Bacterial Fouling in a Model Core System

We have used a sintered glass bead core to simulate the spaces and surfaces of reservoir rock in studies of the bacterial plugging phenomenon that affects waterflood oil recovery operations. The passage of pure or mixed natural populations of bacteria through this solid matrix was initially seen to promote the formation of adherent bacterial microcolonies on available surfaces. Bacteria within these microcolonies produced huge amounts of exopolysaccharides and coalesced to form a confluent plugging biofilm that eventually caused a >99% decrease in core permeability. Aerobic bacteria developed a plugging biofilm on the inlet face of the core, facultative anaerobes plugged throughout the core, and dead bacteria did not effectively plug the narrow (33-μm) spaces of this solid matrix because they neither adhered extensively to surfaces nor produced the extensive exopolysaccharides characteristic of living cells. The presence of particles in the water used in these experiments rapidly decreased the core permeability because they became trapped in the developing biofilm and accelerated the plugging of pore spaces. Once established, cells within the bacterial biofilm could be killed by treatment with a biocide (isothiazalone), but their essentially inert carbohydrate biofilm matrix persisted and continued to plug the pore spaces, whereas treatment with 5% sodium hypochlorite killed the bacteria, dissolved the exopolysaccharide biofilm matrix, and restored permeability to these plugged glass bead cores.     

Reactive Transport Modeling of Induced Selective Plugging by Leuconostoc Mesenteroides in Carbonate Formations

A process-based mechanistic reactive transport model was developed to understand how in-situ coupled processes and operational factors affect selective plugging of reactive carbonate formations by the fermenting bacteria Leuconostoc mesenteroides that produces a plugging polymer dextran. The growth and transport of L. mesenteroides and the associated (bio) geochemical reactions were simulated explicitly with enzyme activity at the field scale over spatial extents of hundreds of meters. Simulations were performed to explore controls on selective bioplugging of high permeability zones in a representative carbonate reservoir, a process that can be used to improve oil sweep efficiency through lower permeability layers. Simulation results indicate that dextran production and the effectiveness of plugging can be largely affected by sucrose and bacteria injection rates. Selective plugging of high permeability zones can only be achieved when the injection rates are high compared to the rates of dextran production. Otherwise, plugging only occurs at the vicinity of injection wells. Due to the dependence of enzyme activity on pH and the reactive nature of carbonate formations, the chemistry of the injection and the formation water is also important. The injection of sucrose and L. mesenteroides at the optimum pH for dextran production (5.2) leads to the dissolution of calcite and an increase in pH levels. However, the resulting pH does not suppress plugging with dextran. Lactic acid and CO₂ formed during the growth of L. mesenteroides buffers the pH of water to levels between 5.2 and 7.0 for continued dextran production. At neutral and basic pH levels, induced precipitation of calcite does not significantly modify the permeability profile at carbonate concentrations typically found in oilfield formation waters. This is the first work that examines the controlling parameters that affect selective plugging of carbonate formations at the field scale within the context of enhanced oil recovery. The demonstrated approach can be used to identify optimal operational conditions for enhanced oil recovery and other applications where selective plugging can be beneficial.     

Effect of biological activity on the movement of fluids through porous rocks and sediments and its application to enhanced oil recovery

Movement of fluids through geological porous media is an important factor in ore genesis, crude oil genesis, and in oil recovery from oil reservoirs. Such a movement may be beneficially affected by biological in situ activity. We have shown that once bacteria have been introduced into a porous medium, they are able to penetrate into their environment and multiply without decreasing significantly the rock's permeability. The limiting bacterial concentration, which may cause plugging, depends on the bacterial size, pore size, and pore size distribution of the reservoir rock. Microorganisms may, under anaerobic conditions, affect the composition of crude oil by increasing its asphaltenic fraction. In simulated systems microorganisms increased the recovery of the oil in place approximately five times. A bacterial consortium was more effective than a single pure bacterial strain.     

Evaluation of bioemulsifier mediated Microbial Enhanced Oil Recovery using sand pack column

Bacillus licheniformis K125, isolated from an oil reservoir, produces an effective bioemulsifier. The crude bioemulsifier showed 66% emulsification activity (E(24)) and reduced the surface tension of water from 72 to 34 mN/m. It contains substantial amount of polysaccharide, protein and lipid. This bioemulsifier is pseudoplastic non-Newtonian in nature. It forms oil in water emulsion which remains stable at wide range of pH, temperature and salinity. It gave 43+/-3.3% additional oil recovery upon application to a sand pack column designed to simulate an oil reservoir. This is 13.7% higher than that obtained from crude lipopeptide biosurfactants produced by the standard strain, Bacillus mojavensis JF2 and 8.5% higher than hot water spring isolate, Bacillus licheniformis TT42. The increased oil recovery obtained by using the crude bioemulsifier can be attributed to its combined surface and emulsification activity. Its mechanism of oil recovery must be similar to the mechanism exhibited by surfactant-polymer flooding process of chemical enhanced oil recovery.     

Effects of oil reservoir conditions on the production of water‐insoluble Levan by Bacillus licheniformis

Bacillus licheniformis produced a water‐insoluble levan which has potential application as a selective plugging agent in microbial enhanced oil recovery (MEOR). The microorganism grew on sucrose, glucose, and fructose but produced levan only on sucrose. Plugging may thus be selectively controlled in the reservoir by substrate manipulation. B. licheniformis and a crude preparation of its extracellular enzymes were evaluated for their ability to produce levan under reservoir conditions. Oil reservoirs which have a temperature of less than 55°C, a pH between 6 and 9, a pressure less than 500 atm, and a salt concentration of 4% or less are potentially suitable. Examples of such reservoir conditions are found in Lloydminster on the Alberta‐Saskatchewan border, one of the largest Canadian oil reserves.     

Response of microbial community structure to microbial plugging in a mesothermic petroleum reservoir in China

Microbial plugging, a microbial enhancement of oil recovery (MEOR) technique, has been applied in a candidate oil reservoir of Daqing Oil Field (China). The goal of this study is to monitor the survival of injected bacteria and reveal the response of microbial communities in field trial of microbial plugging through injection of selected microbial culture broth and nutrients. Culture-dependent enrichment and culture-independent 16S rDNA clone library methods were used. The results show that it was easy to activate targeted biopolymer-producing bacteria in a laboratory environment, and it was difficult for injected exogenous bacteria to survive. In addition, microbial communities in the oil reservoir also changed before and after the field trial. However, microbial communities, activated by fermentative medium for biopolymer-producing bacteria, appeared to show greater differences in the laboratory than in the natural reservoir. It was concluded that microbial populations monitoring was important to MEOR; results of response of microbial communities could provide a guide for the future field trials.     

Alteration of the growth rate and lag time of Leuconostoc mesenteroides NRRL-B523.

Bacterial profile modification is an important enhanced oil recovery technique used to direct injected water into a reservoir's low permeability zone containing trapped crude oil. During water flooding, the use of bacteria to plug the high permeability water zone and divert flow into the oil-bearing low-permeability zone will have a significant economic impact. However, during the field implementation of bacterial profile modification, the rapid growth of bacteria near the injection well bore may hinder the subsequent injection of growth media so that profile modification of the reservoir occurs only in the immediate vicinity of the well bore. By slowing the growth rate and prolonging the lag phase, the onset of pore-space plugging may be delayed and the biologically active zone extended deep into the reservoir. High substrate loading, high pH values, and the addition of the growth inhibitors sodium dodecylsulfate and sodium benzoate have been used in combination to alter the growth characteristics of Leuconostoc mesenteroides NRRL-B523 grown in batch conditions. The highest sucrose concentration used in these studies, 500 g/L, produced lag times 12-fold greater than the slowest lag times achieved at low sucrose concentrations. When L. mesenteroides was grown in media containing 500 g/L sucrose, an alkaline pH value threshold was found above which bacteria did not grow. At this threshold pH value of 8.1, an average lag time of 200 h was observed. Increasing the concentration of sodium benzoate had no effect on lag time, but reduced the growth rate until the threshold concentration of 0.6%, above which bacteria did not grow. Last, it was found that a solution of 0.075 mM sodium dodecylsulfate in media containing 15 g/L sucrose completely inhibited bacterial growth.     

Effects of biocide treatment and backflow pressure on the permeability of microbially fouled model cores

Summary Sintered glass bead cores were plugged until the permeability was reduced to 1% or less of the original permeability by the injection of a slime-producing bacterium isolated from produced water. Scanning electron microscopy of fractured core sections showed that the bacteria were predominantly located in the uppermost sections, around the core inlet. Killing the bacterial cells in the plugging biofilm, using a biocide, had little effect on core permeability. The dead cells were only removed when backflow pressure, simulated by inversion of the test core followed by fluid injection, was applied and maintained at 55–69 kPa. Backflow of plugged cores containing live bacteria produced transient pressure-dependent increases in permeability that were proportional to the backflow pressure applied. We conclude that only sustained backflow procedures reduced permeability: such operations are not effective for oil recovery in field conditions.     

Foreshore sand as a source of Escherichia coli in nearshore water of a Lake Michigan beach

Swimming advisories due to excessive Escherichia coli concentrations are common at 63rd Street Beach, Chicago, Ill. An intensive study was undertaken to characterize the source and fate of E. coli in beach water and sand at the beach. From April through September 2000, water and sand samples were collected daily or twice daily at two depths on three consecutive days per week (water samples, n = 1,747; sand samples, n = 858); hydrometeorological conditions and bird and bather distributions were also recorded. E. coli concentrations in sand and water were significantly correlated, with the highest concentration being found in foreshore sand, followed by those in submerged sediment and water of increasing depth. Gull contributions to E. coli densities in sand and water were most apparent on the day following gull activity in a given area. E. coli recolonized newly placed foreshore sand within 2 weeks. Analysis of variance, correlation, cluster analyses, concentration gradients, temporal-spatial distribution, demographic patterns, and DNA fingerprinting suggest that E. coli may be able to sustain population density in temperate beach sand during summer months without external inputs. This research presents evidence that foreshore beach sand (i) plays a major role in bacterial lake water quality, (ii) is an important non-point source of E. coli to lake water rather than a net sink, (iii) may be environmentally, and perhaps hygienically, problematic, and (iv) is possibly capable of supporting an autochthonous, high density of indicator bacteria for sustained periods, independent of lake, human, or animal input.     

Effect of solid bed-materials on vegetative cells of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis>

Nostoc flagelliforme was cultivated on three solid bed-materials which have different wettability. The results show that the wettability of solid bed-materials is very important to the need for water in N. flagelliforme, and also greatly affects photosynthetic activity, polysaccharide secretion and the morphology of the cells. Glass residue has the best wettability, followed by sand, and then nylon6 (PA6). We show that only sand is suitable for the growth of N. flagelliforme. When cultivated on sand for 100 days, the cells maintained a typical rosary-like shape. On glass residue, two to three vegetative cells formed a group with slime. On PA6, cells were in the form of loose group with slimes. N. flagelliforme is applicable to desert control as it cannot only grow on the sand, but also adhere to sand to form a biological crust.     

Legacy Effects of Oil Road Reclamation on Soil Biology and Plant Community Composition

Following the unprecedented oil drilling presently occurring in western North Dakota, thousands of kilometers of oil roads must be reclaimed to an acceptable post‐extraction condition. This study assessed the soil biological and plant communities of nine decommissioned oil roads reclaimed during three periods between 1983 and 2002 in the Little Missouri National Grasslands of western North Dakota. We hypothesized that time‐since‐reclamation would positively affect soil biological and plant communities and, consequently, success of reclamation at older sites. To assess this hypothesis, we measured soil enzyme activity, soil microbial community composition, plant community composition, and soil physical and chemical properties along a gradient extending from road‐center to adjacent native prairie for the nine roads. Time‐since‐reclamation did not affect soil and plant properties measured, indicating that older reclamations are not more similar to native prairie than reclamations occurring more recently. A strong gradient between samples from road‐center and native prairie was identified with univariate and ordination analyses, indicating that soil and plant communities of reclaimed oil roads do not resemble those of the surrounding prairie. Soil organic matter (SOM) was identified as the most significantly affected soil property, being 30% lower on reclaimed roads than prairie. The relationship between SOM, microbial community, and plant community suggests that incorporating additional SOM could hasten reclamation as a result of improving the physical environment for plants and providing a labile carbon and energy source for the soil microbial community which, in turn, will enhance the nutrient and physical conditions for plant growth.     

Preliminary in vitro observations on the bacteriology of the black plug layer phenomenon associated with the biofouling of golf greens

Nine sand columns were developed and subjected to various conditions thought to be associated with the bacterial generation of a black plug layer. Five axenic cultures which were originally isolated from a natural black plug layer (BPL) and a clay-like plug layer (CLPL) were used. These cultures were applied individually and in various combinations to biofoul and possibly synthesize a BPL or CLPL in sterile vertical sand columns. Biofilm formation was monitored by gravity water flow impedance and through an observation of visual changes.
Combinations (consortia) of the isolated cultures were found to form a synthetic BPL rather than where individual strains were used. All strains caused some flow impedance biofouling. Rapid plugging of the sand columns occurred in the first 3 d of the experiment. This plugging tended to occur at the top and bottom of the sand column around the upper in- and lower out-flow ports to the medium circulation system. There was no relationship between the black colour associated with BPL and the development of the initial plugging. A clear biofilm developed initially in all cases to cause plugging. When sulphur-reducing bacteria were incorporated into the column inoculum, visible black sulphide deposits were observed to develop after 3 d to form the BPL. Insoluble gases were produced in all of the columns and could be observed through the generation of fracture cavities and pockets within the sand column with associated increases in internal pressures resulting in back pressures and port disruption.     

Plugging of a Model Rock System by Using Starved Bacteria

The effects of starvation on bacterial penetration through artificial rock cores were examined. Klebsiella pneumoniae was starved in a simple salts solution for a duration of up to 4 weeks. These cell suspensions were injected into sintered glass bead cores, and the resulting reductions in core permeabilities were recorded. Vegetative cell cultures of K. pneumoniae grown in a sodium citrate medium were injected into other, similar cores, and the reductions in core permeabilities were recorded. The starved cell suspensions did not completely block the core pores, whereas the vegetative cultures reduced core permeability to less than 1%. Scanning electron microscopy of core sections infiltrated with either vegetative or starved cells showed that the former produced shallow “skin” plugs and copious amounts of glycocalyx at the inlet face, whereas the latter produced very little glycocalyx and the cells were distributed evenly throughout the length of the core. The use of a DNA assay to produce a cell distribution profile showed that, compared with the vegetative cells, starved bacteria were able to penetrate deeper into the cores. This was due to the smaller size of the cells and the reduction in biofilm production. This ability of starved bacteria to penetrate further into cores than the normal-size vegetative cells can be usefully applied to selective plugging for enhanced oil recovery. To further test the suitability of starved cells for use in selective plugging, the activities of starved cells present within cores were monitored before and after nutrient stimulation. Our data indicate that with nutrient stimulation, the starved cells lose their metabolic dormancy and produce reductions in core permeability due to cell growth and polymer production.     

Tree swallows (Tachycineta bicolor) nesting on wetlands impacted by oil sands mining are highly parasitized by the bird blow fly Protocalliphora spp

Oil sands mining is steadily expanding in Alberta, Canada. Major companies are planning reclamation strategies for mine tailings, in which wetlands will be used for the bioremediation of water and sediments contaminated with polycyclic aromatic hydrocarbons and naphthenic acids during the extraction process. A series of experimental wetlands were built on companies' leases to assess the feasibility of this approach, and tree swallows (Tachycineta bicolor) were designated as upper trophic biological sentinels. From May to July 2004, prevalence and intensity of infestation with bird blow flies Protocalliphora spp. (Diptera: Calliphoridae) were measured in nests on oil sands reclaimed wetlands and compared with those on a reference site. Nestling growth and survival also were monitored. Prevalence of infestation was surprisingly high for a small cavity nester; 100% of the 38 nests examined were infested. Nests on wetlands containing oil sands waste materials harbored on average from 60% to 72% more blow fly larvae than those on the reference site. Nestlings on reclaimed sites suffered mean parasitic burdens about twice that of those on the reference site; and for comparable parasitic load, they exhibited greater pathologic effects (e.g., decreased body mass) than control nestlings. The heavy blow fly infestation on oil sands-impacted wetlands suggests that oil sands mining disturbs several components of the local ecosystem, including habitat characteristics, blow fly predators, and host resistance to parasites.     

Partial mechanical impedance can increase the turgor of seedling pea roots

Roots of 3-d-old pea seedlings (Pisum sativum L.) were mechanically impeded using a sand core apparatus, which allowed mechanical impedance to be varied independently of aeration and water status. Turgor of root cortical cells was then measured using a pressure probe. In seedlings grown in sand cores for 1 d, impedance had little effect on turgor, but in seedlings grown in the sand cores for 2 d, impedance increased turgor by 0.18 MPa in the apical 6 mm.     

Influence of benthic organisms on solute transport in lake sediments

Increased inputs of nutrients into the waters of Lake Okeechobee has raised concern that the lake is becoming hypereutrophic. One aspect in understanding the overall cycling and dynamics of the nutrients in the system is the effect of benthic organisms on solute transport. Various diffusional models have been used to approximate the effect of benthic organisms on solute transport within sediments using diffusion coefficient values which represent the pooled contributions of molecular diffusion (D _s ) and enhanced solute mixing due to macrobenthos activity (D _i ). The objective of this study was to investigate the impact of benthic activity on solute transport by measuringD _s (i.e., no benthic activity) and an apparent-dispersion or mixing coefficientD _m (i.e., with benthic activity) for the four major sediment types of Lake Okeechobee, Florida using a reservoir technique. This method involved monitoring the depletion of a conservative tracer (tritiated water) from the overlying water (reservoir) resulting from transport into sediments using disturbed sediments repacked in cores (3.2 cm diam.) and undisturbed cores (3.2 to 12 cm diam.) obtained from the lake. Additional estimates ofD _m andD _s were also obtained by measuring tracer concentration profiles in the sediment cores at the end of a specified diffusion period. Molecular diffusion coefficients (D _s ) measured forrepacked cores of sand, littoral, mud and peat sediments ranged from 0.90 to 1.29 cm² d⁻¹, and estimates ofD _s were slightly higher in undisturbed cores without benthic organisms.D _m values for undisturbed cores of mud, sand and littoral sediments having macrobenthic populations ranged from 2.09 to 24.78 cm² d⁻¹; values that were 1.6 to 15 times higher than those in sediments without benthic activity. Undisturbed cores of varying diameter from mud sediments had similar estimates ofD _m for tritium; however, the undisturbed cores with larger diameters from littoral sediments yielded larger estimates ofD _m , reflecting the heterogeneity of benthic population densities and activity. Therefore,D _s estimates may not adequately represent transport processes for mud, sand and littoral sediments of Lake Okeechobee; hence careful consideration should be given to the role of benthic organisms in the overall transport of solutes across the sediment-water interface. A contribution of the Florida Agricultural Experiment Station Journal Series No. R-01150. A contribution of the Florida Agricultural Experiment Station Journal Series No. R-01150.     

Foreshore Sand as a Source of Escherichia coli in Nearshore Water of a Lake Michigan Beach

Swimming advisories due to excessive Escherichia coli concentrations are common at 63rd Street Beach, Chicago, Ill. An intensive study was undertaken to characterize the source and fate of E. coli in beach water and sand at the beach. From April through September 2000, water and sand samples were collected daily or twice daily at two depths on three consecutive days per week (water samples, n = 1,747; sand samples, n = 858); hydrometeorological conditions and bird and bather distributions were also recorded. E. coli concentrations in sand and water were significantly correlated, with the highest concentration being found in foreshore sand, followed by those in submerged sediment and water of increasing depth. Gull contributions to E. coli densities in sand and water were most apparent on the day following gull activity in a given area. E. coli recolonized newly placed foreshore sand within 2 weeks. Analysis of variance, correlation, cluster analyses, concentration gradients, temporal-spatial distribution, demographic patterns, and DNA fingerprinting suggest that E. coli may be able to sustain population density in temperate beach sand during summer months without external inputs. This research presents evidence that foreshore beach sand (i) plays a major role in bacterial lake water quality, (ii) is an important non-point source of E. coli to lake water rather than a net sink, (iii) may be environmentally, and perhaps hygienically, problematic, and (iv) is possibly capable of supporting an autochthonous, high density of indicator bacteria for sustained periods, independent of lake, human, or animal input.     

In situ biosurfactant production by Bacillus strains injected into a limestone petroleum reservoir

Biosurfactant-mediated oil recovery may be an economic approach for recovery of significant amounts of oil entrapped in reservoirs, but evidence that biosurfactants can be produced in situ at concentrations needed to mobilize oil is lacking. We tested whether two Bacillus strains that produce lipopeptide biosurfactants can metabolize and produce their biosurfactants in an oil reservoir. Five wells that produce from the same Viola limestone formation were used. Two wells received an inoculum (a mixture of Bacillus strain RS-1 and Bacillus subtilis subsp. spizizenii NRRL B-23049) and nutrients (glucose, sodium nitrate, and trace metals), two wells received just nutrients, and one well received only formation water. Results showed in situ metabolism and biosurfactant production. The average concentration of lipopeptide biosurfactant in the produced fluids of the inoculated wells was about 90 mg/liter. This concentration is approximately nine times the minimum concentration required to mobilize entrapped oil from sandstone cores. Carbon dioxide, acetate, lactate, ethanol, and 2,3-butanediol were detected in the produced fluids of the inoculated wells. Only CO(2) and ethanol were detected in the produced fluids of the nutrient-only-treated wells. Microbiological and molecular data showed that the microorganisms injected into the formation were retrieved in the produced fluids of the inoculated wells. We provide essential data for modeling microbial oil recovery processes in situ, including growth rates (0.06 +/- 0.01 h(-1)), carbon balances (107% +/- 34%), biosurfactant production rates (0.02 +/- 0.001 h(-1)), and biosurfactant yields (0.015 +/- 0.001 mol biosurfactant/mol glucose). The data demonstrate the technical feasibility of microbial processes for oil recovery.     

Starvation survivability of Thermococcus strains isolated from Japanese oil reservoirs

The starvation survivability of seven Thermococcus strains isolated from four Japanese oil reservoirs was compared with that of Thermococcus strains from marine hydrothermal fields. 16S rDNA analyses showed the isolates to be closely related to Thermococcus litoralis. Growth of the isolates was dependent on amino acids, which were present at low concentrations in the oil reservoirs. At 80 degrees C in the formation water, strain CKU-1 from the oil reservoir showed a higher starvation survivability than strain KS-1 from the marine hydrothermal field. Crude oil did not affect the starvation survivability of strain CKU-1, but it reduced that of strain KS-1. These results indicate that strain CKU-1 could survive longer than stain KS-1 under the conditions of an oil reservoir. At 90 degrees C in artificial seawater without organic nutrients, the half-lives of the isolates were between 7.7 and 25.1 days. However, those of the strains from marine hydrothermal fields, except Thermococcus litoralis and Thermococcus chitonophagus, were less than 1.0 day. The higher starvation survivability is probably important for the hyperthermophiles to continue to exist in a hot subterranean oil reservoir where the supply of nutrients seems to be limited.     

Relationship between Transport of Bacteria and Their Clogging Efficiency in Sand Columns

In an earlier article, we reported that, under conditions in which neither exopolymers nor bacterial mats were produced, Arthrobacter sp. strain AK19 was an effective plugging agent in sand columns, whereas the bacterial strain SLI^- had no significant effect on the permeability of the medium. A laboratory experiment with sand columns was carried out to elucidate the causes of this difference in behavior. Measured values of the saturated hydraulic conductivity of the sand were explained in terms of biomass accumulation, which was estimated by solving a mass balance equation. The relationship between the saturated hydraulic conductivity and the biomass density within the sand was exponential, although two different exponential coefficients were needed to fit the data for biomass densities above or below 13 mg (wet weight) per cm³, suggesting that two different clogging mechanisms may be involved in different ranges of biomass densities. The experimental results suggest that the SLI^- strain was a poor clogging agent partly because of its lower yield coefficient relative to the limiting nutrient (oxygen) and partly because 60% of the biomass produced in situ was washed out from the column, compared with only 1.2% in the case of Arthrobacter sp. strain AK19.