Influence of a nonaqueous phase liquid (NAPL) on biodegradation of phenanthrene

A series of batch reactor experiments was carried out to examine the effect of a nonaqueous phase liquid (NAPL) on the biodegradation of a hydrophobic solute. A mathematical program model that describes physical processes of solute solubilization and partitioning between the NAPL and aqueous phases as well as microbial degradation and oxygen utilization was used to analyze the test data. The model calculates the cumulative changes in concentration of substrate, cell mass, carbon dioxide, and dissolved oxygen as a function of time. The equations incorporate the effects of solute solubilization, partitioning, biodegradation, as well as oxygen availability. Hexadecane was used as the model NAPL and was not biodegraded in the timeframe of the experiments performed. The model solute was the polyaromatic hydrocarbon, phenanthrene. In agreement with several previous studies, experimental measurements showed that hexadecane increased rates of mineralization of 15 mg phenanthrene when present at low mass but decreased rates at high mass. Model results suggest that partitioning of the phenanthrene into the hexadecane phase limits bioavailability at high NAPL mass. Further the model suggests that mineralization rates were higher with the low NAPL mass because aqueous phenanthrene concentrations were higher in those treatments from ca. 20 to 40 h than in other treatments. Finally, experiments showed that the presence of hexadecane, at all masses tested, resulted in a lower cell yield, effectively increasing the amount of CO₂ produced during the experiment. Model results suggest that this is due to changes in phenanthrene metabolism that are induced by the presence of the hexadecane phase. Model studies aimed at increasing rates of biodegradation by modifying operating conditions are described along with practical approaches to implementing these modifications.     

The effect of inorganic and organic supplements on the microbial degradation of phenanthrene and pyrene in soils

The effects of several bioremediation stimulants, including potentialmetabolism pathway inducers, inorganic/organic nutrients, and surfactants onthe metabolism of phenanthrene and pyrene, as well as the populationdynamics of PAH degrading microorganisms was examined in five soils withdiffering background PAH concentrations, exposure histories and physicalproperties. Most of the supplements either had no significant effect ordecreased the mineralization of [¹⁴C]-phenanthrene and[¹⁴C]-pyrene in soil slurry microcosms. The effect of aparticular supplement, however, was often not uniform within or acrosssoils. Decreased mineralization of [¹⁴C]-phenanthrene and[¹⁴C]-pyrene was usually due to either preferential use of thesupplement as carbon source and/or stimulation of non-PAH degradingmicroorganisms. Many of the supplements increased populations ofheterotrophic microorganisms, as measured by plate counts, but did notincrease populations of phenanthrene degrading microorganisms, as measuredby the [¹⁴C]-PAH mineralization MPN analysis or cellularincorporation of [¹⁴C]-PAH. These results suggest that the PAHdegrading community at each site may be unique in their response tomaterials added in an attempt to stimulate PAH degradation. Thecharacteristics of the site, including exposure history, soil type, andtemporal variation may all influence their response.     

Annual changes of nitrogen and phosphorus in two aquatic macrophytes (Nymphaea Tuberosa and Ceratophyllum Demersum)

The nitrogen and phosphorus content of Nymphaea tuberosa and Ceratophyllum demersum, in Lake Onalaska, Wisconsin, was studied for a year. On a yearly basis, N. tuberosa exhibited nitrogen and phosphorus differences among seasons and among plant parts. Variation among plant parts was also evident in C. demersum. However, within individual plant structures, no seasonal differences were observed.Funds were provided by the River Studies Center, University of Wisconsin-La Crosse, Thomas O. Claflin, Director.Funds were provided by the River Studies Center, University of Wisconsin-La Crosse, Thomas O. Claflin, Director.     

Enhanced biodegradation of phenanthrene in a biphasic culture system

Degradation of phenanthrene byPseudomonas aeruginosa AK1 was examined in (i) an aqueous mineral salts medium to which phenanthrene particles of varying size (i.e. diameter) were added, and (ii) an aqueous/organic biphasic culture system consisting of mineral salts medium supplemented with 2,2,4,4,6,8,8-heptamethylnonane (HMN) as the phenanthrene-carrying organic phase. In both systems, the rate of phenanthrene biodegradation could be significantly enhanced by manipulations leading to improved phenanthrene mass transfer into the aqueous phase. With crystalline phenanthrene, the rate of biodegradation was found to be directly correlated to the particle surface area, whereas in the biphasic system the rate of biodegradation of the dissolved phenanthrene was mainly governed by the HMN/water interface area. In the latter system, exponential growth with a doubling time t_d of 6–8 hours has been achieved under conditions of intensive agitation of the medium indicating that phenanthrene degradation by strain AK1 is limited mainly by physicochemical parameters. Addition of selected surfactants to the culture medium was found to accelerate phenanthrene degradation by strain AK1 only under conditions of low agitation (in the presence of HMN) and after pretreatment of phenanthrene crystals by ultrasonication (in the absence of HMN). Evidence is presented that the stimulating effect of the surfactants was primarily due to improved dispersion of phenanthrene particle agglomerates (in the aqueous mineral salts medium supplemented with phenanthrene crystals) or of the phenanthrene-carrying lipophilic solvent drops (in the aqueous/organic biphasic culture system) whereas the solubilizing activity towards phenanthrene was neglectible. Under conditions of intensive mixing of the culture medium (i.e. if a high particle surface area or HMN/water interface area, respectively, is provided), the addition of surfactants did not enhance phenanthrene biodegradation.     

Naphthalene,phenanthrene and surfactant biodegradation

The impact of surfactants on naphthalene and phenanthrene biodegradation and vice versa after surfactant flushing were evaluated using two anionic surfactants: sodium dodecyl sulfate (SDS) and sodium dodecyl benzene sulfonate (SDBS); and two nonionic surfactants: POE (20) sorbitan monooleate (T-maz-80) and octylphenol poly(ethyleneoxy) ethanol (CA-620). Naphthalene and phenanthrene biodegradation varied differently in the presence of different surfactants. Naphthalene biodegradation was not impacted by the presence of SDS. In the presence of T-maz-80 and CA-620, naphthalene biodegradation occurred at a lower rate (0.14 d^-1 for T-maz-80 and 0.19 d^-1 for CA-620) as compared to un-amended control (0.29 d^-1). Naphthalene biodegradation was inhibited by the presence of SDBS. In the presence of SDS, phenanthrene biodegradation occurred at a lower rate (0.10 d^-1 as compared to un-amended control of 0.17 d^-1) and the presence of SDBS, CA-620 and T-maz-80 inhibited phenanthrene biodegradation. The surfactants also responded differently to the presence of naphthalene and phenanthrene. In the presence of naphthalene, SDS biodegradation was inhibited; SDBS and T-maz-80 depleted at a lower rate (0.41d^-1 and 0.12 d^-1 as compared to 0.48 d^-1 and 0.22 d^-1). In the absence of naphthalene, CA-620 was not degradable, while in the presence of naphthalene, CA-620 began to degrade at a comparatively low rate (0.12 d^-1). In the presence of phenanthrene, SDS biodegradation occurred at a lower rate (1.2 d^-1 as compared to 1.68 d^-1) and a similar trend was observed for T-maz-80. The depletion of SDBS and CA-620 did not change significantly. The choice of SDS for naphthalene-contaminated sites would not adversely affect the natural attenuation of naphthalene, in addition, naphthalene was preferentially utilized to SDS by naphthalene-acclimated microorganisms. Therefore, SDS was the best choice. T-maz-80 was also found to be usable in naphthalene-contaminated sites. For phenanthrene contaminated sites, SDS was the only choice.     

Gull contributions of phosphorus and nitrogen to a Cape Cod kettle pond

Nutrient excretion rates and the annual contribution of P from the feces of the gulls Larus argentatus and L. marinus (and of N from L. argentatus) to the nutrient budget of Gull Pond (Wellfleet), a soft water seepage lake, have been estimated. Intensive year-round gull counts by species were combined with determinations of defecation rate and the nutrient content of feces to quantitatively assess the P loading rates associated with regular gull use of this coastal pond on a seasonal and annual basis. Total P loading from gulls was estimated to be 52 kg yr^–1, with 17 kg from L. argentatus and 35 kg from L. marinus, resulting from about 5.0 × 10⁶ h yr^–1 and 1.7 × 10⁶ h yr^–1 of pond use. This compares with P loading estimates of 67 kg yr^–1 from upgradient septic systems, 2 kg yr^–1 from precipitation and 3 kg yr^–1 from unpolluted ground water. Fifty-six percent of annual gull P loading was associated with migratory activity in late fall. Estimated annual N loading by L. argentatus was 14 kg TKN, 206 g NO₃-N, and 1.85 g g NH₃-N.     

普通小球藻对养殖污水脱氮除磷的效果研究

随着我国养殖业的不断发展,养殖污水排放量的日益增加,养殖污水的高氮、磷含量导致水体富营养化问题日趋严重。小球藻是光能自养生物,能有效同化氮、磷,使污水中的氮、磷减少。本研究通过在实验室模拟不同氮、磷含量的养殖污水环境,分析小球藻对氮、磷的去除效果;在此基础上,用小球藻处理某养殖场污水;并联合膨润土与小球藻,探究两者脱氮除磷的协同作用能力及膨润土对小球藻细胞沉降的效果。结果表明,小球藻对模拟污水的氨氮去除率可达80%,对磷酸根的最高去除率接近100%;对养殖污水中的氮、磷也有一定的去除效果;但养殖污水成分复杂,小球藻的生长被抑制。膨润土与小球藻的结合,能够提高污水中的氮磷去除率并帮助藻细胞快速沉降,为污水处理后藻细胞的收集处理提供了有效方法。     

Simultaneous removal of phosphorus and nitrogen in sequencing batch reactor

In this research, investigations were made on material transfer mechanisms and optimum operation mode for sequencing batch reactor system removing phosphorus and nitrogen simultaneously. Phosphorus release characteristics were expressed in the Monod equation, in which the reaction rate was replaced with specific phosphorus release (SPR) rate. The rate of SPR was increased during the first 80 days, but increased sharply to reach 0.003 hr^-1 afterwards. Phosphorus removal efficiencies were about 60% in the first 80 days, 75% after 80 days, and above 95% after 120 days. After 120 days, phosphorus concentration in effluent was below 0.5 mgl^-1 when 8 mgl^-1 was in the influent and the released phosphorus after 3-hour-anaerobic period was 60 mgl^-1. In the proposed optimum operation strategy (2-hour anaerobic react, 3-hour aerobic react, 4-hour anoxic react, and 3-hour settle and draw), phosphorus reappeared if the oxidized nitrogen was completely denitrified. In order to prevent this undesirable phosphorus release, anoxic period should be reduced to the extent of which the minimal concentration of the oxidized nitrogen existed. Phosphorus removal efficiency was stable under shock load as 5 times high as normal phosphorus concentration.Abbreviations dP/dt Phosphorus release rate (mgl^-1 hr^-1) - K Phosphorus release yield constant (mg P mg TOC^-1) - dS/dt Substrate utilization rate (mgl^-1 hr^-1) - X Mixed liquor suspended solid (MLSS, mgl^-1) - S Soluble TOC (mgl^-1) - k-q_max (Y_max)^-1 Maximum substrate utilization rate - Y Yield coefficient (mg mg^-1) - K_s Saturation constant (mgl^-1) - P_max kK-Maximum phosphorus release rate (hr^-1) - P_rel Total released phosphorus (mgl^-1) - P_o Phosphorus in influent (mgl^-1) - P_e phosphorus in effluent (mgl^-1) - t Anaerobic period (hr)     

Genetics of naphthalene and phenanthrene degradation by Comamonas testosteroni

Naphthalene and phenanthrene have long been used as model compounds to investigate the ability of bacteria to degrade polycyclic aromatic hydrocarbons. The catabolic pathways have been determined, several of the enzymes have been purified to homogeneity, and genes have been cloned and sequenced. However, the majority of this work has been performed with fast growing Pseudomonas strains related to the archetypal naphthalene-degrading P. putida strains G7 and NCIB 9816-4. Recently Comamonas testosteroni strains able to degrade naphthalene and phenanthrene have been isolated and shown to possess genes for polycyclic aromatic hydrocarbon degradation that are different from the canonical genes found in Pseudomonas species. For instance, C. testosteroni GZ39 has genes for naphthalene and phenanthrene degradation which are not only different from those found in Pseudomonas species but are also arranged in a different configuration. C. testosteroni GZ42, on the other hand, has genes for naphthalene and phenanthrene degradation which are arranged almost the same as those found in Pseudomonas species but show significant divergence in their sequences. Received 10 August 1997/ Accepted in revised form 15 August 1997     

Effects of nitrogen: phosphorus supply ratios on nitrogen fixation in agricultural and pastoral ecosystems

An analysis of data compiled from the literature confirms a strong inverse relationship between annual rates of nitrogen fixation and the soil nitrogen content in agricultural and pastoral ecosystems. However, this inverse relationship is strongly modified by the rate of application of phosphorus fertilizer, which strongly influences the activities of both symbiotic and non-symbiotic nitrogen fixing organisms. In the case of symbiotic legumes, the response of N-fixation to N and P is in part a result of changes in legume dominance within the plant community. These results, as well as supporting data presented from a review of experiments on nitrogen fixation in a variety of other terrestrial and aquatic ecosystems, provide important support for the hypothesis that phosphorus availability is a key regulator of nitrogen biogeochemistry. Published as Paper No. 9950, Journal Series, Nebraska Agricultural Research Division, University of Nebraska, Lincoln, NE, USA.     

Indirect bioremediation: biodegradation of hydrocarbons on a commercial sorbent

Urea-formaldehyde polymer is currently used as asorbent for containment and clean up of hydrocarbons. The aerobic biodegradability of this polymer andhydrocarbons sorbed to the polymer were tested. Soilmicroorganisms readily grew on the polymer, and twoorganisms, a bacterium and a fungus, capable of growthon the polymer were isolated. However, biodegradationof the polymer was very slow and possibly incomplete. Biodegradation of the polymer was evident as a changein appearance of the polymer, but disappearance of thepolymer was not detectable in liquid culturesincubated for six months or soil cultures incubatedfor one month. Destruction of the polymer by soilmicroorganisms at ambient temperature does not appearto be practical. Degradation of ¹⁴C-labeledhexadecane and phenanthrene mixed with crude oil inliquid cultures inoculated with soil microorganismswas used as an estimate of general hydrocarbondegradation. When nitrogen was not limiting, therates of hexadecane and phenanthrene degradation werethe same, whether those hydrocarbons were sorbed tothe polymer or not sorbed. When nitrogen waslimiting, the polymer stimulated the rate ofhexadecane degradation but not the rate ofphenanthrene degradation. The polymer may stimulatehexadecane degradation by serving as a source ofnitrogen. However, optimal degradation of sorbedhydrocarbons requires nitrogen addition. The resultssuggest that it may be feasible to decontaminate spentpolymer by biodegradation of sorbed hydrocarbons.     

Effect of carbon:nitrogen ratio on kinetics of phenol biodegradation byAcinetobacter johnsonii in saturated sand

In polluted soil or ground water, inorganic nutrients such as nitrogen may be limiting, so that Monod kinetics for carbon limitation may not describe microbial growth and contaminant biodegradation rates. To test this hypothesis we measured¹⁴CO₂ evolved by a pure culture ofAcinetobacter johnsonii degrading 120 µg¹⁴C-phenol per ml in saturated sand with molar carbon:nitrogen (CN) ratios ranging from 1.5 to 560. We fit kinetics models to the data using non-linear least squares regression. Phenol disappearance and population growth were also measured at CN1.5 and CN560.After a 5- to 10-hour lag period, most of the¹⁴CO₂ evolution curves at all CN ratios displayed a sigmoidal shape, suggesting that the microbial populations grew. As CN ratio increased, the initial rate of¹⁴CO₂ evolution decreased. Cell growth and phenol consumption occurred at both CN1.5 and CN560, and showed the same trends as the¹⁴CO₂ data. A kinetics model assuming population growth limited by a single substrate best fit the¹⁴CO₂ evolution data for CN1.5. At intermediate to high CN ratios, the data were best fit by a model originally formulated to describe no-growth metabolism of one substrate coupled with microbial growth on a second substrate. We suggest that this dual-substrate model describes linear growth on phenol while nitrogen is available and first-order metabolism of phenol without growth after nitrogen is depleted.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Dynamics of nitrogen and phosphorus retention during wetland ecosystem succession

We compared the mechanisms of nitrogen (N) and phosphorus (P) removal in four young (<15 years old) constructed estuarine marshes with paired mature natural marshes to determine how nutrient retention changes during wetland ecosystem succession. In constructed wetlands, N retention begins as soon as emergent vegetation becomes established and soil organic matter starts to accumulate, which is usually within the first 1–3 years. Accumulation of organic carbon in the soil sets the stage for denitrification which, after 5–10 years, removes approximately the same amount of N as accumulating organic matter, 5–10 g/m²/yr each, under conditions of low N loadings. Under high N loadings, the amount of N stored in accumulating organic matter doubles while N removal from denitrification may increase by an order of magnitude or more. Both organic N accumulation and denitrification provide for long-term reliable N removal regardless of N loading rates. Phosphorus removal, on the other hand, is greatest during the first 1–3 years of succession when sediment deposition and sorption/precipitation of P are greatest. During this time, constructed marshes may retain from 3 g P/m²/yr under low P loadings to as much as 30 g P/m²/yr under high loadings. However, as sedimentation decreases and sorption sites become saturated, P retention decreases to levels supported by organic P accumulation (1–2 g P/m²/yr) and sorption/precipitation with incoming aqueous and particulate Fe, Al and Ca. Phosphorus cycling in wetlands differs from forest and other terrestrial ecosystems in that conservation of P is greatest during the early years of succession, not during the middle or late stages. Conservation of P by wetlands is largely regulated by geochemical processes (sorption, precipitation) which operate independently of succession. In contrast, the conservation of N is controlled by biological processes (organic matter accumulation, denitrification) that change as succession proceeds.     

Effects of fertility management strategies on phosphorus bioavailability in four West African soils

Low phosphorus (P) in acid sandy soils of the West African Sudano-Sahelian zone is a major limitation to crop growth. To compare treatment effects on total dry matter (TDM) of crops and plant available P (P-Bray and isotopically exchangeable P), field experiments were carried out for 2 years at four sites where annual rainfall ranged from 560 to 850 mm and topsoil pH varied between 4.2 and 5.6. Main treatments were: (i) crop residue (CR) mulch at 500 and 2000 kg ha^–1, (ii) eight different rates and sources of P and (iii) cereal/legume rotations including millet (Pennisetum glaucum L.), sorghum [Sorghum bicolor (L.) Moench], cowpea (Vigna unguiculata Walp.) and groundnut (Arachis hypogaea L.). For the two Sahelian sites with large CR-induced differences in TDM, mulching did not modify significantly the soils' buffering capacity for phosphate ions but led to large increases in the intensity factor (C_P) and quantity of directly available soil P (E _1min). In the wetter Sudanian zone lacking effects of CR mulching on TDM mirrored a decline of E _1min with CR. Broadcast application of soluble single superphosphate (SSP) at 13 kg P ha^–1 led to large increases in C _P and quantity of E _1min at all sites which translated in respective TDM increases. The high agronomic efficiency of SSP placement (4 kg P ha^–1) across sites could be explained by consistent increases in the quantity factor which confirms the power of the isotopic exchange method in explaining management effects on crop growth across the region.     

Fate and effects of phosphorus additions in soils under N2-fixing red alder

Soil phosphorus (P) dynamics are controlled by the interaction of geochemical, biochemical and biological processes. Changes in species composition or management could alter the relative importance of these processes. We examined soil P dynamics in two plantations of N₂-fixing red alder (Alnus rubra) by determining the fate and effects of added fertilizer P. History of the plantations varied such that sites were previously occupied by 60-yr-old stands of alder or non-fixing Douglas-fir (Pseudotsuga menziesii). Without fertilization, the soil with a longer period of alder influence had more organic P (P_o) and less sorbed inorganic P (Hydroxide- and Bicarb-extractable P_i). Fertilization increased soil total P, and 88% of the fertilizer was accounted for in the surface mineral soil (0–15 cm). Sorbed P_i was the major sink for fertilizer P (55–60%), independent of site history. Although P_o was 35–70% of soil P in unfertilized plots, added P did not accumulate as P_o. Neither site history nor P addition influenced phosphatase activity. Fertilization increased decomposition during incubation of the organic horizon, suggesting that late-stage decomposition is P-limited in these N-rich soils. On the time-scale of a few years, geochemical sorption and desorption of inorganic P were the most important processes controlling the distribution of added P. Organic P accumulation is expected to occur over a longer time frame, linked to the production and turnover of organic matter.     

Kinetics of nitrogen and phosphorus release in varying food supplies byDaphnia magna

Rates of nitrogen and phosphorus release from individualDaphnia magna were determined by measuring ammonia and soluble reactive phosphorus in successive 10-min incubations in small (0.05 ml) vessels after the animals were removed from their food. Release rates of both nutrients were generally highest initially and decreased with time after removal. The ratio of nitrogen to phosphorus released increased with time after animals were removed from an artificial detritus/bacterial food; ratios were lower and changed with time less for animals fed algae. These data suggest errors may be introduced by assumptions of constant stoichiometry for nutrient release in varying environments.GLERL Contribution No. 268     

Sources of organic nitrogen,phosphorus and carbon in antarctic streams

Dissolved and particulate organic materials were analysed in 14 streamwaters of the McMurdo Sound region of Antarctica. These streams are fed by glacial meltwaters and pass through catchments largely devoid of terrestrial vegetation. Nonetheless they contained measurable amounts of organic material in both dissolved and particulate form. Most of the dissolved organic carbon (DOC) values lay in the range 1–3 g C m^–3. Higher values were recorded close to penguin rookeries on the coast. Dissolved organic nitrogen (DON) concentrations were generally two orders of magnitude less than DOC and in flowing waters with rich blue-green algal growth DON increased with distance downstream. Dissolved organic phosphorus levels were generally much lower than DON, but highly variable. Particulate organic carbon concentrations (both fine and coarse) were unexpectedly high. Five sources of organic matter were identified: birdlife (only near the coast), autochthonous algal production (especially important for DON), streambed soils (important at first flows), lacustrine and marine sediments, through which certain streams and glaciers cut, and the glacial ice, which received organic input from wind-blown particulates, snowfall and the underlying bedrock of sedimentary origin. Highest organic levels were recorded in the first melt down the glacier face, suggesting that winter deposition of organic materials may be especially important.     

Combined effects of pH and biosurfactant addition on solubilization and biodegradation of phenanthrene

Phenanthrene solubilization and biodegradation with a biosurfactant (rhamnolipid) solution were investigated as a function of pH. Batch phenanthrene solubilization experiments were performed in the pH range 4–8 and the highest solubilities with the biosurfactant were detected around a pH of 4.5–5.5. The apparent solubility at pH 5.5 was 3.8 times greater than at pH 7 in the presence of 240 ppm rhamnolipid, probably due to the rhamnolipid—an anionic surfactant—forming different pH-dependent structures. Biodegradation experiments using Pseudomonas putida CRE 7 were performed in the absence and the presence of the rhamnolipid solution. Without the biosurfactant, the specific growth rate () at pH 6 was higher than at other pH values, and analysis for the total phenanthrene loss confirmed the trends in , with the greatest phenanthrene removal at pH 6. In presence of the rhamnolipid, the maximum value shifted to around pH 5, which showed maximum enhancement of solubility in the abiotic experiment. Although there was an increase in the observed specific growth rate with the biosurfactant, this increase was not as great as the increase in solubilization. For example, the 1.44 times increase in the value at pH 5 was lower than the 3.8 times enhancement in the solubility at the same pH. Thus, as observed by others, not all of the solubilized phenanthrene was bioavailable to the microorganisms. Interestingly, the results of a size distribution experiment showed that a large portion of the phenanthrene-rhamnolipid aggregates existed at a molecular weight of >300,000. Furthermore, this fraction appeared to be the most available for biodegradation, although not all the phenanthrene was bioavailable.