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An important consideration concerning novel organic compounds is their “inherent biodegradability,” i.e., their propensity to undergo ultimate bio-degradation resulting in C02, H20, minerals and microbial biomass. Inherent biodegradability is typically tested using C-labeled compounds under conditions favorable for microbial activity. This chapter shows that liposome packaging of test compounds offers a way to differentiate true recalcitrance from mass transfer and permeability problems. Alternately, liposomes may be prepared by extrusion through a small pore size filter. This method of preparation was introduced by F. Olson etal. and developed further by M. J. Hope etal. Using a 100 nm pore diameter filter, the average diameter of the resulting liposomes is 70-80 nm. Slightly smaller diameter liposomes can be prepared by the use of a 30 nm pore diameter filter. The extrusion process through a 100-nm pore size filter yielded phosphatidylcholine liposomes containing either 5% pyrene or 1.07% n-octadecane content.

Pseudomonas aeruginosa produces rhamnolipids which are tenso-active compounds with potential industrial and environmental applications. There are two main types of rhamnolipids produced in liquid cultures, rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (mono-rhamnolipid) and rhamnosyl-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxyd ecanoate (di-rhamnolipid). In this work we report the selective isolation of a rhamnolipid deficient mutant (IBT8), which does not accumulate mono-rhamnolipid while still producing di-rhamnolipid. IBT8 was selected after random mutagenesis with Tn501; yet, its mono-rhamnolipid deficiency was found associated neither with its Tn501 insertion nor with a possible alteration in the rhlABRI genes for rhamnosyl-transferase 1 synthesis. Different possibilities to explain IBT8 phenotype are discussed.

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG ) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.

The effectiveness of in situ bioremediation in many systems may be constrained by low contaminant bioavailability due to limited aqueous solubility or a large magnitude of sorption. The objective of this research was to evaluate the effect of hydroxypropyl-β-cyclodextrin (HPCD) on phenanthrene solubilization and biodegradation. Results showed that analytical-grade HPCD can significantly increase the apparent solubility of phenanthrene. The increase in apparent solubility had a major impact on the biodegradation rate of phenanthrene. For example, in the presence of 105 mg L-1 HPCD, the substrate utilization rate increased from 0.17 mg h-1 to 0.93 mg h-1 while the apparent solubility was increased from 1.3 mg L-1 to 161.3 mg L-1. As a result, only 0.3% of the phenanthrene remained at the end of a 48 h incubation for the highest concentration of HPCD tested (105 mg L-1). In contrast, 45.2% of the phenanthrene remained in the absence of HPCD. Technical-grade HPCD, which contains the biodegradable impurity pro...

A drop-collapse method has been refined for use as both a qualitative assay to screen for surfactant-producing microbes, and as a quantitative assay to determine surfactant concentration. The assay is rapid, easy to perform, reproducible and requires little specialized equipment. The assay is performed in a 96-microwell plate, where each well is thinly coated with oil. A 5 μL sample droplet is added to the center of a well and observed after 1 min. The droplet will either bead up, spread out slightly or collapse, depending on the amount of surfactant in the sample. The basis for this method is the type of oil used to coat each well. In the qualitative method, each well is coated with 1.8 μL of Pennzoil® and either the drop collapses, indicating the presence of surfactant (a positive result), or the drop remains beaded, indicating the absence of surfactant (a negative response). In the quantitative method, each well is coated with 2 μL of mineral oil, and a dissecting microscope is used to measure the diameter of the droplet at 1 min. Results with both a test biosurfactant (rhamnolipid) and a test synthetic surfactant (sodium dodecyl sulfate) indicate a direct linear correlation between droplet diameter and surfactant concentration. The drop-collapse method has several advantages over commonly used methods that measure surface tension, such as the du Nouy ring method; a smaller volume is required (5 μL vs. 20 mL), the effective range of measurement is greater and it does not require specialized equipment.

This study investigated the effect of cation type, ionic strength, and pH on the performance of an anionic monorhamnolipid biosurfactant for solubilization and removal of residual hexadecane from sand. Three common soil cations, Na+, Mg2+, and Ca2+, were used in these experiments and hexadecane was chosen to represent a nonaqueous phase liquid (NAPL) less dense than water. Results showed that hexadecane solubility in rhamnolipid solution was significantly increased by the addition of Na+ and Mg2+. Addition of up to 0.2 mM Ca2+ also increased hexadecane solubility. For Ca2+ concentrations greater than 0.2 mM there was little effect on hexadecane solubility due to competing effects of calcium-induced rhamnolipid precipitation and enhanced hexadecane solubilization. Efficiency of NAPL solubilization can be expressed in terms of molar solubilization ratios (MSR). The results showed that MSR values for hexadecane in rhamnolipid solutions increased 7.5-fold in the presence of 500 mM Na+, and 25-fold in the presence of 1 mM Mg2+. The presence of cations also reduced the interfacial tension between rhamnolipid solutions and hexadecane. For example, an increase in Na+ from 0 to 800 mM caused a decrease in interfacial tension from 2.2 to 0.89 dyn cm−1. Similarly, decreasing pH caused a reduction in interfacial tension. The lowest interfacial tension value observed in this study was 0.02 dyn cm−1 at pH 6 in the presence of 320 mM Na+. These conditions were also found to be optimal for removal of hexadecane residual from sand columns, with 58% of residual removed within three pore volumes. The removal of residual NAPL from the packed columns was primarily by mobilization, even though solubilization was significantly increased in the presence of Na+.

The efficiency of biosurfactant-facilitated removal of soil-bound metals is affected by biosurfactant sorption to soil. In this study, batch and column experiments were performed to minimize rhamnolipid biosurfactant sorption and to optimize rhamnolipid application for removal of cadmium from four soils. In batch studies, rhamnolipid sorption to a model coarse loamy soil was found to vary with applied rhamnolipid and K+ concentration of the rhamnolipid matrix. The presence of solution-phase biosurfactant was correlated to the release into solution of a soil-bound metal (cadmium). A series of column experiments was performed to evaluate whether rhamnolipid could remove cadmium from soil under saturated flow conditions. Four different soils were contaminated with cadmium and treated first with an KNO3 electrolyte solution (3.5 or 7 mM K+) and then with a rhamnolipid-containing solution (5 or 10 mM). Results showed that between 15 and 36% of the cadmium was removed by the initial electrolyte treat ment and a...

Biodegradation rates for polycyclic aromatic hydrocarbons (PAH) in the environment are limited by their low solubility and sorption to solid surfaces. The purpose of this study was to quantify the effect of biosurfactants on the dissolution, bioavailability, and biodegradation of a slightly soluble PAH, phenanthrene, in a series of batch solution studies. A mathematical model that describes the combined effects of solubilization and biodegradation, including description of bioavailability within surfactant micelles, was used to analyze the experimental results. Two forms of the biosurfactant, a monorhamnolipid and a dirhamnolipid, were tested; it was found that both surfactants increased the solubility and enhanced the rate of phenanthrene biodegradation. Monorhamnolipid was more effective than dirhamnolipid for solubilization; however, overall rates of mineralization were essentially the same. This seems to result from variable bioavailability of substrate: phenanthrene within monorhamnolipid micelles w...

Column studies were used to investigate the fate of a representative nonaqeous-phase liquid (NAPL), hexadecane, with specific regard to (1) the effect of attached bacteria on the formation of residual saturation and (2) the role of biodegradation and biosurfactants on the removal of residual NAPL. Residual saturation of hexadecane was determined using sterile sand (40/50 mesh) columns and was found to be 19.0 ± 4.8% of the pore volume. Columns loaded with bacterial biomass (Pseudomonas aeruginosa ATCC 15442, 109 cells g-1) showed no difference in residual hexadecane formation as compared to sterile sand columns. In further column studies examining the effect of ATCC 15442 and biosurfactants on the removal of hexadecane residual, results showed that biodegradation alone removed approximately 50% of the [14C]hexadecane, in the form of 14CO2 and undefined cellular metabolites, during elution with at least 200 pore volumes of mineral salts medium. The columns were then eluted with 1 mM rhamnolipid biosurfacta...

Agricultural practices may cause contamination of soil and ground water with a combination of organic compounds (pesticides and fuel) and nitrogen fertilizers. In coupled microcosm studies that monitored the mineralization of naphthalene and the nitrification of ammonia, it was observed that the solvent (dichloromethane) used to apply naphthalene to the soil inhibited nitrification, although there was no effect on naphthalene mineralization. Further studies were performed with a series of application solvents: methanol, acetonitrile, trichloromethane, and dichloromethane. Soil and solvent were allowed to equilibrate with ambient air for various times before capping and incubation of microcosms. Results indicated that dichloromethane equilibrated for 5 mins inhibited nitrification for at least 3 weeks relative to the control (water). Acetonitrile and trichloromethane similarly inhibited nitrification. Methanol and dichloromethane equilibrated for 60 mins also significantly delayed nitrification, although to a lesser extent. Inhibition of nitrification was not permanent, and nitrification activity was eventually restored in all systems tested. None of the solvents inhibited mineralization of the added carbon source. These results indicate that special care must be taken to ensure that applications solvents do not affect the activity of sensitive microbial populations, such as the nitrifiers, that may be part of a study.

An anionic monorhamnolipid biosurfactant produced by Pseudomonas aeruginosa was investigated for its potential to remove residual hexadecane from sand columns. In a series of column experiments, residual hexadecane saturation was established by pumping 14C-hexadecane into water-saturated sand columns and then flushing with water at a velocity of 25 cm h−1. Monorhamnolipid solutions of varying concentration were then applied to the columns at a velocity of 15 cm H−1 to remove the residual hexadecane. Of the rhamnolipid concentrations tested, which ranged from 40 to 1500 mg l−1, the optimal concentration for residual removal was 500 mg l−1, approximately ten times the critical micelle concentration (cmc). Approximately 84% of the residual was removed from the column packed with 20 30 mesh sand, and 22% was removed from the 40 50 mesh column. The primary mechanism for residual removal was mobilization (displacement and dispersion), whereas solubilization was found to be insignificant. The performance of monorhamnolipid was compared with that of two synthetic surfactant solutions on a mass basis (500 mg l−1) for the 40 50 mesh sand. Sodium dodecyl sulfate (0.2 X cmc), and polyoxyethylene (20) sorbitan monooleate (38 × cmc), removed 0% and 6.1% of the residual saturation, respectively.

This study describes methodology for the detection and quantitation of PCR amplified DNA. Specifically we report the estimation of the prevalence of E. coli in marine waters and other environmental samples from Mamala Bay, Hawaii. High performance liquid chromatography (HPLC) was used to quantitate PCR products containing between 1 and 250 ng DNA. PCR was used to amplify E. coli DNA through the use of lamB primers. A standard curve was generated that related initial cell template concentrations to amplified product DNA concentrations within a template range of 520 to 5.2×107 cells. The standard curve was used to determine initial template concentrations of the lamB gene sequence present within 11 different environmental samples. Quantified PCR analyses were most useful when samples contained only low numbers of target organisms, and when environmental samples contained few PCR inhibitory substances, as for example in marine water samples. Quantitation of amplified DNA and comparison with culture data also suggested the presence of viable but nonculturable organisms in some environmental samples. Overall these data are unique in that they indicate the successful use of HPLC to quantitate PCR amplifications with concomitant estimation of PCR template within environmental samples.

Approximately 15000 L of unleaded gasoline werereleased into the surrounding vadose zone from aleaking underground storage tank. Initialremediation was by soil vapor extraction andcombustion which soon became cost prohibitive, asadded propane was required to reach the combustionlimit of the extracted vapors. As a cost effectivealternative, a field-scale compost based biofilterwas used in conjunction with soil vapor extractionto remediate the vadose zone. The biofilter wasconstructed on site using 4:1 diatomaceousearth:composted horse manure. Results of a fivemonth study showed that the biofilter removedapproximately 90% of total petroleum hydrocarbons(TPH) and >90% of the BTEX compounds (benzene,toluene, ethylbenzene, xylene), achieving thestringent permit requirements set at either 90% TPHreduction or less than 1.36 kg per day of volatileorganic compounds (VOC's) released to theatmosphere. The biofilter showed the capacity toreadily adapt to changing environmental conditionssuch as increased contaminant loading, andvariations in temperature and moisture. Thebacterial population in the biofilter was uniformlydiverse throughout the biofilter, suggesting that aconsortium of bacteria was needed for efficientbiodegradation. The cost of biofilter set up andoperation saved 90% in the first year alone of theoperating expenses incurred by soil vapor extractionand combustion.

Arbitrarily primed polymerase chain reaction (AP-PCR) has been used extensively for genetic mapping, and the identification of bacterial isolates. To ensure that the results will be reproducible and due to true genetic variations, the AP-PCR reaction conditions must be optimized. In this study, three cultured bacterial isolates were screened with 100 arbitrary primers. Of these, five were chosen for the optimization study. The parameters optimized included: the operating conditions of the thermal cycler, the agarose gel concentration, the annealing temperature, and the concentrations of Taq polymerase enzyme, magnesium chloride, primer, and template. The final optimized PCR reaction conditions were 1 × buffer (3.5 mM MgCl 2 , 10 mM Tris-HCl, 50 mM KCl and 0.1 mg ml −1 gelatin), 200 μM dNTP, 0.4 μM primer, 2.5 U AmpliTaq® (Perkin-Elmer Cetus) polymerase enzyme, and 5 μl of template (at least 10 6 lysed bacterial cells). The Perkin-Elmer Gene-Amp™ 9600 PCR System was used with the following cycling conditions; a 94°C 15 s denaturation step, a 45°C 15 s annealing step, and 72°C 30 s extension step for a total of 35 cycles. Reproducible, unique fingerprints were generated for the three isolates using each of the five arbitrary primers.

A rhamnolipid biosurfactant produced by Pseudomonas aeruginosa ATCC 9027 is reported to increase the aqueous dispersion and biodegradation of petroleum hydrocarbons and to complex heavy metals. These reports indicate the potential for application of rhamnolipids in remediation of contaminated sites. Effective use of rhamnolipids will require understanding of rhamnolipid morphology and the effects of pH and organic and inorganic contaminants on that morphology. We used cryo-transmission electron microscopy to investigate the morphology of vitrified, frozen hydrated suspensions of rhamnolipid over a pH range of 5.5 to 8.0, and to determine the effect of a model alkane, octadecane, and a model heavy metal, cadmium, on rhamnolipid morphology. Micrographs clearly showed that rhamnolipid morphology was a function of pH, changing from lamellar, to vesicular, to micellar as pH increased. The effect of cadmium and octadecane on rhamnolipid morphology was determined at pH 6.8 and 7.0, where maximum cadmium complexation and maximum octadecane dispersion occurs. Cadmium seemed to stabilize rhamnolipid vesicle structures as shown by an increase in vesicle number and a decrease in vesicle diameter. In contrast, octadecane favored the micellar structure as shown by the complete absence of vesicles.

Complexation of cadmium, lead, and zinc (singly and in a mixture) by a monorhamnolipid biosurfactant produced by Pseudomonas aeruginosa ATCC 9027 was studied in batch solution and soil experiments. Conditional stability constants (log K L ) for metalrhamnolipid complexation in a buffered medium (0.1 M Pipes, pH 6.8) were determined in duplicate using an ion-exchange technique and averaged 6.5 (Cd 2+ ), 6.6 (Pb 2+ ), and 5.4 (Zn 2+ ) ; these values are similar or slightly higher than literature values for Cd 2+ and Pb 2+ complexation with fulvic acid and activated sludge solids. To determine the ability of rhamnolipid to desorb soil-bound metals, rhamnolipid solutions (12.5, 25, 50, and 80 mM) were added to soil containing sorbed Cd 2+ (1.46 mmol kg -1 ), Pb 2+ (1.96 mmol kg -1 ), or a mixture of Pb 2+ -Cd 2+ -Zn 2+ (3.4 mmol kg -1 ). At 12.5 and 25 mM rhamnolipid, rhamnolipid sorption to soil exceeded 78%, and less than 11% of soil-bound Cd 2+ and Zn 2+ was desorbed. However, ion exchange of bound metals with K + present in the rhamnolipid matrix could account for the removal of between 16 and 48% of the sorbed Cd 2+ and Zn 2+ . At 50 and 80 mM rhamnolipid, rhamnolipid sorption to soil decreased to between 20 and 77%, and the removal of Cd 2+ and Zn 2+ could exceed the removal by ion exchange by as much as 3-fold. The behavior of Pb 2+ was quite different. Less than 2% of soil-bound Pb 2+ was desorbed due to ion exchange, although up to 43% was desorbed by 80 mM rhamnolipid.

The potential of microbially-produced surfactants (biosurfactants) to complex heavy metals was investigated in this study. Batch solution studies using a model metal, cadmium, and an anionic monorhamnolipid biosurfactant produced by Pseudomonas aeruginosa ATCC 9027 showed that complexation of the metal and biosurfactantwas rapid and stable and achieved high reductions in the free Cd 2+ concentration. For example 92% of Cd 2+ (0.72 mM) was complexed by 7.3 mM rhamnolipid, and 97% of Cd 2+ (0.36 mM) was complexed by 3.9 mM rhamnolipid. Under the experimental conditions used, the maximum complexation capacity of the rhamnolipid was 0.2 Cd 2+ /rhamnolipid on a molar basis

Using the radiolabeled model pollutants 2,4-dichlorophenol (DCP) and 2,4,5-trichlorophenol (TCP) the authors demonstrated that the brief UV (300-nm) photolysis greatly facilitates the removal of the two chlorophenols from sewage through accelerated mineralization and binding of polar products. The addition of 0.1 M H/sub 2/O/sub 2/ strongly accelerated the photolysis process resulting in the half-lives of 1.68 and 0.87 min for DCP and TCP, respectively. In natural sunlight, half-lives of the chlorophenols were less than 1 day when H/sub 2/O/sub 2/ was present. During 4 days of incubation in activated sewage sludge, only 3% of unphotolyzed DCP and 1% of unphotolyzed TCP were mineralized. Mineralization rose to 79 and 59%, respectively, after photolysis in the presence of H/sub 2/O/sub 2/. Photolysis without H/sub 2/O/sub 2/ resulted in removal of chlorophenols from solution chiefly by binding. Increased mineralization and binding were observed also upon incubation of photolyzed chlorophenols in soil. Disruption of carbon-halogen bonds by brief photolysis followed by traditional biological effluent treatment offers an alternative to activated charcoal treatment for removal of xenobiotics from industrial effluents.

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.

Miscible-displacement experiments were conducted to compare the effects of aqueous soil solutions with ethyl alcohol, ethylene glycol, diethylene glycol, and triethylene glycol on the movement of metals through soils. Aqueous or alcohol solutions containing 1 mM each Cd, Ni, and Zn and 5 mM Ca were perfused through columns containing River Sand, Canelo loam (Canelo 1) or Mohave sandy clay loam (Mohave scl) until effluent metal concentrations (C) equaled influent concentrations (C 0 ) or CC 0 −1 = 1. In general, the order of sorption was Zn > Ni > Cd in aqueous-perfused columns, while in alcohol-perfused columns sorption of Ni ⪢ Cd ≥ Zn. In comparison to aqueous solutions, alcohols reduced total metal sorption by at least 25%. Metal sorption was best correlated to cation exchange capacity of the soil, sorption of metals being greatest in the Mohave scl and least in the River Sand. After CC 0 −1 = 1 was reached, columns were leached with deionized water. While leaching did not affect the sorption of metals in columns which had been perfused with aqueous solvents, sorption behavior of metals changed significantly in columns which had been perfused with alcohol solvents. Leaching caused desorption of 5 to 30% of the sorbed Ni. In general, Cd was desorbed (up to 45%) from the soils tested. The exceptions were River Sand columns perfused with diethylene and triethylene glycol in which additional Cd was sorbed to the soil from the soil solution. Additional Zn was sorbed in all columns tested with the exception of the Canelo 1 column perfused with ethyl alcohol.

The S-thiolated proteins phosphorylase b (Phb) and carbonic anhydrase III (CAIII) were prepared with [3H]glutathione in a reaction initiated with diamide. These substrates were used to measure the rate of reduction (dethiolation) of protein mixed-disulfides by enzymes with properties similar to those of thioredoxin and glutaredoxin. This enzyme activity is termed a dethiolase since the identities of the enzymes are still unknown. The dethiolation of either S-[3H]glutathiolated Phb or S-[3 H]glutathiolated CAIII was employed in tissue assays and for study of two partially purified dethiolases from cardiac tissue. NADPH-dependent dethiolase activity was most abundant except in rat liver and muscle. Total dethiolase activity was approximately 10-fold higher in neutrophils, 3T3-L1 cells, and Escherichia coli than in other sources. Rat skeletal muscle had 3- to 4-fold higher dethiolase activity than rat heart or liver. These data indicate that protein dethiolase activity is ubiquitous and that normal expression of the two dethiolase activities varies considerably. A partially purified cardiac NADPH-dependent dethiolase acted on Phb approximately 1.5 times faster than CAIII, and a glutathione (GSH)-dependent dethiolase acted on Phb 3 times faster than CAIII. The Km for glutathione for the GSH-dependent dethiolase was 15 μ m with Phb as substrate and 10 μ m with CAIII. Thus, the GSH-dependent dethiolase is probably not affected by normal changes in the cardiac glutathione content (normally approximately 3 m m ). Partially purified cardiac NADPH-dependent dethiolase was inactivated by BCNU (N,N′-bis(2-chloroethyl)-N-nitrosourea) and the GSH-dependent dethiolase was unaffected under similar conditions. In a soluble extract from bovine heart, 200 μ m BCNU inhibited NADPH-dependent dethiolase by more than 60% but did not affect GSH-dependent activity. These results demonstrate that BCNU is a selective inhibitor of the NADPH-dependent dethiolase.

The objective of this research was to evaluate the effect of low concentrations of a rhamnolipid biosurfactant on the in situ biodegradation of hydrocarbon entrapped in a porous matrix. Experiments were performed with sand-packed columns under saturated flow conditions with hexadecane as a model hydrocarbon. Application of biosurfactant concentrations greater than the CMC (the concentration at which the surfactant molecules spontaneously form micelles or vesicles [0.03 mM]) resulted primarily in the mobilization of hexadecane entrapped within the sand matrix. In contrast, application of biosurfactant concentrations less than the CMC enhanced the in situ mineralization of entrapped hexadecane; however, this effect was dependent on the choice of bacterial isolate. The two Pseudomonas isolates tested, R4 and ATCC 15524, were used because they exhibit different patterns of biodegradation of hexadecane, and they also differed in their physical response to rhamnolipid addition. ATCC 15524 cells formed extensive multicell aggregates in the presence of rhamnolipid while R4 cells were unaffected. This behavior did not affect the ability of the biosurfactant to enhance the biodegradation of hexadecane in well-mixed soil slurry systems but had a large affect on the extent of entrapped hexadecane biodegradation in the sand-packed-column system that was used in this study.

The objective of this study was to investigate the influence of an anionic rhamnolipid biosurfactant on the transport of bacterial cells through soil under saturated conditions. Three cell types with various hydrophobicities, i.e., Pseudomonas aeruginosa ATCC 9027, ATCC 27853, and ATCC 15442, were used in this study. In a series of experiments, columns packed with sterile sand were saturated with sterile artificial groundwater for 15 h, and then 3 pore volumes of (sup3)H-labeled bacterial suspensions with various rhamnolipid concentrations was pumped through the column. This was followed by 4 pore volumes of the rhamnolipid solution alone. The measured bacterial cell breakthrough curves were optimized by using an advection-dispersion transport model incorporating two-domain reversible sorption (instantaneous and rate limited) and with two first-order sink terms for irreversible adsorption. The influence of the rhamnolipid on the surface charge densities of the bacteria and the porous medium was also investigated. The results show that the rhamnolipid enhanced the transport of all cell types tested. For example, the rhamnolipid increased the recovery of the most hydrophilic strain, ATCC 9027, from 22.5 to 56.3%. Similarly, the recovery of ATCC 27853 increased from 36.8 to 49.4%, and the recovery of ATCC 15442, the most hydrophobic strain, increased from 17.7 to 40.5% in the presence of the rhamnolipid. The negative surface charge density of the porous medium was increased, while the surface charge density of the bacteria was not changed in the presence of the rhamnolipid. The model results suggest that the rhamnolipid predominantly affected irreversible adsorption of cells.

A microbial surfactant (biosurfactant) was investigated for its potential to enhance bioavailability and, hence, the biodegradation of octadecane. The rhamnolipid biosurfactant used in this study was extracted from culture supernatants after growth of Pseudomonas aeruginosa ATCC 9027 in phosphate-limited proteose peptone-glucose-ammonium salts medium. Dispersion of octadecane in aqueous solutions was dramatically enhanced by 300 mg of the rhamnolipid biosurfactant per liter, increasing by a factor of more than 4 orders of magnitude, from 0.009 to > 250 mg/liter. The relative enhancement of octadecane dispersion was much greater at low rhamnolipid concentrations than at high concentrations. Rhamnolipid-enhanced octadecane dispersion was found to be dependent on pH and shaking speed. Biodegradation experiments done with an initial octadecane concentration of 1,500 mg/liter showed that 20% of the octadecane was mineralized in 84 h in the presence of 300 mg of rhamnolipid per liter, compared with only 5% octadecane mineralization when no surfactant was present. These results indicate that rhamnolipids may have potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited water solubility.

The reaction of glycogen phosphorylase b and creatine kinase with glutathione disulfide, cystine, and cystamine was compared by direct analysis on electrofocusing gels. This method was useful for individual proteins or for mixtures of the proteins. Millimolar concentrations of glutathione disulfide were required for both proteins and the rate of modification of each protein was similar. The reaction of glutathione disulfide with creatine kinase was inhibited by reduced glutathione (GSH), but the effect on the reaction with phosphorylase was minimal. Cystine and cystamine were required in micromolar amounts to effectively form the disulfide adducts. Both proteins were modified by cystine but cystamine reacted only with phosphorylase. Cystamine (10 μ m ) was an effective inhibitor of the reaction of phosphorylase b with 2 m m glutathione disulfide. S-thiolation of creatine kinase inactivated the enzyme and a direct assay of the enzyme activity could be used to quantitate S-thiolation of this protein by each of the disulfides. The effect of each disulfide on enzyme activity confirmed the results obtained by gel electrofocusing. Glutathione disulfide and cystine both inactivated the enzyme while cystamine had no effect on the activity. S-thiolation of phosphorylase had no observable effect on any activity parameter, but it effectively prevented binding of phosphorylase to high-molecular-weight glycogen, probably at the glycogen storage site of phosphorylase. The rate of S-thiolation of a mixture of phosphorylase and creatine kinase by thiol-disulfide exchange with glutathione disulfide was compared to the rate of S-thiolation of these proteins by a xanthine oxidase-initiated process (presumably due to protein sulfhydryl activation by reactive oxygen species). The xanthine oxidase-initiated mechanism was somewhat faster than thiol-disulfide exchange with both proteins. It was shown that GSH inhibited S-thiolation of creatine kinase by this mechanism as well as by thiol-disulfide exchange. It is suggested that both mechanisms may play a role in protein S-thiolation in vivo . For proteins that are typified by creatine kinase, the concentration of GSH in the cells may determine whether the S-thiolated form of the protein accumulates. For proteins typified by phosphorylase b , the accumulation of S-thiolated forms may be more independent of GSH.

Bioremediation of metal-contaminated wastestreams has been successfully demonstrated. Normally, whole cells or microbial exopolymers are used to concentrate and/or precipitate metals in the wastestream to aid in metal removal. Analogous remediation of metal-contaminated soils is more complex because microbial cells or large exopolymers do not move freely through the soil. The use of microbially produced surfactants (biosurfactants) is an alternative with potential for remediation of metal-contaminated soils. The distinct advantage of biosurfactants over whole cells or exopolymers is their small size, generally biosurfactant molecular weights are less than 1500. A second advantage is that biosurfactants have a wide variety of chemical structures that may show different metal selectivities and thus, metal removal efficiencies. A review of the literature shows that complexation capacities of several bacterial exopolymers was similar to the complexation capacity of a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa ATCC 9027.

Substantial reductions in municipal water use are feasible without reducing the quality of life. If conservation measures are preplanned, properly engineered, and coordinated, reductions in utility bills for water, waste water, and energy are estimated at $30+per person per year. Installation of devices to reduce water use, engineering plans to prevent malfunctioning of collecting sewers, and engineered process modifications of treatment facilities must be coordinated to achieve full benefits of water conservation. Pollutant discharges to the aquatic environment are reduced in direct proportion to the reduction in water use.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.

The recalcitrance of xenobiotics may be caused by an absence of transforming enzymes or by their inability to enter microbial cells. A nondestructive method for differentiating between these two possibilities is described. The solid n -alkanes octadecane (C 18 ) and hexatriacontane (C 36 ) were encapsulated into phosphatidylcholine bilayers (liposomes). The uptake and metabolism rates of encapsulated and unencapsulated substrates were then compared. During 1 h at 25°C, a Pseudomonas isolate took up 1.3% of radiolabeled and unencapsulated C 18 (solid state) versus 23.5% of labeled and encapsulated C 18 . Growth at 25°C occurred with an apparent k s of 2453 ± 148 mg/liter. Liposome encapsulation decreased this K s to 60 ± 12 mg/liter. At 34°C, growth on C 18 (liquid state) occurred with an apparent K s of 819 ± 83 mg/liter and on the readily available carbon source succinate, K s values were 80 ± 10 and 13 ± 7 mg/liter at 25 and 34°C, respectively. At 25°C, the isolate grew on C 36 with an apparent K s of 2,698 ± 831 mg/liter. Liposome encapsulation decreased the K s more than 60-fold to 41 ± 7 mg/liter, resulting in the complete utilization of 400 mg of C 36 per liter in 16 h. Since controls excluded the metabolic utilization of phosphatidylcholine, the results clearly identify transport limitation as the cause for C 36 recalcitrance.

In this study, the effect of a purified rhamnolipid biosurfactant on the hydrophobicity of octadecane-degrading cells was investigated to determine whether differences in rates of octadecane biodegradation resulting from the addition of rhamnolipid to four strains of Pseudomonas aeruginosa could be related to measured differences in hydrophobicity. Cell hydrophobicity was determined by a modified bacterial adherence to hydrocarbon (BATH) assay. Bacterial adherence to hydrocarbon quantitates the preference of cell surfaces for the aqueous phase or the aqueous-hexadecane interface in a two-phase system of water and hexadecane. On the basis of octadecane biodegradation in the absence of rhamnolipid, the four bacterial strains were divided into two groups: the fast degraders (ATCC 15442 and ATCC 27853), which had high cell hydrophobicities (74 and 55% adherence to hexadecane, respectively), and the slow degraders (ATCC 9027 and NRRL 3198), which had low cell hydrophobicities (27 and 40%, respectively). Although in all cases rhamnolipid increased the aqueous dispersion of octadecane at least 10(4)-fold, at low rhamnolipid concentrations (0.6 mM), biodegradation by all four strains was initially inhibited for at least 100 h relative to controls. At high rhamnolipid concentrations (6 mM), biodegradation by the fast degraders was slightly inhibited relative to controls, but the biodegradation by the slow degraders was enhanced relative to controls. Measurement of cell hydrophobicity showed that rhamnolipids increased the cell hydrophobicity of the slow degraders but had no effect on the cell hydrophobicity of the fast degraders. The rate at which the cells became hydrophobic was found to depend on the rhamnolipid concentration and was directly related to the rate of octadecane biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)