Microbial enhanced oil recovery method
10227853 ยท 2019-03-12
Assignee
Inventors
- William J. Kohr (Gig Harbor, WA, US)
- David J. Galgoczy (San Francisco, CA, US)
- Zhaoduo Zhang (Pleasanton, CA, US)
Cpc classification
C12P7/6463
CHEMISTRY; METALLURGY
C12P19/06
CHEMISTRY; METALLURGY
International classification
C12N1/26
CHEMISTRY; METALLURGY
C12P7/64
CHEMISTRY; METALLURGY
Abstract
The present invention provides methods for increasing the viscosity of the drive fluid for displacing oil from a subterranean formation by the use of microorganisms selected or modified for the ability to produce cell free polymers without the formation of any significant bioplugging biofilm or capsule.
Claims
1. A method of enhancing oil recovery, the method comprising: (a) introducing into an oil reservoir a microorganism capable of growing in an environment of a waterflood drive fluid; wherein said oil reservoir comprises indigenous bioplug-forming microorganisms; and wherein said microorganism is genetically modified (i) to be deficient in its ability to produce a flow-restricting bioplug and (ii) to produce soluble cell free biopolymers in situ within the oil reservoir; and (b) water-flooding said reservoir with a waterflood drive fluid having a pH or a temperature that kills or suppresses the growth of said indigenous bioplug-forming microorganisms to reduce the concentration of said indigenous bioplug-forming microorganisms in the oil reservoir.
2. The method of claim 1 wherein the oil reservoir is selected from the group consisting of underground reservoirs, producing wells, non-producing wells, experimental wells, exploratory wells, oil sands and other sources of heavy oil.
3. The method of claim 1 wherein the microorganism is an archaeon or a bacterium.
4. The method of claim 1 wherein the microorganism is present in a culture of microorganisms comprising a plurality of microorganisms that are genetically modified to be deficient in their ability to produce said flow-restricting bioplug, and to produce said soluble cell free biopolymers in situ within the oil reservoir.
5. The method of claim 4 wherein said microorganism is present in a culture of microorganisms comprising a plurality of microorganisms that are able to produce surfactants.
6. The method of claim 1 wherein said microorganism is able to produce surfactants.
7. The method of claim 1 wherein said microorganism is able to utilize simple carbons selected from the group comprising glucose, sucrose, mannose, starch, glycerin, organic acids, and other simple sugars.
8. The method of claim 1 wherein said soluble cell free biopolymers comprise a polysaccharide.
9. The method of claim 1 wherein said microorganism is further characterized by (i) containing functional genes for carbohydrate metabolism and conversion of carbohydrates into high molecular weight polysaccharides; (ii) lacking functional genes for formation of a biofilm; (iii) containing functional genes for production of surfactants; and (iv) being regulated to express the functional genes of steps (i) and (iii), and grow in the waterflood drive fluid.
10. The method of claim 1 further comprising a step of injecting a nutrient mixture into said reservoir.
11. The method of claim 1, wherein said microorganism is further naturally deficient in its ability to produce said flow restricting bioplug.
12. The method of claim 1, wherein said microorganism is selected for its ability to penetrate 0.2 m filters or sandstone cores of 50 mD or less permeability.
13. The method of claim 1, wherein the pH of the waterflood drive fluid is alkaline.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1)
(2)
(3)
DETAILED DESCRIPTION OF THE INVENTION
(4) Definitions
(5) Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
(6) It must be noted that as used herein and in the appended claims, the singular forms a, and, and the include plural referents unless the context clearly dictates otherwise.
(7) Throughout this specification and claims, the word comprise, or variations such as comprises or comprising, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
(8) All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Publications cited herein are cited for their disclosure prior to the filing date of the present application. Nothing here is to be construed as an admission that the inventors are not entitled to antedate the publications by virtue of an earlier priority date or prior date of invention. Further the actual publication dates may be different from those shown and require independent verification.
(9) The term biofilm means a complex structure comprising biopolymers, proteins, DNA and bacteria in a biomass layer of microorganisms. Biofilms are often embedded in extracellular polymers, which adhere to surfaces submerged in, or subjected to, aquatic environments. Biofilms consist of a matrix of a compact mass of microorganisms with structural heterogeneity, which may have genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances.
(10) The term plugging biofilm means a biofilm that is able to alter the permeability of a porous material, and thus retard the movement of a fluid through a porous material that is associated with the biofilm.
(11) The term bioplugging refers to making permeable material less permeable due to the biological activity, particularly by a microorganism.
(12) The term oil reservoir is used herein in the broadest sense and includes all forms of hydrocarbon deposits, including, without limitation, underground reservoirs, producing wells, non-producing wells, experimental wells, exploratory wells, oil sands and other sources of heavy oil and the like, which may be accessible by any means, such as, for example, one or more wellbores.
(13) The term crude oil refers to a naturally occurring, flammable liquid found in rock formations and comprises a complex mixture of hydrocarbons of various molecular weights, plus other organic compounds. Without limitation, the crude oil may contain, for example, a mixture of paraffins, aromatics, asphaltenes, aliphatic, aromatic, cyclic, polycyclic and/or polyaromatic hydrocarbons. The crude oil may be generic or may be from a reservoir targeted for enhanced oil recovery in accordance with the present invention.
(14) The terms well and reservoir may be used herein interchangeably and refer to a subterranean or seabed formation from which oil may be recovered. The terms well and reservoir include the physical/chemical composition of the soil-rock-sediment structure of the reservoir below the surface.
(15) The term environmental sample means any substance exposed to hydrocarbons, including a mixture of water and oil comprising microorganisms. As used herein, environmental samples include water and oil samples that comprise indigenous microorganisms and/or populations of microorganisms of varying genus and species. The environmental samples may comprise a microbial consortium unique to a geographic region or target reservoir, or, alternatively the microbial consortium may be adaptable to other environment sites, geographics and reservoirs.
(16) The terms microorganism and microbe are used interchangeably and in the broadest sense, including all types of microorganisms, including bacteria, fungi, archaea, and protists, and microscopic animals, such as plankton, planarian and amoeba. Preferred microbes for the purpose of the present invention are bacteria and archaea.
(17) The term microbial consortium is used herein to refer to multiple interacting microbial populations. Members of a consortium communicate with one another. Whether by trading metabolites or by exchanging dedicated molecular signals, each population or individual detects and responds to the presence of others in the consortium. This communication enables a division of labor within the consortium. The overall output of the consortium rests on a combination of tasks performed by constituent individuals or sub-populations.
(18) Archaea comprise one of the three distinct domains of life, with bacteria and eukaryotes. For a review, see, e.g. Makarove and Koonin, Genome Biology 4:115 (2003).
(19) The term halophile is used herein to refer to an extremophile that thrives in environments with very high concentrations, typically at least about 5% (50,000 ppm), or at least about 10%, or at least about 15% of salt.
(20) The term obligatory halophile is used herein to refer to an extremophile whose growth is obligatory dependent on high salt concentrations, typically at least about 5% (50,000 ppm), or at least about 10%, or at least about 15% of salt.
(21) The terms repression and inhibition with reference to gene expression are used herein interchangeably and refer to any process which results in a decrease in production of a gene product, regardless of the underlying mechanism. A gene product can be either RNA or protein. Gene repression includes processes which decrease transcription of a gene and/or translation of mRNA. Thus, specifically included in this definition are processes that inhibit the formation of a transcription initiation complex along with those that decrease transcription rates and those that antagonize transcriptional activation is gene repression. These repressions can be either reversible or irreversible, both of which are specifically included in this definition.
(22) The term lateral gene transfer is used herein in the broadest sense and refers to the transmission of genetic information from one genome to another.
(23) The term surfactant as used herein means microbially produced surface-active agents, including, but not limited to, glycolipids (e. g. sophorose lipid or rhamnose lipid), lipoproteins, polysaccharide-fatty acid complexes, mono- and diglycerides, lipoheteropolysaccharides, peptidolipids, neutral lipids, corynomycolic acids, trehalose dimycolates and polysaccharide-protein complexes.
(24) The term hydrocarbon is used herein in the broadest sense to describe any organic compound that contains only carbon and hydrogen. The term specifically includes, without limitation, straight and branched chained saturated hydrocarbons (alkanes), straight and branched chained unsaturated hydrocarbons (including alkenes and alkynes), cycloalkanes, and aromatic hydrocarbons (arenes).
(25) A short chained alkane, as defined herein, contains 1 to 4 carbon atoms.
(26) A high molecular weight hydrocarbon, as defined herein, is a hydrocarbon having at least about 40 carbons, for example, a hydrocarbon having between about 40 and about 60, or between about 40 and about 80, or between about 40 and about 100, or between about 40 and about 120 carbons.
DETAILED DESCRIPTION
(27) A process is disclosed for better and more reliable microbial enhanced oil recovery (MEOR) that makes use of a species of microorganism or group of microorganisms that produces little or no biofilm. In the preferred mode, the selected microbes remain in the planktonic state, but still produce soluble (non cell attached) exopolymer. Biofilms, which are often comprised of significant amounts of multiple types of polymer, are inhibited or prevented from being formed. The synthesis of biopolymers is highly regulated because bacterial survival in a natural wild environment will be compromised if an unnecessary expenditure of metabolic energy synthesizing such large molecules occurs at a time, for example, when energy must be directed to growth. Moreover, the regulation of exopolysaccharide synthesis and assembly is very complex because a large number of proteins are required to create and export these macromolecules. A typical biofilm is comprised of several individually produced biopolymers. In addition, the complex structure of the biofilm may require proteins and DNA polymers that strengthen the structure and facilitate the attachment to the rock surface. Each of these biofilm elements are coded for and controlled by groups of sequences of DNA or genes in the cell's genome. Therefore, by a number of gene deletions or changes, biofilm producing microorganisms that may have been used for MEOR in prior art methods can now be converted to a new strain of microorganism with attenuated biofilm formation capabilities. Furthermore, by use of a laboratory screening method, each of these gene and gene cluster changes can be evaluated for its effect on the cells' ability to increase viscosity of the media, adherence to rock surfaces, or formation of a biofilm that plugs porous material such as a sand pack column or a reservoir core sample.
(28) A useful step in the selection of a wild-type, randomly mutated or engineered site-directed mutations deletions and gene additions for improved oil recovery (IOR) is a fast laboratory scale screening test. In a preferred mode, the screening assay selects for soluble polymer production, migration of cells through porous media, lack of attachment to solid surfaces and lack of biofilm formation. A preferred screening test selects cells that have undergone change in phenotype related to biofilm/bioplugging and soluble biopolymer production. Preferably, the screening test is fast and able to screen a large number of mutants. It is expected that relatively few altered cells will be able to grow, produce soluble biopolymer and not produce a full strength biofilm.
(29) One commonly used laboratory method is to spread a small volume of bacteria out on an agar plate so that each colony formed is the result of a single cell. Cell colonies that are producing extracellular polymers (mucoid) will have a different phenotype (domed and shining or glistening) than those cells that do not produce extracellular polymers (non-mucoid and dense). Colonies of biofilm forming cells also have a wrinkled appearance and grow to more height than non-biofilm forming microorganisms (see Aamir Ghafoor, et al. (2011) AEM 77(15):5238-5246). By this method or similar colonies screening methods known to those skilled in the art of microbiology, one can isolate a few single strain colonies that can produce extracellular polysaccharides and not lull biofilms from hundreds or thousands of single cell colonies.
(30) In this mode microbes are selected or are modified at the genetic level to reduce their ability to build a biofilm or to form a bioplug in porous material. A new and non-bioplugging microbe that can grow without producing a biofilm allows for penetration of the microbe deeper into the reservoir than previously reported for biofilm capable microbes. This is an advantage for its use in MEOR because of its ability to penetrate more deeply will still in active growth. The non-bioplugging microbe is selected or genetically modified such that soluble and cell free polymers are continually formed or regulated in its production within the reservoir. In situ polymer production will improve mobility control by increasing the viscosity of the drive fluid as it becomes diluted by the resident water within the reservoir.
(31) An important element of this invention is production of cell free and soluble polymers within the reservoir and not the production of a biofilm or capsule which can unduly restrict the flow of fluid into the formation. The benefits of production of biopolymers within a reservoir are described in MEOR prior art, but in the context of biofilms/bioplugging. Most microorganisms that can form a biofilm will also make cell free and soluble polysaccharides. However, the soluble polysaccharides and biopolymers can become associated with the biofilm structure. In that mode, the bound biopolymers are not as effective at increasing the viscosity of the drive fluid. In the preferred mode the soluble polymers flow just ahead of the bacteria with the drive fluid to displace the oil and form an oil bank (a zone of displaced oil at the front edge of the waterflood). The invention presented here makes a distinction between biopolymer formation in biofilms that can result in plugging and the decrease of flow in a high flow zone and the production of soluble (cell-free) biopolymer for increase of drive fluid viscosity.
(32) From a practical standpoint, it may be difficult to inject sufficient quantities of actively growing microorganisms into a formation to effect appreciable change. Sand filters are highly efficient in removing polysaccharide producing microorganisms in water purification plants, and the sand in a typical reservoir formation would probably filter out a large portion of the microorganisms at or near the formation face at the wellbore. However, microorganisms that are spores or are attenuated in the ability to form a biofilm and have increased motility due to lack of attached biofilm are more likely to penetrate deeper into a reservoir than biofilm forming contaminating microorganisms. Therefore microorganisms that have been selected or mutated to not produce a biofilm have an advantage in that they can penetrate further into a reservoir while being given sufficient quantities of nutrient for growth. For example, it is believed that the first step in the transition from motile or planktonic state to a biofilm is an attachment to a solid surface. Therefore, some of the genes that are needed for the change from a planktonic state to a sessile state code for proteins or biopolymers that mediate attachment to solid surfaces. If these genes are inactivated a mutant form of the microorganism could be less able to attach to the sand surface and would stay in the supernatant or flow through a small sand pack column. The propensity of individual cells to adhere to solid surfaces can be screened in a fast and high through-put mode by a small scale mixing with sand or passing through a small sand pack column. This method or a procedure similar to this could be used to enrich for non-adherent mutants.
(33) However, indigenous microorganisms that are biofilm producers can pose a problem in the method of the present invention. The injection of new fluids may cause introduced growth due to the injection of growth nutrients. Oil-degrading microorganisms are capable of sustaining a minimal population in most underground petroleum formations but do not flourish. Crude oil provides adequate carbon and energy, but is nutritionally deficient in both nitrogen and phosphorus. Most microorganisms are known to occur in microbial populations which are in a state of starvation. In the case of microorganisms existing in a subterranean hydrocarbon-bearing formation, the nutritional deficiencies are primarily nitrogen- and phosphorus-containing compounds, not carbonaceous materials. The injection of those compounds in amounts that is nutritionally sufficient for the injected attenuated biofilm soluble biopolymer producing microbial population can result in the growth of the indigenous microorganisms. This is a potential problem because the indigenous microbes are generally able to produce biofilms. The production of a biofilm mass can form a permeability reducing bioplug regardless of whether it is from indigenous microbe or injected microbes. Microbial utilization of crude oil by indigenous microbes can also chemically change the nature of hydrocarbon constituents of the crude oil and thereby change the properties of the oil. The depletion of short chain aliphatic compounds, for example, may increase the viscosity of the oil.
(34) Therefore, to prevent the interference of indigenous microorganisms that are capable of producing plugging biofilms and/or degrading short chain hydrocarbons and increasing the viscosity of the oil, a step can be added to kill or suppress the indigenous microbes. One method of suppressing growth of indigenous microbes that will favor the growth of injected microbes is disclosed in U.S. Pat. No. 8,316,933 (Kohr). Another method is disclosed by in U.S. Patent Application Publication No. 20130062053 (Kohr et al.). Both methods are hereby incorporated by reference. These methods rely on a drive fluid that is chemically different than aqueous fluid that has existed in the oil reservoir prior to oil production. Furthermore, the high salinity, pH or concentration of toxic chemical elements will kill or suppress the growth of the indigenous microorganisms. The objects of those inventions are to give the host or recipient organism a competitive advantage for the specially modified environment of the hydrocarbon resource reservoir before and/or during a waterflood oil recovery process. This process kills indigenous microorganisms that can interfere with the injected microbes. The injected microorganisms are adapted to the injected fluid and benefit from the environmental and chemical changes that result from introducing a new fluid into the oil containing formation. That is, the modified microorganisms are designed to thrive in the new reservoir environment of chemical waterflooding not in the preexisting environment. In a non-limiting example, the new conditions can be a higher pH, salinity, or temperature or a compound that is toxic to most of the indigenous bacteria but that is not toxic to the injected microbes.
(35) For the purposes of this invention, the exact identity of the specific microorganisms is unimportant as long as they have been selected, mutated and/or genetically altered to produce soluble polymers and/or surfactants without the ability to form cell restricting bioplugs and enhanced adhesion to the rock surface. Representative microorganisms, which may be present either singly or in combination, are represented by microorganisms that comprise classes of aerobes, facultative anaerobes, obligate anaerobes and denitrifiers. Various species of microorganisms (bacteria and fungi) that can be used to improve sweep efficiency and enhance oil recovery include, but are not limited to, the genera: Pseudomonas, Bacillus, Geobacillus, Sphingomonas, Actinomycetes, Acinetobacter, Arthrobacter, Halomonas, Diomarina, Schizomycetes, Clostridium, Corynebacteria, Achromobacteria, Arcobacter, Enterobacteria, Nocardia, Saccharomycetes, Schizosaccharomyces, Vibrio, Shewanella, Thauera, Petrotoga, Microbulbifer, Marinobacteria, Fusibacteria, and Rhodotorula. The terms genus and genera, as used herein, refer to the category of microorganisms ranking below a family and above a species in the hierarchy of taxonomic classification of microorganisms. The term species refers to a group of microorganisms that share a high degree of phenotypic, biochemical and genotypic similarities. The oil-degrading microorganisms utilized in the experiments reported herein were isolated from crude oil environments or other environments using conventional microbiological techniques.
(36) Isolation of a small fraction of a diverse population of microorganisms can be done by relying on a phenotypic or functional difference that the subpopulation displays. In one non-limiting example microorganisms are isolated that lack the ability to form a strong adhesion to a solid sand surface. In this example, a strain of Bacillus or Pseudomonas that is known to be capable of forming a biofilm is treated under conditions that are known to produce mutations. A 100 to 500 ml culture of log phase microbes is added to 10 to 50 gm of washed, clean, fine material sand. The mixture is rolled slowly in a roller bottle to allow for good mixing and attachment of cells to the sand surface. Samples of culture solution are removed at several time points, from one hour to three days. Total cell concentration is determined by counting with a microscope and a Petroff-Hausser Counting Chamber (Electron Microscopy Sciences) and or a viable cell concentration is determined by plating out diluted samples on agar plates and subsequently counting colonies. Another method is reported by E. A. Robleto et al. in J. of Bacteriology Vol. 185 No. 2 p 453-460 (2003). In this referenced method bacterial strains grown overnight in LB broth were inoculated into 12 ml of minimal medium broth and grown to an A.sub.530 of 0.4 to 0.5. Cells were centrifuged for 5 min at 6,000 rpm (Sorvall RC5b Plus), and the pellets were washed twice in PBS. Cells were resuspended in 4 ml of PBS and drawn into a syringe containing 12 g of Ottawa sand (Fisher Scientific catalog no. S23-3). An aliquot of the inoculum was used to determine cell density and allow an estimate of percent attachment. The sand column was allowed to equilibrate for 5 min, and the volume contained in the column was estimated by subtracting the amount that drained out of the column in the first 5 min. Columns were incubated for 1 to 1.5 h and washed with 1 equilibration volume. Washes were serially diluted, plated onto LB agar with appropriate antibiotics, and incubated at 28 C. Viable-cell counts from washes were used to estimate percent attachment on the basis of the initial number of viable cells in the inoculums.
(37) By either of these methods, the media suspension or the cells first eluted off the small columns will be enriched in cells that do not attach to the sand particles. Following repeated iterations of this enrichment process, the percentage of cells that do not attach will increase in the media suspension. The culture of enriched non-adherent microorganisms can be plated out on growth agar. Single colonies that show a shiny or glistening, mucoid phenotype, which are believed to be polymer producers, will be selected for further analysis. The selected colonies will be transferred to multi-well microtiterplates (#353047, BD Biosciences) or to test tubes containing growth media to screen for isolates that to not form biofilms or pellicles. The test tubes containing 5 to 10 ml of media will be incubated for four days without shaking as described by Friedman and Kolter 2004 Mol. Microbiol. 51:675-690. Several isolates will be screened in test tubes and microtiter plates to identify those that grow, but do not form pellicles or biofilms.
(38) The invention is further illustrated by the following non-limiting Examples.
Example 1
(39) Screening for Polymer Production
(40) Plate assays are carried out for polymer production, in which cells are treated under mutagenic conditions, plated on rich agar plates, and incubated to allow colonies to form. Colonies are screened for a visible mucoid appearance. (as described in Mathee et al., 1999 Microbiology 145:1349-1357). In an earlier reference Darzins and Chakrabarty (1984) in J. Bacteriol Vol. 164 p 516-524 reported isolating 21 non-mucoid mutants of Pseudomonas aeruginosa strain 8821 by screening 4 thousand colonies that had been mutagenized with EMS. The non-mucoid producing mutants were recognized by their inability to produce a typical mucoid colony on solid media. The 21 colonies were tested by growth on a media with a simple carbohydrate as the only carbon source to make sure that the lack of alginate synthesis was not due to genes controlling all carbohydrate metabolisms. This lead to cloning the genes controlling alginate biosynthesis from mucoid cystic fibrosis isolates of Pseudomonas aeruginosa.
(41) A similar screening procedure can be used to isolate mutants that do not adhere to sand from the above sand adhesion isolation, but still produce soluble polymers to give the colony a mucoid appearance. In this mode mutant strains can be isolated that produce soluble and unattached exopolysaccharides, but that do not form a strong adhesion to a sand surface. These mutants would be useful in moving through the formation with the waterflood drive fluid while producing viscosity increasing soluble polymers. In another mode, this screening procedure could be used to isolate the genes that control attachment of cells to the sand surface and the start of biofilm formation and the genes which may be different that control the production of cell free and unattached soluble polymers. Identifications of these genes can be used to further modify the wild type bioplugging biofilm producing microorganisms into planktonic soluble polymer and or surfactant producing strains by site directed mutagenesis and other gene manipulation techniques.
Example 2
(42) Screening for Biofilm Forming Microorganisms with Glass Beads
(43) Screening of isolated strains for their ability to form biofilms on silicate surfaces under aerobic and anaerobic conditions was disclosed by Keeler et al. in U.S. Pat. No. 8,357,526. Sterile glass beads (3 mm, #11-312A, Fisher Scientific, Hampton, N.H.) were placed into the wells of a 24-well microtiter plate. Aliquots (1.0 mL) of either the Injection Water or the PPGAS medium (20 mM NH. sub. 4Cl, 20 mM KCl, 120 mM Tris-Cl, 1.6 mM MgSO. sub. 4, 1% peptone, 0.5% glucose, pH 7.5) were added to each well. Samples (10 u.L) of overnight microbial cultures were then added, and the plates were incubated at room temperature for up to one week. Glass beads were examined by microscopy directly in the microtiter wells. To quantify the anaerobic formation of biofilms across different strains, single colony isolates were grown anaerobically in 1.0 mL Injection Water supplemented with 1600 ppm sodium nitrate. Silica beads were added into the wells of a 96-well microtiter plate (#353070, BD Biosciences). After eleven days of anaerobic incubation, the beads were removed from the wells, rinsed in sterile water, and transferred to a new microtiter plate. Crystal violet dye (75 u.L, 0.05%) was added to each well, and the plate was incubated at room temperature for 5 min. The dye was then removed by washing each bead (times. 4) with 200 u.L sterile water. To remove the bacteria from the heads and solubilize the remaining dye, 100 u.L of 95% ethanol was added and samples were incubated at room temperature for 20 min with intermittent mixing. Aliquots (10 u.L) were removed and added into 90 u.L sterile water in a new microtiterplate. Absorbance of each sample at OD. sub. 590 was measured in a Victor3 (Perkin Elmer, Waltham, Mass.) plate reader to quantify the dye reflecting the relative concentrations of microorganisms that were attached to the silica beads.
Example 3
(44) Determination and Assay of Amount of Biofilm Formed
(45) Biofilm formation in microtiter plates was determined essentially as described previously (O'Toele, G. A., and R. Kolter. 1998. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28:449-461). Cells were grown in liquid cultures in microtiter plates (0.2 ml) for 18 h in M9Glu/sup at 30 C. The liquid culture was removed, and the cell optical density at 600 nm (OD.sub.600) was determined spectrophotometrically. Cells attached to the microtiter plates were washed with 0.1 M phosphate buffer (pH 7.0) and then stained for 20 min with 1% crystal violet (CV) in ethanol. The stained biofilms were thoroughly washed with water and dried. CV staining was visually assessed, and the microtiter plates were scanned. For semiquantitative determination of biofilms, CV-stained cells were resuspended in 0.2 ml of 70% ethanol, and their absorbance was measured at 600 nm and normalized to the OD.sub.600 of the corresponding liquid culture.
(46) Isolation and purification of extracellular biopolymers can be done by growth of cells to stationary phase followed by centrifugation of cells to form a cell free supernatant that can be precipitated with the slow addition of ethanol to an equal volume of each. After overnight chilling to 18 degrees Celsius, the polymer mixture is centrifuged at 13,000 g for 30 minutes to make a pellet. The pellet can then be washed with cold ethanol and then dissolved in hot water and then dialyzed against distilled water. The lyophilized polymer can be weighed or further purified for analysis.
Example 4
(47) Method of Screening for Microbial Penetration, In-Situ Polymer Production or Biofilm Formation by Means of Sandpack Columns
(48) Small sandpack columns can be used to screen a number of plate isolated genetically modified or wild type bacteria. This method evaluates the strain isolates for their ability to travel through porous media, form a stable biofilm in porous media and increase the viscosity of the transport fluid. The change in pressure at constant flow is used to determine the decrease in permeability caused by bacterial growth. A glass column 8150 mm (#5813 Ace Glass) is fitted with a #7 Ace Glass Teflon end fitting (35801-7 Ace Glass). A small plug is made out of stainless steel wool as a sand filter for each end fitting. The column is filled with fine sand while tapping to compact the sand. The dry-packed column is then filled with water while evacuating with a vacuum to remove any air pockets. The water is pumped into the column at about 0.5 ml per minute by means of a peristaltic pump with a tee connection to a pressure gauge. Sterile media is then pumped into the column to replace the water. After at least two pore volumes of media have been pumped into the column, the pressure and the flow rate are recorded. A culture of microorganisms to be tested is then pumped into the column. The volume is at least one pore volume and the number of cells per ml is estimated by counting with a microscope in a Petroff-Hausser Counting Chamber (Electron Microscopy Sciences) or by plating out dilutions to determine the viable cell count. If the cultures of microorganisms for testing are anaerobes or facultative anaerobes, the columns are allowed to stand without flow for 3 to 15 days to allow for cell growth. After the incubation period, one pore volume of sterile media is pumped into the column. The pressure and flow rate are recorded and used to calculate change in permeability. The volume of liquid replaced from pumping the column with fresh media is collected and analyzed for viscosity and cell concentration (number of cells per ml). This process is repeated with several new pore volumes of media to determine the change in permeability with time and to determine the viscosity and the number of cells in the liquid eluted off of the sand column. The degree of plugging can be determined by the change in pressure with a constant flow rate or by the change in flow rate if flow rate at a constant pressure. If the viscosity of the pore volumes of fluid eluted from the column as measured separately the change in permeability as a result of the cell growth and biofilm formation in the porous media can be determined by Darcy's law. The number of pore volumes that are needed to be pumped through the column to increase the permeability back to the original state is a function of how stable and strong of a bioplug the strain produced. The rate at which the cells are washed out of the column is a function of how adherent the microorganism is to the sand. This method can be used to rate the various strains or mutations in regards to their ability to migrate through porous media, form a strong bioplugging biofilm or increase the viscosity of the drive fluid by producing soluble polymers that are not attached to the sand, cells or biofilm.
(49) In particular, sandpack columns can be used to screen a number of isolated genetically modified, mutated or wild type bacteria. This method evaluates various microbial strains for their ability to aid in oil recovery by one of several mechanisms including: penetrating through porous media, forming a biofilm plug in a highly porous zone or increasing the viscosity of the transport fluid by secreting cell free soluble polymers. Columns can be made of varying permeability by packing a column with sand of various size distributions. The permeability can be decreased by addition of finer sand or by grinding the sand to finer size profile in a Drum Rotary Tumbler (Chicago Electric Power Tools #67632 sold by Harbor Freight Tools). The pressure at constant flow is used to determine the permeability constant in Darcys by the Darcy equation:
Q=kA (pressure drop)/uL
(50) Where Q is flow in milliliter (ml) per second, A is area in square centimeters, L is the length in centimeters, the pressure drop is in atmospheres, u is the viscosity in centipoises. The permeability constant k is in Darcys.
(51) A simple test column is constructed from inch PVC (polyvinyl chloride) schedule 40 tubing which is available at home building supply centers such as Home Depot. To construct a column, the ten foot PVC tube was cut into 5 foot in length and then capped with threaded PVC end caps using PVC glue. This simple column will withstand 20 to 30 atmospheres of pressure. The column can also be cut into small sections for analysis of the packed sand after the microbial elution and plugging tests. A small sand filter is made out of 200 mesh/75 micron stainless steel screen (owfb store on Ebay) and placed into a threaded PVC female end cap which has a threaded Swagelok inch compression fitting. The column is filled first with water, fine sand is poured in while tapping to compact the sand. The threaded end cap is replaced onto the column to prevent the loss of water or sand to prevent voids for forming in the sand packed column. The sandpack column can be used for experiments testing microbial penetration, in-situ polymer generation, the formation of a flow restricting bioplug or biofilm.
(52) Microbial Penetration:
(53) The ability of microorganisms to penetrate porous material is a function of the microbe's size, motility and affinity for the surface of the porous material. The microbe's surface affinity is a function of the surface nature of the porous material, such as clean sand vs. oil covered sand. The cells attraction to the porous material's surface is also a function of the growth state of the microbe from vegetative to starved or sporulated cells. Therefore, various strains of microbes should be compared in the same growth state on columns of similar surface nature and permeability.
(54) Sterile water or brine is pumped into the column at about 0.5 ml per minute by means of a peristaltic pump (Manostat cassette pump model 72-500-000), or a higher pressure pump (Millipore-Waters M-45 lab chromatography pump, HPLC solvent delivery system), with a tee connection to a pressure gauge to measure any change in pressure. Sterile media to maintain the cells in the selected growth state is then pumped into the column to replace the water. After at least one or more volumes of media have been pumped into the column, the pressure and the flow rate are recorded and used to calculate the permeability. A culture of microorganisms to be tested is then pumped into the column. The volume is at least one pore volume and the number of cells per ml is estimated by plating out dilutions to determine the viable cell count. Volumes of column effluent are collected analyzed by plating and colony counting. This method is used to estimate the number of pore volumes needed to elute the cells and also an estimate of the approximate or relative recovery of cell that can pass all the way though the length of sandpack column. In
(55) After eluting the sandpack column with several pore volumes, the number of microbes retained by the sand or oil-sand surface can be estimated by cutting the PVC column into small sections and removing the oil-sand-fluid and bacteria mixture from each section as shown in
(56) Procedure for Quantifying Adhered Biofilm on Sandpack Columns
(57) About 25 grams of sand containing the bacteria, biofilm along with the liquid is removed from each section of the cut PVC sandpack column. The sand mixture is then transferred to a 50 ml tube. 30 ml of brine or water is added to each tube and gently mixed to remove unattached cells. The 30 ml wash process is repeated three times. The first wash is used to plate out viable cells. For oil containing sand, the residual oil can be extracted from the wet sand with 30 ml of toluene. If the sand still contains oil a second toluene extraction can be done. The oil is removed to prevent possible interference with the biofilm assay. After the toluene layer is removed, the sand is placed in an open dish and allowed to dry for about one hour. A fraction of the sand of about 1.0 ml volume is removed and placed in a tube for Crystal Violet biofilm analysis. A 0.5 ml volume of a 0.1% Crystal Violet solution is added to each tube containing sand and gently mixed with the sand. The mixture is incubated for about 15 minutes. After that, 3 ml of water is added to each tube with gentle mixing. After the sand settles out the liquid above the sand is removed. The 3 ml wash process is repeated three times to remove the excess Crystal Violet solution. After as most of the final wash water is removed from the sand in the tube, 3 ml of 95% ethanol is added to each tube to extract the Crystal Violet dye from the biofilm attached to the sand. After vigorous mixing, the ethanol sand mixture was allowed to settle out for 10 minutes. Then 1.0 ml from the liquid layer was removed and the absorbance at 600 nm was measured. The absorbance of the ethanol extracted dye is a function of the amount of biofilm adhered to the sand in each section of the sandpack column. The absorbance of each section can be plotted verses the distance from the injection of bacteria to determine the amount of bio-plugging that occurs near the point of entry or injection.
(58) By these methods of analysis microbial strains can be compared by their ability to penetrate through porous sand without a large percentage of the cells being retained on the sand surface near the point of introduction, or forming a bio-plug near the point of entry known as face plugging. In addition, in the case of culture believed to contain a number of mutated cells, mutants that have lost the ability to adhere to the oil-sand surface will be enriched in the breakthrough volume of eluted cells. Therefore this procedure can be useful to select mutants for deeper penetration into an oil formation without forming a bioplug near the injection well.
(59) In-Situ Production of Cell Free Polymers to Increase the Viscosity of the Drive Fluid:
(60) Microbes that produce and secrete cell free or soluble polymers can increase the viscosity of the fermentation broth. This ability to increase the viscosity of the broth is a function of the genetics of the microbial strain, media and carbon source, the oxygen level and shear force due to mixing. Therefore, to evaluate the ability of microbial strains to increase the viscosity of the drive fluid as it moves through the formation, the growth of microbes should be in a sandpack column or a sandstone core. The PVC sandpack column described can be used to screen strains for this ability. If the cultures of microorganisms for testing are anaerobes or facultative anaerobes, the columns are allowed to stand without flow for 3 to 15 days to allow for cell growth. This will test cell growth and polymer secretion in a static no flow situation. Alternatively a slow flow condition similar to the rate of propagation of drive fluid through a formation can be approximated with several 5 foot columns connected in series, so that the fluid eluting of the end multi-column system has had 5 to 10 days for the growth of microbes and production of polymer as the fluid migrates at one to two feet per day. After the incubation period, a small volume of sterile media is pumped into the column. The pressure and flow rate are recorded and used to calculate the total change in permeability. The production of polymer by the microorganism will increase the pressure when pumping the column. This method can be used to compare various strains to cause plugging of a high permeability zone.
(61) The volume of liquid replaced as a result of pumping the column with fresh media is collected and analyzed for viscosity and cell concentration (number of cells per ml). If microbial cells are present, the fluid can be centrifuged to remove cells so that the viscosity of the cell free polymer can be measured without the presence of cells. A new permeability constant can be calculated by the Darcy equation using the new viscosity of the eluded fluid. If the permeability constant is calculated to be the same, then most of the increase in backpressure is from the increased viscosity from the secreted polymers. If the calculated permeability constant remains lower than the original value of permeability after the viscous fluid is eluted, then the decrease in permeability is the result of microbial growth within the column and the formation of a biofilm. By this means, strains of microbes can be selected for their ability to produce cell free polymer that can increase the viscosity of the drive fluid, or to select strains that form biofilms and block flow to a zone.
(62) The degree of plugging can be determined by the change in pressure with a constant flow rate, or by the change of flow rate at a constant pressure. The viscosity of the pore volumes of fluid eluted from the column as measured separately is a different property of cell growth than the change in permeability as a result of the cell growth and biofilm formation in the porous media. The number of pore volumes that are needed to be pumped through the column to increase the permeability back to the original state is a function of how stable and strong of a bioplug the strain produced. The rate at which the cells are washed out of the column is a function of how adherent the microorganism is to the sand. This method can be used to rate the various strains or mutations in regards to their ability to migrate through porous media, to form a strong bioplugging biofilm, to increase the viscosity of the drive fluid by producing soluble polymers that are not attached to the sand, cells or biofilm.
(63) A similar sandpack column test can be used for aerobic microorganisms. In the aerobic mode a slow flow rate of media is circulated through the column to maintain a higher dissolved oxygen level. Sterile, filtered air is used to aerate the circulating media. Alternatively a mixture of air and liquid can be pumped through the column to maintain a high level of dissolved oxygen. Aerobic microbial oil recovery process have been disclosed by L. W. Jones in U.S. Pat. No. 3,332,487 issued in 1967 and by E. Sunde in U.S. Pat. No. 5,163,510 issued in 1992 and U.S. Pat. No. 6,546,962 issued in 2003. The introduction of air and or oxygen will affect the type of bacteria that predominates the process and the amount of soluble extracellular polysaccharides and the amount of biofilm that is formed. Therefore the microorganisms should be screened and selected for high soluble production without excessive bioplugging under aerobic conditions unless it is desirable to form a stable biofilm or bioplug within a high flow zone to stop the flow of drive fluid into a watered-out or thief zone.
(64) By use of the above screening process with sandpack columns, various strains and mutants of strains or genetically modified strains can be evaluated for the ability to penetrated far into a formation and then alter being over taken by the right media, carbon source or oxygen level produce cell free or soluble polymer that increases the viscosity of the drive fluid for mobility profile modification. Alternatively, strains can be selected for their ability to penetrate into a high flow zone and then be triggered into producing a biofilm or bioplug by following and overtaking the cells with a media that causes a biofilm to form and plug the zone.
Example 5
(65) Making Mutant Microorganisms that Only Secrete Soluble Polymer and are Deficient in Forming Biofilm by the Process of Random Mutagenesis
(66) Because the ability to form a biofilm is an advantage to most wild type microorganisms, it may be difficult to isolate otherwise useful bacteria that cannot make stable biofilms. However, because biofilms are comprised of microorganisms embedded in a self-produced matrix of extracellular polymeric substance (EPS) which contains significant amounts of polysaccharides (polymer) of several types as well as proteins and DNA, it may be possible to disrupt the stable biofilm formation process. The formation of a stable biofilm may be disabled or disrupted by randomly destroying genes within a microorganism's genome followed by isolating mutated cells that can still function and grow but that are unable to produce all of the needed components of a stable biofilm. The soluble and cell-free polymer in the biofilm has great potential application in enhanced oil recovery (EOR) because it can move ahead of the growing cells and create a high viscosity fluid to push the oil off of the formation rock. Therefore, the goal is to select a few cells that have lost the ability to make complete stable biofilms, but that still can produce soluble cell free polymer.
(67) Random mutated cells can be generated by a number of methods known to microbiologists such as the use of Acridine Mutagen ICR 191, chemical Ethyl methanesulfonate (EMS), and Ultraviolet (UV) to treat biofilm producing microbes (Pseudomonas, Bacillus) to disable their biofilm formation. In this example, the goal is to generate and isolate mutants able to produce water soluble polymer but no biofilm. The new microbial mutants will be used in an oil reservoir for in situ polymer production for increasing the viscosity of the drive fluid in a process of enhanced oil recovery (EOR). Some non limiting examples for cell mutagenesis are given below.
(68) Acridine Mutagen ICR 191 Mutagenesis
(69) Grow cells to log phase, count cell and dilute cells to 10^3-10^4 cells/mL, aliquot 10 mL cells in 6 conical tubes, add ICR 191 (stock 5 mg/mL in 0.1N HCl) to final concentration: 0 ug/mL, 1 ug/mL, 2 ug/mL, 3 ug/mL, 4 ug/mL, 5 ug/mL. Treat the 10 mL cells in a rotator (250 rpm) for 7-10 days, then proceed to the screening process.
(70) EMS (Ethyl Methanesulfonate) Mutagenesis
(71) Dilute the log phase cells to 10^5 cells/mL, aliquot 10 mL, cells in 6 conical tubes, add EMS to final 200 M, treat the 10 mL cells in a rotator (250 rpm) for 0, 20, 40, 60, 80, 100 minutes, then proceed to the screening process.
(72) UV (Ultraviolet) Mutagenesis
(73) Dilute the log phase cells to 10^5 cells/mL, aliquot 30 mL cells to 6 petri dishes and expose the cells to following doses of ultraviolet in a Stratalinker UV crosslinker: none (control), 2.510^4 J/cm2, 5.010^4 J/cm2, 7.510^4 J/cm2, 1010^4 J/cm2, 1510^4 J/cm2. Proceed to the screening process.
(74) Screening Process
(75) Collect cells of each treatment by centrifugation, then wash the cells three times using the culture medium, and resuspend the cells in 2 mL medium. Spread 0.1-0.5 mL on medium agar plates and incubate at appropriate conditions (eg, aerobic, anaerobic, temperature) for 7-10 days.
(76) Choose mucoid colonies (as described above) and transfer them to liquid medium, incubate for 7-10 days. Choose mutant cells that form no biofilms in liquid culture for further viscosity measurement and polymer quantification. Cells selected from this process should produce a very viscous (high yield of polymer) solution when grown in larger culture with no biofilm formation.
Example 6
(77) Site Directed Mutating of Genes Coding for Biofilm Formation.
(78) Construction of Biofilm Deficient Pseudomonas Strains
(79) Defects in the mucA gene in P. aeruginosa PAO1 result in an overproduction of the soluble exopolysaccharide alginate (Mathee, K., et al. Microbiology 1999 June: 145 (pt6): 1349-57) and a decrease in some biofilm characteristics including surface attachment (Hay, I. D., et al. Appl Environ Microbiol. 2009 September; 75(18): 6022 6025). Moreover, PAO1 strains containing deletions in the psI gene cluster have defects in attachment to a microtitre dish well and do not form robust biofilm biomass (Colvin, K. M., et al. Environ Microbiol. 2012 August; 14(8):1913-28). It has been observed that a PAO1 isolate containing a defect in the mucA gene (strain PDO300) in which the pelF and pslA genes have also been deleted produces high levels of alginate but does not produce the Pel or Psl polysaccharides that are important in biofilm formation.
(80) To generate a Pseudomonas strain with a defect in biofilm formation, but with enhanced cell-free soluble polymer production, knockout constructs were generated for P. aeruginosa PAO1 (ATCC BAA-47) that were designed to implement an unmarked deletion strategy (as described in Colvin above). Flanking regions of each gene targeted for deletion were synthesized and introduced into the suicide vector pEX100T. The flanks were synthesized as two separate 750 bp DNA fragments (Integrated DNA Technologies). One fragment, partGFF28 (SEQ ID NO: 2), included sequence matching 19 bp from vector pEX100T immediately upstream of the SmaI restriction site, followed by 716 bp of sequence upstream of the mucA gene (716 to 1 nt before the start of mucA), followed by sequence matching 1 to 15 bp after the end of the mucA ORF. The second fragment, partGFF29 (SEQ ID NO: 3), included 16 bp matching 16 to 1 bp before the beginning of the mucA ORF, followed by sequence matching 716 bp downstream of the mucA gene (1 to 716 nt after the end of mucA), followed by sequence matching 18 bp immediately following the SmaI restriction site in pEX100T. The two fragments were combined with SmaI cut pEX100T in an isothermal assembly reaction using Gibson Assembly Master mix (New England Biolabs), at 50 C. for 1 hour, according to the manufacturer's instructions. Assembly of the correct sequence was verified in one isolated plasmid, pGFF173, by DNA sequencing. pGFF173 will be transformed into P. aeruginosa PAO1 by electroporation or conjugal transfer from E. coli, and single recombination integrants will be selected on LB plates containing 300 g/ml carbenicillin (and 25 g/ml irgasan in cases of conjugal transfer). Double recombination mutants will be selected on LB plates containing 10% sucrose, to generate a mucA knockout strain.
(81) Knockout plasmids were generated to knock out the pslD and pelF genes in P. aeruginosa PAO1, using a markerless knockout design in the pEX100T vector similar to pGFF173 (above). To generate the pslD knockout construct, a DNA fragment including 525 bp upstream of the pslD ORF was generated by PCR amplification from P. aeruginosa PAO1 genomic DNA using DNA primers prGFF695 (SEQ ID NO: 62) and prGFF697 (SEQ ID NO: 64), and a DNA fragment including 527 bp immediately downstream of the pslD ORF was generated by PCR amplication from the P. aeruginosa PAO1 genomic DNA using DNA primers prGFF696 (SEQ ID NO: 63) and prGFF698 (SEQ ID NO: 65). Primer sequences included sequences that matched the ends of the SmaI cut pEX100T vector and the adjacent fragment (as with partGFF28 (SEQ ID NO: 2) and partGFF29 (SEQ ID NO: 3), above). The two fragments were combined with SmaI cut pEX100T in an isothermal assembly reaction using Gibson Assembly Master mix (New England Biolabs), at 50 C. for 1 hour, according to the manufacturer's instructions. Assembly of the correct sequence was verified in an isolated plasmid, pGFF174, by DNA sequencing. A pelF knockout construct, pGFF175, was generated in the same manner, using primers prGFF699 (SEQ ID NO: 66) and prGFF700 (SEQ ID NO: 67) for the upstream fragment (480 base pairs) and prGFF701 (SEQ ID NO: 68) and prGFF702 (SEQ ID NO: 69) for the downstream fragment (497 base pairs) at pelF. Subsequent knockouts will be made by the same method employed for the mucA knockout, generating a mucApslD, a mucApelF, a pslDpelF, and a mucApslDpelF strain.
(82) A cluster of genes that are homologous to the Pseudomonas aeruginosa algUmucABCD cluster of genes is present in multiple sequenced P. stutzeri genomes, including ATCC 17588, CCUG 29243, DSM 10701, and DSM 4166, with mucA homologs present in several sequenced P. stutzeri genomes (DSM 10701, A1501, DSM 4166, ATCC 17588, CCUG 29243, RCH2). Likewise, pslA is present in at least five sequenced P. stutzeri genomes and significant conservation of the psl gene cluster is readily apparent (See Tables 2 and 3). However, pelA does not have a close sequence homolog (BLAST E-value <1.0) in sequenced P. stutzeri strains.
(83) Primers were designed based on existing P. stutzeri genomic sequences with homology to Pseudomonas aeruginosa PAO1 mucA sequence. prGFF605 (SEQ ID NO: 20) and prGFF612 (SEQ ID NO: 22), which occur approximately 725 to 705 bp upstream and 997 to 978 bp downstream of mucA in P. stutzeri, were used to amplify the mucA sequence from P. stutzeri PTA-8823. The resulting PCR product was separated by agarose gel electrophoresis, gel purified, and sequenced with primers prGFF599 (SEQ ID NO: 18), prGFF600 (SEQ ID NO: 19), prGFF605 (SEQ ID NO: 20), prGFF610 (SEQ ID NO: 21), and prGFF612 (SEQ ID NO: 22).
(84) For detecting and sequencing genes involved in biopolymer synthesis and biofilm formation in P. alcaliphila AL15-21 (DSM17744) and P. toyotomiensis HT-3 (JCM15604), primers were designed based on existing P. alcaliphila 34 genomic sequencing project sequences available in Genbank (GI:484034433). prGFF643 (SEQ ID NO: 23) and prGFF653 (SEQ ID NO: 33) were used to amplify sequences homologous to mucA and flanking regions from P. alcalphila and P. toyotomiensis. The resulting PCR products were treated with ExoSap-IT (USB) and sequenced with primers prGFF643-653 (SEQ ID NOs: 23-33), yielding sequence reads that yielded single DNA sequence contigs spanning the mucA gene and flanks. For the pelA-G gene cluster, eleven pairs of primers were used to amplify, or to attempt to amplify, sequences. These included prGFF662 (SEQ ID NO: 34) and prGFF663 (SEQ ID NO: 35), prGFF664 (SEQ ID NO: 36) and prGFF668 (SEQ ID NO: 38), prGFF665 (SEQ ID NO: 37) and prGFF669 (SEQ ID NO: 39), prGFF672 (SEQ ID NO:) and prGFF675 (SEQ ID NO: 43), prGFF673 (SEQ ID NO: 41) and prGFF674 (SEQ ID NO: 42), prGFF676 (SEQ ID NO: 44) and prGFF678 (SEQ ID NO: 46), prGFF676 (SEQ ID NO: 44) and prGFF680 (SEQ ID NO: 47), prGFF677 (SEQ ID NO: 45) and prGFF678 (SEQ ID NO: 46), prGFF724 (SEQ ID NO: 71) and prGFF725 (SEQ ID NO: 72), prGFF727 (SEQ ID NO: 74) and prGFF729 (SEQ ID NO: 76), and prGFF728 (SEQ ID NO: 75) and prGFF730 (SEQ ID NO: 77). Products were sequenced with primers prGFF676 (SEQ ID NO: 44), prGFF680 (SEQ ID NO: 47), prGFF677 (SEQ ID NO: 45), prGFF678 (SEQ ID NO: 46), prGFF723-728 (SEQ ID NOs: 70-75), and prGFF730 (SEQ ID NO: 77). Products were assembled into contigs that matched portions of the pelA-G gene cluster in P. aeruginosa PAO1.
(85) A blast search was carried out in the P. alcaliphila 34 genome sequencing project sequences in Genbank using the pslA-O gene cluster from P. aeruginosa PAO1 as a search query. No close matches were found, suggesting that genes from that cluster are not present in the P. alcaliphila 34 genome. Blast searches carried out against other Pseudomonas genomes identified matches in P. mendocina ymp, P. mendocina DLHK, P. pseudoalcaligenes CECT5344, and P. alcaligenes MRY13. Based on these sequences, primers were designed to detect pslA-O sequences in P. alcaliphila AL15-21 (DSM17744) and P. toyotomiensis HT-3 (JCM15604). PCR amplifications were attempted from genomic DNA derived from these two strains with nine pairs of DNA primers: prGFF683 (SEQ ID NO: 50) and prGFF684 (SEQ ID NO: 51), prGFF681 (SEQ ID NO: 48) and prGFF682 (SEQ ID NO: 49), prGFF685 (SEQ ID) NO: 52) and prGFF689 (SEQ ID NO: 56), prGFF685 (SEQ ID NO: 52) and prGFF690 (SEQ ID NO: 57), prGFF686 (SEQ ID NO: 53) and prGFF689 (SEQ ID NO: 56), prGFF686 (SEQ ID NO: 53) and prGFF690 (SEQ ID NO: 57), prGFF687 (SEQ ID NO: 54) and prGFF688 (SEQ ID NO: 55), prGFF691 (SEQ ID NO: 58) and prGFF693 (SEQ ID NO: 60), and prGFF692 (SEQ ID NO: 59) and prGFF694 (SEQ ID NO: 61). Of these PCR reactions, three gave products for each strain that were sequenced. These PCR products sequences did not contain homology to the pslA-O gene cluster from P. aeruginosa PAO1 when searched in Genbank using BLAST. The failure to detect pslA-O homologous sequences in AL15-21 and HT-3 after several PCR and sequencing attempts suggests that these sequences are not present in these genomes. To generate a Pseudomonas strain with a defect in biofilm formation, but with enhanced cell-free soluble polymer production, knockout constructs were generated for P. alcaliphila AL15-21 (DSM17744), P. toyotomiensis HT-3 (JCM15604), and P. stutzeri PTA-8823 that implement an unmarked deletion strategy (as described in Colvin above). A markerless mucA knockout construct was generated for P. stutzeri PTA-8823 by assembly of upstream and downstream sequences, synthesized in a single fragment of DNA, partGFF27 (SEQ ID NO: 1), into SmaI cut pEX100T. A markerless mucA knockout construct was generated for P. alcaliphila AL15-21 (DSM17744) by assembly of upstream and downstream sequences, synthesized as two overlapping fragments of DNA, partGFF30 (SEQ ID NO: 4) and partGFF31 (SEQ ID NO: 5) respectively, into SmaI cut pEX100T. A markerless mucA knockout construct was generated for P. toyotomiensis HT-3 (JCM15604) by assembly of upstream and downstream sequences, synthesized as two overlapping fragments of DNA, partGFF32 (SEQ ID NO: 6) and partGFF33 (SEQ ID NO: 7) respectively, into SmaI cut pEX100T. For each assembly, the one or two fragments were combined with SmaI cut pEX100T in an isothermal assembly reaction using Gibson Assembly Master mix (New England Biolabs), at 50 C. for 1 hour, according to the manufacturer's instructions. Assembly of the correct sequences into the plasmid vector were verified for each of these constructs; plasmid pGFF167 was isolated containing the mucA markerless knockout construct for P. stutzeri generated from partGFF27 (SEQ ID NO: 1). Plasmid pGFF170 was isolated containing the mucA markerless knockout construct for P. alcaliphila generated from partGFF30 (SEQ ID NO: 4) and partGFF31 (SEQ ID NO: 5). Plasmid pGFF171 was isolated containing the mucA markerless knockout construct for P. toyotomiensis generated from partGFF32 (SEQ ID NO: 6) and partGFF33 (SEQ ID NO: 7). pGFF167 will be transformed into P. stutzeri PTA-8823 by electroporation or conjugal transfer from E. coli, and single recombination integrants will be selected on LB plates containing 300 g/ml carbenicillin (and 25 g/ml irgasan in cases of conjugal transfer). pGFF171 will be transformed into P. toyotomiensis HT-3 (JCM15604) by electroporation or conjugal transfer from E. coli, and single recombination integrants will be selected on LB plates containing 300 g/ml carbenicillin (and 25 g/ml irgasan in cases of conjugal transfer). Double recombination mutants will be selected on LB plates containing 10% sucrose. mucA knockout strains will be generated for P. stutzeri PTA-8823 and P. toyotomiensis HT-3 by this approach.
(86) Plasmid pGFF170 was introduced into P. alcaliphila AL15-21 (DSM17744) by triparental mating of P. alcaliphila with dH5alpha E. coli containing plasmid pRK2013 and dH5alpha E. coli containing plasmid pGFF70. Isolates was selected on a minimal salts medium VBMM containing 500 g/ml carbenicillin. Isolates were then passaged in the absence of selection and plated onto LB plate medium containing 10% sucrose. Isolates from the sucrose plate that were no long carbenicillin resistant were tested for the markerless knockout by using primers prGFF643 (SEQ ID NO: 23) and prGFF653 (SEQ ID NO: 33), which were external to the construct, upstream and in the P. alcaliphila genome in a PCR. Isolates were also tested with primers prGFF645 (SEQ ID NO: 25) and prGFF651 (SEQ ID NO: 31), occurring 163 nt upstream and 172 nt downstream of the mucA ORF. One isolate, GFF419, contained a single PCR product for both primer pairs of a size that indicated that the mucA ORF had been removed from the P. alcaliphila genome. The PCR product amplified by prGFF643 (SEQ ID NO: 23) and prGFF653 (SEQ ID NO: 33) was sequenced, and verified the removal of the mucA ORF at the appropriate sequence in GFF419. Sequencing of a PCR product generated with 16S rDNA PCR primers prGFF123 (SEQ ID NO: 8) and 124 (SEQ ID NO: 9) verified that GFF419 is identical to P. alcaliphila AL15-21 (DSM17744) at that locus.
(87) Defects in the mucA gene in P. aeruginosa PAO1 result in an overproduction of the soluble exopolysaccharide alginate (Mathee, K., et al. Microbiology 1999 June: 145 (pt6): 1349-57) and a decrease in some biofilm characteristics including surface attachment (Hay, I. D., et al. Appl Environ Microbiol. 2009 September; 75(18): 6022 6025). To test for overproduction of soluble polymer in the mucA strain in P. alcaliphila, GFF419, saturated overnight cultures (approx. OD600 3.0) for the wildtype P. alcaliphila AL15-21 parent strain and GFF419 were diluted to the equivalent OD600 of 0.01 in 30 mls Luria Broth (LB)+5% glucose in 250 ml shake flasks. 0.03 Units of Neutrase (Sigma) were added to the medium to prevent the action of potential alginate degrading enzymes, such as alginate lyase, in the cultures. Cultures were grown for four days at 32 C. with shaking. 10 ml of each culture was withdrawn and the viscosities measured using an Ostwald (U-tube) viscometer (Table 3). The viscosity of the culture from the wildtype culture (1.18 centipoise) was measured to be similar to that of water (1.00 centipoise). By contrast, the mucA strain, GFF419, culture was clearly viscous, with a measurement of 7.55 centipoise. Subsequently, 25 mls of each culture was centrifuged at 6000 g for 15 min. The supernatant was removed, and both the supernatant and the cell pellet saved. 25 ml isopropanol was added to the culture supernatant and mixed. The mixture was then placed at 20 C. for 3 days. The cell pellet was washed in 1 ml LB medium and centrifuged, and the cell pellet was stored at 80 C. The supernatant was spun at 6000 g for 15 min and the supernatant was removed. The precipitated pellet was washed in 0.5 ml 70% ethanol and centrifuged. The supernatant was removed from the precipitate. As a control, a 25 ml sample of LB+5% medium was also carried through the precipitation regime. The control, supernatant precipitates and the cell pellets were placed in a lyophilizer overnight and were dried completely. The samples were weighed. The supernatant from the wildtype P. alcaliphila culture gave a small pellet (7 mg), which was only slightly larger than the control from medium alone. The supernatant from the mucA P. alcaliphila isolate, GFF419, yielded a markedly larger pellet (34 mg) (Table 1). The higher viscosity of the GFF419 culture and the higher amount of isopropanol precipitate from the culture supernatant, versus the wild type strain, indicates production of viscous soluble polymer.
(88) P. aeruginosa PAO1 strains in which the mucA gene is defective exhibit defects in the formation of biofilms. To test a mucA disruption for a similar defect in P. alcaliphila, wildtype P. alcaliphila AL15-21 (DSM17744) and GFF419 were inoculated from saturated overnight cultures at 1/200 concentration in 5 mls YC-Alk10 medium (per liter: MgSO4.7H2O, 0.2 g; NaCl, 25 g; KCl, 1 g; KH2PO4, 1 g; NH4Cl, 1 g; Na glutamate, 1 g; Yeast extract, 5 g; Casamino acids (Oxoid), 5 g; Na2CO3, 5 g; FeCl2.4H2O, 36 mg; MnCl2.4H2O, 0.36 mg; adjusted to pH10 with NaOH) supplemented with 5% glucose and grown 5 days at 32 C. in a six well plate without shaking. Both of the AL15-21 and GFF419 cultures grew and became turbid. However, whereas the wild type strain exhibited significant formation of typical biofilms within the culture no biofilms were observed for the GFF419 mucA strain.
(89) Based on the conservation of biofilm biosynthesis and regulatory genes, gene knockouts of mucA and psl gene homologs in P. stutzeri likely impact biofilm architecture in a similar manner. The conservation of sequence between the P. stutzeri genomes allow PCR primers to be designed to generate targeted gene knockout constructs. Knockouts will be generated using an unmarked deletion strategy (as described in Colvin above). Flanking regions of each gene targeted for deletion will be PCR amplified from P. stutzeri ATCC PTA-8823 genomic DNA and ligated into the suicide vector pEX18Gm. Plasmid inserts will be verified by sequencing. Plasmids will be transformed into P. stutzeri ATCC PTA-8823 by electroporation or conjugal transfer from E. coli, and single recombination integrants will be selected on LB containing 100 g/ml gentamycin (and 25 g/ml irgasan in cases of conjugal transfer). Double recombination mutants will be selected on LB plates containing 10% sucrose. By this approach, mucA and mucApslA strains will be constructed such that the gene deletions will remove the first to last nucleotides of the mucA and pslA ORFs.
Example 7
(90) Transfer of Xanthan Gum Biosynthesis Gene Cluster into Pseudomonas Strains
(91) Xanthan gum is a soluble acidic heteropolysaccharide produced by Xanthomonas species (typically Xanthomonas campestris) that has a high molecular weight, ranging from 500 to 2000 kDa. Xanthan gum is produced for a wide range of commercial applications by industrial fermentation, and the genetics underlying its biosynthesis has been described. Xanthan gum production is directed by a cluster of at least twelve genes coding for assembly, acetylation, pyruvylation, polymerization, and secretion. These genes consist of gumBCDEFGHIJKLM, and may include the gumA and gumN genes at either end of the gene cluster, though it appears that they do not function in Xanthan gum synthesis (Pollock T. J., et al. J. of Ind. Microbiology and Biotechnology (1997) 19, 92-97. Moreover, these genes appear to be controlled by a single promoter (Katzen, F., et al., J. of Bacteriology, July 1996 vol. 178 No. 14 p 4313-18). The gum gene cluster (including gumBCDEFGHIJKLM) has previously been cloned into a plasmid and transferred into a Sphingomonas strain, from which production of Xanthan gum production was subsequently detected (Pollock above).
(92) The Xanthan gumBCDEFGHIJKLM cluster was PCR amplified from Xanthomonas campestris DSM 19000 genomic DNA using rTth DNA polymerase (Life Technologies) to generate a PCR product corresponding to nucleotides 11 to 16054 in Genbank sequence U22511 (Xanthomonas B1459 gum cluster) flanked by homologous sequence at the cloning site in pBBR1MCS. The primers used for amplification of the gum gene cluster included (prGFF533) gaattcctgcagcccaaatGAGGCGGTAACAGGGGAT (SEQ ID NO: 10) and (prGFF534) gcggtggcggccgctctagaCCTTGCTGACCTTCCACAG (SEQ ID NO: 11) and contained the SwaI half site and a full XbaI site. pBBR1MCS was cut with XbaI and SmaI and purified. The cut vector was assembled with the gum gone cluster DNA fragment by isothermal assembly using the Gibson Assembly Master Mix (New England Biolabs). The assembly product was transformed into DH10-beta competent E. coli cells and selected on LB medium containing chloramphenicol. Individual clone isolates were selected, screened by PCR for assembly, and shotgun sequenced. The sequencing reads were assembled into a single contig, and the resulting assembled sequence verified the fidelity of the gum gene cluster insert. Plasmid pGFF155, a successful DNA clone containing the gum gene cluster, was transformed into Pseudomonas alcaliphila AL15-21 (DSM17744) and P. toyotomiensis HT-3 (JCM15604) by electroporation and selected on medium containing 200 and 400 g/ml chloramphenicol, respectively. Presence of pGFF155 in P. alcaliphila and P. toyotomiensis was verified by PCR, and the P. alcaliphila and P. toyotomiensis transformant isolates were named GFF375 and GFF377 respectively.
(93) GFF257 is an isolate of P. toyotomiensis HT-3 transformed with the original pHBR1MCS vector by electroporation. Strains GFF257 and GFF377 were grown overnight at 28 C. in 10 mls MhYC-alk medium (Per liter: MgSO.sub.4.7H.sub.2O, 0.2 g; NaCl, 25 g; KCl, 1 g; KH.sub.2PO.sub.4, 1 g; NH.sub.4Cl, 1 g; Na glutamate, 1 g; Yeast extract, 5 g; Casamino acids (Oxoid), 5 g; FeCl.sub.2.4H.sub.2O, 36 mg; MnCl.sub.2.4H.sub.2O, 0.36 mg; Na.sub.2CO.sub.3 added to pH7.0) containing 400 g/ml chloramphenicol. Saturated overnight cultures were diluted 200-fold into 1000 mls Pmn medium (Per liter: glucose, 50 g; (NH.sub.4).sub.2HPO.sub.4, 1.25 g; K.sub.2HPO.sub.4, 1.25 g; CaCl.sub.2.2H.sub.2O, 0.1 g; MgSO.sub.4.7H.sub.2O, 0.4 g; Citric acid, 0.99 g; Yeast extract (Oxoid), 0.2 g; MnSO.sub.4.4H.sub.2O, 1.1 mg; ZnSO.sub.4.7H.sub.2O, 0.2 mg; CoSO.sub.4.7H.sub.2O, 0.28 mg; CuSO.sub.4.5H.sub.2O, 0.25 mg; H.sub.3BO.sub.3, 0.06 mg; FeSO.sub.4.7H.sub.2O, 3.6 mg; pH 7.0) containing 300 g/ml chloramphenicol, and grown in 2.8 L flasks at 28 C. for 7 days. Pmn medium is a nitrogen limiting medium based on a medium described to grow a Pseudomonas mendocina strain for alginate production (Anderson, A. J., et al. Journal of General Microbiology. 1987. 133: 1045-1052). 900 mls of each culture was centrifuged (30 min, 5000 RPM). Supernatant was taken and an equal volume (900 mls) 100% ethanol was added to each supernatant, to precipitate soluble polymer. The mixtures were place at 20 C. overnight. The mixtures were centrifuged (30 min, 5000 RPM) and washed twice in 70% ethanol. The liquid was removed and the pellets allowed to air dry overnight. The precipitated and dried pellets were weighed. The GFF257 pellet weighed 0.0 g and the GFF377 pellet weighed 0.9 g. Each pellet was resuspended in 25 mls water. The viscosities were measured with an Uberholde viscometer (TABLE 4). Results indicated the presence of viscous polymer, which could be isolated by precipitation, in the culture medium in the P. toyotomiensis strain containing the Xanthan gene cluster.
(94) Plasmid pGFF155 will also be transformed into wildtype P. aeruginosa PAO1 and into the P. aeruginosa PAO1 pslBCDpelA biofilm deficient strain by electroporation and Pseudomonas transformants will be selected on LB medium containing chloramphenicol. Uptake of the vector and gum gene cluster in the P. aeruginosa PAO1 strains will be verified by PCR.
Example 8
(95) Construction of Biofilm Deficient Bacillus Strains
(96) Biofilms in Bacillus subtilis are complex structures that have been studied extensively and may consist of several components such as structural neutral polymers (Ievan and exopolysaccharides generated by the eps operon), charged polymers (poly-gamma-glutamic acid), amphiphilic molecules (surfactin), active EPS enzymes, and the amyloid fiber-forming TasA protein (reviewed in Marvasi, M., et al., 2010 FEMS Microbiology letter, 313:1-9).
(97) The biofilm-forming B. subtilis strain NCIB3610 forms biofilms on the surface of solid agar medium plates and robust floating biofilms (termed pellicles) on standing cultures at the air-liquid interlace. The biofilm structure is dependent on the extracellular matrix, formed largely by exopolysaccharide and the TasA protein (Branda, S. S., et al, Mol Microbiol. 2006 February; 59(4):1229-38). The TasA protein is 261 amino acids in length and polymerizes to form amyloid fibers that have been shown to be essential for the structural integrity of the extracellular matrix in biofilms and which are resistant to breakdown (Chu, F. et al., Mol Microbiol. 2006 February; 59(4):1216-28, Romero, D., et al., Proc Natl Acad Sci USA. 2010 February 2; 107(5): 2230 2234). Strains with defects in the tasA gene fail to form bundles of cells typical in biofilm pellicles, form less extracellular material in biofilms, and form biofilms that are thinner and weaker (Branda 2006, Dogsa, I, et al. PLoS One. 2013; 8(4): e62044. Published online 2013 Apr. 26. doi: 10.1371/journal.pone.0062044).
(98) To generate a Bacillus strain deficient in biofilm formation, a tasA::cat PCR construct was generated for B. subtilis strain NCIB3610 in two PCR steps by the long-flanking homology PCR (LFH-PCR) technique (Kim and Kim, Biotechnol Bioprocess Eng 2000 Kim, J and Kim, B, Biotechnol. Bioprocess Eng. 2000 5(5): 327-331.). The 1200 bp sequences immediately upstream and downstream of the tasA ORF were PCR amplified from NCIB3610 genomic DNA using primer pairs prGFF556 (SEQ ID NO: 12) and prGFF557 (SEQ ID NO: 13) and prGFF558 (SEQ ID NO: 14) and prGFF559 (SEQ ID NO: 15), respectively. The cat gene, conferring chloramphenicol resistance, was PCR amplified from pNW33n (BGSC ECE136) using primers prGFF560 (SEQ ID NO: 16) and prGFF561 (SEQ ID NO: 17), which contain sequences at the 5 ends that match the 35 bp immediately upstream and 35 bp immediately downstream of the tasA ORF, respectively. The three PCR products were combined in a subsequent PCR reaction with primers prGFF556 (SEQ ID NO: 12) and prGFF559 (SEQ ID NO: 15). The product of the second PCR step was resolved on a 1% agarose gel, and a product of 2257 bp was detected and purified from the gel. This product will be introduced into B. subtilis strain NCIB3610 directly (Kim, J and Kim, B, Biotechnol. Bioprocess Eng. 2000 5(5): 327-331), which will result in a deletion of the tasA ORF from the first nucleotide to the last.
(99) A homolog of the Bacillus subtilis 168 tasA gene was identified in the genome sequenced Bacillus mojavensis strain RO-H-1; the corresponding protein (GI:498020761) shares 98% sequence identity with Bacillus subtilis 168 tasA at the amino acid level. Bacillus mojavensis JF2 (ATCC 39307) is a well-studied strain isolated from an oil field that has been described and tested as an effective strain for MEOR applications. The tasA gene will be deleted in Bacillus mojavensis JF2 by the pMAD markerless knockout system (Arnaud, M. et al., Appl Environ Microbiol. 2004 November 70 (11) 6887-, as described in Durand, S et al., PLoS Genetics 8(12): e1003181. doi: 10.1371/journal.pgen.1003181. Epub 2012 Dec. 27)
(100) 500 nt sequences upstream and downstream of the tasA gene will be PCR amplified with overlapping oligonucleotides from B. mojavensis JF2. The two flanking sequences will be assembled in a subsequent PCR reaction to generate a 1000 nt PCR product containing a deletion of the tasA ORF. The assembled product will subsequently be digested and ligated into pMAD. The integrative plasmid will then be transformed into Bacillus mojavensis JF2 by electroporation, by a method previously described for several B. mojavensis strains (Olubajo, B and Bacon, C. J Microbiol Methods. 2008 August; 74(2-3):102-105).
(101) TABLE-US-00001 TABLE 1 algU, mucA, mucB, mucC, and mucD (P. aeruginosa PAO1) BLASTP match results in P. stutzeri protein sequences in Genbank (E-value <1e{circumflex over ()}40) bit Definition % identity % positives evalue score # Query: gi|15595959|ref|NP_249453.1| RNA polymerase sigma factor AlgU [Pseudomonas aeruginosa PAO1] >gi|518172025|ref|WP_019342233.1| RNA polymerase sigma 93.26 97.41 3.00E132 373 factor AlgU [Pseudomonas stutzeri] >gi|387969827|gb|EIK54107.1| RNA polymerase sigma factor 92.75 97.41 2.00E131 371 AlgU [Pseudomonas stutzeri TS44] >gi|397686068|ref|YP_006523387.1| RNA polymerase sigma 92.23 97.41 3.00E131 370 factor AlgU [Pseudomonas stutzeri DSM 10701] >gi|146281607|ref|YP_001171760.1| RNA polymerase sigma 91.71 97.41 8.00E131 369 factor AlgU [Pseudomonas stutzeri A1501] # Query: gi|15595960|ref|NP_249454.1| anti-sigma factor MucA [Pseudomonas aeruginosa PAO1] >gi|431928191|ref|YP_007241225.1| negative regulator of 68.53 81.22 2.00E73 223 sigma E activity [Pseudomonas stutzeri RCH2] >gi|452006822|gb|EMD99087.1| anti-sigma factor MucA 67.51 81.22 3.00E73 223 [Pseudomonas stutzeri NF13] >gi|392420216|ref|YP_006456820.1| anti-sigma factor 67.51 81.22 2.00E71 219 MucA [Pseudomonas stutzeri CCUG 29243] >gi|409781090|gb|EKN60694.1| anti-sigma factor MucA 65.48 79.19 7.00E69 212 [Pseudomonas stutzeri KOS6] >gi|379064449|gb|EHY77192.1| anti-sigma factor MucA 67.01 80.71 1.00E65 204 [Pseudomonas stutzeri ATCC 14405 = CCUG 16156] >gi|397686069|ref|YP_006523388.1| anti-sigma factor 65.5 77 1.00E65 204 MucA [Pseudomonas stutzeri DSM 10701] >gi|146281608|ref|YP_001171761.1| anti-sigma factor 68.18 80.3 2.00E64 201 MucA [Pseudomonas stutzeri A1501] >gi|386019815|ref|YP_005937839.1| anti-sigma factor 68.18 80.3 3.00E64 200 MucA [Pseudomonas stutzeri DSM 4166] >gi|518172026|ref|WP_019342234.1| sigma factor AlgU 66.5 79.5 2.00E63 198 negative regulatory protein [Pseudomonas stutzeri] >gi|339493209|ref|YP_004713502.1| anti-sigma factor 67.17 79.29 5.00E62 194 MucA [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|387969826|gb|EIK54106.1| anti-sigma factor MucA 67.17 79.8 6.00E57 181 [Pseudomonas stutzeri TS44] # Query: gi|15595961|ref|NP_249455.1| negative regulator for alginate biosynthesis MucB [Pseudomonas aeruginosa PAO1] >gi|392420217|ref|YP_006456821.1| sigma E regulatory 60.19 76.05 3.00E129 375 protein MucB/RseB [Pseudomonas stutzeri CCUG 29243] >gi|431928190|ref|YP_007241224.1| negative regulator of 60.52 75.08 1.00E128 374 sigma E activity [Pseudomonas stutzeri RCH2] >gi|146281609|ref|YP_001171762.1| negative regulator for 59.03 75.16 3.00E125 365 alginate biosynthesis MucB [Pseudomonas stutzeri A1501] >gi|386019816|ref|YP_005937840.1| negative regulator for 59.03 75.16 3.00E125 365 alginate biosynthesis MucB [Pseudomonas stutzeri DSM 4166] >gi|518237074|ref|WP_019407282.1| sigma factor AlgU 58.71 74.84 3.00E124 362 regulatory protein MucB [Pseudomonas stutzeri] >gi|515814934|ref|WP_017245687.1| sigma factor AlgU 58.71 74.84 3.00E124 362 regulatory protein MucB [Pseudomonas stutzeri] >gi|339493210|ref|YP_004713503.1| negative regulator for 58.39 74.19 3.00E123 360 alginate biosynthesis MucB [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|409781089|gb|EKN60693.1| sigma E regulatory 57.28 73.14 2.00E122 358 protein MucB/RseB [Pseudomonas stutzeri KOS6] >gi|379064450|gb|EHY77193.1| sigma E regulatory 61.35 76.24 7.00E121 353 protein, MucB/RseB [Pseudomonas stutzeri ATCC 14405 = CCUG 16156] >gi|516319045|gb|EPL59790.1| sigma E regulatory protein 60.78 76.33 2.00E118 347 MucB/RseB [Pseudomonas stutzeri B1SMN1] >gi|518172027|ref|WP_019342235.1| sigma factor AlgU 57.56 72.99 2.00E116 342 regulatory protein MucB [Pseudomonas stutzeri] >gi|397686070|ref|YP_006523389.1| sigma E regulatory 59.09 70.98 7.00E114 335 protein MucB/RseB [Pseudomonas stutzeri DSM 10701] >gi|387969825|gb|EIK54105.1| sigma E regulatory protein 58.16 73.05 6.00E112 330 MucB/RseB [Pseudomonas stutzeri TS44] >gi|452006824|gb|EMD99089.1| sigma E regulatory 62.15 74.77 4.00E89 269 protein MucB/RseB [Pseudomonas stutzeri NFis] # Query: gi|15595962|ref|NP_249456.1| positive regulator for alginate biosynthesis MucC [Pseudomonas aeruginosa PAO1] >gi|387969824|gb|EIK54104.1| positive regulator for 61.64 75.34 5.00E56 176 alginate biosynthesis MucC [Pseudomonas stutzeri TS44] >gi|379064451|gb|EHY77194.1| positive regulator for 57.43 73.65 5.00E54 171 alginate biosynthesis MucC [Pseudomonas stutzeri ATCC 14405 = CCUG 16156] >gi|409781088|gb|EKN60692.1| positive regulator for 57.72 72.48 2.00E53 169 alginate biosynthesis MucC [Pseudomonas stutzeri KOS6] >gi|452006825|gb|EMD99090.1| positive regulator for 57.43 71.62 7.00E53 168 alginate biosynthesis MucC [Pseudomonas stutzeri NF13] >gi|392420218|ref|YP_006456822.1| positive regulator for 58.11 72.3 1.00E50 162 alginate biosynthesis MucC [Pseudomonas stutzeri CCUG 29243] >gi|397686071|ref|YP_006523390.1| positive regulator for 63.51 77.7 3.00E49 158 alginate biosynthesis MucC [Pseudomonas stutzeri DSM 10701] >gi|431928189|ref|YP_007241223.1| Positive regulator of 56.76 70.95 4.00E49 158 sigma E activity [Pseudomonas stutzeri RCH2] >gi|146281610|ref|YP_001171763.1| positive regulator for 58.78 71.62 1.00E44 147 alginate biosynthesis MucC [Pseudomonas stutzeri A1501] >gi|339493211|ref|YP_004713504.1| positive regulator for 58.78 71.62 1.00E44 147 alginate biosynthesis MucC [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|518172028|ref|WP_019342236.1| positive regulator for 51.03 66.9 2.00E41 138 alginate biosynthesis MucC [Pseudomonas stutzeri] # Query: gi|15595963|ref|NP_249457.1| serine protease MucD [Pseudomonas aeruginosa PAO1] >gi|387969823|gb|EIK54103.1| serine protease MucD 73.84 86.5 0 699 [Pseudomonas stutzeri TS44] >gi|409781087|gb|EKN60691.1| serine protease MucD 73.05 85.47 0 695 [Pseudomonas stutzeri KOS6] >gi|452006826|gb|EMD99091.1| serine protease MucD 72.84 85.05 0 692 [Pseudomonas stutzeri NF13] >gi|379064452|gb|EHY77195.1| serine protease MucD 72 84.21 0 691 [Pseudomonas stutzeri ATCC 14405 = CCUG 16156] >gi|392420219|ref|YP_006456823.1| serine protease 72.84 84.63 0 690 MucD [Pseudomonas stutzeri CCUG 29243] >gi|431928188|ref|YP_007241222.1| periplasmic serine 72.63 85.05 0 688 protease, Do/DeqQ family [Pseudomonas stutzeri RCH2] >gi|146281611|ref|YP_001171764.1| serine protease MucD 71.91 85.12 0 682 [Pseudomonas stutzeri A1501] >gi|397686072|ref|YP_006523391.1| serine protease 72.36 85.23 0 681 MucD [Pseudomonas stutzeri DSM 10701] >gi|339493212|ref|YP_004713505.1| serine protease MucD 71.49 84.7 0 676 [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|518169326|ref|WP_019339534.1| serine peptidase 71.34 85.99 0 674 [Pseudomonas stutzeri] >gi|387969988|gb|EIK54268.1| HtrA-like protease AlgW 46.13 67.68 4.00E83 265 [Pseudomonas stutzeri TS44] >gi|409782192|gb|EKN61759.1| HtrA-like protease AlgW 43.11 64.07 8.00E83 262 [Pseudomonas stutzeri KOS6] >gi|338800400|gb|AEJ04232.1| HtrA-like protease AlgW 46.13 68.35 2.00E82 263 [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|431928383|ref|YP_007241417.1| trypsin-like serine 46.26 68.37 3.00E82 263 protease with C-terminal PDZ domain [Pseudomonas stutzeri RCH2] >gi|452006561|gb|EMD98833.1| HtrA-like protease AlgW 45.79 68.01 1.00E81 261 [Pseudomonas stutzeri NF13] >gi|392422348|ref|YP_006458952.1| HtrA-like protease 45.79 68.01 1.00E81 261 AlgW [Pseudomonas stutzeri CCUG 29243] >gi|397685896|ref|YP_006523215.1| HtrA-like protease 42.99 64.48 2.00E81 260 AlgW [Pseudomonas stutzeri DSM 10701] >gi|409779972|gb|EKN59617.1| HtrA-like protease AlgW 45.79 67.34 3.00E81 260 [Pseudomonas stutzeri KOS6] >gi|379063956|gb|EHY76699.1| HtrA-like protease AlgW 45.45 68.01 7.00E81 259 [Pseudomonas stutzeri ATCC 14405 = CCUG 16156] >gi|518172423|ref|WP_019342631.1| 2-alkenal reductase 45.45 67.34 2.00E80 259 [Pseudomonas stutzeri] >gi|516323248|gb|EPL63964.1| 2-alkenal reductase 38.56 55.88 3.00E53 186 [Pseudomonas stutzeri B1SMN1] >gi|339496248|ref|YP_004716541.1| hypothetical protein 37.5 55.59 3.00E50 178 PSTAB_4171 [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|386021023|ref|YP_005939047.1| hypothetical protein 37.5 55.59 5.00E50 177 PSTAA_2421 [Pseudomonas stutzeri DSM 4166]
(102) TABLE-US-00002 TABLE 2 pslA (P. aeruginosa PAO1) BLASTP match results in P. stutzeri protein sequences in Genbank (E-value <1e{circumflex over ()}40) Definition # Query: gi|15597427|ref|NP_250921.1| protein PslA bit [Pseudomonas aeruginosa PAO1] % identity % positives evalue score >gi|387967350|gb|EIK51654.1| capsular polysaccharide 65.69 81.17 0 653 biosynthesis protein [Pseudomonas stutzeri TS44] >gi|452008339|gb|EME00580.1| capsular polysaccharide 65.48 80.54 0 649 biosynthesis protein [Pseudomonas stutzeri NF13] >gi|516320548|gb|EPL61274.1| capsular polysaccharide 64.85 80.54 0 649 biosynthesis protein [Pseudomonas stutzeri B1SMN1] >gi|392421696|ref|YP_006458300.1| capsular 65.06 80.54 0 648 polysaccharide biosynthesis protein [Pseudomonas stutzeri CCUG 29243] >gi|339494468|ref|YP_004714761.1| capsular 64.85 80.54 0 648 polysaccharide biosynthesis protein [Pseudomonas stutzeri ATCC 17588 = LMG 11199] >gi|146282832|ref|YP_001172985.1| capsular 64.85 80.33 0 646 polysaccharide biosynthesis protein [Pseudomonas stutzeri A1501] >gi|386021197|ref|YP_005939221.1| capsular 64.64 80.54 0 646 polysaccharide biosynthesis protein [Pseudomonas stutzeri DSM 4166] >gi|431926982|ref|YP_007240016.1| undecaprenyl- 64.23 79.29 0 642 phosphate glucose phosphotransferase [Pseudomonas stutzeri RCH2] >gi|379063599|gb|EHY76342.1| capsular polysaccharide 63.18 79.5 0 639 biosynthesis protein [Pseudomonas stutzeri ATCC 14405 = CCUG 16156] >gi|395808331|gb|AFN77736.1| capsular polysaccharide 64.09 80.79 0 619 biosynthesis protein [Pseudomonas stutzeri DSM 10701] >gi|409780607|gb|EKN60234.1| capsular polysaccharide 65.9 80.75 0 616 biosynthesis protein [Pseudomonas stutzeri KOS6] >gi|518171415|ref|WP_019341623.1| capsular 63.6 80.75 0 614 polysaccharide biosynthesis protein [Pseudomonas stutzeri] >gi|516319785|gb|EPL60518.1| sugar transferase 42.68 62.5 2.00E78 256 [Pseudomonas stutzeri B1SMN1]
(103) TABLE-US-00003 TABLE 3 Wildtype P. alcaliphila AL15-21 (DSM17744) and GFF419 (mucA) viscosity measurements and soluble polymer detection after 4 days' growth. Cell pellet Supernatant time Viscosity DCW Precipitate Strain Medium (sec) (cP) (mg) (mg) H2O 11 1.00 LB + 5% 0 5 glucose AL15-21 LB + 5% 13 1.18 95 7 (wildtype) glucose GFF419 LB + 5% 83 7.55 68 34 (mucA) glucose
(104) TABLE-US-00004 TABLE 4 Isolation and viscosity testing of soluble polymer produced by P. toyotomiensis with an empty vector (GFF257) and with a vector containing the Xanthan gum cluster (GFF377). Supernatant time Viscosity Precipitate Strain Medium (sec) (cP) (g) H2O 12 1.00 GFF257 Pmn 12 1.00 0 (pBBR1MCS) GFF377 Pmn 697 58.08 0.9 (pGFF155)
(105) TABLE-US-00005 TABLE5 SynthesizedDNAsequences SEQ Sequence ID name Sequence Notes Length NO: partGFF27 CCTGTTATCCCTACCCcatctggtggcgcgc partGFF27_GFF39 749 1 ggcagacgcccgccggatagtgatgtgagtt 0mucAdeletion ccgaggacgccgagttttatgagggcgatca constructfor cgctctcaaggacatcgagtcaccggaacgc Isothermal tcgctgctcagggatgagattgaagataccg assemblyinto ttcatcgaaccattcaacttttgccagaaga pEX100T;5prime tttgcgtacggctctaacactgcgtgaattt 358ntplus3prime gatggtcttagttatgaagacattgcgagcg 358ntplusflanking tcatgcagtgtccggtgggcacagtgcgttc sequenceonends ccggatatccgggcacgtgaagccatagata aagcattgcaacccttgttgcatgaatcctg agacagcggcgacagccaagagaggaaccgc cGGAGAAACATGCGCGTATTACCGCTGTATG TGGTGATGGGGCTGCTGGCTATCGATGCCGG CCCTTGCTGCTGATGCCGGGTCCTGGATGGA GCGACTTGCGGCGGCAGAGCAGAAACAGAGT TATACCGGTACGTTCGTCTACGAACCTCAAT GGCACTCTTTTCCAGTCATGCCGTTTGGCAG CAGGTCGAAGAAGGTCAAGTGCAGGAGCGAT TGCTTCAGCTTGACGGAGCTCCGGCTGAAGT TCTGCTGGTAAATGGTCAGATGCAATGCGCT ACCGATGACCTCGCGGCGCAAGTGCGTGAAG CGCAGGCTTGGCACGGGCAGCGTCTCGATCC GAAAGCACTCTCCGAGTGGTAgggattaccc tgttatc partGFF28 CCTGTTATCCCTACCCGGGcgcatgcttgga partGFF28PAO1 750 2 ggggagaacttttgcaagaagcccgagtcta mucA5prime tcttggcaagacgattcgctgggacgctcga 716ntflankfor agctcctccaggttcgaagaggagcatcatg assemblywith ctaacccaggaacaggatcagcaactggttg partGFF29fora aacgggtacagcgcggagttcaagcgggctt seemlessko tcgatctgctggtactgaaataccagcacaa construct gatactgggattgatcgtgcggttcgtgcac intoPEX100T gacgcccaggaagcccaggacgtagcgcagg aagccttcatcaaggcataccgtgcgctcgg caatttccgcggcgatagtgctattatacct ggctgtatcggatcgccatcaacaccgcgaa gaaccacctggtcgctcgcgggcgtcggcca ccggacagcgatgtgaccgcagaggatgcgg agttcttcgagggcgaccacgccctgaagga catcgagtcgccggaacgggcgatgttgcgg gatgagatcgaggccaccgtgcaccagacca tccagcagttgcccgaggatttgcgcacggc cctgaccctgcgcgagttcgaaggtttgagt tacgaagatatcgccaccgtgatgcagtgtc cggtggggacggtacggtcgcggatcttccg cgctcgtgaagcaatcgacaaagctctgcag cctttgagcgagaagcctgacacagcggcaa atgccaagagaggtatcgctGGAGAGACATG CGCA partGFF29 CAAGAGAGGTATCGCTggagagacatgcgca partGFF29_PAO1 750 3 ccacctccctgttgcttttgcttggcagcct mucA3prime gatggcggttcccgccactcaggctgccgac 716ntflankfor gcttccgactggctgaatcgtctcgccgagg assemblywith ccgatcgccagaacagtttccaaggcacctt partGFF28fora cgtctacgagcgcaatggcagcttctccacc seemlessko catgagatctggcatcgcgtggagagcgatg construct gtgcggttcgcgagcgcctgctccagctcga intoPEX100T cggcgcgcgccaggaagtggtccgggtcgac gggcgcacccagtgcatcagcggcggccttg ccgaccaactggccgatgcccagctgtggcc ggtgcgcaagttcgatccctcccagctggct tcctggtacgacctgcgcctggtcggcgaat cccgtgtcgccggccgcccggcagtggtcct tgcggtgactccgcgcgaccagcatcgctac ggcttcgagctgcacctggaccgcgacaccg gcctgccgttgaagtcgctgctgctgaacga gaaggggcagttgctcgagcgcttccagttc acccagttgaataccggcgcggcacctgccg aagaccagttgcaggcgggcgccgaatgcca ggtcgtcggcccggccaaggccgacggcgag aagaccgtggcctggcgctcggaatggctgc cgccaggatcaccctgacccgcagtttcatg cgtccatccggtcaccCGGGATTACCCTGTT ATC partGFF30 cctgttatccctacccgggAGCTTGCTTGGA partGFF30GFF238_ 749 4 GGGGAGAACTTTTGCGTAAGACCCGAGTCTA mucA_5prime TCTTGGCAAGCTGATTCGCTTACGGGCGCAA 715ntflank GCCTCCTCCAAGCGTTACGAGGAGAATGCAT ForAssembly GCTAACCCAGGAAGATGATCAGCAACTGGTC IntopEX100T GAGCGAGTGCAGCGTGGTGACAAGCGTGCCT TCGATCTGTTGGTGCTGAAGTATCAGCACAA GATCCTCGGTCTGATCGTGCGATTCGTGCAC GACACCCACGAGGCTCAGGATGTCOCTCAGG AGGCGTTCGTAAAAGCCTACCGAGCGCTTGG AAACTTTCGCGGTGACAGTGCGTTCTATACA TGGCTGTACCGCATCGCCATCAACACGGCGA AGAATTATCTGGTGTCCCGCGGTCGGCGGCC GCCAGATAGTGATGTCAGTAGCGATGACGCG GAGTTCTATGATGGCGATCACGGCCTCAAGG ACATCGAGTCACCGGAGCGGGCATTGCTGCG CGACGAGATCGAAGCCACCGTGCATCGAACC ATCGCCCAACTGCCGGATGATTTGCGCACGG CCCTGACCCTGCGTGAGTTCGAAGGCTTGAG TTACGAGGACATTGCAGGCGTCATGCAATGC CCGGTAGGCACGGTGCGTTCGCGGATATTCC GTGCACGTGAGGCAATTGATAAGTCCCTGCA ACCTCTGTTGCAGGAAACCTAAGGCAGCGGC GACAGCCAAGAGAGGAACACCgcgctaagga gtcac partGFF31 ccaagagaggaacaccGCGCTAAGGAGTCAC partGFF31_GFF238_ 750 5 ATGCGCGCGATTCCCCTCTACCTTCTCGGTG mucA_3prime GTCTGCTGGCGTTGCCGGTTCAGGCCTCCGA 714ntflank GGTGCAGGACCTGCTCGGGCGTCTCGCTGCG ForAssembly GCGGAGCGCCAGCAAAGCTTCCAGGGCACGT IntopEX100T TCATCTATGAGCGTAATGGGAGTTTTTCTAC CCATGCCGTGTGGCATCGGGTGGAGGAGGGG GGCGCAGTTCGCGAACGCCTTCTGCAACTCG ATGGGCCTGCTCAGGAAGTGCTGAAAGTCGA TGGTCAGGCTCAGTGCGTCACTGGCGCGTTG GCCGACCAGGTCAGTGAAGGGCAGGCATGGC CTGCTCGCCAGCTGGATGCCGAGCAACTGAG CGACTGGTATGACATTCGTGTCGCTGGCAAG TCGCGCATTGCCAATCGTCCAGCGGTCGTTC TGGTGTTGGCCCCCAAGGACCAGCATCGCTA CGGCTTCGAATTGCATCTGGATCGTGAGACC GGGCTGCCGCTGAAGTCCCTGCTGTTGAACG AGCGCGGCCAGCTTCTGGAACGCTTCCAGTT CGCCCAACTGGATACTTCTGTACCGGCTGAA AATGCCATGCAGCCTAGCTCCAGCTGCAGGC CGGTGCGGTTCCGCGCTGCCGACAGCATGGA CGAAGGCAGTTGGCGATCCGACTGGTTGCCG CCGGGTTTCACTCTGACCACTGCGCAGGTGC GTCGCGGGCCTGCCGCTcccggattaccctg ttatc partGFF32 cctgttatccctacccgggAGCTTGCTTGGA partGFF32_GFF248_ 749 6 GGGGAGAACTTTTGCGTAAGACCCGAGTCTA mucA_5prime TCTTGGCAAGCTGATTCGCTTACGGGCGCAG 715ntflank ACCTCCTCCGAGCGTTATGAGGAGAGTGCAT ForAssembly GCTAACCCAGGAAGATGATCAGCAGCTGGTC IntopEX100T GAGCGAGTGCAGCGCGGTGACAAGCGTGCCT TCGATCTGTTGGTGCTGAAGTATCAGCACAA GATCCTCGGTTTGATCGTGCGATTCGTGCAC GACACCCACGAGGCTCAGGATGTCGCTCAGG AGGCTTTCGTAAAAGCCTACCGAGCGCTTGG AAACTTTCGCGGTGACAGCGCGTTCTATACA TGGCTGTACCGCATCGCCATCAACACGGCGA AGAATTATCTGGTGTCACGCGGTCGGCGGCC GCCAGATAGTGATGTCAGTAGCGATGACGCG GAGTTCTATGACGGCGACCACGGCCTGAAGG ACATCGAGTCACCGGAGCGGGCATTGCTGCG CGACGAGATCGAAGCCACCGTGCATCGAACC ATCGCCCAGTTGCCGGATGATTTGCGCACGG CCCTGACCTTGCGTGAGTTCGAAGGCTTGAG TTACGAGGACATTGCCGGCGTCATGCAGTGT CCGGTAGGTACGGTGCGTTCGCGGATCTTCC GTGCGCGTGAGGCAATTGATAAGTCCCTGCA GCCTCTGTTGCAGGAAACCTAAGGCAGCGGC GACAGCCAAGAGAGGAACACCgcgctaagga gtcac partGFF33 ccaagagaggaacaccGCGCTAAGGAGTCAC partGFF33_GFF24 750 7 ATGCGCGCGATTCCCCTCTACCTTCTCGGTG 8_mucA_3prime GTCTGCTGGCGTTGCCGGTTCAGGCCACTGA 714ntflank GGTGCAAGACTTGCTCGGGCGCCTCGCTGCG forAssembly GCAGAGCGCCAGCAAAGCTTCCAGGGCACGT IntopEX100T TCATCTATGAGCGCAATGGAAGTTTTTCCAC CCATCTCCGTGTGGCATCGCGTAGAGGAGGG GGGCGAAGTTCGCGAGCGCCTTCTGCAGCTC GATGGGCCTGCCCAGGAGGTGCTGAAAGTCG ATGGCCAGGCTCAGTGCGTCACTGGCGCGTT GGCTGACCAGGTCAGTGAAGGGCAGGCTTGG CCTGCTCGTCAGTTGGCTGTCGAGCAATTGA GCAACTGGTATGACATTCGTGTCGTGGGTCA GTCGCGCATAGCCAATCGTCCGGCAGTCGTT CTGGTGCTGGCGCCCAAGGACCAGCATCGCT ACGGCTTCGAATTGCATCTGGACCGGGAGAC CGGTCTGCCGTTGAAATCCCTCCTGTTGAAC GAGCCTTGGCCACTCTACTGGAGCGCTTCCA GTTCGCTCAGCTCTGATACCTCTGTACCCGT TGAGGATGCCATGCAGCCGAGTTCGAGCTGC AGGCCGGTGCGTTTTCGTGCTGCCGACAGCA TGGCCGAAGGTACCTGGCGATCCGACTGGCT GCCGCCGGGCTTTACTCTGACTACTGCGCAG GTACGCCGCGTGCCTTCCGCTcccagggatt accagttatc
(106) TABLE-US-00006 TABLE6 DNAoligonucleotides SEQID Primername Sequence Description NO: prGFF123 CAGGCCTAACACATGAAG 63Fprimerforbacterial 8 TC 16S(Yamane,2008) prGFF124 GGGCGGWGTGTACAAGGC 1387Rprimerfor 9 bacterial16S(Yamane, 2008) prGFF533 gaattcctgcagcccaaa FwdOverlappingprimer 10 tGAGGCGGTAACAGGGGA forGibsonassemblyof T GumAtoM(ConstructI) intopGFF80cutwith SmaIandXbaI prGFF534 gcggtggcggccgctcta RevOverlappingprimer 11 gaCCTTGCTGACCTTCCA forGibsonassemblyof CAG GumAtoM(ConstructI) intopGFF80cutwith SmaIandXbaI prGFF556 ttcagcatacagataaaa 1200to1177ntbefore 12 actgcc B.subtilisNCIB3610 TasA(forward orientation) prGFF557 ggtaagctccccttttat 1to25ntbefore 13 tgaatga B.subtilisNCIB3610 TasA(rc) prGFF558 taacagcaaaaaaaagag 1to26ntafterendof 14 acggccca B.subtilisNCIB3610 TasA prGFF559 tattgtcactaggtcttc 1200to1175ntafterend 15 acctagtc ofB.subtilisNCIB3610 TasA(rc) prGFF560 CGATATAAAATCATTCAA -35to-1upstreamat 16 TAAAAGGGGAGCTTACCt B.subtilisNCIB3610 tcaacaaacgggattgac TasAplus-106to-77nt ttttaaaaaag atcatORFinpNW33N prGFF561 TATGAATACTGGGCCGTC 35to1ntdownstreamof 17 TCTTTTTTTTGCTGTTAg B.subtilisNCIB3610 tcgggaaacctgtcgtgc TasAORF(rc)plus100 cag to78ntaftercatORFin pNW33N(213to235in pNW33N) prGFF599 ggcggttcctctcttggc -1to-25ntatmucAin 18 tgtcgcc PstutzeriDSM 10701_wellconserved prGFF600 TTAGGGAGAAACATGCGC 1beforeendto20nt 19 GTA aftermucAinPstutzeri A1501,DSM4166, ATCC17588, CCUG29243_notethat stopdiffersfrom10701, butthissequence conserved prGFF605 TTGCAGGATAGCTTGCTA CHECK_PRIMER_-725 20 GGA to-705atmucAin PstutzeriDSM10701_Int differencefromother stutzerigenomes prGFF610 GCTCGTGCGGCCAGCATG 29to6ntbeforeendof 21 GAAGA mucAinPstutzeri A1501,DSM4166, ATCC17588, CCUG29243notethat stopdiffersfrom10701, butthissequence conserved prGFF612 TCCAGCGCAACGACCCGC 997to978ntsafter 22 CC mucAinPstutzeri A1501,DSM4166, ATCC17588_notethat thisdiffersfrom10701 prGFF643 ctggaagtttggctcatg Forwardprimeratnt5 23 tc inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF644 agtctatcaggcaagctg Forwardprimeratnt323 24 attc inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF645 gtgagttcgaaggctgag Forwardprimeratnt837 25 ttac inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF646 gcttctccggtactctcg Forwardprimeratnt 26 gta 1355inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF647 gtgctgaaagtcgatggt Forwardprimeratnt 27 cag 1823inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF648 tctgaaaggtcgcgtcgt Forwardprimeratnt 28 2353inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF649 taatcttatcgccgctca Reverseprimeratnt222 29 tttcc inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF650 cgtcatagaactccgcgt Reverseprimeratnt713 30 cat inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF651 gcttccattgcgctcata Reverseprimeratnt 31 gatg 1735inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF652 cggatcgccaggtacctt Reverseprimeratnt 32 c 2228inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF653 ctcgagcgccactacacg Reverseprimeratnt 33 2568inP.alcaliphila34 sequence_mucAplus 1kbflanks prGFF662 CTTGCTGGCCGGGCGCAT 9076to9096inPelAtoG 34 CGC geneclusterin P.alcaliphila34(note thatnumberingisbased onPAO1sequence);224 to204ntbeforePelF prGFF663 GGGCCGGAACTGATCAGG 10981to10959(rc 35 CCGGC orientation)inPelAtoG inP.alcaliphila34(note thatnumberingisbased onPAO1sequence); 101tont78ntafterend ofPelF prGFF664 GAGGGAACCTGGCCCTAT 9355to9374inPelAtoG 36 GT geneclusterin P.alcaliphila34(note thatnumberingisbased onPAO1sequence);In PelF prGFF665 TTCGTCAATTACTTCTGG 9766to9785inPelAtoG 37 AC inP.alcaliphila34(note thatnumberingisbased onPAO1sequence);In PelF prGFF668 TCGACCAGCTCGCGACAC 10601to10578(rc 38 GAGCC orientation)inPelAtoG inP.alcaliphila34(note thatnumberingisbased onPAO1sequence);In PelF prGFF669 TCGATGCCATTGGGGATC 10202to10182(rc 39 ACC orientation)inPelAtoG inP.alcaliphila34(note thatnumberingisbased onPAO1sequence);In PelF prGFF672 CTGTCGGTMGGGGAGTTC 289to312in 40 GACGGC P.aeruginosaPelA; alignmentwith P.protogensmatches wasusedtogenerate consensussequencehere prGFF673 CAGGGCTACGCCGGCCTG 454to476in 41 TTCCT P.aeruginosaPelA; alignmentwith P.protogensmatches wasusedtogenerate consensussequencehere prGFF674 AAGGCGAACGGRTCGAGG 1496to1471(rc)in 42 ATCCAGCG PAO1PelA;alignment withP.protogens matcheswasusedto generateconsensus sequencehere prGFF675 GGCCCGACYTCGCCTTCG 1712to1693(rc)in 43 AT PAO1PelA;alignment withP.protogens matcheswasusedto generateconsensus sequencehere prGFF676 ATGGCGGGTATAGGCTTT 10826to10849in 44 GAACTG PelAtoGinP.alcaliphila 34(notethatnumbering isbasedonPAO1 sequence);Beginningof PelG prGFF677 ATGGCGGTGTTCCTGGTG 11678to11697in 45 CG PelAtoGinP.alcaliphila 34(notethatnumbering isbasedonPAO1 sequence);InPelG prGFF678 TCGAACTCGAGATCGTCC 12174to12152(rc 46 AGCGC orientation)inPelAtoG inP.alcaliphila34(note thatnumberingisbased onPAO1sequence);In PelG prGFF680 TCAGCGATTGAGCATGAA 12196to12173(rc 47 GGTTTC orientation)inPelAtoG inP.alcaliphila34(note thatnumberingisbased onPAO1sequence); Notethatfirst8ntsare fromPAO1sequence (missinginblastmatch sequencefromPa34); EndofPelG prGFF681 TTGATCTGCGCCCAGCCG 1286to1263(rc)in 48 GTGATG PAO1pslA prGFF682 TTCAAGTTCCGCTCGATG 1003to1023inPAO1 49 TAC pslA prGFF683 ATGCGTCAGCTTTTGCAC 1to23inP.mendocina 50 GGTAG pslA;ympandDLHK sequencesareidentical prGFF684 TCAGTAGGCCTCGCGGGT 1434to1412(rc)in 51 GAAGA P.mendocinapslA;ymp andDLHKsequences areidentical prGFF685 GACGTGATCGGCAGCGAG InP.mendocinapslA-O 52 GACGCCTA blastmatchat3431to 3456,accordingto alignmentwithPAO1 P.aeruginosanumbering prGFF686 CCCGAGGACGTGATCGGC InP.mendocinapslA-O 53 AGCGA blastmatchat3425 3447,accordingto alignmentwithPAO1 P.aeruginosanumbering prGFF687 CCATCTACGAGCTGACGC InP.mendocinapslA-O 54 TGT blastmatchat4067to 4087,accordingto alignmentwithPAO1 P.aeruginosanumbering; inPslDORF prGFF688 AGATAGAGGTCGACGCCC InP.mendocinapslA-O 55 TCGAT blastmatchat4544to 4522(rc),accordingto alignmentwithPAO1 P.aeruginosanumbering; inPslDORF prGFF689 CGGAATGTACGAATTTCG InP.mendocinapslA-O 56 ATCATG blastmatchat4650to 4528(rc),accordingto alignmentwithPAO1 P.aeruginosanumbering; afterPslDORF prGFF690 CGAAAGGAACGAATTTCG InPseudomonas 57 ATCATG pseudoalcaligenesstrain CECT5344pslA-O blastmatchat4650to 4528(rc),accordingto alignmentwithPAO1 P.aeruginosanumbering; afterPslDORF prGFF691 ACAAGCTCGCCATCCCGC InPseudomonas 58 CCGCCTA alcaligenesstrain MRY13pslA-Oblast matchat17569to 17593,accordingto alignmentwithPAO1 P.aeruginosanumbering; inPslNORF prGFF692 CGCAAGCAGTACCGCTAC InPseudomonas 59 CACCCGC alcaligenesstrain MRY13pslA-Oblast matchat17661to 17685,accordingto alignmentwithPAO1 P.aeruginosanumbering; inPslNORF prGFF693 CGCAGGTGCTCCAGGGCC InPseudornonas 60 AG alcaligenesstrain MRY13pslA-Oblast matchat18196to18177 (rc),accordingto alignmentwithPAO1 P.aeruginosanumbering; inPslNORF prGFF694 GCCCAGGTGCGGTAGTCC InPseudomonas 61 TTGGCG alcaligenesstrain MRY13pslA-Oblast matchat18166to18143 (rc),accordingto alignmentwithPAO1 P.aeruginosanumbering; inPslNORF prGFF695 cctgttatccctacccgg pslDupstreamflank 62 gCAGCAAGCGCCTGGCCG knockoutprimer;19nts AC frompEX100T (upstream)atSmaIsite plus3319to3337in pslAtoOsequence(525 to507ntbeforepslD); basedonByrd2009 knockoutconstruct prGFF696 CAGGAAGTGCTCCCTCAT pslDdownstreamflank 63 GAAAcgctgaggagcgac knockoutprimer22nt atcgccatgatag beforepslDandfirst6nt ofpslDplusLast6ntof pslDand21ntafterpslD prGFF697 ctatcatggcgatgtcgc pslDupstreamflank 64 tcctcagcgTTTCATGAG knockoutprimer; GGAGCACTTCCTG Reversecomplementof pslDOLF prGFF698 gataacagggtaatcccg pslDdownstreamflank 65 ggGATCTCCATCACCGTC knockoutprimer;20nts GAG frompEX100T (downstream)atSmaI siteplus5141to5123in pslAtoOsequence(527 to509afterpslDend) prGFF699 cctgttatccctacccgg pelFupstreamknockout 66 gCGACGTCCGCCTGCTGG primer;19ntsfrom CCTAC pEX100T(upstream)at SmaIsiteplus8821to 8842;480to459before startofpelForf;based onColvin12 prGFF700 CATGCAATCTcggtgtgt pelFupstreamflank 67 tcggtcatgtcc knockoutprimer;1to10 ntbeforeendofPelF (rc)plus+16to-4ntat PelFstart(rc) prGFF701 cgaacacaccgAGATTGC pelFdownstreamflank 68 ATGACATGGCCG knockoutprimer;6to 16ntafterstartofPelF pluslast11ntofPelF and8ntafterPelF prGFF702 gataacagggtaatcccg pelFdownstream 69 ggCCAGCAGGATGCGTTT knockoutprimer; GTAGG 20ntsfrompEX100T (downstream)atSmaI siteplus11321to 11301inpelAtoG; 497to477afterendof pelForf prGFF723 ATCAGGCCGCCATACAGG 10914to10892in 70 TAGGC PelAtoG(rcorientation) 89to67inPelGin P.alcaliphila34, GFF238,andGFF248 (matchallthree)based onnumberinginPAO1 sequencePelAtoG prGFF724 AGCATCACGCTGATGATC 10956to10935in 71 GACA PelAtoG(rcorientation) 131to1110inPelGin P.alcaliphila34, GFF238,andGFF248 (matchallthree)based onnumberinginPAO1 sequencePelAtoG prGFF725 TCCTGGCCTACTCGATGC 8832to8853inPAO1 72 TCGA PelAtoG;518to539in PelE(Pao1):Matches P.alcaliphila34 sequence prGFF726 CTGGCGCGTTGGTACTGG 8948to8970in 73 GAACT P.alcaliphila34PelAtoG (numberingbasedon PAO1);634to656in PelE(numberingbased onPAO1) prGFF727 GTCCAGAAGTAATTGACG 9785to9766inPelAtoG 74 AA (rcorientation)485to 466inPelFinPAO1; identicalmatchto P.alcaliphila34 prGFF728 TCCTTCTGACCGCCGATG 9464to9445inPelAtoG 75 AA (rcorientation)in P.alcaliphila34 (numberingbasedon PAO1);164to145in PelF;1nucleotide mismatchwithPAO1 prGFF729 CAGCGTCTGCTGGAAGGC 8044to8067inPAO1 76 AGCCAG PelAtoG;271to248 beforePelE(Pao1); MatchesP.alcaliphila34 sequence prGFF730 TGAATGATCAGCAAGTGG 8312to8331inPAO1 77 CT PelAtoG;-3to+17at PelE;Matches P.alcaliphila34 sequence
(107) TABLE-US-00007 TABLE7 Summaryofsequencingdata gene/ SEQ Strain locus contig IDNO: Sequence P. mucA 1 78 ctggaagtgttggctcatgtccgagttccgcgtgaaagcgagccctagtatatagagag alcaliphila gggcagcggcacaatagccagcttatgaccgaccgcggtgctcgggaacatcttagtca AL15-21; ttaaggttccatagagggtcgcccatataagggagagcgcatgcgcgtgcctgcatctg GFF238 aagtcagcggagggtgtggaaatgagcggcgataagattatttgcgcgagcggaaaggt tccgctggcgccattccaggcagagggaagaaaactctgcgggattgcttgcttggagg ggagaacttttgcgtaagacccgagtctatcttggcaagctgattcgcttacgggcgct tagcctcctccaagcgttacgttggagaatgcatgctaacccaggaagatgatcagcaa ctggtcgagcgagtgcagcgtggtgacttagcgtgccttcgatctgttggtgctgaagt atcagcacaagatcctcggtctgatcgtgcgartcgtgcacgacacccacgaggctcag gatgtcgctcaggaggcgttcgtaaaagcctaccgagcgcaggattacatcgcggtgac agtgcgttctatacatggctgtaccgcatcgccatcaacacggcgaagaattatctggt gtcccgcggtcggcggccgccagatagtgatgtcagtagcgatgacgcggagttctatg atggcgatcacggcctcaaggacatcgagtcaccggagcgggcattgctgcgcgacgag atcgaagccaccgtgcatcgaaccatcgcccaactgccggatgatttgcgcacggccct gaccctgcgtgagttcgaaggcttgagttacgaggacattgcaggcgtcatgcaatgcc cggtaggcacggtgcgttcgcggatattccgtgcacgtgaggcaattggaagtccctgc aacctctgttgcaggaaacctaaggcagcggcgacagccaagagaggaacaccatgagt cgtgaaaccctgcaggaatcgctgtccgcggtgatggataacgaagcggacgaactgga actgcggcgtgtgctcgcagccagcgaggatggcgagctgcgtggcacctggcgcgtta ccaggtcgcccgtgcagccatgcatcgtgaactgttggtgccgcaactggacatcgcat ctgcggtctccgcggcgctggccgacgaagccgttccggcacgcaaggcgccgatctgg cgtagtgtcggtcgcgtagccgtggcagcatcggtgaccgttgcagtgctggcgggtgt gcgatctacaatcaggatgacctgagcggcgctcaactggcccagcaggagacttctcc ggtactctcggtacctcaggtccagggtcctgcactgctcgctggttacaacagcagcg aggaagccggcgaagccgccgaagcaggcactgccagctggcatgagcagcgcctgccg aactacctgcgtcaacatgcgcaggaagccgtgatgggtaccggtgaaaccgctctgcc ttatgctcgggctgcgagtctggattaaccgctaagcgctttaggagtcacatgcgcgc gattcccctctacatctcggtggtctgctggcgagccggacaggcctccgaggtgcagg acctgctcgggcgtctcgctgcggcggagcgccagcaaagcttccagggcacgttcatc tatgagcgtaatgggagtattctacccatgccgtgtggcatcgggtggaggaggggggc gcagttcgcgaacgccttctgcaactcgatgggcctgctcaggaagtgctgaaagtcga tggtcaggctcaggcgtcactggcgcgaggccgaccaggtcagtgaagggcaggcatgg cctgctcgccagctggatgccgagcaactgagcgactggtatgacattcgtgtcgctgg cttagtcgcgcattgccaatcgtccagcggtcgttctggtgttggcccccaaggacctt gcatcgctacggcacgaattgcatctggatcgtgagaccgggagccgctgaagtccctg ctgttgaacgagcgcggccagcttctggaacgcttccagttcgcccaactggatacttc tgtaccggctgaaaatgccatgcagcctagctccagctgcaggccggtgcggttccgcg ctgccgacagcatggacgaaggcagttggcgatccgactggttgccgccgggtttcact ctgaccactgcgcaggtgcgtcgcgggcctgccgctgatgactccgtcacctatctgat gtacggcgatggcctggtgcgattctcggtttttctcgagcctcttaaaggtcgcgtcg tcgaagacgcgcgcagtcagttgggtccaaccgtcgccgtttcgcgacggatgagcacc gatgcgggtgacgtgatggttaccgtggtcggtgagattcctctggggactgccgagcg catagccctgtcgatgcgcgccggagtgcctgaacaggctagccaatgatcgaagagca ggggcgtgtagtggcgctcga P.toyotomie mucA 1 79 tggctcatgtccgagttccgcgtgaaagcgagccctagtatatagagaggggcagcggc nsis1IT-3; acaataggtggcttatgaccgaccgcggtgctcgggaactttcttagtcgttaaggttc GFF248 catagagggtcgcccatataagggagagcgcagcgcgtgcctgcatctgaagtcagcgg agggtgtggaaatgagcggcgataagattatttgcgcgagcggaaaggttccgctggcg ccattccaggcagagggaagaaaactctgcgggaagcttgcttggaggggagaactatg cgtaagacccgagtctatcttggcaagctgattcgcttacgggcgcttgacctcctccg agcgttatgaggagagtgcggctaacccaggaagatgatcagcagctggtcgagcgagt gcagcgcggtgacaagcgtgccttcgatctgttggtgctgaagtatcagcacaagatcc tcggtttgatcgtgcgattcgtgcacgacacccacgaggctcaggatgtcgctcaggag gctttcgtaaaagcctaccgagcgcttggaaactttcgcggtgacagcgcgttctatac atggctgtaccgcatcgccatcaacacggcgaagaattatctggtgtcacgcggtcggc ggccgccagatagtgatgtcagtagcgatgacgcggagactatgacggcgaccacggcc tgaaggacatcgagtcaccggagcgggcattgctgcgcgacgagatcgaagccaccgtg catcgaaccatcgcccagttgccggatgatttgcgcacggccctgaccttgcgtgagtt cgaaggcttgagttacgaggacattgccggcgtcatgcagtgtccggtaggtacggtgc gttcgcggatcttccgtgcgcgtgaggcaattgataagtccctgcagcctagttgcagg aaacctaaggcagcggcgacagccaagagaggaacaccatgagtcgtgaaaccctgcag gaatcgctgtccgcggtgatggataacgaagcggacgaactggaactgcggcgtgtgct cgcagccagcgatgatggcgagagcgcggcacctggtcgcgttaccagatcgcccgtgc agccatgcatcgtgagctgtggtgccgcaactggacatcgcatctgcggtttccgcggc gctggccgacgaagccgtcccggcacgcaaggcgccgatctggcgtagtgtcgggcgcg tagccgtcgcagcatcggtgaccgttgcagtgctggcgggtgtgcgcactacaatcagg atgacctgagcggcgcccaattggcacagcaggaggcactccggtactctctgtaccgc aggtgcaaggtcctgcgctgctcgctggttacaacagcagcgaggaagccggcgaagcc gccgaagcaggcactgccagctggcatgagcagcgtttgccgaactacctgcgtcaaca tgcgcaggaagccgtgatgggtaccggtgaaaccgctctgccttatgacgggctgcaag tctggaaaaccgctaagcgctaaggagtcacatgcgcgcgattcccctctaccttctcg gtggtctgctggcgagccggttcaggccactgaggtgcaagacttgctcgggcgcctcg ctgcggcagagcgccagcaaagcttccagggcacgttcatctatgagcgcaatggaagt tatccacccatgccgtgtggcatcgggtagaggaggggggcgaagttcgcgagcgcctt ctgcagctcgatgggcctgcccaggaggtgctgaaagtcgatggccaggctcagtgcgt cactggcgcgttggctgaccaggtcagtgaagggcaggcttggcctgctcgtcagttgg ctgtcgagcaattgagcaactggtatgacattcgtgtcgtgggtcagtcgcgcatagcc aatcgtccggcagtcgttctggtgctggcgcccaaggaccagcatcgctacggcttcga attgcatctggaccgggagaccggtctgccgttgaaatccctcctgttgaacgagcgtg gccagctactggagcgcttccagttcgctcagctggatacctctgtacccgttgaggat gccatgcagccgagttcgagctgcaggccggtgcgttacgtgctgccgacagcatggcc gaaggtacctggcgatccgactggctgccgccgggattactctgactactgcgcaggta cgccgcgtgccttccgctgatgatcccgtcacctatctcatgtatggcgatggcctggc gcgattctcggtttttctcgaacccctgaaaggtcgcgtcgtcgaggatgcacgcagcc agctgggcccaaccgtcgcggtttcgcggcggatgagtaccgactctggtgacgtcatg gtgaccgtggtgggtgagatccccttggggactgccgaacgcatcgccctgtccatgcg cgccggagtgcctgaacaggctagccaatgatcgaggagcaggggcgtg P.stutzeri mucA 1 80 ttgctaggaggggggagaacttagcgtaaagoccgggtctattaggcaggtcggttcgc PTA-8823; tggtgtgagcgacgctactccgctaccgaggaggagcgttcatgttgactcaggagcag GFF390 gaccagcagctggagaacgagtgcagcgtggtgacaagcgggcgtttgatctgaggtaa tgaaataccapacaagatccttgggttgatcgtgcggttcgtgcatgactctcatgaag ctcaggatgttgcccaagaggcttttatcaaagcctaccgtgcactagccaattttcgc ggtgacagcgctttctacacctggctgtaccgcatcgccatcaatacggcgaagaatca tctggtggcgcgcggcagacgcccgccggatagtgatgtgagaccgaggacgccgagtt ttatgagggcgatcacgctctcaaggacatcgagtcaccggaacgctcgctgctcaggg atgagattgaagataccgttcatcgaaccattcaacttagccagaagatttgcgtacgg ctctaacactgcgtgaatttgatggtcttagttatgaagacattgcgagcgtcatgcag tgtccggtgggcacagtgcgacccggatcttccgggcacgtgaagccatagataaagca ttgcaacccttgttgcatgaatcctgagacagcggcgacagccaagagaggaaccgcca tgagtcgtgaagccctgcatgaatcgctgtccgcggtgatggataacgaagcggacgag aggaattacgtcgcatgctcgcangcgacaacccggagctacgtgctacctggtcgcgt tatcaacttgcccgtgccgccatgcacaaggagttgatcgagccgcgcctggatatcgc ttctgcggtatcggctgcg 2 81 cagcatggaagaacgttagggagaaacatgcgcgtattaccgctgtatgtggtgatggg gggaggctatcgatgccggcccttgctgctgatgccgggtcctggatggagcgacttgc ggcggcagagcagaaacagagttataccggtacgttcgtctacgaacgcaatggcagct tttccagtcatgccgtttggcagcaggtcgaagaaggtcaagtgcaggagcgattgatc agcttgacggagctccggctgaagttctgctggtaaatggtcagatgcaatgcgctacc gatgacctcgcggcgcaagtgcgtgaagcgcaggcttggcacgggcagcgtctcgatcc gaaagcactctccgagtggtacgaattccgtgagatcggggattcacgagttgctggcc gccccgccgtgggctggctgtcgtgccgaaggatcagcaccgttacggcttcgaactct gcatctcgaccaagataccgcattgcccctcaagtcgctgatgctgaacgagaaagggc agctgctcgagcgtaccaattcacccagttcacggctggcagtgtatctgccgagcaac tgaagcccggcgccgattgcaatccagtgaccgtgaaccggcgcgaggcgaatcctaca tcgccctggcgctccgactggttgccttccggcttcacgctactggatgccaacgaacg acctagtcccgcctatccgaaactgtttcctggttgtcctacggtgatggtctagcgaa gttttccgtgtttctggagccgctgcgcggcgccttggttgaggacgcgcgaagccaga tggggcctaccgtcgcggtctctaagcgcatcagtactgcggatggcgatgtcatggtg accgtggtgggcgagattccacttggtacggctgagcgggttgccctgtccatgcgagc cagttcggaacaggcacaacgatgatcga P.alcaliphila PelE 1 82 caggtacctcaggccgcgttgcatatcctcttctcatggcttgggaagcgccatgctgg AL15-21; toEnd ctgcagggatctggctgctgctgccgcgccgctatcgctaccccttgccctggagcccc GFF238 of tgttcattttcagcgtctcgacttcattcccttgattgggatgatcggcgtagcgctgg PelG cgctgtttccggccactacttgccgcgcaagcgcaaggtgcagtcctgggaggctactg ccgttcccgagctgcctttccggccgcgcgagcgcaagcgtgagctgatgttcagcgat ggcgggagcaggatgtgttacgccatgcgogcgaccccgatcagcgtctgacggccatc ttcgcgacacgacgcatgcgcagcaaggaggccatcccgattctcaagctggcgctgcg cgatccatctgacgatgttcgcctgctggcctattcgatctcgatcagcgtgaaagccg aatcaaccagcgtatcgagcgtgcgaggcagatatggagagcgccagcacggaccgtaa gttcgccctgcatgggcaactggcgcgctggtactgggagctcgcctatgtcggcctgg cccagggcagtgtgttggagcacgtgctgcagcaggctggagccatgtgatggcggcgc tgcagggtggctcgggtggcgaactgcacttgcttgccgggcgcatcgccatggagcag ggcaatctcgatgaggcgttggcgcagttcgaccagtcggcgctggccgggatggatgc ggtgcaattggcgccgtatcgggctgaaatcgcgtattgcgtcaacgctatgaggaaat tccagaaatgctggcgacgatgccggccgaactgttgcaacgtccccccttcgcggcca ggctagatattggttatgagtgagaaatctgctgttcctgacgtgcagagcgtcgatgt ctgcctgttgctcgagggaacctggccgtacgtgcgcgggggggtatcgagctggatca accaactgatcctggggctgccggaactgaccttctcggtgctgttcatcggcggtcag aagga 2 83 gcctacctgtatgccggcctgatcagttccggcccttgggtattgtcgatcatcagcgt gatgctcatcggcgtactgagcctgggggcggtgctgccggaaacgctgatcggtcagt tcctggtcaccgtgacgtacctgatggccacgtcgctgattctcaccggcggactgcag ctgttcttcacccgcttcgtctccgaccggctgttcgagcggcgcctggacctgatcct cccaatctgatcgggatactgctgctggtgacgatcttctcggggctgctggcggtctg tgtgatgggcctgctgttcgatcagtcgtttacctatcgcctgctggtgatggccaact tcgtggtgctgtgcaacctctggctggtgatcatcttcctgtcggggatgaaggcttat aaccgcatcctgggggtgatgtttctcggctactcgctgatggtcgcctcggcctatct gctgcgctttctgaatatcgacggcttgctgctgggcctgctgatcggtcattccagcc tgctgttcatcttcctgttcgacatcctgcgcgagtacccggccgagcgcctggtcgcg ttcgattttctcaagcgtcgccaggtgttcggcagcctgctgctgacaggactttgcta caacctgggcatctggatcgacaagttcatcttctggtcaacccctcgacttccgaagc cgtgatcggcccgttgcgggcctcgatcctctatgacctgccgatcttccttgcctacc tgtcgatcatccccggcatggcggtgttcctggtgcgtatcgaaacggacttcgccgaa tggtacgagcgggtctatgacgcgatccgcggcggtgaaaccctgcagcacatcggctg gctcaaggagcagatgatcctggcgattcgccagggcctgatggagatctgcaaggttc aggggctgaccctggttctgctattcctgctcgcgccgcagttgttgtcctggctgggc atctcgcactactacctgccgctgttctacatcgacgtgataggcgtgagcattcaggt ggtgttcatggccttgctcaacgtgttcttctatctggacaagcgtgccatcgtcctcg aactctgcgttctcttcgtcctggcaaacggcgcgttgaccctgttcagccagatgctt ggcccgaccttcttcggctatggcttcaccctgtcgctgctgctgtgcgtactcctcgg gttgtatcgtctcaacgaggcgcttgatgatctcgagttcgaaaccttcatgctcaatc gctga P.toyotomie PelE 1 84 atgatcagcaagtggctgtttagcggcgctgcgctgctcgaggtcgggagctgggccag nsisHT-3; toEnd tgcggtcagcgatctcccgattcatcaggccgcgttgctctatgcctccgcgcatggcc GFF248 of tgggcagtgcgatgctggctgccgggatctggctgctgctgccgcgccgctatcgctac PelG cccttgccttggagcctgctgttcattttcagcatctcgttcttcatccccctgatcgg natgatcggcgtggcgctggcgttgtttcccgccctctacttgccgcgaaagggcaagg tgcagtatgggaggcgaccgccgacccgaactgcctttccgaccgcgtgagcgcaagca ggagttgatgacagcgatggtggccttcaggacgtgttgcgtcatgcgcgtgaccccga tcagcgcctgacggcgatcttcgccacacgacgcatgcgcagcaaggaggccatcccga ttctcaagctggcgctgcgcgatccatcngacgacgtgcgtttgctggcntactcgatg ctcgatcagcgtgaaagccgaatcaaccagcgtatcgagcgcgctctggcagatatgga gagcgccagcacggaccgccagttcgccctgcatgggcaactggcgcgttggtactggg agcttgcctatgtcggcctggcccagggcagtgttctggagcacgtgctgcagcaggcc tggagccatgtgatggcggcgctgcagggcaactcgggtggtgagctgcacttgttggc cgggcgtatcgccatggagcagggcaatctcgacgaggcgttggcgcagttcgaccagt cggcacaggccgggatggatgcggtgcagttggcgccgtatcgggccgagatcgcgttt ttgcgtcaacgctatgaggattattccagacatgaggcgacgatgccggccgagctgtt gcaacgtccccccttcgcggctttggcaagatactggttatgagtgagaaatccgctgt tcctgacgtacagagcgtcgatgtctgtctgttgctagaggggacctggccctatgtgc gtgggggcggtctagctggatcaaccagctgatcctggggctgcctgagctgactttct cggtgctgttcatcggcggtcagaaggaggcctacggcaagcgccattacgctatcccg gacaacgtggtgcacattcaggagcatttcctcgaagactcctggagttcgattcccac caccagtacccgtgcgagcgctgagctggccgagctgatgctggatgtgcaccgtttcc tgcacaacccggaggaacccagcactgagcagggcgatgcattcgtcgataccctggct gccggacgcatcggccgtgaggccttcct 2 85 tcagctacatccgtagattgtggatccgcttcttcgagcgtattgggctgatcacctac cgatctgcgcactccatcatcgcgctgtacgagggcaacaggcggcgtcaggttctcga tggcgctcctgaggagcgtactagggtcattoccaatggcatcgatctggccagttggg atcaggcgctgctcagccggccagttaggcgtgccgccggtggccggcctggttgggcg ggtggtaccgatcaaggacgtcaaaaccttcatccgcgccatccgtggtgtggtcagcg tcatacccgaagccgagggctggatcgtcgggccggaagaagaagatccggattacgcg gccgagtgccacagcctggtggccagcctggggctgcaggacaaggtgcgttttctcgg cttccgccaggtgcgcgaagtcgtaccgcaactgggcgtgatggtgctgacgtcgatca gtgaggcgcagccgctggtggtacttgaggcctgggcggccggcacgccagtggtgacc agtgacgtcggctcgtgtcgcgagctggtcgaaggctccacgcaggaggatcgccagct cggtacggcgggggaggtggtagcaattgccgacccgcaggccacctcgcgcgccattc tctcgctgttgcgcaacccggagcgctggaaggctgcgcaggctgtcggccttgagcgt gtgcggcgttactacaccgaagaactgatgctcgcgcgctatcgcgagagtatcgcgaa ggcacggagagcgcgtaatggcgggtatagctttgaactgaggaagatcctgtccaagg 3 86 gatgcgcgcctacctgtatgccggcctgatcagaccggcccttgggtgttgtcgatcat cagcgtgatgctcatcggtgtactgagcctgggggcggtgctgccggaaacgctgatcg gccagttcctggtcaccgtgacctacctgatggccacgtcgctgattctcaccggcggg ctgcagctgttcttcacccgcttcgtctccgaccggttgttcgagaaacgcctggacct gatcctgcccaacctggtgggcatactgctgctggtaacgatcgtctcggggctgctgg caatctgcgtgttgggcttgctgttcgatcagtcgttttcctatcgcctgctggtgatg gccaacttcgtggtgctgtgcaacctgtggctggtgatcatcttcctgtcggggatgaa ggcctacaaccgcattctcggcgtgatgttcctcggctactcgctgatggtcgcctcgg cctatctctgcgattctcaatatcgacggcctctgctgggcctgctgatcggtcactcc agcctgctgttcatcttcctcttcgacatccttcgcgagtacccggccgagcgcctggt cgcgttcgatttcctcaagcgccgtcaggtattcggcagcctgctgctgacagggctgt gctacaacctgggtatctggatcgacaagttcatcttctggttcaacccctcgacttcc gaagccgtgatcggcccattgcgcgcatcgatcctctatgacctgccgatatcctcgcc tatctgtcgatcatccccggcatggcggtgtttctggtacgtatcgaaaccgacttcgc cgagtggtacgagcgggtctatgacgcgatccgcggcggggaaaccctgcagcacatcg gctggctcaaggagcagatgatcctggcgattcgccagggtctgatggaaatctgcaag gtccaggggctgaccctggttctgctgtttctgctggcgcccagttgttgtcctggctc ggcatctcgcactactacctgccgctgactatatcgacgtgatcggcgtgagcattcag gtggtattcatggccttgctcaacgtgttcttctatctggacaagcgcgccatcgtcct cgaactctcgttctatcgtcctggcaaacggtgcgctgaccctgttcagccagatgctt ggcccgacattatcggctatggcttcaccctgtcgctgctgcigtgcgtgctcctgggg ttgtatcgtct
(108) TABLE-US-00008 TABLE 8 DNA plasmid vectors Plasmid Alternative Vector E. coli Name name backbone Insert--Gene Notes marker pGFF80 pBBR1MCS pBBR1MCS Broad host plasmid; From Cam CBS pGFF93 pNW33N; pNW33N From BGSC; Gram positive Cam BGSC expression vector; E. coli; ECE136 shuttle vector pGFF155 pGFF80 gumABCDEFGHIJKLM cloned by isothermal Cam (X. campestris) assembly; construct I (generated with oligos prGFF533&534); verified by NGS sequencing pGFF157 ATCC pEX100T From ATCC; Gram negative Amp 87436 knockout vector; Amp, SacB, Ori (pMB1), OriT, LacZalpha pGFF167 pGFF157 mucAdelta knockout vector; cloned mucA Amp (P. stutzeri seamless knockout construct GFF390) (GFF390) into SmaI site in pGFF157 by isothermal assembly of partGFF27 (5prime 358 nt plus 3prime 358 nt); Orientation is forward in vector (reverse in LacZ gene) pGFF170 pGFF157 mucAdelta knockout vector; cloned mucA Amp (P. alcaliphila seamless knockout construct GFF238) (GFF238) into SmaI site in pGFF157 by isothermal assembly of partGFF30 & 31 (5prime 715 nt plus 3prime 714 nt); Orientation is forward in vector (reverse in LacZ gene) pGFF171 pGFF157 mucAdelta knockout vector; cloned mucA Amp (P. toyotomiensis seamless knockout construct GFF248) (GFF248) into SmaI site in pGFF157 by isothermal assembly of partGFF32 & 33 (5prime 715 nt plus 3prime 714 nt); Orientation is forward in vector (reverse in LacZ gene) pGFF173 pGFF157 mucAdelta knockout vector, cloned mucA Amp (P. aeruginosa seamless knockout construct PAO1) (GFF233) into SmaI site in pGFF157 by isothermal assembly of partGFF28&29 product (5prime 716 nt plus 3prime 716 nt); Orientation is forward in vector (reverse in LacZ gene) pGFF174 pGFF157 pslDdelta knockout vector; cloned pslD Amp (P. aeruginosa seamless knockout construct PAO1) (GFF233) into SmaI site in pGFF157 by isothermal assembly of PCR products prGFF695/697 & prGFF696/698 (5prime 525 nt plus 3prime 527 nt); Orientation is forward in vector (reverse in LacZ gene) pGFF175 pGFF157 pelFdelta knockout vector; cloned pelF Amp (P. aeruginosa seamless knockout construct PAO1) (GFF233) into SmaI site in pGFF157 by isothermal assembly of PCR products prGFF699/700 & prGFF701/702 (5prime 480 nt plus 3prime 497 nt); Orientation is forward in vector (reverse in LacZ gene)
(109) TABLE-US-00009 TABLE 9 Strains Strain Strain Plasmid name Species Background Name Insert--Gene Genotype GFF233 Pseudomonas ATCC aeruginosa PAO1 BAA-47 GFF238 Pseudomonas DSM alcaliphila 17744; AL15-21 GFF248 Pseudomonas JCM15604 toyotomiensis GFF257 Pseudomonas GFF248; pGFF80 toyotomiensis JCM15604 GFF373 Bacillus subtilis BGSC 1A1; 168 GFF374 Bacillus subtilis BGSC 3A1; NCIB3610 GFF375 Pseudomonas GFF238; pGFF155 gumABCDEFGHIJKLM alcaliphila DSM17744 (X. campestris) GFF377 Pseudomonas GFF248; pGFF155 gumABCDEFGHIJKLM toyotomiensis JCM15604 (X. campestris) GFF390 Pseudomonas stutzeri ATCC PTA-8823; LH4:15 GFF419 Pseudomonas GFF238; mucAdelta alcaliphila DSM17744
Example 9
(110) Isolating and Screening Biosurfactant and Biopolymer Producing Alkaliphilic Microbes from Mono Lake
(111) 1. Isolation of Biosurfactant and Biopolymer Producing Microbes from Mono Lake
(112) Water samples were collected at Marina Beach, Navy Beach and South Tofa, as well as Hot Springs on Paoha Island of Mono Lake using plastic bottles, wrapped in foil and stored in an ice containing cooler. YCAB10, a pH 10 artificial medium similar to the chemistry of Mono Lake water, containing 0.7% boric acid, 5% NaCl, 2% NaHCO3 was prepared and filtered (0.2 M) sterile. 100 L of each Mono Lake water sample was spread on YCAB10 agar plates, incubated at 30-50 C for 16 hours. Large colonies formed by various fast growing microbes were transferred to new fresh plates for screening biosurfactant production microbes. After 16 hours' incubation at 30 C, a thin layer of Paraffin oil was sprayed onto the plates using an airbrush (Central Pneumatic Oil-less Air brush Compressor kit model #95630 sold by Harbor Freight Tools). A microbial colony surrounded by uniform round droplets of Paraffin oil suggested that the microbe secreted biosurfactant, which migrated outward from the colony and changed the chemistry around it on plate. Biosurfactant secreting colonies were chosen and cultured in YCAB10 liquid medium, surfactant production was further confirmed by oil spreading, i.e., a drop of culture on a thin layer of petroleum formed on top of water in a petri dish. A drop of surfactant containing culture would replace an area of the petroleum layer. In addition, biosurfactant was isolated from cultures by acid precipitation, i.e. the culture supernatant was adjust to pH2.0 by hydrochloric acid, the biosurfactant was precipitated by centrifugation at 12,000 g for 15 min, quantified, analyzed by HP LC-mass spectrometry.
(113) In water flooding or MEOR, biosurfactant increases oil recovery by emulsifying petroleum components and enhancing the interaction of oil and water. Biopolymer is another critical component in microbial enhanced oil recovery (MEOR). Biopolymer increases oil recovery by increasing the viscosity of the flooding fluid. Therefore, an ideal microbe applied in microbial enhanced oil recovery should be able to produce either biosurfactant or biopolymer, or produce both of them in situ. Mucoid colony phenotype was typical for a microbe capable of producing biopolymer. To isolate microbes able to produce biosurfactant and biopolymer, biosurfactant producing colonies selected above were placed on YACB10 agar plates, mucoid colonies were biopolymer producing microbes and chosen for further characterization and penetration laboratory testing.
(114) Biopolymer produced by microbes in culture can also increased the viscosity of the culture. To test microbial biopolymer production, candidate colonies were cultured in YCAB10 medium, the culture viscosity was measured using a No. 2 A627 viscometer tube, suggested those isolated alkaliphilic microbes from Mono Lake did produce biopolymer, increased the viscosity of the culture two to three fold. Biopolymers was isolated from the cultures, and dissolved in an appropriate solution, to quantitatively analyzed the viscosity of the biopolymer.
(115) After a series of screening processes, more than a hundred microbial isolates were isolated from Mono Lake. Some of these isolates are facultative anaerobes, able to grow in the absence of oxygen if nitrate is supplied. Some of isolates from Hot Springs in Paoha Island were able to grow at 55 C.
(116) 16S rRNA gene sequences were PCR amplified and sequenced from these alkaliphilic microbes isolated from Mono Lake. Sequences analysis and BLAST search revealed that microbes isolated from Mono Lake beach belonged to Halomonas genus, while those isolated from hot springs of Paoha Island were in genus Bacillus such as Caldalkalibacillus uzonensis and Bacillus halodurans. The 16S rDNA gene sequences data is provided as FASTA format in Table 10.
(117) TABLE-US-00010 TABLE10 AlkaliphilicIsolates16SrDNA SEQ Isolate Species IDNO: Sequence ML2-9 Halomonas 87 GGGGATAACCTGGGGAAACCCAGGCTAATACCGCATAC 16SrDNA campisalis GTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTGCG (1170bp) CTATCGGATGAGCCCATGTCGGATTAGCTTGTTGGTGA GGTAATGGCTCACCAAGGCGACGATCCGTAGCTGGTCT GAGAGGATGATCAGCCACATCGGGACTGAGACACGGCC CGAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGAC AATGGGCGAAAGCCTGATCCACTCCATGCCGCGTGTGT GAAGAAGGCCCTCGGGTTGTAAAGCAGTTTCAGTGGGG AAGAAAGCCTTGAGGTTAATACCTTCGAGGAAGGACAT GACCCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCA GCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAAT TACTGGGCGTAAAGCGCGCGTAGGCGGTCTGATAAGCC GGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATC CGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGA ATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAG GAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACT GACGCTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATT AGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACT AGCCGTTGGGGTCCTTGAGACCTTTGTGGCGCAGTTAA CGCGATAAGTCGACCGCCUGGGGAGTACGGCCGCAAGG TTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAC CTTACCTACCCTTGACATCGAGAGAACTTGGCAGAGAT GCCTTGGTGCCTTCGGGAACTCTCAGACAGGTGCTCCA TCTGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTA AGTCCCGTAACGAGCGCAACCCTTGTCCTTATTTGCCA GCGCGTAATGGCGGGAACTCTAAGGAGACTGCCGGTGA CAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCAT GGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGA CGGTACAAAGGGTTGCAAAGCCGCGAGGTGGAGCTAAT CCCATAAAGCTGTTCTCAGTCCGGATCGGAGT HS2 Caldalkalib 88 AGCCTGATGGAGCACGCCGCGTGAGCGAGGAAGGTCTT 16SrDNA acillus CGGATTGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGT (783bp) uzonensis CGTTCGAATAGGGCGGCACCTTGACGGTACCTAACGAG AAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAAT ACGTAGGGGGTCGAGCGTTGTCCGGAATTATTGGGCGT AAACTCGCGCGCAGGCGGTCTCTTAAGTCTGATGTGAA AGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGG GAGACTTGAGTGCAGGAGAGGGAAGCGGAATTCCACGT GTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAG TGGCGAAGGCGGCTTCCTGGCCTGTAACTGACGCTGAG GCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT GGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGG GGGTTTCAACACCCTCAGTGCTGAAGTTAACACATTAA GCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACT CAAAGGAATTGNCGGGGGCCCGCACAAGCGGTGGAGCA TGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAG GACTTGACATCCTCTGACCGCCCTAGAGATAGGGTCTT CCCCTTCGGGGGACAGAGTGACAGGTGGTGCATGGTTG TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG CAACGACCGCAACCCTTGACCTTAGTTGCCAGCATTCA GTTGGGCACTCTAAGGTGACTGCC HS9 Bacillus 89 TATACAAGGAAAGGCCGCTGAAAAGCGACCTCTTTTTT 16SrDNA halodurans CATCAATCAATGTCCAGAGGCCTTGTACACNCCGCCCC (1168bp) 99%match NNGGTTTCGGCTATCACTTACAGATGGGCCCGCGGCGC ATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAAC GATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA GTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAG CAACGCCGCGTGAGTGATGAACGTTTTCGGATCGTAAA ACTCTGTTGTTAGGGAAGAACAAGTGCCGTTCGAAAGG GCGGCACCTTGACGGTACCTAACGAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCA AGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAG GCGGTCTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAA CCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTACA GAAGAGCAGAGTGGAATTCCACGTGTAGCGGTGAAATG CGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACT CTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGG GGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGATGAGTGCTAGGTGTTAGGGGTTTCGACGCCT TTAGTGCCGAAGTTAACACATTAAGCACTCCGCCTGGG GAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCAGTGGAGCATGTGGTTTAATTCG AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTT TGACCACCCTAGAGATAGGGCTTTCCCCTTCGGGGGAC AAAGTGACAGGTGGTCTCATGGTTGTCGTCAGCTCGTG TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTTGACCTTAGTTGCCAGCATTCAGTTGGGCACTCTA AGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACA CACGTGCTNCTCNGGCNTAACACANGNANTCGAAGCCG CGAAGGNGCANCCGATACCTNAAAGCCCT BF1 Idiomarin 90 GAGGGTTAAGCTATCTACTTCTGGTGCAGCCCACTCCC 16SrDNA sp ATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGT (1395bp) ATTCACCGTGGCATTCTGATCCACGATTACTAGCGATT CCGACTTCACGGAGTCGAGTTGCAGACTCCGATCCGGA CTACGACGCGCTTTTTGAGATTCGCTTGCTATCGCTAG CTTGCTGCCCTTTGTGCGCGCCATTGTAGCACGTGTGT AGCCCATCCCGTAAGGGCCATGATGACTTGACGTCGTC CCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCCTAG AGTTCCCACCATTACGTGCTGGCAACTAAGGATAAGGG TTGCGCTCGTTGCGGGACTTAACCCAACATCTCACAAC ACGAGCTGACGACAGCCATGCAGCACCTGTCTCAGAGT TCCCGAAGGCACTAATCCATCTCTGGAAAATTTTCTGG ATGTCAAGGGATGGTAACTGTTCTTCGCGTTGCATCGA ATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGT CAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCC AGGCCCTTCAACTTAGTGCGTTAGCTGCGTTACTCACA TCATAATGACACGAACAACTAGTTGACATCGTTTACGG CGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCA CGCTTTCGCTCCTCAGCGTCAGTTTTTGACCAGGTGGC CGCCTTCGCCACTGGTATTCCTTCCAATATCTACGCAT TTCACCGCTACACTGGAAATTCTACCACCCTCTTCAAA ACTCTAGCCTGCCAGTTCAAAATGCTATTCCAAGGTTG AGCCCTGGGCTTTCACATCTTGCTTAACAGGCCGCCTA CGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTCGC ACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGC CGGTGCTTCTTCTGTGGCTAACGTCAATCCTTTGCCGC TATTAACGACAACGCCTTCCTCACCACTGAAAGTGCTT TACAACCCGAAGGCCTTCTTCACACACGCGGCATGGCT GGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTG CTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCA GTCTTGGCTGATCATCCTCTCAGACCAGCTAGAGATCG TCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATC TCGCTTGGGTTCATCTGATGGCGTATGGCCCGAAGGTC CCATACTTTGCTCCGAAGAGATTATGCGGTATTAGCCA CCGTTTCCAGTGGTTGTCCCCCGCCATCAGGCAGATCC CCAAGTATTACTCACCCGTCCGCCGCTCGTCATCATCT AGCAAGCTAGATATGTTACCGCTCGACTGCA
(118) 2. Isolation of Alkaliphilic Ultramicrobacteria (0.2 M) from Mono Lake
(119) To isolate ultramicrobacteria (0.2 M), Mono Lake water was collected in September from Marina Beach. The water was filtered by a 0.2 M filter to remove any particles and microbes larger than 0.2 micron. The filtered water was spread on YCAB10 agar plates, and incubated at 30 C for 72 hours. Two types of colonies, mucoid and non-mucoid, were isolated. The 16S ribosomal RNA gene of mucoid ultramicrobe was amplified using universal primers, and 16 rRNA gene sequences were obtained. 16S RNA Gene sequences analysis using BLAST and BioEdit, identified the isolated ultramicrobacteria was similar to gamma-proteobacteria Alteromonadales bacterium and Idiomarina sp. To isolate additional ultramicrobacteria slightly larger than 0.2 micron, membrane filter with 0.45 M pores was also used to filter Mono lake water, the same procedure described above was used and a number of colonies were isolated. However, 16S RNA sequences analysis suggested they were identical with those ultramicrobacteria isolated from 0.2 M filtered Mono Lake water, belonged to genus Idiomarina.
(120) 3. Sandpack Column and Berea Sandstone Core Penetration of Microbes Isolated from Mono Lake
(121) The alkaliphilic microbes isolated from Mono Lake were cultured, and used for sand pack columns and Berea sandstone columns penetration tests. A number of strains of Bacillus and Pseudomonas obtained from microbial collection center were used as control, such as biosurfactant producing Bacillus mojavensis JF-2, biofilm producing Bacillus subtilis 3610, alkaliphilic B. circulans, and P. stutzeri. All these microbes were able to penetrate sand pack column, the recovery rate of these microbes varied, ranging from 5% (ML2-9) to 95% (Bacillus mojavensis JF-2).
(122) Additionally, our penetration experiments showed that starved cells, vegetative cells or spores of these microbes were unable to penetrate low permeability Berea Sandstone Core (50 mili Darcy). However, they were able to penetrate high permeability Berea Sandstone Core (>100 mili Darcy), viable cells were recovered at elute at 5% (Bacillus JF-2 starved cells) to 6% (Bacillus mojavensis JF-2 spores). The control Sand Stone Core penetration experiments showed that about 80% was recovered for ultramicrobacterium Sphingomonas alaskensis, and about 50% for Mono Lake isolate gamma-proteobacterium BF1 (Idiomarin sp).
(123) The alkaliphilic microbial cultures were also used to recover the residual oils in sandpack cells and columns. In comparison with control microbe Bacillus and Pseudomonas culture, alkaliphilic microbial cultures recovered residual oil 15-50% more than that recovered by the control microbes.
(124) The penetration of microbes through the formation or sandstones remains as an unsolved problem, a challenge and an obstacle for MEOR application. To test the penetration of ultramicrobacteria in sand stone cores, Berea Sandstone cores (purchased from Cleveland Quarries 5270 Devon Dr. Vermilion, Ohio 44089 with estimated permeability in the range of 50 to 1000 mD) and Stone Cores from oil field in New Mexico (a gift from HEYCO Energy Group Inc. permeability estimated less than 50 mD) were used for ultramicrobacterial penetration test. Three pore volumes of sterile water were flooded through the cores, followed by flooding Mono Lake Water filtered by 0.2 micron filter and untramicrobacteria cultures. To check untramicrobacterial capability to penetrate the stone cores, elutes were collected and spread on agar plates. Our results showed that after 10 to 12 pore volume flooding, the Alkaliphilic untramicrobacteria isolated from Mono Lake penetrated the stone cores, and the recovered rate of viable cells was around 2%, suggesting the untramicrobacteria was able to penetrate Berea sandstones and the formation from oil field.