In another embodiment, a bacterial strain for use in the
present invention is a strain of Myxococcus spp., e.g., Myxococcus
fulvus or Myxococcus xanthus, and combinations thereof.
[0068] In another embodiment, a bacterial strain for use in the
present invention is a strain of Nocardiodes spp., e.g.,
Nocardiodes oleivorans and combinations thereof.
[0069] In another embodiment, a bacterial strain for use in the
present invention is a strain of Oceanospirillum spp., e.g.,
Oceanospirillum linum and combinations thereof.
[0070] In another embodiment, a bacterial strain for use in the
present invention is a strain of Pediococcus spp., e.g.,
Pediococcus acidilactici or Pediococcus pentosaceus and
combinations thereof.
[0071] In another embodiment, a bacterial strain for use in the
present invention is a strain of Photobacterium spp., e.g.,
Photobacterium damsela or Photobacterium phosphoreum and
combinations thereof.
[0072] In another embodiment, a bacterial strain for use in the
present invention is a strain of Planctomyces spp., e.g.,
Planctomyces brasiliensis or Planctomyces maris and combinations
thereof.
[0073] In another embodiment, a bacterial strain for use in the
present invention is a strain of Polyangium spp., e.g., Polyangium
cellulosum and combinations thereof.
[0074] In another embodiment, a bacterial strain for use in the
present invention is a strain of Pseudoalteromonas spp., e.g.,
Pseudoalteromonas atlantica or Pseudoalteromonas nigrifaciens and
combinations thereof.
[0075] In another embodiment, a bacterial strain for use in the
present invention is a strain of Pseudonorcardia spp., e.g.,
Pseudonorcardia autotrophic and combinations thereof.
[0076] In another embodiment, a bacterial strain for use in the
present invention is a strain of Paenibacillus spp., e.g.,
Paenibacillus alvei; Paenibacillus amylolyticus; Paenibacillus
azotofixans; Paenibacillus cookii; Paenibacillus macerans;
Paenibacillus polymyxa; or Paenibacillus validus, and combinations
thereof.
[0077] In another embodiment, a bacterial strain for use in the
present invention is a strain of Paracoccus spp., e.g., Paracoccus
alcaliphilus; Paracoccus denitrificans; Paracoccus kocurii; or
Paracoccus pantotrophus, and combinations thereof.
[0078] In another embodiment, a bacterial strain for use in the
present invention is a strain of Pseudomonas spp., e.g.,
Pseudomonas anitmiicrobica; Pseudomonas aureofaciens; Pseudomonas
chlororaphis; Pseudomonas corrugata; Pseudomonas fluorescens;
Pseudomonas marginalis; Pseudomonas nitroreducens; or Pseudomonas
putida, and combinations thereof.
[0079] In another embodiment, a bacterial strain for use in the
present invention is a strain of Rhodococcus spp., e.g.,
Rhodococcus coprophilus; Rhodococcus erythropolis; Rhodococcus
marinonascens; Rhodococcus rhodochrous; Rhodococcus ruber, or
Rhodococcus zopfii, and combinations thereof.
[0080] In another embodiment, a bacterial strain for use in the
present invention is a strain of Rhodospirillum spp., e.g.,
Rhodospirillum rubrum and combinations thereof.
[0081] In another embodiment, a bacterial strain for use in the
present invention is a strain of Salmonella spp., e.g., Salmonella
bongori; or Salmonella enterica, and combinations thereof.
[0082] In another embodiment, a bacterial strain for use in the
present invention is a strain of Sphingomonas spp., e.g.,
Sphingomonas adhaesiva, and combinations thereof.
[0083] In another embodiment, a bacterial strain for use in the
present invention is a strain of Stackebrandtia spp., e.g.,
Stackebrandtia nassauensis, and combinations thereof.
[0084] In another embodiment, a bacterial strain for use in the
present invention is a strain of Streptomyces spp., e.g.,
Streptomyces aureofaciens or Streptomyces griseus, and combinations
thereof.
[0085] In another embodiment, a bacterial strain for use in the
present invention is a strain of Thiobacillus spp., e.g.,
Thiobacillus halophilus or Thiobacillus thioparus, and combinations
thereof.
[0086] In another embodiment, a bacterial strain for use in the
present invention is a strain of Vibrio spp., e.g., Vibrio fischeri
or Vibrio logei, and combinations thereof.
[0087] In another embodiment, a fungal strain for use in the
present invention is a strain of Penicillium spp., e.g.,
Penicillium aurantiogriseum; Penicillium bilaiae; Penicillium
camemberti; Penicillium candidum; Penicillium chrysogenum;
Penicillium claviforme; Penicillium commune; Penicillium crustosum;
Penicillium digitatum; Penicillium expansum; Penicillium
funiculosum; Penicillium glabrum; Penicillium glacum; Penicillium
italicum; Penicillium lacussarmientei; Penicillium marneffei;
Penicillium purpurogenum; Penicillium roqueforti; Penicillium
stoloniferum; Penicillium ulaiense; Penicillium verrucosum; or
Penicillium viridicatum, and combinations thereof.
[0088] In another embodiment, a microorganism for use in the
present invention is a strain of Agrobacterium spp., e.g.,
Agrobacterium atlanticum; Agrobacterium rubi; Agrobacterium
tumefaciens; or Agrobacterium vitis, Arthrobacter spp., e.g.,
Arthrobacter oxydans; Arthrobacter aurescens; Arthrobacter
globiformis; Arthrobacter ramosus; or Arthrobacter viscosus,
Bacillus spp., e.g., Bacillus amyloliquefaciens; Bacillus
atrophaeus; Bacillus azotoformans; Bacillus brevis; Bacillus
cereus; Bacillus circulans; Bacillus clausii; Bacillus coagulans;
Bacillus firmus; Bacillus flexus; Bacillus fusiformis; Bacillus
globisporus; Bacillus glucanolyticus; Bacillus infermus; Bacillus
laevolacticus; Bacillus licheniformis; Bacillus marinus; Bacillus
megaterium; Bacillus mojavensis; Bacillus mycoides; Bacillus
pallidus; Bacillus parabrevis; Bacillus pasteurii; Bacillus
polymyxa; Bacillus popiliae; Bacillus pumilus; Bacillus sphaericus;
Bacillus subtilis; Bacillus thermoamylovorans; or Bacillus
thuringiensis, Bacteriodes spp., e.g., Bacteriodes cellulosolvens;
Bacteriodes galacturonicus; Bacteriodes pectinophilus; or
Bacteriodes vulgates, Beggiatoa spp., e.g., Beggiatoa alba,
Beijerinckia spp., e.g., Beijerinckia derxia; Beijerinckia
fluminensis; Beijerinckia indica; or Beijerinckia mobilis,
Bifidobacterium spp., e.g., Bifidobacterium animalis;
Bifidobacterium inducum; Bifidobacterium magnum; Bifidobacterium
minimum; or Bifidobacterium subtile, Brachybacterium spp., e.g.,
Brachybacterium alimentarium; Brachybacterium nesterenkovii; or
Brachybacterium rhamnosum, Bradyrhizobium spp., e.g.,
Bradyrhizobium elkanii; Bradyrhizobium japonicum; or Bradyrhizobium
liaoningense, Brevibacillus spp., e.g., Brevibacillus brevis;
Brevibacillus formosus; Brevibacillus laterosporus; or
Brevibacillus parabrevis, Burkholderia spp., e.g., Burkholderia
andropogonis; Burkholderia sacchari; or Burkholderia vandii,
Carnobacterium spp., e.g., Carnobacterium divergens; Carnobacterium
funditum; Carnobacterium mobile; or Carnobacterium pleistocenium,
Caulobacter spp., e.g., Caulobacter bacteriodes; Caulobacter
fusiformis; Caulobacter variabilis; or Caulobacter viriodoes,
Cellulomonas spp., e.g., Cellulomonas humilata or Cellulomonas
xylanilitica, Citrobacter spp., e.g., Citrobacter amalonaticus;
Citrobacter koseri; or Citrobacter freundii, Corynebacerium spp.,
e.g., Corynebacterium flavescens or Corynebacterium glutamicum,
Enterobacter spp., e.g., Enterobacter cloacae; Enterobacter
dissolvens; Enterobacter gergoviae; Enterobacter nimipressuralis;
or Enterobacter pyrinus, Escherichia spp., e.g., Escherichia
albertii; Escherichia blattae; Escherichia coli; Escherichia
fergusonii; Escherichia hermannii; or Escherichia vluneris Erwinia
spp., e.g., Erwinia amylovora or Erwinia caratovora, Flavobacterium
spp., e.g., Flavobacterium acidurans or Flavobacterium resinovorum,
Gluconobacter spp., e.g., Gluconobacter oxidans, Halomonas spp.,
e.g., Halomonas elongate or Halomonas salinas, Hyphomicrobium spp.,
e.g., Hyphomicrobium facilis or Hyphomicrobium indicum,
Lactobacillus spp., e.g., Lactobacillus casei; Lactobacillus
helveticus; Lactobacillus johnsonii; or Lactobacillus paracasei,
Lactococcus spp., e.g., Lactococcus lacti, Leuconostoc spp., e.g.,
Leuconostoc citreum or Leuconostoc mesenteroides, Lysobacter spp.,
e.g., Lysobacter antibioticus; Lysobacter brunescens; or Lysobacter
enzymogenes, Methylobacterium spp., e.g., Methylobacterium
organophilum or Methylobacterium rhodesianum, Microbacterium spp.,
e.g., Microbacterium laevaniformans, Myxococcus spp., e.g.,
Myxococcus fulvus or Myxococcus xanthus, Nocardiodes spp., e.g.,
Nocardiodes oleivorans, Oceanospirillum spp., e.g., Oceanospirillum
linum, Pediococcus spp., e.g., Pediococcus acidilactici or
Pediococcus pentosaceus, Photobacterium spp., e.g., Photobacterium
damsela or Photobacterium phosphoreum, Planctomyces spp., e.g.,
Planctomyces brasiliensis or Planctomyces marls, Polyangium spp.,
e.g., Polyangium cellulosum, Pseudoalteromonas spp., e.g.,
Pseudoalteromonas atlantica or Pseudoalteromonas nigrifaciens,
Pseudonorcardia spp., e.g., Pseudonorcardia autotrophic,
Paenibacillus spp., e.g., Paenibacillus alvei; Paenibacillus
amylolyticus; Paenibacillus azotofixans; Paenibacillus cookii;
Paenibacillus macerans; Paenibacillus polymyxa; or Paenibacillus
validus, Paracoccus spp., e.g., Paracoccus alcaliphilus; Paracoccus
denitrificans; Paracoccus kocurii; or Paracoccus pantotrophus,
Pseudomonas spp., e.g., Pseudomonas anitmiicrobica; Pseudomonas
aureofaciens; Pseudomonas chlororaphis; Pseudomonas corrugata;
Pseudomonas fluorescens; Pseudomonas marginalis; Pseudomonas
nitroreducens; or Pseudomonas putida, Rhodococcus spp., e.g.,
Rhodococcus coprophilus; Rhodococcus erythropolis; Rhodococcus
marinonascens; Rhodococcus rhodochrous; Rhodococcus ruber, or
Rhodococcus zopfii, Rhodospirillum spp., e.g., Rhodospirillum
rubrum, Salmonella spp., e.g., Salmonella bongori; or Salmonella
enterica, Sphingomonas spp., e.g., Sphingomonas adhaesiva,
Stackebrandtia spp., e.g., Stackebrandtia nassauensis, Streptomyces
spp., e.g., Streptomyces aureofaciens or Streptomyces griseus,
Thiobacillus spp., e.g., Thiobacillus halophilus or Thiobacillus
thioparus, Vibrio spp., e.g., Vibrio fischeri or Vibrio logei, and
Penicillium spp., e.g., Penicillium aurantiogriseum; Penicillium
bilaiae; Penicillium camemberti; Penicillium candidum; Penicillium
chrysogenum; Penicillium claviforme; Penicillium commune;
Penicillium crustosum; Penicillium digitatum; Penicillium expansum;
Penicillium funiculosum; Penicillium glabrum; Penicillium glacum;
Penicillium italicum; Penicillium lacussarmientei; Penicillium
mameffei; Penicillium purpurogenum; Penicillium roqueforti;
Penicillium stoloniferum; Penicillium ulaiense; Penicillium
verrucosum; or Penicillium viridicatum, and combinations
thereof.
[0089] In an embodiment, the one or more bacterial strains are
selected from the group consisting of:
[0090] the Bacillus megaterium strain having the deposit accession
number ATCC 14581;
[0091] the Bacillus pumilus strain having the deposit accession
number ATCC 700385;
[0092] the Paenibacillus azotofixans strain having the deposit
accession number ATCC 35681;
[0093] the Bacillus licheniformis strain having the deposit
accession number NRRL B-50014;
[0094] the Bacillus licheniformis strain having the deposit
accession number NRRL B-50015;
[0095] the Bacillus pumilus strain having the deposit accession
number NRRL B-50016;
[0096] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50017;
[0097] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50018;
[0098] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50136;
[0099] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50141;
[0100] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50304;
[0101] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50349;
[0102] the Bacillus megaterium strain having the deposit accession
number PTA-3142;
[0103] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7541;
[0104] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7542;
[0105] the Bacillus atrophaeus strain having the deposit accession
number PTA-7543;
[0106] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7544;
[0107] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7545;
[0108] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7546;
[0109] the Bacillus subtilis strain having the deposit accession
number PTA-7547;
[0110] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7549;
[0111] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7790;
[0112] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7791;
[0113] the Bacillus atrophaeus strain having the deposit accession
number PTA-7792; and
[0114] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7793; or a mixture of at least two of the
above deposited strains, including more than two, such as, at least
three of the above strains, at least four of the above strains, at
least five of the above strains, at least six of the above strains,
at least seven of the above strains, up to an including all of the
above strains.
[0115] The terms "effective amount", "effective concentration" or
"effective dosage" are defined herein as the amount, concentration
or dosage of one or more bacterial strains that can reduce and/or
prevent biofilm formation caused by undesired microorganisms on a
membrane. The actual effective dosage in absolute numbers depends
on factors including: the undesired microorganism(s) in question;
whether the aim is prevention or reduction; the contact time
between the strain(s) or composition comprising said strain(s);
other ingredients present, and also the membrane in question. In an
embodiment an effective dosage of bacteria, e.g., of the strain
NRRL B-50017, would be introduced to the membrane surface at a
final concentration of 1.times.10.sup.4-1.times.10.sup.11
CFU/cm.sup.2, with a preferred range of
1.times.10.sup.6-1.times.10.sup.7 CFU/cm.sup.2. Typically this
would result in the introduction of these bacterial strains in the
membrane-containing vessel of 1.times.10.sup.3-1.times.10.sup.10
CFU/ml, with a preferred range of 1.times.10.sup.5-1.times.10.sup.6
CFU/ml.
[0116] The "effective amount", "effective concentration" or
"effective dosage" is ultimately achieved by subjecting the water
and/or membrane to the one or more microorganisms described herein
for use in reducing and/or preventing biofilm formation, as
described herein.
[0117] In general, environments that receive high loads of
undesirable microorganisms and nutrients require high doses of
mitigating bacterial strains, while environments with low loads of
undesirable organisms require lower doses of mitigating bacterial
strains. Further, for instance, preventing biofilm formation on
membranes, in general, require lower doses of the concerned
bacterial strain(s) than reducing biofilm formation on the
corresponding membrane.
[0118] Consequently, a method of the invention can be used for
inhibiting growth (i.e., leading to reduced biofilm formation) of
one or more undesired microorganisms, preferably bacteria already
present on a membrane or surface. In another embodiment the
invention relates to preventing and/or significantly retarding
biofilm formation on an essentially clean membrane (i.e., membrane
with essentially no undesired microorganisms). In other words, the
concerned bacterial strain(s) protect(s) the membrane against
future growth of one or more undesired microorganisms. A method of
the invention may result in the reduction of undesired
microorganisms. The concerned bacterial strain(s) may in a
preferred embodiment be applied to the membrane in question.
Periodically means that the method of the invention may be
reiterated or repeated over a period of time, e.g., every minute,
hour, day, week, month, etc. As mentioned above, the effect may not
last for a long period of time. It may require redosing of the
bacterial strain(s).
[0119] According to the invention, the bacterial strains can be
introduced to the membrane before the membrane is employed in the
process, immediately following cleaning of the membrane after it
has been employed in the process, at any time during the process,
or any combination thereof.
Undesired Microorganisms
[0120] In the context of the invention the term "undesired
microorganisms" means microorganisms that may result in an effect
considered to be negative on the membrane in question. For example,
the negative effect may be fouling of the membrane by such
undesired microorganisms. Undesired microorganisms can also include
pathogenic microorganisms, especially pathogenic bacteria. In order
to determine if a bacterial strain is undesirable, a comparison is
made with Pseudomonas aeruginosa PAO1 (ATCC 47085). In particular,
a bacterial strain is undesirable and cannot be used in the
compositions and methods of the present invention if the strain
causes fouling of the membrane (as measured by flux reduction, in
accordance with Example 1). In another embodiment, a bacterial
strain is undesirable and cannot be used in the compositions and
methods of the present invention if the strain causes fouling of
the membrane (as measured by flux reduction, in accordance with
Example 1) at least as much as Pseudomonas aeruginosa PAO1 (ATCC
47085). A bacterial strain is also undesirable if the bacterial
strain does not reduce fouling caused by Pseudomonas aeruginosa
PAO1 (ATCC 47085). Thus, different strains of the same species may
have opposite effects on the flux.
[0121] By using one or more of the isolated bacterial strains
concerned herein in an effective amount, biofilm formation on
membranes can be reduced and/or prevented.
[0122] In a preferred embodiment the membrane in question prone to
biofilm formation may be subjected to one or more of the bacterial
strains as a preventative measure prior to any biofilm
formation/buildup. This results in the formation of significantly
less biofilm or in the formation of a biofilm which is
significantly less conducive to membrane fouling.
[0123] Examples of undesired microorganisms include those disclosed
below.
[0124] Undesired microorganisms include, but are not limited to,
aerobic bacteria or anaerobic bacteria, gram-positive and
gram-negative bacteria, fungi (yeast or filamentous fungus), algae,
and/or protozoa. Undesirable bacteria include bacteria selected
from the group consisting of Acetobacter, Aeromonas, Azotobacter
vinelandii, Betabacterium, Burkholderia, Clostridium botulinum,
Corynebacterium diphteriae, Escherichia coli, Flavobacterium,
Leuconostoc, Legionella spp., Listeria spp., Mycobacterium
tuberculosis, Pneumococcus, Pseudomonas spp., including Pseudomonas
aeruginosa, Salmonella, Staphylococcus, Streptococcus spp., and
Vibrio spp.
[0125] In one embodiment, the undesired microorganism is an aerobic
bacterium. In another embodiment, the aerobic bacterium is an
Aeromonas strain. In another embodiment, the aerobic bacterium is a
Burkholderia strain. In another embodiment, the aerobic bacterium
is a Flavobacterium strain. In another embodiment, the aerobic
bacterium is a Microbacterium strain. In another embodiment, the
aerobic bacterium is a Pseudomonas strain. In another embodiment,
the aerobic bacterium is a Salmonella strain. In another
embodiment, the aerobic bacterium is a Staphylococcus strain. In
another embodiment, the aerobic bacterium is from the family
Enterobacteriaceae (including, e.g., Escherichia coli).
[0126] In another embodiment, the aerobic bacterium is Burkholderia
cepacia. In another embodiment, the aerobic bacterium is a
Microbacterium imperiale or Mycobacterium tuberculosis. In another
embodiment, the aerobic bacterium is Pseudomonas aeruginosa. In
another embodiment, the aerobic bacterium is Pseudomonas
fluorescens. In another embodiment, the aerobic bacterium is
Pseudomonas oleovorans. In another embodiment, the aerobic
bacterium is Pseudomonas pseudoalcaligenes. In another embodiment,
the aerobic bacterium is Salmonella enteritidis. In another
embodiment, the aerobic bacterium is Staphylococcus aureus. In
another embodiment, the aerobic bacterium is Staphylococcus
epidermidis.
[0127] In another embodiment the bacterium is Listeria
monocytogenes.
[0128] In another embodiment the bacterium is Legionella
adelaidensis. In another embodiment the bacterium is Legionella
pneumophila. In another embodiment the bacterium is Legionella
feeleii. In another embodiment the bacterium is Legionella
moravica.
[0129] In another embodiment the bacteria is Vibrio harveyi, Vibrio
fischerii, and/or Vibrio alginolyticus.
[0130] In another embodiment, the microorganism is an anaerobic
bacterium. In another embodiment, the anaerobic bacterium is a
Desulfovibrio strain. In another embodiment, the anaerobic
bacterium is Desulfovibrio desulfuricans.
Quorum Sensing and Other Microbial Signaling Mechanisms
[0131] Quorum sensing is a mechanism that allows bacteria to
"communicate" and affect phenotypic aspects of the bacterial
population such as pigmentation, motility, pathogenicity and
biofilm formation. Quorum sensing is believed to be achieved
through secretion of small signaling molecules called autoinducers.
Quenching or inactivating these autoinducers can prevent biofilm
formation of undesirable microorganisms. Thus, quorum sensing
inhibition is a mode of action for biofilm control. In one
embodiment, the bacterial strains of the present invention prevent
membrane fouling by preventing biofilm formation by quorum sensing
inhibition. In another embodiment, the quorum sensing inhibition is
through the inhibition of acyl homoserine lactone (AHL) inhibition.
In another embodiment, the quorum sensing inhibition is due to
acylase activity of the microorganism(s). In another embodiment,
the quorum sensing inhibition is due to lactonase activity of the
microorganism(s). In another embodiment, the quorum sensing
inhibition is due to racemase activity of the microorganism(s).
[0132] Another embodiment of the present invention includes a
method of screening microorganisms for use in the methods and
compositions of the present invention based on the ability of the
bacteria to prevent biofilm formation by quorum sensing inhibition.
In another embodiment of the present invention, the quorum sensing
inhibition is through the inhibition of acyl homoserine lactone
inhibition (AHL). In yet another embodiment, of the present
invention, the quorum sensing inhibition is due to acylase activity
of the microorganism(s). The quorum sensing may be effective
achieved due to the action of one microorganism or a combination of
microorganisms.
Membranes
[0133] A variety of membrane types and configurations can be used
in water or wastewater treatment processes. Types of membrane
configurations include capillary tube, tubular, hollow fiber,
multi-tube, plat-and-frame/flat sheet, pleated cartridge filter,
spiral wound, and ceramic including ceramic disc. Membranes can be
made from one or more materials including, for example, chlorinated
polyethylene, polyacrylonitrile, polysulfone, polyethersulfone,
polyvinylalcohol, cellolose acetate, regenerated cellulose,
polyvinylidene difluoride, polyethlysulphone, polyethylene,
polypropylene, and ceramic material. Other characteristics of the
membranes that can vary based on the application include, for
example, the membrane pore size. The size of the membrane pores may
be larger or smaller depending upon the size of particulate or
impurity being removed from the water or wastewater. Membrane
types, according to the present invention, include those utilized
for ultrafiltration, microfiltration, and nanofiltration.
Membrane Bioreactor Systems
[0134] Membrane bioreactor (MBR) systems typically combine two
basic processes: biological degradation and membrane separation,
into a single process where suspended solids and microorganisms
responsible for biodegradation are separated from the treated water
by a membrane filtration unit. See, for example, Water Treatment
Membrane Processes, McGraw-Hill, 1996, p. 17.2. The entire biomass
is confined within the system, providing for both control of the
residence time for the microorganisms in the reactor (sludge age)
and the disinfection of the effluent.
[0135] In a typical MBR unit, influent wastewater is pumped or
gravity fed into an aeration tank where it is brought into contact
with the biomass which biodegrades organic material in the
wastewater. Aeration means such as blowers provide oxygen to the
biomass. The resulting mixed liquor is pumped or gravity fed from
the aeration tank into the membrane module where it is mechanically
or gravitationally filtered through a membrane under pressure or is
drawn through a membrane under low vacuum. In some systems, the
aeration tank and the membrane tank are the same tank. The effluent
is discharged from the system while the concentrated mixed liquor
is returned to the bioreactor. Excess sludge is pumped out in order
to maintain a constant sludge age, and the membrane is regularly
cleaned by backwashing, chemical washing, air scouring, or any
combination of these mechanisms.
[0136] MBR systems have multiple configurations. Two main MBR
process configurations include submerged/immersed and sidestream.
There are also two primary mechanisms of hydraulic operation
including pumping and airlifting. These configurations and bulk
liquid transfer modes are typically referred to as conventional
biomass rejection MBRs. Other configurations include extractive and
diffusive process modes which employ membranes for purposes other
than separating biomass from the treated water. All of these
process configurations include one or more membrane units
comprising membranes such as those described in the "Membranes"
section above.
[0137] In one embodiment, the membranes are present in a membrane
bioreactor. In another embodiment, the wastewater treatment process
occurs in a membrane bioreactor in which the membrane flat-sheet
cassette unit, or hollow-fiber unit, itself is typically
immersed.
[0138] In one embodiment, the wastewater is pretreated prior to
entering the membrane bioreactor. Pretreatment can occur at the
source of the wastewater, at a pretreatment plant, or as part of
the overall MBR system. Such pretreatments can include a bar
screen, grit chamber, or rotary drum screen to achieve coarse
solids removal. Other pretreatments may include removal of
substances such as harmful pollutants, oils or fuels, or other
toxic substances.
Water Treatment Processes
[0139] One or more water treatment processes are contemplated by
the present invention. Such water treatment processes include, but
are not limited to, reverse osmosis, water desalination and
drinking water purification, and wastewater treatment processes.
The water or wastewater, according to the present invention, can be
from any source including human waste, cesspit leakage, septic tank
discharge, sewage plant discharge, washing water such as greywater
or sullage, collected rainwater, groundwater, surplus manufactured
liquids, seawater, river water, manmade liquid disposal, highway
drainage, storm drains, blackwater, industrial waste, industrial
site wastewater or drainage such as cooling or process waters, and
agricultural wastewater or drainage.
Compositions of the Invention
[0140] The invention also relates to a composition comprising one
or more of the microorganisms, including deposited strains, as
described herein. It is to be understood that a composition of the
invention may comprise one or more of the bacterial strains
concerned herein as single strains or blends of two or more strains
and further comprises one or more additional ingredients mentioned
below.
[0141] The invention also relates to a composition comprising one
or more microorganisms or one or more of the bacterial strains,
including deposited strains, as described herein in a form suitable
for application to a water treatment process and/or membrane, such
as, in a form and in an effective amount for reducing or preventing
undesirable biofilm formation on the membrane. The microorganisms
are preferably in the form of stable spores.
Additional Ingredients
[0142] The composition may comprise one or more additional
ingredients and/or enzymes. Examples of contemplated enzymes are
mentioned in the "Enzymes" section below.
[0143] Other ingredients may be active ingredients that also reduce
or prevent biofilm formation or inactive ingredients and include,
but are not limited to, cis-2-decanoic acid, dispersants,
stabilizers, fragrances, dyes, and biocides. The other ingredients
can be made via traditional chemistry or biologically, such as the
case may be.
Enzymes
[0144] One or more enzymes may be present in a composition of the
invention. For example, the composition may comprise an acylase
and/or lactonase. In one embodiment, the enzyme(s) are from a
fungal or bacterial source. In another embodiment, the enzyme(s)
may also be produced "in situ" by one or more of the bacterial
strains of the present invention that have been genetically
modified to express such enzyme(s). In another embodiment, the
enzyme(s) are native to the bacterial strains of the present
invention and may be naturally expressed or the bacterial strain(s)
may be genetically modified to alter the level of expression of the
naturally occurring enzyme(s).
EXAMPLES
Example 1
Method for Screening Candidate Strains Capable of Reducing or
Preventing Anti-Fouling in MBR Systems
[0145] Candidate strains were grown and cultured over an
approximately 16 hour period subject to shaking at 25.degree. C. in
1.times. Lysogeny Broth (10 g Tryptone; 5 g yeast extract; 1 g
NaCl, and deionized water to 1 liter). Candidate strains were then
counted using a hemocytometer and then serial diluted to a
concentration of 1.times.10.sup.3 cells/ml. Each well of a PVDF
(poly(vinylidene fluoride))-bottomed 96-well plate (Millipore.RTM.
no.: MSGVS2210) was filled with 100 microliters sterile 0.1.times.
Lysogeny Broth. 100 microliters of the diluted candidate strains
were added to the well. Those wells not including the addition of
candidate strains were filled with 100 microliters sterile 1.times.
Lysogeny broth. The 96-well plate was sealed with Breathe Easy.RTM.
plate sealing film and placed on a plate shaker for approximately
16 hours at 25.degree. C.
[0146] Pseudomonas aeruginosa PAO1 was selected as a
biofilm-forming strain and grown and cultured over an approximately
16 hour period subject to shaking at 200 rpm in 1.times. Lysogeny
Broth (10 g Tryptone; 5 g yeast extract; 1 g NaCl, and deionized
water to 1 liter) at 25.degree. C. P. aeruginosa cultures were
counted using a hemocytometer and then serial diluted to a
concentration of 1.times.10.sup.3 cells/ml.
[0147] Following culture of the biofilm-forming strain, the Breathe
Easy.RTM. plate sealing film was removed from the 96-well plate and
100 microliters of the diluted bio-film forming strain, P.
aeruginosa, was added to the wells containing candidate strains.
Those wells not including the addition of biofilm-forming strains
were filled with 100 microliters sterile 1.times. Lysogeny broth.
The 96-well plate was re-sealed with a new Breathe Easy.RTM. plate
sealing film and placed on a plate shaker at 200 rpm for a 24 hour
period at 25.degree. C.
[0148] After 24 hours the Breathe Easy.RTM. plate sealing film was
removed from the 96-well plate. 10 microliters was removed from
each well and placed into the corresponding well of a new sterile
96-well plate for plating or optical density measurements at 590
nm. An additional 990 microliters of phosphate buffered saline
solution (PBS) was added to each well to bring the volume of each
well to approximately 1 ml. The 96-well plate was then inverted
onto a Wypall* (Kimberly-Clark) for removing any planktonic cells
and then excess media was removed using a pipettor. Each well was
subsequently rinsed with 250 microliters of PBS and then inverted
for a second time onto a Wypall* (Kimberly-Clark). Remaining PBS
was removed using a pipettor then 250 .mu.l of 0.25% Brilliant
Green Dye in PBS was added to each well. The 96-well plate was then
placed on top of a Millipore vacuum manifold (Millipore no.:
MSVMHTS00) over a 96-well clear bottomed collection plate. The
vacuum was applied to the 96-well plate at -0.5 bar for 2 mins. The
vacuum was powered off and the 96-well collection plate was
recovered for flow-through evaluation.
[0149] Specifically, the 96-well collection plate was placed on a
plate reader (BioTek Synergy HT) and the absorbance of each well
was measured at .lamda.=610 nm (Abs.sub.610). The volume of
flow-through collected in each well was determined by applying the
equation V=Abs.sub.610.times.88.997+15.334 where V=the volume of
0.25% brilliant green in a well. This equation was derived by
measuring the A.sub.610 of 0.25% Brilliant Green in a 96 well plate
and plotting this against the known volumes in each well. Wells
having a higher absorbance had a higher volume than those wells
with a lower absorbance. Accordingly, those wells with high
absorbance were selected as containing likely candidate strains
capable of reducing or preventing biofilm formation.
[0150] Results of the 96-well screening method are found in Table
1. 38 strains belonging to 16 genera within 11 families have been
tested for their ability to protect flux through a PVDF membrane in
the presence of a biofilm forming Pseudomonas aeruginosa strain,
PAO1, using a 96-well based method. A strain is considered a
candidate strain if it is capable of maintaining >25% of the
flow allowed by a sterile, uninoculated PVDF membrane of the same
size under the same conditions. The results show that a
phylogenetically broad range of bacteria and a fungus (Penicillium
sp) are capable of transmembrane flux maintenance in the presence
of biological fouling agents.
TABLE-US-00001 TABLE 1 Family Strain (genus, species) Number %
Protection Acetobacteraceae Gluconacetobacter SB3779 16.14
diazatrophicus (DSMZ 5601) Bacillaceae Bacillus megaterium SB3112
20.22 (PTA-3142) Bacillaceae Bacillus licheniformis SB3181 4.87
(NRRL B-50015) Bacillaceae Bacillus pumilus SB3182 19.35 (NRRL
B-50016) Bacillaceae Bacillus amyloliquefaciens SB3448 16.63
(PTA-7791) Bacillaceae Bacillus pumilus SB3002 7.81 (ATCC 700385)
Bacillaceae Bacillus megaterium SB3059 6.74 (ATCC 14581)
Bacillaceae Bacillus subtilis SB3086 31.52 (NRRL B-50136)
Bacillaceae Bacillus megaterium SB3112 12.96 (PTA-3142) Bacillaceae
Bacillus licheniformis SB3131 12.96 (ATCC 12713) Bacillaceae
Bacillus amyloliquefaciens SB3195 15.02 (NRRL B-50017) Bacillaceae
Bacillus subtilis SB3259 7.79 Bacillaceae Bacillus
amyloliquefaciens SB3615 31.15 (NRRL B-50349) Bacillaceae Bacillus
subtilis SB3223 (A164) 12.67 (ATCC 6051A) Burkholderiaceae
Burkholderia sp GW5 19.37 Corynebacteriaceae Corynebacterium
mucifaciens C.muc 10.75 (ATCC 700355) Corynebacteriaceae
Corynebacterium diphtheriae C.dip 12.51 (ATCC 11913)
Corynebacteriaceae Corynebacterium xanthophilus C.xp10 -0.17 (ATCC
373) Enterobacteriaceae Citrobacter sp SB3257 25.82
Enterobacteriaceae Enterobacter cloacae SB3255 34.63
Enterobacteriaceae Enterobacter gergoviae SB3258 26.02
Enterobacteriaceae Enterobacter cloacae SB3103 -10.99 (ATCC 31482)
Enterobacteriaceae Enterobacter disolvens SB3013 -2.35 (NRRL
B-50257) Enterobacteriaceae Eschericia coli SB3254 53.17
Enterobacteriaceae Salmonella enterica SAL 40.02 (ATCC 167)
Nocardiaceae Rhodococcus erythropolis SB3100 16.79 Paenibacillaceae
Brevibacillus epidermidis Brevi 3.78 (ATCC 35514) Paenibacillaceae
Brevibacillus parabrevis SB3187 25.06 (ATCC 10068) Paenibacillaceae
Paenibacillus azotofixans SB3054 38.09 (ATCC 35681)
Paenibacillaceae Paenibacillus validus SB3263 36.14
Pseudomonodaceae Pseudomonas aeruginosa SB3088 4.43
Pseudomonodaceae Pseudomonas aeruginosa SB3259 26.04
Pseudomonodaceae Pseudomonas monteilii BL44 9.28 Rhodospirillaceae
Azospirillum sp SB3772 10.20 Sphingobacteriaceae Mucilaginibacter
sp GW6 43.74 Staphylococcaceae Staphylococcus epidermidis Staph
10.11 (ATCC 14990) Trichocomaceae Penicillium sp Peni1 35.18
Example 2
Lab Scale MBR Model (PVDF)
[0151] Lab-scale MBR systems were prepared using 0.5.times.
Lysogeny Broth (5 g Tryptone; 2.5 g yeast extract; 0.5 g NaCl, and
deionized water to 1 liter) flowing via gravity feed into an Amicon
8200 stirred cell ultrafiltration unit (Millipore, Billerica,
Mass., USA) fitted with a 63.5 mm diameter (28.7 cm.sup.2 effective
area) PVDF membrane that had been treated with 95% isopropanol
prior to use followed by sterilization with 10% perchlorate. The
filtration devices were inoculated with spores of strains of
interest at a rate of 2.times.10.sup.6 cfu/cm.sup.2 and incubated
for 24 hours at 25.degree. C. with constant stirring at
approximately 125 rpm and a flow rate of 8.5 ml/hr/cm.sup.2. A
control unit was prepared similarly but was not inoculated with a
strain of interest. After 24 hours incubation, the units were
inoculated with 2.times.10.sup.4 cfu/cm.sup.2 Pseudomonas
aeruginosa strain PAO1, a known biofilm forming organism and the
flow-through rates of all concurrently running filter units were
adjusted to approximately 8 ml/hr/cm.sup.2. The filter units
continued to run under the above conditions for a further 50 hours.
Flow rates through the membrane were determined at regular
intervals by measuring the volume of effluent discharge from each
of the filter units over a 5 minute period. At the conclusion of
the experiment, the filter unit was aseptically disassembled and
viable counts were performed on both the media portion and 0.18
cm.sup.2 portions of the membrane to determine the cell density of
both the strain of interest and the Pseudomonas aeruginosa
strain.
[0152] The measurement at the 48 hour timepoint (F.sub.48) was
taken as the best indication point for flow comparison.
(F.sub.0-F.sub.48)*100/F.sub.0=% decrease in flow
[0153] The strains of interest and the flow rates obtained are
provided in Table 2.
TABLE-US-00002 TABLE 2 Flow decrease % Pro- Strain (genus, species)
Number at 48 hours tection Pseudomonas aeruginosa PAO1 52% 0% (ATCC
47085) Bacillus amyloliquefaciens SB3195 10% 81% (NRRL B-50141)
Bacillus amyloliquefaciens SB3232 20% 62% Bacillus
amyloliquefaciens SB3615 26% 50% (NRRL B-50349) Bacillus subtilis
SB3086 9% 83% (NRRL B-50136) Bacillus subtilis SB3223 (A164) 19%
63% (ATCC 6051A) Bacillus megaterium SB3112 21% 60% (PTA-3142)
Bacillus megaterium SB3059 50% 1% (ATCC 14581) Bacillus pumilus
SB3002 48% 2% (ATCC 700385) Bacillus subtilis SB3295 26% 50%
(PTA-7547) Paenibacillus azotofixans SB3054 24% 54% (ATCC 35681)
Brevibacillus parabrevis SB3187 26% 50% (ATCC 10068) Rhodococcus
erythropolis SB3100 18% 65%
[0154] The results show that many of the strains significantly
improved the flow rate through the membrane.
Example 3
Lab Scale MBR Model (PES)
[0155] A lab-scale MBR experiment was constructed similar to that
described in Example 2 utilizing a polyethersulfone (PES) membrane
as opposed to the PVDF membrane. MBR units were inoculated as in
Example 2 with either NRRL B-50141 or NRRL B-50136. A control unit
was prepared similarly but was not inoculated with a strain of
interest. Filter units were allowed to operate for 50 hours under
the conditions specified in Example 2 and flow rates through the
membrane were determined at regular intervals by measuring the
volume of effluent discharge from each of the filter units over a 5
minute period. The measurement at the 48 hour timepoint (F.sub.48)
was taken as the best indication point for flow comparison.
(F.sub.0-F.sub.48)*100/F.sub.0=% decrease in flow
[0156] The efficacy of strains NRRL B-50141 and NRRL B-50136 at
maintaining flow rates through a PES membrane was determined and is
provided in Table 3.
TABLE-US-00003 TABLE 3 Flow decrease % Pro- Strain (Genus, species)
Number at 48 hours tection Pseudomonas aeruginosa PAO1 62% 0% (ATCC
47085) Bacillus amyloliquefaciens SB3195 25% 40% (NRRL B-50141)
Bacillus subtilis SB3086 29% 38% (NRRL B-50136)
[0157] The results show that strains NRRL B-50141 and NRRL B-50136
significantly improved the flow rate through the PES membrane.
Example 4
Pilot Scale Test of Bacillus amyloliquefaciens NRRL B-50141. MBR
Membrane Colonization and Flux Effect with Microbial Inoculation
and with Recycling of Inoculum Water Prior to Operation
[0158] The setup of the MBR system utilized in this example is
described in FIG. 1.
[0159] The MBR system had a total PVDF membrane surface area of 20
m.sup.2 and was run for a total of 241 days. The interval from day
110 through day 150 is considered the reference period. Several
cleaning events using sodium hypochlorite (500 ppm Cl.sub.2) were
employed to chemically decrease the biofouling and raise the
permeability. The cleaning on day 89 resulted in a permeability
rate of 300 l/m.sup.2/hr/bar which persisted during the reference
period until day 150. This permeability rate is the rate that is
typically observed at this treatment plant under these conditions.
The membrane was then once again cleaned by the method described
above, and subsequently inoculated with a spore suspension of
Bacillus amyloliquefaciens, NRRL B-50141. The spore inoculum (NRRL
B-50141) was prepared at approximately 10% (w/w) blend of the NRRL
B-50141 spray-dried spore concentrate with dendritic salt (NaCl).
The final concentration of NRRL B-50141 was 4.11.times.10.sup.10
CFU/g. The inoculum was distributed in 20 g aliquots into sterile
blue-cap conical tubes (50 ml size) for shipment and
application.
[0160] The inoculant was prepared by adding 20 g inoculum into
approximately 400 ml water. The mixture was shaken by hand for
approximately 1 minute, dispersing the spores in the water. The
shaken mixture was poured into a large bucket with approximately
10-15 liters of water and stirred to blend. The entire contents of
the bucket were gradually poured into a 4600 liter aeration tank
over the top of the membrane holder, covering the surface area
relatively evenly. Prior to addition of the inoculum, the
conditions of the MBR system were as follows: water temperature
25.degree. C., pH 7.6, oxygen tension 5.8 mg/l and water flow 900
l/h. Following the addition of the inoculum, the conditions of the
MBR system were as follows: water temperature 25.degree. C., pH
7.35, oxygen tension 0.7 mg/l and water flow 750 l/h. The final
NRRL B-50141 spore concentration was approximately
1.8.times.10.sup.5 CFU/ml in the aeration tank. The inoculant was
allowed to disperse for 20-30 minutes, followed by water
recirculation in the aeration tank for approximately 20 hours, or
about 2.5 passes of the water through the membranes, in order to
enhance the opportunity of the NRRL B-50141 inoculant to interact
with the membrane. The ratio of added microbes to membrane surface
area is thus approximately 3.5.times.10.sup.6 CFU/cm.sup.2.
[0161] The water level in the permeate tank of the MBR system was
regulated to be under the water level in the membrane tank,
resulting in a pressure difference over the membranes (TMP),
driving the water through the membranes. The TMP was controlled at
a relatively constant level in the interval of 250-300 mm water
column by using pressure transmitters to control the influent flow.
Air scouring was continuously employed to prevent buildup of sludge
cake on the membrane. Further, flow through the membrane was
stopped periodically, approximately 10 minutes of flow alternated
with 2 minutes of no flow, to aid in the prevention of sludge and
cake buildup.
[0162] Scraping samples were taken on or about day 190. During
sampling, air scouring was stopped and the surface level of the MBR
fluid was lowered to enable physical access to the upper part of
the membranes. This was achieved by allowing part of the fluid into
a storage tank. Subsequently, scrapings were taken of exposed
membrane surfaces above the fluid surface on the side or the center
portions of the membranes sampled, both before and after a short
water flush of the membrane. Six scrapings (samples 1-6)
corresponding to approximately 10 cm.sup.2 of MBR membrane were
placed into sterile screw-cap tubes and stored cold (4-10.degree.
C.) prior to microbial analysis.
[0163] The scraped material for each of the six samples was
resuspended in a 0.1 M phosphate buffer at pH 7.0 and shaken in a
standard wrist action shaker for 30 minutes at 23.degree. C.
Dilutions were plated by standard techniques on Standard Method
Agar (SMA plates, Smith River Biologicals, Ferrum, Va., USA), and
incubated for 2 days at 35.degree. C.
[0164] An estimate of the percentage of cells recovered from the
membrane scrapings was obtained by quantifying the number of
colonies with the distinct NRRL B-50141 morphology compared with
the number with different morphologies. MBR biofilm sample
information, including the results from the analysis of samples 1-6
are shown in Table 4.
TABLE-US-00004 TABLE 4 Approximate amount Sample Number and
location of NRRL B-50141 1) Before flush, membrane center 1.2% 2)
Before flush, membrane side 21.1% 3) Before flush, membrane center
4.5% 4) Before flush, membrane center 2.5% 5) After H.sub.2O flush
16.5% 6) After flush, membrane center 6.6%
[0165] Colonies from each of the samples with a morphology matching
that of the NRRL B-50141 strain were isolated and assessed for
identity to the known NRRL B-50141 parent strain by purifying DNA
from each isolate and using the DiversaLab RAPD PCR-amplification
procedure (Agilent 2100 Bioanalyzer with DiveraLab Strain typing
software using the Bacillus Kit repPCR materials from bioMerieux,
Inc., Durham, N.C., USA).
[0166] As detailed in Table 5, all 24 isolates chosen (four from
each of the 6 scrapings taken) gave a strong match to the known
parent NRRL B-50141 strain. A strain with a different colony type
(control; Isolate 26) did not match NRRL B-50141 (i.e., it had less
than 90% similarity).
TABLE-US-00005 TABLE 5 Sample origin Similarity to (Table 2) NRRL
B-50141 Match (>90%) Similarity 1 94.5% Match to NRRL B-50141 1
95.8% Match to NRRL B-50141 1 95.9% Match to NRRL B-50141 1 95.8%
Match to NRRL B-50141 2 94.9% Match to NRRL B-50141 2 94.4% Match
to NRRL B-50141 2 95.4% Match to NRRL B-50141 3 95.6% Match to NRRL
B-50141 3 95.4% Match to NRRL B-50141 3 95.5% Match to NRRL B-50141
4 95.7% Match to NRRL B-50141 4 95.7% Match to NRRL B-50141 4 96.6%
Match to NRRL B-50141 4 96.7% Match to NRRL B-50141 5 96.5% Match
to NRRL B-50141 5 97.2% Match to NRRL B-50141 5 97.0% Match to NRRL
B-50141 5 97.3% Match to NRRL B-50141 5 96.8% Match to NRRL B-50141
5 97.6% Match to NRRL B-50141 6 96.6% Match to NRRL B-50141 6 97.0%
Match to NRRL B-50141 6 96.8% Match to NRRL B-50141 Parent 97.7%
Match to NRRL B-50141 3 84.2% No-match
Effect of NRRL B-50141 on MBR Flux in Field Trial Assessments
[0167] The enhanced permeability rate was notably enhanced in the
period after microbial inoculation (days 154-200), with a
persistent permeability level averaging 400 l/m.sup.2/hr/bar. This
represents approximately a 33% increase in overall flow rate
following the microbial inoculum compared with the reference
period, i.e., days 105-152 (without the microbial inoculum) under
virtually identical conditions of temperature and pressure (see
FIG. 2).
[0168] The permeability shown in FIG. 2 is based on daily average
net permeate flows corrected to a standard temperature of
15.degree. C. using the following equation:
F.sub.15.degree. C.=F.sub.temp*e.sup.(-0.0267(T-15))
wherein F is the flow (l/m.sup.2*hr), T is the actual temperature,
and permeability=F/pressure (bars).
[0169] Transmembrane pressure (TMP) was kept constant, and chemical
and biochemical parameters were assessed daily throughout the
reference and trial periods.
Second Inoculation of NRRL B-50141. MBR Membrane Colonization and
Maintained Permeability Enhancement with Microbial Inoculation
without Recycling.
[0170] On or about day 195, NRRL B-50141 was again inoculated into
the MBR tank, except that the water recirculation after the second
inoculation was not performed. High permeability rates of about 375
l/m.sup.2/hr/bar were maintained until day 212.
[0171] After approximately 26 days post-inoculation, on or about
day 211, additional scrapings were collected and analyzed using the
same procedures as described above. Results for percentage of
colonies and strain identity are demonstrated in Tables 6 and 7.
The presence of the inoculated strain (NRRL B-50141) in the
membrane scrapings ranged from 7-52% of the total recovered
microbial strains. The identity of the NRRL B-50141 strain was
again confirmed by high homology sequence analysis of the 1500 bp
segment of the 16S rDNA of strains isolated with similar colony
morphology on standard solid media. This verified the presence of
the inoculated strain during the time of enhanced permeability
across the membrane.
TABLE-US-00006 TABLE 6 Approximate amount Sample number and
location of NRRL B-50141 1a) Membrane center 42% 2a) Membrane
center 52% 3a) Membrane center 13% 4a) Membrane side 17% 5a)
Membrane side 7% 6a) Membrane side 50%
TABLE-US-00007 TABLE 1 Sample Similarity to origin NRRL B-50141
Match (>90%) Similarity 1a 98.9% Match to NRRL B-50141 1a 99.0%
Match to NRRL B-50141 2a 98.8% Match to NRRL B-50141 2a 99.2% Match
to NRRL B-50141 3a 98.8% Match to NRRL B-50141 3a 98.2% Match to
NRRL B-50141 4a 98.5% Match to NRRL B-50141 4a 98.7% Match to NRRL
B-50141 5a 98.5% Match to NRRL B-50141 5a 98.7% Match to NRRL
B-50141 6a 98.1% Match to NRRL B-50141 6a 99.3% Match to NRRL
B-50141 Parent 100.0% Match to NRRL B-50141
Example 5
Disruption of Genes of Interest in Bacillus subtilis Strain A164
(ATCC6051A) for MBR Antifouling Experiments
[0172] The racX gene of Bacillus subtilis A164 (ATCC 6051A) was
disrupted by replacement of most of the racX coding sequence with a
gene conferring resistance to the antibiotic neomycin. The gene
disruption was constructed in vitro using three-way SOE (splicing
by overlap extension) PCR.
[0173] Three DNA fragments were amplified by PCR. A fragment
comprising a region of DNA downstream of the Bacillus subtilis racX
gene was amplified from Bacillus subtilis A164 genomic DNA using
primers 0610964 and 0610965. A neomycin resistance gene (neo) was
amplified from pBEST501 plasmid DNA Maya et al., 1989, Nucl. Acids
Res. 17:4410) using primers 0610966 and 0610967. A fragment
comprising a region of DNA upstream of the Bacillus subtilis racX
gene was amplified from Bacillus subtilis A164 genomic DNA using
primers 0610968 and 0610969.
TABLE-US-00008 Primer 0610964: (SEQ ID NO: 1)
5'-GGATTAACGAGGGCCAAC-3' Primer 0610965: (SEQ ID NO: 2)
5'-AGAATTGATCTGCGGCACATATCTTGCTTATCAAAGCTAG-3' Primer 0610966: (SEQ
ID NO: 3) 5'-ATAAGCAAGATATGTGCCGCAGATCAATTCTGATAATTAC-3' Primer
0610967: (SEQ ID NO: 4) '5-ATCGACCTCGCCGTTTATAGGTCGAGATCAGGGAATG-3'
Primer 0610968: (SEQ ID NO: 5)
'5-CATTCCCTGATCTCGACCTATAAACGGCGAGGTCGAT-3' Primer 0610969: (SEQ ID
NO: 6) 5'-TGCAGCATATCATGGCGT-3'
[0174] The PCRs were performed using Phusion.RTM. Hot Start DNA
Polymerase (New England Biolabs, Inc., Beverly, Mass., USA)
according to the manufacturer's instructions in a PTC-200 Peltier
thermal cycler (MJ Research, Inc., Waltham, Mass., USA) using the
following temperature profile:
1 cycle of 96.degree. C. for 2 minutes; 11 cycles of 94.degree. C.
for 30 seconds; 60.degree. C. for 45 seconds, decreasing by
1.degree. C. per cycle; and 72.degree. C. for 1 minute; 20 cycles
of 94.degree. C. for 30 seconds; 50.degree. C. for 45 seconds; and
72.degree. C. for 1 minutes, increasing by 5 seconds per cycle; 1
cycle of 72.degree. C. for 5 minutes.
[0175] Primers 0610965 and 0610966 were designed to base-pair with
each other so that the downstream racX fragment could be fused to
the neo fragment. Likewise, primers 0610967 and 0610968 were
designed to base-pair with each other so that the neo fragment
could be fused to the upstream racX fragment. The three PCR
products were combined in a single SOE PCR to fuse them into a
single PCR product, as follows.
[0176] The PCR products were purified using a QIAQUICK.RTM. Gel
Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to
the manufacturer's instructions and used as template DNA in an SOE
PCR using primers 0610964 and 0610969. The PCR was performed using
Phusion.RTM. Hot Start DNA Polymerase according to the
manufacturer's instructions in a PTC-200 Peltier thermal cycler
using the following temperature profile:
1 cycle of 96.degree. C. for 2 minutes; 11 cycles of 94.degree. C.
for 30 seconds; 60.degree. C. for 45 seconds, decreasing by
1.degree. C. per cycle; and 72.degree. C. for 3 minutes; 20 cycles
of 94.degree. C. for 30 seconds; 50.degree. C. for 45 seconds; and
72.degree. C. for 3 minutes, increasing by 20 seconds per cycle; 1
cycle of 72.degree. C. for 5 minutes.
[0177] The resulting racX::neo PCR product was purified using a
QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's
instructions. In order to generate a larger quantity of the PCR
product, the purified racX::neo PCR was used as template DNA in a
PCR using primers 0610964 and 0610969. The PCR was performed as
described for the SOE PCR.
[0178] Bacillus subtilis A164 was transformed with the resulting
PCR fragment according to the method of Anagnostopoulos and
Spizizen (J. Bacteriol. 81:741-746 (1961)). Transformants were
selected on TBAB neomycin plates at 37.degree. C. TBAB medium was
composed of Difco Tryptose Blood Agar Base (BD Diagnostics,
Franklin Lakes, N.J., USA). TBAB neomycin plates were composed of
TBAB medium and 6 micrograms of neomycin per ml. One such
transformant was designated Bacillus subtilis MDT361. Disruption of
the racX gene by insertion of the neo gene was confirmed by PCR and
DNA sequencing.
[0179] The ylmE gene of Bacillus subtilis A164 was disrupted by
replacement of most of the ylmE coding sequence with a gene
conferring resistance to the antibiotic spectinomycin. The gene
disruption was constructed in vitro using three-way SOE (splicing
by overlap extension) PCR.
[0180] Three DNA fragments were amplified by PCR. A fragment
comprising a region of DNA upstream of the Bacillus subtilis ylmE
gene was amplified from Bacillus subtilis A164 genomic DNA using
primers 0610970 and 0610971. A spectinomycin resistance gene (spc)
was amplified from pSJ5218 plasmid DNA (PCT Application WO
2002/000907) using primers 0610972 and 0610973. A fragment
comprising a region of DNA downstream of the Bacillus subtilis ylmE
gene was amplified from Bacillus subtilis A164 genomic DNA using
primers 0610974 and 0610975.
TABLE-US-00009 Primer 0610970: (SEQ ID NO: 7)
5'-TATTGGGGAGGAAGTTGG-3' Primer 0610971: (SEQ ID NO: 8)
5'-TTTCACAATTTGTCTACAGCGTAAATTATCAACAACACGC-3' Primer 0610972: (SEQ
ID NO: 9) 5'-TTGTTGATAATTTACGCTGTAGACAAATTGTGAAAGGATG-3' Primer
0610973: (SEQ ID NO: 10)
5'-ACTAACGATGCCACTAATATTAATAAACTATCGAAGGAAC-3' Primer 0610974: (SEQ
ID NO: 11) 5'-TAGTTTATTAATATTAGTGGCATCGTTAGTCGGAAATGAA-3' Primer
0610975: (SEQ ID NO: 12) 5'-CTTCAATCAGCATTTGGAAAC-3'
[0181] The PCRs were performed using Phusion.RTM. Hot Start DNA
Polymerase according to the manufacturer's instructions in a
PTC-200 Peltier thermal cycler using the following temperature
profile:
1 cycle of 96.degree. C. for 2 minutes; 11 cycles of 94.degree. C.
for 30 seconds; 60.degree. C. for 45 seconds, decreasing by
1.degree. C. per cycle; and 72.degree. C. for 1 minute; 20 cycles
of 94.degree. C. for 30 seconds; 50.degree. C. for 45 seconds; and
72.degree. C. for 1 minute, increasing by 5 second per cycle; 1
cycle of 72.degree. C. for 5 minutes.
[0182] Primers 0610971 and 0610972 were designed to base-pair with
each other so that the upstream ylmE fragment could be fused to the
spc fragment. Likewise, primers 0610973 and 0610974 were designed
to base-pair with each other so that the spc fragment could be
fused to the downstream ylmE fragment. The three PCR products were
combined in a single SOE PCR to fuse them into a single PCR
product, as follows.
[0183] The PCR products were purified using a QIAQUICK.RTM. Gel
Extraction Kit according to the manufacturer's instructions and
used as template DNA in an SOE PCR using primers 0610970 and
06109705. The PCR was performed using Phusion.RTM. Hot Start DNA
Polymerase according to the manufacturer's instructions in a
PTC-200 Peltier thermal cycler using the following temperature
profile:
1 cycle of 96.degree. C. for 2 minutes; 11 cycles of 94.degree. C.
for 30 seconds; 60.degree. C. for 45 seconds, decreasing by
1.degree. C. per cycle; and 72.degree. C. for 3 minutes; 20 cycles
of 94.degree. C. for 30 seconds; 50.degree. C. for 45 seconds; and
72.degree. C. for 3 minutes, increasing by 5 seconds per cycle; 1
cycle of 72.degree. C. for 5 minutes.
[0184] The resulting ylmE::spc PCR product was purified using a
QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's
instructions. In order to generate a larger quantity of the PCR
product, the purified ylmE::spc PCR was used as template DNA in a
PCR using primers 0610970 and 06109705. The PCR was performed as
described for the SOE PCR.
[0185] Bacillus subtilis A164 was transformed with the resulting
PCR fragment according to the method of Anagnostopoulos and
Spizizen (J. Bacteriol. 81:741-746 (1961)). Transformants were
selected on TBAB spectinomycin plates at 37.degree. C. TBAB medium
was composed of Difco Tryptose Blood Agar Base (BD Diagnostics,
Franklin Lakes, N.J., USA). TBAB spectinomycin plates were composed
of TBAB medium and 120 micrograms of spectinomycin per ml. One such
transformant was designated Bacillus subtilis MDT362. Disruption of
the ylmE gene by insertion of the spc gene was confirmed by PCR and
DNA sequencing.
[0186] The transformant Bacillus subtilis MDT362 was transformed
with genomic DNA from Bacillus subtilis MDT361 according to the
method of Anagnostopoulos and Spizizen (J. Bacteriol. 81:741-746
(1961)). Transformants were selected on TBAB neomycin plates at
37.degree. C. One such transformant was designated Bacillus
subtilis MDT363. Disruption of the racX gene by insertion of the
neo gene and disruption of the ylmE gene by insertion of the spc
gene were confirmed by PCR and DNA sequencing.
[0187] Wild-type A164 and gene knockouts of the B. subtilis A164
were grown approximately 16 hours in 0.5.times. Lysogeny Broth (LB)
with shaking at 200 rpm. After growth, culture density was
determined by direct counting on a hemocytometer. Membrane discs
were placed in filter holders and treated first with 100%
isopropanol then with 10% sodium perchlorate. Membranes and holders
were rinsed with sterile water then inoculated with 0.1.times.LB
containing the strain to be tested at a rate of 100 cells X
ml.sup.-1 via syringe in a total volume of 1 ml and incubated at
25.degree. C. with shaking at 250 RPM approximately 16 hours.
Membranes were subsequently inoculated with P. aeruginosa strain
PAO1 at a rate of 100 cells/ml.sup.-1 in 1 ml of 0.1.times.LB and
incubated overnight at 25.degree. C. with shaking at 250 RPM. Media
and planktonic cells were removed from the filter holders by
aspirating the contents with a syringe. Flow rates were
subsequently determined by placing the treated and untreated
filters on individual ports of a vacuum manifold with a syringe
containing 3 ml of phosphate buffered saline (PBS) and applying
-2.0 bar vacuum for 5 minutes. Filtrate was recovered for each
filter separately and flow-through volume was determined
gravimetrically. Data presented is the mean volume of flow-through
.+-.1 standard deviation. See Table 8. Significant differences in
flow-through volume were observed for the yFInD disruption mutant
as well as the dual racemace knockout, racX+ylmE. Strains were
tested in triplicate, both unchallenged and challenged with PAO1 as
a biofilm-forming strain
TABLE-US-00010 TABLE 8 Strain No PAO1 With PAO1 A164 (Wild-type)
1217.33 .+-. 55.42 1145.67 .+-. 36.37 A164 .DELTA.racX +
.DELTA.ylmE (MDT 363) 1232.67 .+-. 66.90 1061.33 .+-. 67.34 A164
.DELTA.racX (MDT361) 1261.00 .+-. 41.38 1091.67 .+-. 127.38 A164
.DELTA.ylmE (MDT362) 1240.33 .+-. 36.43 1091.00 .+-. 46.11
Example 6
Dual-Track Large-Scale Laboratory Test of Bacillus subtilis NRRL
B-50136
[0188] MBR membrane flux effect and colonization with microbial
inoculation compared with a parallel non-inoculated MBR
membrane.
[0189] The setup of the MBR system utilized in this example is
described in FIG. 3. For clarity only one of two identical MBR
units is drawn. The process parameters for the MBRs of Example 6
are disclosed in Table 9.
[0190] The applied flat sheet membranes are PVDF microfiltration
membranes, average poresize of 0.2 micro-m, manufactured by Alfa
Laval A/S. The membranes are stacked in a cassette of 10
membranes--the effective membrane area in the current tests was 0.8
m.sup.2. The reactors are both aerobic with a constant aeration for
membrane scouring of 10 L/minm.sup.2. Along the side of the
reactor, aeration (20 L/min total) was likewise established to
avoid sedimentation and secure complete mixing of the reactors.
[0191] The transmembrane pressure (TMP) is established as the
difference in water level of the membrane reactor and the permeate
buffer system. The effective TMP applied was 30 mbar (without the
pressure drop of the flow meter and permeate system). Two minutes
of relaxation was applied after each ten minutes of filtration by
stopping the pump activity. The reactors were initially inoculated
with activated sludge from Aalborg East WWTP (waste water treatment
plant) and acclimatized for about a month before one the MBRs was
inoculated with Bacillus subtilis NRRL B-50136 at the same rate and
concentration as that described in Example 4 for NRRL B-50141.
Sludge was taken out on a daily basis to keep a constant MLSS of 10
g/L. This sludge removal resulted in a sludge age (SRT) of 25-30
days.
TABLE-US-00011 TABLE 9 Parameter {unit} Value Parameter {unit}
Value Reactor Volume {m.sup.3} 0.35 MLSS {g/L} 10 Membrane area
{m.sup.2} 0.8 MLVSS {g/L} 9 Membrane material PVDF SRT {days} 25-30
Avg. Poresize {micro-m} 0.2 HRT{days} 0.9-1.sup. Scouring air
{L/min m.sup.2} 10 F/M {kg BOD/kg 0.07 Mixing air {L/min} 20 MLSS
day} TMP {mBar} 30 F/M {kg COD/kg 0, 12 Relaxation {min/min} 2/10
MLSS day} Bulk pH 7.6 Bulk Conductivity 0.61 {mS/cm} Bulk
Temperature 20.5 {deg. C.}
[0192] The wastewater was composed of a mixture of tap water and
concentrated substrate. The tap water inlet is controlled by a
float valve in the inlet buffer tank. Concentrated substrate was
added from a separate input line and the addition was controlled
after a F/M ratio set point of 0.1 kg BOD/kg MLSSday. The
concentrated substrate was a standard commercial dog feed which is
mixed with demineralized water, blended and sedimented to remove
larger particles and fibers before addition. In addition, fine
commercial fish meal was added to the substrate mixture to increase
the total protein contents. The concentrated substrate composition
is disclosed in Table 10.
TABLE-US-00012 TABLE 10 Parameter {unit} Value Organic fraction 90%
Proteins (% of organic) 50% Carbohydrates (% of organic) 40% Fats
(% of organic) 10% Total N {mg/L} 57.8 NH4--N {mg/L} 14 NO3--N
{mg/L} 4.4 Total P {mg/L} 92.5 o-PO4--P {mg/L} 81.2
[0193] The full results from the 18-Day test period are provided in
FIG. 3. The results show that after an initial period of
approximately 5 days of operation the flow rate of the untreated
reactor decreased rapidly while the reactor treated with NRRL
B-50141 maintained a higher flow rate from this point until
approximately 18 days of operation. At approximately Day 10.5
post-inoculation, the MBR reactor treated with Bacillus subtilis
NRRL B-50136 exhibited a 34% greater flow than the untreated
reactor. A closer view of representative data from a 3-hr period
approximately 9 days post-inoculation is provided in FIG. 4. The
regular pump relaxation events, a standard practice for MBR
operation, lasted for 2 minutes each and occurred at 10 minute
intervals. Results show that in the untreated MBR reactor
relaxation events result in a temporary increase immediately
followed by a drop in flow rate whereas in the treated reactor a
higher flow rate is maintained regardless of relaxation events.
These results indicate that the treated reactor membranes are less
impacted by fouling than the membranes in the untreated
reactor.
[0194] The present invention is described by the following numbered
paragraphs:
1. A method of improving the permeability of a membrane used in a
process or the flux through a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane. 2. The method of paragraph 1, wherein microorganisms
includes one or more bacterial strains capable of reducing or
preventing undesirable biofilm formation on the membrane. 3. The
method of paragraph 1, wherein the one or more microorganisms are
spore forming microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane. 4. The method of
paragraph 2, wherein the one or more bacterial strains are spore
forming bacterial strains capable of reducing or preventing
undesirable biofilm formation on the membrane. 5. The method of
paragraphs 1, wherein microorganisms includes one or more bacterial
strains, one or more fungal strains, or a mixture of one or more
bacterial and fungal strains capable of reducing or preventing
undesirable biofilm formation on the membrane. 6. The method of
paragraphs 1-5, wherein the membrane is subjected to a strain of
Bacillus spp., e.g., Bacillus amyloliquefaciens; Bacillus
atrophaeus; Bacillus azotoformans; Bacillus brevis; Bacillus
cereus; Bacillus circulans; Bacillus clausii; Bacillus coagulans;
Bacillus firmus; Bacillus flexus; Bacillus fusiformis; Bacillus
globisporus; Bacillus glucanolyticus; Bacillus infermus; Bacillus
laevolacticus; Bacillus licheniformis; Bacillus marinus; Bacillus
megaterium; Bacillus mojavensis; Bacillus mycoides; Bacillus
pallidus; Bacillus parabrevis; Bacillus pasteurii; Bacillus
polymyxa; Bacillus popiliae; Bacillus pumilus; Bacillus sphaericus;
Bacillus subtilis; Bacillus thermoamylovorans; or Bacillus
thuringiensis. 7. The method of paragraphs 1-6, wherein the
membrane is subjected to a strain of Bacillus amyloliquefaciens or
Bacillus subtilis. 8. The method of any of paragraphs 1-7, wherein
the membrane is subjected to a strain of Brevibacillus spp., e.g.,
Brevibacillus brevis; Brevibacillus formosus; Brevibacillus
laterosporus; or Brevibacillus parabrevis. 9. The method of any of
paragraphs 1-8, wherein the membrane is subjected to a strain of
Paenibacillus spp., e.g., Paenibacillus alvei; Paenibacillus
amylolyticus; Paenibacillus azotofixans; Paenibacillus cookii;
Paenibacillus macerans; Paenibacillus polymyxa; or Paenibacillus
validus. 10. The method of any of paragraphs 1-9, wherein the
membrane is subjected to a strain of Rhodococcus spp., e.g.,
Rhodococcus coprophilus; Rhodococcus erythropolis; Rhodococcus
marinonascens; Rhodococcus rhodochrous; Rhodococcus ruber; or
Rhodococcus zopfii. 11. The method of any of paragraphs 1-10,
wherein the membrane is subjected to a strain of Escherichia spp.,
e.g., Escherichia albertii; Escherichia blattae; Escherichia coli;
Escherichia fergusonii; Escherichia hermannii; or Escherichia
vluneris. 12. The method of any of paragraphs 1-11, wherein the
membrane is subjected to a strain of Enterobacter spp., e.g.,
Enterobacter cloacae; Enterobacter dissolvens; Enterobacter
gergoviae; Enterobacter nimipressuralis; or Enterobacter pyrinus.
13. The method of any of paragraphs 1-12, wherein the membrane is
subjected to a strain of Citrobacter spp., e.g., Citrobacter
amalonaticus; Citrobacter koseri; or Citrobacter freundii. 14. The
method of any of paragraphs 1-13, wherein the membrane is subjected
to a strain of Salmonella spp., e.g., Salmonella bongori; or
Salmonella enterica. 15. The method of any of paragraphs 1-14,
wherein the membrane is subjected to a strain of Penicillium spp.,
e.g., Penicillium aurantiogriseum; Penicillium bilaiae; Penicillium
camemberti; Penicillium candidum; Penicillium chrysogenum;
Penicillium claviforme; Penicillium commune; Penicillium crustosum;
Penicillium digitatum; Penicillium expansum; Penicillium
funiculosum; Penicillium glabrum; Penicillium glacum; Penicillium
italicum; Penicillium lacussarmientei; Penicillium marneffei;
Penicillium purpurogenum; Penicillium roqueforti; Penicillium
stoloniferum; Penicillium ulaiense; Penicillium verrucosum; or
Penicillium viridicatum. 16. The method of any of paragraphs 1-15,
wherein the improved flux allows for the use of a membrane
apparatus with a smaller cross-sectional area while maintaining
required optimal wastewater flow and volume as provided by the
former larger system. 17. The method of any of paragraphs 1-16,
wherein the improved flux allows for the use of a membrane having a
smaller membrane surface area. 18. The method of any of paragraphs
1-17, wherein the membrane is part of a membrane bioreactor system.
19. The method of any of paragraphs 1-18, wherein the process is a
water treatment process. 20. The method of paragraph 19, wherein
the water treatment process is a wastewater treatment process. 21.
The method of any of paragraphs 1-20, wherein the one or more
microorganisms are capable of preventing or reducing biofilm
formation through quorum sensing inhibition. 22. The method of any
of paragraphs 1-21, wherein the one or more bacterial strains are
capable of preventing or reducing biofilm formation through quorum
sensing inhibition. 23. The method of any of paragraphs 1-22,
wherein the one or more bacterial strains are selected from strains
of the genus Bacillus. 24. The method of paragraph 6, wherein the
one or more strains of Bacillus are selected from the group
consisting of:
[0195] the Bacillus megaterium strain having the deposit accession
number ATCC 14581;
[0196] the Bacillus pumilus strain having the deposit accession
number ATCC 700385;
[0197] the Paenibacillus azotofixans strain having the deposit
accession number ATCC 35681;
[0198] the Bacillus licheniformis strain having the deposit
accession number NRRL B-50014;
[0199] the Bacillus licheniformis strain having the deposit
accession number NRRL B-50015;
[0200] the Bacillus pumilus strain having the deposit accession
number NRRL B-50016;
[0201] the Bacillus subtilis strain having the deposit accession
number ATCC 6051A;
[0202] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50017;
[0203] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50018;
[0204] the Bacillus subtilis strain having the deposit accession
number NRRL B-50136;
[0205] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50141;
[0206] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50304;
[0207] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50349;
[0208] the Bacillus megaterium strain having the deposit accession
number PTA-3142;
[0209] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7541;
[0210] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7542;
[0211] the Bacillus atrophaeus strain having the deposit accession
number PTA-7543;
[0212] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7544;
[0213] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7545;
[0214] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7546;
[0215] the Bacillus subtilis strain having the deposit accession
number PTA-7547;
[0216] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7549;
[0217] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7790;
[0218] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7791;
[0219] the Bacillus atrophaeus strain having the deposit accession
number PTA-7792; and
[0220] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7793; or a mixture of two or more of the
strains.
25. The method of any of paragraphs 1-24, wherein the one or more
bacterial strains are introduced to the membrane at a final
concentration of 1.times.10.sup.3-1.times.10.sup.10 CFU/ml. 26. The
method of any of paragraphs 1-25, wherein the one or more bacterial
strains are introduced to the membrane at a final concentration of
1.times.10.sup.4-1.times.10.sup.11 CFU/cm.sup.2. 27. The method of
any of paragraphs 1-26, wherein the membrane is subjected to one or
more bacterial strains for about 1 minute to about 2 days before
the membrane is subjected to the process that the membrane is used
in. 28. The method of any of paragraphs 18-27, wherein the membrane
bioreactor is a submerged or immersed process configuration. 29.
The method of any of paragraphs 18-29, wherein the wastewater is
from an industrial or agricultural process. 30. A method of
increasing critical flux of a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane. 31. The method of paragraph 30, wherein
microorganisms includes one or more bacterial strains, capable of
reducing or preventing undesirable biofilm formation on the
membrane. 32. The method of paragraph 30, wherein the one or more
microorganisms are spore forming microorganisms capable of reducing
or preventing undesirable biofilm formation on the membrane. 33.
The method of paragraph 31, wherein the one or more bacterial
strains are spore forming bacterial strains capable of reducing or
preventing undesirable biofilm formation on the membrane. 34. The
method of paragraph 30, wherein microorganisms includes one or more
bacterial strains, one or more fungal strains, or a mixture of one
or more bacterial and fungal strains capable of reducing or
preventing undesirable biofilm formation on the membrane. 35. The
method of paragraph 30, wherein the membrane is part of a membrane
bioreactor system. 36. The method of paragraph 30 or 35, wherein
the process is a water treatment process. 37. The method of
paragraph 36 wherein the water treatment process is a wastewater
treatment process. 38. The method of any of paragraphs 30-37,
wherein the one or more microorganisms are capable of preventing or
reducing biofilm formation through quorum sensing inhibition. 39.
The method of any of paragraphs 30-38, wherein the one or more
bacterial strains are selected from strains of the genus Bacillus.
40. The method of any of paragraphs 30-39, wherein the one or more
bacterial strains are introduced to the membrane at a final
concentration of 1.times.10.sup.3-1.times.10.sup.10 CFU/ml. 41. The
method of any of paragraphs 30-40, wherein the one or more
bacterial strains are introduced to the membrane at a final
concentration of 1.times.10.sup.4-1.times.10.sup.11 CFU/cm.sup.2.
42. The method of any of paragraphs 30-41, wherein the membrane is
subjected to one or more bacterial strains for about 1 minute to
about 2 days before the membrane is subjected to the process. 43.
The method of any of paragraphs 30-42, wherein the membrane
bioreactor is a submerged or immersed process configuration. 44.
The method of any of paragraphs 30-43, wherein the wastewater is
from an industrial or agricultural process. 45. A method of
reducing or preventing fouling of a membrane used in a process,
comprising subjecting the membrane to one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane. 46. The method of paragraph 45, wherein
microorganisms includes one or more bacterial strains capable of
reducing or preventing undesirable biofilm formation on the
membrane. 47. The method of paragraph 45, wherein the one or more
microorganisms are spore forming microorganisms capable of reducing
or preventing undesirable biofilm formation on the membrane. 48.
The method of paragraph 46, wherein the one or more bacterial
strains are spore forming bacterial strains capable of reducing or
preventing undesirable biofilm formation on the membrane. 49. The
method of paragraph 45, wherein microorganisms includes one or more
bacterial strains, one or more fungal strains, or a mixture of one
or more bacterial and fungal strains capable of reducing or
preventing undesirable biofilm formation on the membrane. 50. The
method of paragraph 45, wherein the membrane is part of a membrane
bioreactor system. 51. The method of paragraph 45 or 50, wherein
the process is a water treatment process. 52. The method of
paragraph 51, wherein the water treatment process is a wastewater
treatment process. 53. The method of any of paragraphs 45-52,
wherein the one or more microorganisms are capable of preventing or
reducing biofilm formation through quorum sensing inhibition. 54.
The method of any of paragraphs 45-53, wherein the one or more
bacterial strains are capable of preventing or reducing biofilm
formation through quorum sensing inhibition. 55. The method of any
of paragraphs 45-54, wherein the one or more bacterial strains are
selected from strains of the genus Bacillus. 56. The method of any
of paragraphs 45-55, wherein the one or more bacterial strains are
introduced to the membrane at a final concentration of
1.times.10.sup.3-1.times.10.sup.10 CFU/ml. 57. The method of any of
paragraphs 45-56, wherein the one or more bacterial strains are
introduced to the membrane at a final concentration of
1.times.10.sup.4-1.times.10.sup.11 CFU/cm.sup.2. 58. A method of
improving the permeability of a membrane used in a process or the
flux through a membrane used in a process, comprising adding to the
membrane one or more microorganisms capable of reducing or
preventing undesirable biofilm formation on the membrane. 59. A
method of increasing critical flux of a membrane used in a process,
comprising adding to the membrane one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane. 60. A method of reducing or preventing fouling of a
membrane used in a process, comprising adding to the membrane one
or more microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane. 61. The method of
any of paragraphs 45-60, wherein the membrane is subjected to one
or more bacterial strains for about 1 minute to about 2 days before
the membrane is subjected to the process. 62. The method of any of
paragraphs 45-61, wherein the membrane bioreactor is a submerged or
immersed process configuration. 63. The method of any of paragraphs
45-62, wherein the wastewater is from an industrial or agricultural
process. 64. A method of improving MBR system capacity comprising a
method of any of paragraphs 1-63. 65. A method for reducing the
membrane surface area of an MBR system comprising a method of any
of paragraphs 1-64. 66. A method for reducing the cost of
manufacturing a MBR system comprising a method of any of paragraphs
1-65. 67. A method for reducing the number of membranes within a
MBR system comprising a method of any of paragraphs 1-66. 68. A
composition for the use in membrane filtration systems comprising
one or more microorganisms capable of reducing or preventing
undesirable biofilm formation, and one or more additional
ingredients. 69. The composition of paragraph 68, wherein
microorganisms includes one or more bacterial strains capable of
reducing or preventing undesirable biofilm formation. 70. The
composition of paragraph 68, wherein the one or more microorganisms
are spore forming microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane. 71. The composition
of paragraph 68, wherein the one or more bacterial strains are
spore forming bacterial strains capable of reducing or preventing
undesirable biofilm formation on the membrane. 72. The composition
of paragraph 68, wherein microorganisms includes one or more
bacterial strains, one or more fungal strains, or a mixture of one
or more bacterial and fungal strains capable of reducing or
preventing undesirable biofilm formation. 73. The composition of
paragraphs 1-72, wherein the microorganisms are capable of
preventing or reducing biofilm formation through quorum sensing
inhibition. 74. The composition of paragraphs 1-73, wherein the
microorganisms are capable of preventing or reducing biofilm
formation by converting L-tyrosine to D-tyrosine through the
expression of one or more racemases. 75. The composition of
paragraphs 1-74, wherein the microorganisms are capable of
preventing or reducing biofilm formation by converting L-tyrosine
to D-tyrosine through the expression of a ylmE racemase. 76. The
composition of paragraphs 1-75, wherein the microorganisms are
capable of preventing or reducing biofilm formation by converting
L-tyrosine to D-tyrosine through the expression of a racX racemase.
77. The composition of paragraphs 1-76, wherein the one or more
additional ingredients includes surfactants, enzymes, or a
combination thereof. 78. A filtration system comprising:
[0221] an inlet coupled to an outlet having at least one membrane
disposed therebetween; and
[0222] one or more microorganisms, wherein the one or more
microorganisms selected for addition to the filtration system are
one or more microorganisms capable of reducing or preventing
undesirable biofilm formation on the membrane.
79. The system of paragraph 78, wherein the membrane is a flat
sheet microfiltration membrane. 80. The system of any of paragraphs
78-79, wherein the membrane is a polyvinylidene fluoride (PVDF)
membrane or a polyethylsulphone (PES) membrane. 81. The system of
paragraph 79-80, wherein the membrane is part of a membrane
bioreactor system. 82. The system of any of paragraphs 79-81,
wherein the membrane bioreactor is a submerged or immersed system
configuration. 83. The system of paragraph 79-82, wherein the
system is a water treatment system. 84. The system of paragraph
79-83, wherein the water treatment system is a wastewater treatment
system. 85. The system of any of paragraphs 79-84, wherein the
wastewater is from an industrial or agricultural process. 86. The
system of paragraphs 79-85, wherein the one or more microorganisms
capable of reducing or preventing undesirable biofilm formation on
the membrane decreases the total surface area of the membranes
necessary for the operation of the system. 87. The system of
paragraph 79-86, wherein the one or more microorganisms capable of
reducing or preventing undesirable biofilm formation on the
membrane decreases the total number of membranes necessary for the
operation of the system. 88. The system of paragraphs 79-87,
wherein microorganisms includes one or more bacterial strains,
capable of reducing or preventing undesirable biofilm formation on
the membrane. 89. The system of paragraphs 79-88, wherein the one
or more microorganisms are spore forming microorganisms capable of
reducing or preventing undesirable biofilm formation on the
membrane. 90. The system of paragraphs 79-89, wherein the one or
more bacterial strains are spore forming bacterial strains capable
of reducing or preventing undesirable biofilm formation on the
membrane. 91. The system of paragraphs 79-90, wherein
microorganisms includes one or more bacterial strains, one or more
fungal strains, or a mixture of one or more bacterial and fungal
strains capable of reducing or preventing undesirable biofilm
formation on the membrane. 92. The system of paragraphs 79-91,
wherein the includes a strain of Bacillus spp., e.g., Bacillus
amyloliquefaciens; Bacillus atrophaeus; Bacillus azotoformans;
Bacillus brevis; Bacillus cereus; Bacillus circulans; Bacillus
clausii; Bacillus coagulans; Bacillus firmus; Bacillus flexus;
Bacillus fusiformis; Bacillus globisporus; Bacillus glucanolyticus;
Bacillus infermus; Bacillus laevolacticus; Bacillus licheniformis;
Bacillus marinus; Bacillus megaterium; Bacillus mojavensis;
Bacillus mycoides; Bacillus pallidus; Bacillus parabrevis; Bacillus
pasteurii; Bacillus polymyxa; Bacillus popiliae; Bacillus pumilus;
Bacillus sphaericus; Bacillus subtilis; Bacillus thermoamylovorans;
or Bacillus thuringiensis. 93. The system of paragraphs 79-92,
wherein the membrane includes a strain of Bacillus
amyloliquefaciens or Bacillus subtilis. 94. The system of
paragraphs 79-93, wherein the system includes a strain of
Brevibacillus spp., e.g., Brevibacillus brevis; Brevibacillus
formosus; Brevibacillus laterosporus; or Brevibacillus parabrevis.
95. The system of paragraphs 79-94, wherein the system includes a
strain of Paenibacillus spp., e.g., Paenibacillus alvei;
Paenibacillus amylolyticus; Paenibacillus azotofixans;
Paenibacillus cookii; Paenibacillus macerans; Paenibacillus
polymyxa; or Paenibacillus validus. 96. The system of paragraphs
79-95, wherein the system includes a strain of Rhodococcus spp.,
e.g., Rhodococcus coprophilus; Rhodococcus erythropolis;
Rhodococcus marinonascens; Rhodococcus rhodochrous; Rhodococcus
ruber, or Rhodococcus zopfii. 97. The system of paragraphs 79-96,
wherein the system includes a strain of Escherichia spp., e.g.,
Escherichia albertii; Escherichia blattae; Escherichia coli;
Escherichia fergusonii; Escherichia hermannii; or Escherichia
vluneris. 98. The system of paragraphs 79-97, wherein the system
includes a strain of Enterobacter spp., e.g., Enterobacter cloacae;
Enterobacter dissolvens; Enterobacter gergoviae; Enterobacter
nimipressuralis; or Enterobacter pyrinus. 99. The system of
paragraphs 79-98, wherein the system includes a strain of
Citrobacter spp. e.g., Citrobacter amalonaticus; Citrobacter
koseri; or Citrobacter freundii. 100. The system of paragraphs
79-99, wherein the system includes a strain of Salmonella spp.,
e.g., Salmonella bongori; or Salmonella enterica. 101. The system
of paragraphs 79-100, wherein the system includes a strain of
Penicillium spp., e.g., Penicillium aurantiogriseum; Penicillium
bilaiae; Penicillium camemberti; Penicillium candidum; Penicillium
chrysogenum; Penicillium claviforme; Penicillium commune;
Penicillium crustosum; Penicillium digitatum; Penicillium expansum;
Penicillium funiculosum; Penicillium glabrum; Penicillium glacum;
Penicillium italicum; Penicillium lacussarmientei; Penicillium
mameffei; Penicillium purpurogenum; Penicillium roqueforti;
Penicillium stoloniferum; Penicillium ulaiense; Penicillium
verrucosum; or Penicillium viridicatum. 102. The system of
paragraph 92, wherein the one or more strains of Bacillus are
selected from the group consisting of:
[0223] the Bacillus megaterium strain having the deposit accession
number ATCC 14581;
[0224] the Bacillus pumilus strain having the deposit accession
number ATCC 700385;
[0225] the Paenibacillus azotofixans strain having the deposit
accession number ATCC 35681;
[0226] the Bacillus licheniformis strain having the deposit
accession number NRRL B-50014;
[0227] the Bacillus licheniformis strain having the deposit
accession number NRRL B-50015;
[0228] the Bacillus pumilus strain having the deposit accession
number NRRL B-50016;
[0229] the Bacillus subtilis strain having the deposit accession
number ATCC 6051A;
[0230] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50017;
[0231] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50018;
[0232] the Bacillus subtilis strain having the deposit accession
number NRRL B-50136;
[0233] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50141;
[0234] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50304;
[0235] the Bacillus amyloliquefaciens strain having the deposit
accession number NRRL B-50349;
[0236] the Bacillus megaterium strain having the deposit accession
number PTA-3142;
[0237] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7541;
[0238] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7542;
[0239] the Bacillus atrophaeus strain having the deposit accession
number PTA-7543;
[0240] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7544;
[0241] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7545;
[0242] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7546;
[0243] the Bacillus subtilis strain having the deposit accession
number PTA-7547;
[0244] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7549;
[0245] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7790;
[0246] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7791;
[0247] the Bacillus atrophaeus strain having the deposit accession
number PTA-7792; and
[0248] the Bacillus amyloliquefaciens strain having the deposit
accession number PTA-7793; or a mixture of two or more of the
strains.
[0249] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
Sequence CWU 1
1
12118DNABacillus subtilis 1ggattaacga gggccaac 18240DNABacillus
subtilis 2agaattgatc tgcggcacat atcttgctta tcaaagctag
40340DNABacillus subtilis 3ataagcaaga tatgtgccgc agatcaattc
tgataattac 40437DNABacillus subtilis 4atcgacctcg ccgtttatag
gtcgagatca gggaatg 37537DNABacillus subtilis 5cattccctga tctcgaccta
taaacggcga ggtcgat 37618DNABacillus subtilis 6tgcagcatat catggcgt
18718DNABacillus subtilis 7tattggggag gaagttgg 18840DNABacillus
subtilis 8tttcacaatt tgtctacagc gtaaattatc aacaacacgc
40940DNABacillus subtilis 9ttgttgataa tttacgctgt agacaaattg
tgaaaggatg 401040DNABacillus subtilis 10actaacgatg ccactaatat
taataaacta tcgaaggaac 401140DNABacillus subtilis 11tagtttatta
atattagtgg catcgttagt cggaaatgaa 401221DNABacillus subtilis
12cttcaatcag catttggaaa c 21
* * * * *