On-line estimation of biodegradation in an unsaturated soil

The objective of this study was to develop a model-based estimator of biodegradation in unsaturated soil. This would allow real-time assessment of the efficiency of treatment bioprocesses, such as bioventilation and biopile, and eventually permit optimization through the implementation of control strategies. Based on a reduced-order model, an asymptotic observer was designed to estimate on-line the contaminant concentration, using carbon dioxide measurement. Two observer-based estimators were built to approximate: (1) the specific microbial growth rate; and (2) the biocontact kinetics representing the soil resistance to contaminant biodegradation. State observers and parameter estimators were confronted with the experimental results of biodegradation in microcosms. Hexadecane was used as the model compound, representing petroleum hydrocarbons. Three water contents, corresponding to 20%, 50% and 80% of the water-holding capacity, were tested. The asymptotic observer is able to predict hexadecane depletion with an error on the overall time trajectories of 13%, 8% and 4% for the dry, intermediate and wet soils, respectively, which is acceptable given that all the biokinetic parameters were identified from a biodegradation experiment in liquid phase. The observer-based estimator of the specific microbial growth rate, based on the CO₂ measurement, was successfully calibrated using the off-line measurements of hexadecane as validation data, and allowed estimation of the time when biodegradation switched from a microbial to a biocontact limitation. The biocontact kinetics was also identified on-line, using an estimator based on the hexadecane not in biocontact. These results are very encouraging with respect to the potential for on-line assessment of the performance of treatment bioprocesses in unsaturated soils.     

Modeling the Kinetics of Contaminant Biodegradation by a Mixed Microbial Consortium Based on Carbon Mass Balance

The fate of contaminant carbon was monitored during aerobic biodegradation in the presence of a mixed indigenous microbial consortium in order to calibrate a microbial-growth-based biokinetic model. The methodology simultaneously monitored mineralization, substrate depletion and microbial population evolution in biomass extract spiked with¹⁴C-labeled hexadecane. Hexadecane depletion and hexadecane-degrader population were monitored using sacrificed microcosms by centrifuging the extract so that the supernatant and the residue contained residual hexadecane and microbial population, respectively. This methodology allowed verification of the carbon mass balance (average¹⁴C-carbon recovery of 90.33 ± 1.62% for biotic microcosms) and calibration of a biokinetic model. Four biokinetic parameters and three yield coefficients were identified (Haldane kinetic parameters:μ_S = 1.3639 d^-1, K_s = 0.4295 mg-C, KI = 6.6457 mg-C; decay kinetic parameter:μ_d = 1.3.10² d^-1; substrate/biomass, carbon dioxide/ biomass during growth and carbon dioxide/biomass during decay yield coefficients: Y_s = 1.5948 mg-C/mg-C, Y_P ^g = 0.4554 mg-C/mg-C, Y_P ^d = 1.3263 mg-C/mg-C) and compared with the literature data. The methodology can facilitate the identification of biodegradation models by decoupling the intrinsic ability of microorganisms to degrade contaminant from restrictions imposed by limiting conditions.     

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.     

Relationships of respiratory quotient to microbial biomass and hydrocarbon contaminant degradation during soil bioremediation

This work focused on monitoring respiratory quotient, RQ (defined as a ratio of CO₂ production to O₂ uptake rates), microbial growth and residual hydrocarbon concentration during bioremediation experiments performed on laboratory soil microcosms. The aim of the study was to determine if the time course biodegradation profile of the contaminant can be related to the RQ evolution and to investigate the effect of the water content on RQ measurements. A natural soil was artificially contaminated with hexadecane and adjusted with inorganic nutrients to stimulate biodegradation. Microbial growth, CO₂ production, O₂ uptake and residual hexadecane were periodically monitored at different soil water contents ranging from 0.15 to 0.35 g water g⁻¹ of dry soil. Results showed that microbial activity and contaminant degradation were strongly dependent on soil water content. Maximal growth and hexadecane depletion were obtained at a water content of 0.20 g water g⁻¹ of dry soil, which corresponded to 46.6% of the water holding capacity. Hexadecane degradation was considerably reduced with increasing soil water content. RQ values fluctuated as a function of the hexadecane biodegradation phases. The lowest RQs corresponded to the highest hexadecane depletion and microbial growth. The water content variation did not significantly affect the shape of the RQ evolution curves as a function of time. It only modified the magnitude of RQ values. This study indicates that additional biological and chemical analyses are needed to support RQ data when monitoring contaminant degradation to have an accurate understanding of all the biotic processes, which may occur simultaneously.     

Biodegradation kinetics of naphthalene in nonaqueous phase liquid-water mixed batch systems: comparison of model predictions and experimental results

A model is formulated to describe dissolution of naphthalene from an insoluble nonaqueous phase liquid (NAPL) and its subsequent biodegradation in the aqueous phase in completely mixed batch reactors. The physicochemical processes of equilibrium partitioning and mass transfer of naphthalene between the NAPL and aqueous phases were incorporated into the model. Biodegradation kinetics were described by Monod's microbial growth kinetic model, modified to account for the inhibitory effects of 1,2-naphthoquinone formed during naphthalene degradation under certain conditions. System parameters and biokinetic coefficients pertinent to the NAPL-water systems were determined either by direct measurement or from nonlinear regression of the naphthalene mineralization profiles obtained from batch reactor tests with two-component NAPLs comprised of naphthalene and heptamethylnonane. The NAPLs contained substantial mass of naphthalene, and naphthalene biodegradation kinetics were evaluated over the time required for near complete depletion of naphthalene from the NAPL. Model predictions of naphthalene mineralization time profiles compared favorably to the general trends observed in the data obtained from laboratory experiments with the two-component NAPL, as well as with two coal tars obtained from the subsurface at contaminated sites and composed of many different PAHs (polycyclic aromatic hydrocarbon compounds). The effects of varying the NAPL mass and the naphthalene mole fractions in the NAPL are discussed. It was observed that the time to achieve a given percent removal of naphthalene does not change significantly with the initial mass of naphthalene in a fixed volume of the NAPL. Significant changes in the mineralization profiles are observed when the volume (and mass) of NAPL in the system is changed.     

Rhamnolipid (biosurfactant) effects on cell aggregation and biodegradation of residual hexadecane under saturated flow conditions.

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.     

Biodegradation by an Arthrobacter Species of Hydrocarbons Partitioned into an Organic Solvent

An Arthrobacter strain mineralized naphthalene and n-hexadecane dissolved in 2,2,4,4,6,8,8-heptamethylnonane. The extent of mineralization increased with greater volumes of solvent. Measurements under aseptic conditions of the partitioning of naphthalene into the aqueous phase from the solid phase or from heptamethylnonane showed that the rates were rapid and did not limit mineralization. The rate of mineralization of hexadecane was rapid, although partitioning of the compound into aqueous solution was not detected. The Arthrobacter sp. grown in media with or without heptamethylnonane did not excrete products that increased the aqueous solubility of naphthalene and hexadecane. Measurements of the number of cells in the aqueous phase showed that the Arthrobacter sp. attached to the heptamethylnonane-water interface, but attachment was evident even without a substrate in the heptamethylnonane. Tests with small inocula of the Arthrobacter sp. demonstrated that at least a portion of naphthalene or hexadecane dissolved in heptamethylnonane was degraded by cells attached to the solvent-water interface. The cells did not adhere in the presence of 0.1% Triton X-100. The surfactant prevented mineralization of the hexadecane initially dissolved in heptamethylnonane, but it increased the rate and extent of mineralization of naphthalene initially dissolved in heptamethylnonane. The data show that organic solvents into which hydrophobic compounds partition affect the biodegradation of those compounds and that attachment of microorganisms to the organic solvent-water interface may be important in the transformation.     

Distribution of Hydrocarbon-Utilizing Microorganisms and Hydrocarbon Biodegradation Potentials in Alaskan Continental Shelf Areas

Hydrocarbon-utilizing microorganisms were enumerated from Alaskan continental shelf areas by using plate counts and a new most-probable-number procedure based on mineralization of ¹⁴C-labeled hydrocarbons. Hydrocarbon utilizers were ubiquitously distributed, with no significant overall concentration differences between sampling regions or between surface water and sediment samples. There were, however, significant seasonal differences in numbers of hydrocarbon utilizers. Distribution of hydrocarbon utilizers within Cook Inlet was positively correlated with occurrence of hydrocarbons in the environment. Hydrocarbon biodegradation potentials were measured by using ¹⁴C-radiolabeled hydrocarbon-spiked crude oil. There was no significant correlation between numbers of hydrocarbon utilizers and hydrocarbon biodegradation potentials. The biodegradation potentials showed large seasonal variations in the Beaufort Sea, probably due to seasonal depletion of available nutrients. Non-nutrient-limited biodegradation potentials followed the order hexadecane > naphthalene pristane > benzanthracene. In Cook Inlet, biodegradation potentials for hexadecane and naphthalene were dependent on availability of inorganic nutrients. Biodegradation potentials for pristane and benzanthracene were restricted, probably by resistance to attack by available enzymes in the indigenous population.     

Biodegradability continuum and biodegradation kinetics of natural organic matter described by the beta distribution

We followed a long-term (up to 503 days) microbial mineralization of dissolved organic carbon (DOC) from lake water in a bioassay and described the kinetics of biodegradation with a new model based on a reactivity continuum approach. The biodegradability of DOC was expressed as the probability of biodegradation, which was assumed to follow a beta distribution. We compared the performance of our beta model to five earlier models: the simplest first order kinetic model, two G models, the power model and the gamma model. The simplest first order kinetic model described the decreasing microbial mineralization of DOC poorly (r ² = 0.73), but the other models explained the observed kinetics of biodegradation well (r ² > 0.95). When we assessed the extrapolation power of models beyond the length of the bioassay by reducing the amount of data, the predictive power of the G models was poor. Instead, the beta model predicted the biodegradation kinetics consistently and correctly based on even only three observations in time. The beta model provided also long-term predictions (up to 5,000 years) along the observed long-term mineralization trajectory of organic carbon in sediments. Additionally, the beta model formulated the biodegradability continuum of DOC, which was skewed towards low biodegradability. During the bioassay, the skew towards low biodegradability increased as the most biodegradable parts of DOC were consumed. The beta model describes the biodegradability continuum quantitatively and can predict biodegradation in a realistic manner, thus, improving our understanding about the biodegradability and the role of natural organic matter in the environment.     

Impact of an oil field effluent on microbial activities in a Wyoming river

The survival, functions, and physiological diversity of autochthonous sediment microbiota were examined in situ at five stations along the Little Popo Agie River, WY; one station above, one at, and three below a discharge point for oil wastewater from Union Oil Company's Dallas Field. Below the oil wastewater discharge point there were increases in electron-transport activity, carbon dioxide production, and microbial populations of heterotrophs, ammonifiers, hexadecane degraders, starch hydrolyzers, protein hydrolyzers, and sulfate reducers. At a station 1420 m below the discharge point, however, overall sediment microbial activities and all of the physiological groups of bacteria, except hexadecane-degrading microbiota, were at levels comparable with those at the control station above the discharge point. Similarly, mineralization of glucose, amino acids, hexanoic acid, and hexadecane was elevated at stations directly below the discharge point, but appeared to subside rapidly. Xenobiotic biodegradation potential of the sediments varied with the chemical and the sample location and was not directly related to oil residue levels in the sediment. Microorganisms thus appeared to maintain physiological diversity and increased in numbers and activity in a riverine environment that contained petroleum hydrocarbon concentrations known to be deleterious to freshwater fish and macrobenthic communities.     

Structured modeling and state estimation in a fermentation process: Lipase production by Candida rugosa

A simple structured mathematical model coupled with a methodology of state and parameter estimation is developed for lipase production by Candida rugosa in batch fermentation. The model describes the system according to the following qualitative observations and hypothesis: Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell where it is consumed, transformed, and stored. Lipase is excreted to the medium where it is distributed between the available oil-water interphase and aqueous phase. Cell growth is modulated by the intracellular substrate concentration. Model parameters have been determined and the whole model validated against experiments not used in their determination. The estimation problem consists in the estimation of three state variables (biomass, intra- and extracellular substrate) and two kinetic parameters by using only the on-line measurement provided by exhaust gas analysis. The presented estimation strategy divides the complex problem into three subproblems that can be solved by stable algorithms. The estimation of biomass (X) and the specific growth rate (mu), is achieved by a recursive prediction error algorithm using the on-line measurement of the carbon dioxide evolution rate. mu is then used to perform an estimation of intracellular substrate and the other kinetic parameter related to substrate transport (A) by an adaptive observer. Extracellular substrate is then evaluated by means of the estimated values of intracellular substrate and biomass through the material balance of the reactor. Simulation and experimental tests showed good performance of the developed estimator, which appears suitable to be used for process control and monitoring. (c) 1995 John Wiley & Sons, Inc.     

Hexadecane mineralization activity in ornithogenic soil from Seabee Hook,Cape Hallett,Antarctica

Ornithogenic soils that form in penguin rookeries contain high levels of organic carbon and nitrogen. On Seabee Hook, Cape Hallett, Antartica, ornithogenic soil was contaminated with hydrocarbons following establishment of a scientific research station. In these soils hydrocarbon biodegradation could be supported by available soil nitrogen. Hexadecane mineralization activity was detected in vitro in ornithogenic soil when incubated at 5 or 15°C. At 5°C the extent of hexadecane mineralization was higher in hydrocarbon-contaminated soil than in uncontaminated soil. Alkane-degrading bacteria isolated from Seabee Hook soil were identified as Rhodococcus or Gordonia spp. or an unclassified Corynebacterineae. The alkane degraders grew on n-alkanes from heptane (C8) to eicosane (C20) and pristane, and utilized uric acid or ammonium nitrate as nitrogen source. All of the isolates possessed urease activity. Results of this study indicate biodegradation of hydrocarbons may contribute to the natural attenuation of oil spills in ornithogenic surface soils in summer.     

Biodegradation of N-Nitrosodimethylamine in Aqueous and Soil Systems

N-Nitrosodimethylamine (NDMA) was mineralized by microorganisms in aqueous and soil systems. Initial rates of mineralization (micrograms per milliliter per day) were calculated for a wide range of initial concentrations of NDMA (micrograms per milliliter to picograms per milliliter). Log-log plots of the data were fitted with both linear and nonlinear least-squares analyses; however, linear models provided better fits for the kinetic data in all cases. The slopes of the linear fits were not significantly different than 1.0 (P < 0.05); thus, first-order reaction kinetics were in effect over the range of concentrations tested, and saturation kinetics were not achieved. Rate constants (day⁻¹) and total percent mineralized increased with decreasing initial concentrations of NDMA. Rates of mineralization were reduced in aqueous systems when supplemental carbon was available, whereas in soils, percentages of organic matter and supplemental carbon had little effect on rates of mineralization. Implications of these results for predictions of rates and threshold limits of mineralization activity in natural systems are discussed. A laboratory scale simulated trickling filter containing an activated charcoal bed provided a suitable environment for mineralization of NDMA at concentrations of 50 and 100 μg/ml on a continuous basis. NDMA was not toxic to natural populations of microorganisms at concentrations up to 10 mg/ml. Using high-pressure liquid chromatography coupled with radioactivity detection, we identified formaldehyde and methylamine as intermediates produced during the biodegradation of NDMA.     

Rate of Contaminant Bioavailability in Artificial Soil-Water Column Experiments

Bioavailability of organic contaminants is a key parameter in estimating the fate and transport of contaminants in the environment. Its quantification is necessary for assessing the bioremediation potential of a contaminated site or for evaluating the hazard associated with the presence of the contaminant. However, there is a lack of methods that quantify biovailability under flow-through conditions. Therefore, a method was developed based on mass-balance analysis of contaminant breakthrough curves to quantify a bioavailability rate. The method was performed on an eight-column study where pentachlorophenol (PCP) was injected through saturated columns packed with a blend of sterile or inoculated sand and PCP-sorbing resin. Columns varied in sorption capacity, pore-water velocity and biodegradation activity. Breakthrough curves were modeled using a classic approach that includes advection-dispersion, equilibrium and first-order sorption, as well as biodegradation. Results showed that the bioavailability rates (non-dimensional numbers ranging from 0.02 for a sorptive column to 0.32 for a non-sorptive one) were directly related to the limiting kinetic mechanism. It was also demonstrated that the contaminant aqueous phase concentration does not adequately represent its bioavailability under flow-through conditions.     

Biodegradation of RDX in Unsaturated Soil

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a military explosive that is a common soil and groundwater contaminant at facilities that manufacture, handle, and dispose of munitions. One such facility is the U.S. Department of Energy Pantex Plant, the focus of this research in which the feasibility of in situ bioremediation of contaminated soil in the vadose zone was assessed. A batch technique using ¹⁴C-RDX was developed to investigate the degradation of RDX under aerobic, microaerobic, and anaerobic conditions. In addition, the effect of nutrients (organic carbon and phosphorus) on biodegradation rates was studied. The extent of mineralization was quantified by monitoring the production of ¹⁴CO₂, and RDX biodegradation rates were estimated for each environmental condition. The results showed that RDX degraders were indigenous to the contaminated soil and degraded RDX to a significant extent under anaerobic conditions. Little biotransformation was observed under aerobic conditions. The addition of a biodegradable organic carbon source significantly increased the RDX biodegradation rate. Under appropriate environmental conditions, significant mineralization of RDX also was observed. The half-lives for the degradation of RDX under anaerobic conditions were approximately 60 days and decreased to approximately 40 days with nutrient addition. In contrast, the half-life for aerobic degradation was on the order of 1000 days, with an upper 95% confidence interval approaching infinity.     

Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

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.     

Hexadecane mineralization in oxygen-controlled sediment-seawater cultivations with autochthonous microorganisms.

Laboratory studies investigated the influence of dissolved oxygen tension (DOT) on microbial degradation of hexadecane in cultures with sediment-seawater suspensions. With a fermentor system, it was possible to adjust and regulate different oxic conditions (DOTs between 0.4 and 80% of oxygen saturation) as well as anoxia. The effects of DOT reduction on the amount and rate of hexadecane degraded and on the degree of mineralization and on the production of biomass were investigated. When the DOT was reduced from 80% to 5%, no dependence of the investigated parameters on the oxygen concentration was found. The amount of hexadecane degraded was constant, with an average value of 86% of the initially applied amount. The degradation rate was constant even down to 1% DOT, with an average value of 0.15 mg of hexadecane per g of sediment per h (16.2 mg liter-1 h-1). The mean degree of mineralization was 70% of the initially applied hexadecane, and biomass production reached a value of about 1.5 g per g of hexadecane consumed. A significant influence on the degradation process was detected only with DOTs below 1%. The degree of mineralization and the amount of degraded hexadecane decreased, whereas the degradation rate was still unaffected. Under anoxic conditions, no hexadecane degradation occurred within 190 h. The fact that the hexadecane biodegradation rate was constant down to at least 0.04% DOT shows that the actual oxygen concentration is of minor importance as long as the oxygen supply is high enough to guarantee the oxygen-dependent degradation step.