Advances in Environmental Research 7 (2003) 767782

A case study of bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site
B.K. Gogoia, N.N. Duttaa, P. Goswamia, T.R. Krishna Mohanb,*
b

Regional Research Laboratory, Jorhat 785 006, India CSIR Centre for Mathematical Modelling and Computer Simulation (C-MMACS), Bangalore 560 037, India Accepted 10 March 2002

Abstract Laboratory and field pilot studies were carried out on the bioremediation of soil contaminated with petroleum hydrocarbons in the Borhola oil fields, Assam, India. The effects of aeration, nutrients (i.e. nitrogen and phosphorus) and inoculation of extraneous microbial consortia on the bioremediation process were investigated. The beneficial effects of these parameters on the bioremediation rate were realised equally in laboratory and field pilot tests. The field tests revealed that up to 75% of the hydrocarbon contaminants were degraded within 1 year, indicating the feasibility of developing a bioremediation protocol. A complementary computer simulation study was carried out to enhance the understanding of the basic processes and the rate determining factors for bioremediation under the practically relevant conditions of Borhola oil fields. The simulations indicated that due to the high initial contaminant concentrations, the bioremediation process was restricted mostly to the macropores of the system within the period of 1 year and had not penetrated into the soil aggregates sufficiently. Certain shortcomings of the model have been identified and possible refinements suggested. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Biodegradation; Fuel contamination; Field pilot study; Aeration; Nutrients; Extraneous microbial consortia; Modeling and simulation

1. Introduction Borhola oil fields, under the Damodar Valley project (DVP) of Eastern (India) Regional Business Centre (ERBC), Oil and Natural Gas Corporation Limited (ONGCL), India, with an operational area of approximately 300 acres, have been producing crude oil at an estimated capacity of two million tonnes per annum since 1972. During normal operation, leakage and spillage of crude oil result in soil contamination at such locations as oil wells, sumps and pits, tank batteries, gathering lines and pump stations. Depending on the
*Corresponding author. Tel.: q91-80-527-4649; fax: q9180-526-0392. E-mail address: kmohan@cmmacs.ernet.in (T.R. Krishna Mohan).

site location, the level of oil contaminants in the soil may be as high as 10% wyw. Furthermore, an appreciable amount of waste crude oil is generated during field operations and is collected in a waste pit constructed near the drilling site. The upper layer of oil in the waste pit is removed and transferred to Group Gathering Stations (GGS) and the combustible portion of the remaining contaminated soil in the pit is removed manually to an isolated place and subjected to open dump burning. Finally, there is some contaminated soil still remaining in the waste pit which is left to be degraded by natural processes. This practice of disposal has limitations. During the rainy season, flooding andy or accidents, crude oil from the waste pit may spread to the surrounding fields causing pollution. Although open dump burning may be simple and easily adaptable, this technique has undesirable health and safety hazards

1093-0191/03/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 0 9 3 - 0 1 9 1 0 2 . 0 0 0 2 9 - 1

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from air pollution. Furthermore, due to incomplete combustion, the residual hydrocarbons may gradually percolate into acquifers, causing long-term environmental problems. Bioremediation is an alternative technology capable of achieving permanent remediation at waste sites without such associated problems, as recognised by the US EPA for implementation under the Superfund Amendments and Reauthorisation Act (SARA) of 1986 (Sims et al., 1990); acceptance by the general public is another major advantage of this technology (Skladney and Metting, 1993). To achieve managed in situ bioremediation, nutrients and air are supplied to the subsurface and the indigenous bacterial mix is often augmented to obtain enhanced bioremediation. The design of an efficient bioremediation system requires a set of careful studies of the local conditions. Accordingly, this paper reports on a systematic laboratory treatability and field pilot scale study conducted towards the development of a bioremediation protocol for the oil contaminated soil at Borhola oil fields. Also, we report on a mathematical model that was employed for simulation purposes for forming a better insight into the rate limiting steps involved in the remediation processes. The laboratory exercises require periods of the order of 1 year which makes it difficult to design an optimal system by tuning the parameters in the laboratory. Computer simulations, hand in hand with the laboratory exercises, provide the achievable solutions. 2. Experimental study 2.1. Sampling and site assessment Development of a bioremediation protocol requires a complete site characterisation to define the subsurface geology and the distribution of contaminants. The magnitude of soil contamination in the drill site of CS area of Borhola oil fields is very high and the soil variability in this zone appears to be minimal. In addition, this zone is more prone to cause oil spillage to the nearby paddy fields than are the oil well head sites. The well heads are distributed throughout the oil fields in different sites of which the geological and hydrological conditions vary. Representative soil samples were collected from different locations following appropriate statistical procedures reported in the literature (Huesemann, 1994a). In the selected site of 0.25 acres, the contaminant (crude oil) distribution on the surface was observed to be uniform. Accordingly, eight non-overlapping square grids were selected of equal, 4=4 ft. surface area and samples collected from the centre of each grid. Visual observation and random estimates of the oil content indicated uniformity of infiltration rates upto a depth of 2 ft. (0.6 m). Accordingly, three samples, weighing 3 kg each, were collected from the centre of each grid,

from different depths going upto a maximum of 2 ft. Samples were stored in plastic buckets and homogenised in a mixer before selecting a subsample for analysis. 2.2. Contaminant characterisation While a number of different methods of soil analysis for compounds like BTEX (benzene, toluene, ethylbenzene and xylene) or PNAS (polynuclear aromatic hydrocarbons) may be required by regulatory agencies, only oil and grease (O&G) and total petroleum hydrocarbons (TPH) concentrations are required for the design of optimal land treatment conditions. The success of bioremediation (i.e. TPH removal) depends largely on the contaminant characteristics. Therefore, it is essential to have a comprehension of the same in respect of total oil content and its constituents in terms of the different hydrocarbons present. Usually, the hydrocarbons present comprise saturates (alkanes and cycloalkanes) and aromatics (mono- and polynuclear). The polar fraction of the petroleum containing nitrogen, sulfur and oxygen is comprised mostly of ashphaltenes and resins. Moisture content in the soil is important because it has relevance to the biodegradation process. 2.2.1. Oil and moisture content in soil samples Dried, crushed and sieved contaminated soil samples were subjected to Soxhlet extraction using freonychloroform for 8 h. The extracted oil was concentrated in a vacuum rotary evaporator and weighed. The extract was treated with solice gel to remove the polar compounds. The resulting TPH was then collected in a pre-weighed beaker, dried at 60 8C for 18 h, and weighed according to EPA methods 413.1 (USEPA, 1979a) and 418.1 (USEPA, 1979b). For determination of moisture content, the contaminated soil samples, 50100 g each, were dried at 60 8C overnight and then kept in a dessicator. The samples were then crushed in a mortar, sieved from a 36-mesh sieve, redried and weighed. The loss in weight was calculated as the moisture content. Typical values of oil and moisture contents in five samples are shown in Table 1; samples 68 were almost identical in colour, consistency, etc., and were omitted from further investigations. All analyses were carried out in triplicate and reproducibility was found to be "5%. 2.2.2. Oil component analysis Component analyses of the samples were used to draw inferences regarding natural biodegradability of the crude oil. For the component analysis, the ashphaltenes were precipitated from the extracted oil samples and Borhola crude oil using n-pentane as the antisolvent. After the separation of ashphaltenes, the extracted oil and Borhola oil were further fractionated into saturated

B.K. Gogoi et al. / Advances in Environmental Research 7 (2003) 767782 Table 1 Oil (on a dry weight basis) and moisture content of collected soil samples Sample no. 1 2 3 4 5 Oil content (% wyw) 13.35 8.55 2.97 9.86 6.77 Moisture content (% wyw) 7.88 18.80 4.54 3.14 25.22

769

and aromatic hydrocarbons and resins by column chromatography on an activated alumina column by elution with petroleum ether (4060 8C boiling range) benzene mixture (2:1 vyv) and methanol in sequence. Table 2 shows the various fractions of petroleum compounds present in this extracted oil in samples 15 as well as in the crude. The lower percentage of saturates in the samples, with a relative increase in their aromatic content, may be attributed to natural biodegradation of the crude. It is known that saturates are degraded faster than aromatics and others, so that aromatic contents increase in the weathered samples over that found in the crude oil. Increases in non-hydrocarbon content (as compared to the crude oil) in the samples are also indicative of natural biodegradation phenomena as these compounds are difficult to degrade by microorganisms (Bossert and Partha, 1984). Sample nos. 1, 2 and 5, collected from a fresh spill, are characterised by a high level of saturated hydrocarbon content that may be attributed to the sandy matrix of the sample, which poorly adsorbs ashphaltenes and resins (Weissenfels et al., 1992). Component analysis of sample nos. 3 and 4 shows substantial degradation while sample nos. 1, 2 and 5 exhibit relatively little degradation. In order to draw further inferences regarding natural biodegradation, chromatographic analysis of the saturate fraction was conducted with a Shimadzu GC RIA gas chromatograph, with a metallic capillary column (30
Table 2 Types of components in extracted oil samples and crude Sample no. 1 2 3 4 5 Crude % wyw of Saturates 50.88 58.60 30.29 31.10 58.80 69.38 Aromatics 12.56 19.04 27.16 39.14 19.10 18.89 Resins 27.83 18.97 37.80 19.14 13.29 5.07

m=0.32 mm i.d.) coated with OV-101 and temperature programmed from 100 to 280 8C at 4 8Cymin. Chromatograms of sample nos. 1 and 3 are shown in Fig. 1 while a chromatogram for the fresh crude oil saturate fraction is shown in Fig. 2. As evident from the chromatograms, sample no. 1 is undegraded since it was a fresh sample; its chromatogram is quite similar to that of the crude oil sample. Sample no. 3 was taken from an approximately 10-year-old waste pit and at a depth of 1 ft. (0.3 m) below the surface. It shows substantial degradation as evident from the presence of a high concentration of non-hydrocarbon components. This may be attributed to the low oil content in the sample (2.9% wyw) leading to reduced substrate inhibition effects and a consequent high biodegradation rate. Thus, two general inferences could be drawn: i. Component analysis of the extracted oil samples gives a fair idea of the state of degradation, and ii. chromatographic analysis of the saturate fraction of the extracted oil samples provides a semi-quantitative estimate of the extent of biodegradation. A similar chromatographic approach has been reported elsewhere to explain biodegradation capability of contaminated soil during an in situ study (Gruiz and Kriston, 1995). 2.3. Estimation of soil physico-chemical properties Though at present details are not known about the influence of soil type on biodegradation kinetics, it is likely that the highly sorptive surfaces of some clay and organic matter fractions limit the bioavailability of petroleum hydrocarbons to soil microorganisms (Huesemann, 1994b; Tang et al., 1998). This may be especially the case for intensely weathered soils where the contaminants have had time to migrate into the micropores, which are less accessible to microbial attack. In general, bioavailability of hydrocarbons declines with ageing. The rate and extent of sequestration as measured by the extent of mineralisation of phenanthrene by an added bacterium has been shown to be appreciable in soil samples with )2.0% organic carbon (Nam et al.,

Ashphaltenes 8.73 3.39 4.75 10.05 8.79 6.66

Hc 63.44 77.64 57.45 70.81 77.92 88.27

Non-Hc 36.56 22.36 42.55 29.19 22.08 11.73

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Fig. 1. G C of saturate fractions of extracted oil samples.

1998). In other words, soils from various sources and locations exhibit differences in both rate and extent of sequestration. We have not conducted a study to determine the levels of sequestration at Borhola oil fields. Given the high levels of contaminant concentration in the field, it is expected that there should be significant levels of bioavailable contaminants. In the case of intensely weathered soils, the kinetics are not limited by the number of hydrocarbon degraders or the intrinsic petroleum hydrocarbon biodegradability, but rather by mass transport (desorption, diffusion and convection) phenomena. It is known that the PNA biodegradation rates are affected by the fraction of fines (-0.075 mm) in the soil. Soil characterised by more

than 10% fines exhibited lower PNA biodegradation rates and the extent of bioremediation during land treatment was lower than that of soils with smaller fines fractions, i.e. -10% (Huesemann, 1994b). The increased sorptive surface area of soil with larger fines fractions may affect the bioavailability of certain hydrocarbon contaminants. The soil chemistry is equally important in developing a biodegradation potential for contaminated soil (Rogers et al., 1993). For instance, the soil pH should be adjusted to within the range 68 to enhance microbial activity (Hicks and Caplan, 1993). The levels of nitrogen and phosphorus in the soil may also be very critical as these may limit the biodegradation rates because of an inter-

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nutrient application or injection. Air permeability of the contaminated soil was determined by a method based on Darcys law for steady flow in a packed bed as described in the literature (Johnson et al., 1990). The permeability value so determined was found to be 159 Darcy as compared to the reported value of 145270 Darcy for hydrocarbon contaminated soil (Dupont, 1993). The experimental value obtained here is indicative of clean sand and gravel and thus permeability may not be the limiting factor at the beginning of bioremediation. This nature of the soil bed is also substantiated by the observed values of soil porosity, density and particle size (see below). It is expected that permeabilities under growth conditions in presence of nutrients may decrease by 20% from the above value. The porosity and particle density of soil samples, determined by the usual gravimetric methods, were found to be 38% and 2.6 gycm3, respectively. The average soil particle size determined by sieve analysis was found to be 0.5 mm. The sand and clay-silt content of the sand were in the ranges of 6879% and 2531%, respectively. 2.4. Estimation of soil microbiological properties The soil microbial properties are, perhaps, the vital factors determining natural biodegradability. Microbial characterisation of soil includes enumeration of total microbes and contaminant-specific degraders. Soils usually contain large numbers of native or indigenous microorganisms that are able to degrade petroleum hydrocarbons. Microbial inhibition may occur in the presence of high salt concentration and heavy metals, i.e. Ni, Cr, Pd, Cd, As, etc. (Balrich and Stotsky, 1985). In addition, hydrocarbon levels higher than 10% wt. are associated with varying degrees of inhibitory effects on soil microbes (Huesemann, 1994b). Total microbial content was determined by the Agar Plate Method for Total Microbial Count as described in the literature (Clark, 1965). The total microbial count was compared with an estimate of the population that will degrade the contaminant. The preliminary screening of the contaminant-specific degraders was performed in a medium
Table 3 Soil chemical properties Parameter PH Ammonia-N, mgykg Orthophosphate, mgykg Water soluble potassium, mgykg Water soluble iron, mgykg Water soluble Mg, mgykg Water soluble Ca, mgykg Measured values 4.50 300.00 9.80 127.00 61.40 8.00 8.60

Fig. 2. G C of saturate fraction of Borhola oil.

active process occurring between the nutrients (Walworth and Reynolds, 1995). The levels of N, P, K, Fe, Mg and C, etc., that determine the soil physico-chemical properties along with the pH were estimated by standard text book procedures (Partha and Bossert, 1984) and are shown in Table 3. It is apparent that concentrations of inorganic nutrients, i.e. N, P, Fe and Mg, are low and this could limit the metabolism of the existing microorganisms capable of degrading hydrocarbons in the soil environment; application of extraneous nutrients may be required for developing a feasible bioremediation method. Major soil physical characteristics that may influence the bioremediation process are porosity, bulk density and air permeability. The permeability determines the rate of transfer of electron acceptors to the contaminated soil. It is believed that the reduction of permeability because of microbial biofilms in the soil macrovoids, as well as in the smaller pores of the soil matrix (microvoids), is a major hurdle in managed in situ bioremediation; the problem may be more acute at the point of

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that provides only the necessary inorganic nutrients required for microbial growth, but with no intrinsic carbon source; the contaminant supplement serves as the sole source of carbon. Cultures of selected potential biodegraders were prepared in supplemented and unsupplemented (control) media. Following incubation, the cultures were evaluated for the presence and relative abundance of microbial growth. Isolates demonstrating little or no growth were judged to be poor or non-degraders of the contaminants and eliminated from further studies. The remaining isolates were then evaluated for the effects of environmental parameters and for performance in a bench scale bioremediation test as discussed in a subsequent section. The information on microorganisms and contaminant-specific degraders provides an indication of microbial activity of the soil for existing (unamended) site conditions. The basal medium used for isolation of hydrocarbon degrading microbes had the following composition (gy l): 4.74 K2HPO4; 0.56 KH2PO4; 0.50 MgSO47H2O; 1 NaCl; 0.01 FeCl3; 0.01 CaCl22H2 O; 2.5 NH4NO3; 1 ml of a trace element solution consisting of (gyl) 10 CuSO45H2O; 10 H3BO3; 10 MnSO45H2O; 70 ZnSO47H2O; 10 MOO3 with a medium pH of 7.0. Fifty millilitres of the medium taken in a 250-ml flask was sterilised by autoclaving at 120 8C for 15 min. The degradation capacity of the indigenous microorganisms was assessed qualitatively from the observation of growth and colour change of the enriched culture media. One gram each of the oil contaminated soil samples was then added to 50 ml of medium and 1% vyv crude oil in a flask. The flask was then incubated in a shaker at 30 8C and 280 revymin for 12 days. Thereafter, 1 ml of culture broth from each flask was transferred to a fresh medium of the same composition and incubated under the same conditions for another 12 days. The growth of bacterial population (consortia) and the extent of degradation of crude oil in each flask was assessed visually. A comparative assessment of the potential of bacterial consortia to utilise individual classes of hydrocarbon compounds was made by using n-hexadecane, BTEX and phenanthrene as representatives for the groups of aliphatic, aromatic and PNA hydrocarbons, respectively. The medium and cultivation conditions were the same as that used for the isolation of the hydrocarbon degrading consortia except that crude (as carbon and energy source) was replaced by hexadecane (1% v yv), BTEX (1% vyv) and phenanthrene (0.05% wyv). Each of the consortia was tested for its capacity to degrade aliphatic, aromatic and PNA hydrocarbons. Flasks were inoculated with 1 ml of culture broth from each of the 12-day-old consortia, which were maintained by subculturing every 12th day. Growth and hydrocarbon degradation in all cases were assessed from visual observation of hydro-

carbon dispersion in the aqueous medium, turbidity and colour formation of the culture broth. The effectiveness of the microbial consortia obtained from different oil-contaminated soil samples varied from sample to sample in their capabilities to degrade different types of hydrocarbons. Consortia developed from sample no. 4 had the best crude oil degrading capacity. Regarding degradation capacity for individual hydrocarbons, all the consortia were found to degrade hexadecane and the BTEX fractions of crude oil; however, the consortium from soil sample no. 4 was better than the others. Consortia from soil sample nos. 4 and 5 degrade phenanthrene (PNA) preferentially. It may be inferred that aliphatic degrading microorganisms are more ubiquitous than PNA hydrocarbon degraders. As the aromatic and PNA hydrocarbons are more resistant to microbial degradation, they may accumulate in soil, and in time they may contaminate the aquifers (Huesemann, 1994b). In the contaminated soil samples, alkane degrading microorganisms were more prevalent than aromaticypolyaromatic hydrocarbon degraders. 3. Laboratory evaluation of bioremediation potential Biodegradation potential can be assessed by performing a laboratory treatability study or extensive waste characterisation combined with the simulation of bioremediation potential based on biodegradability data for a given type of compound. In this section, isolation of pure cultures and their efficacy in contaminant degradation, surfactant effect in contaminant solubilisation and biodegradation determined from laboratory shake flask experiments have been documented. Results from a complementary laboratory microcosm study are also presented in order to draw inferences regarding biodegradation potential from a technological perspective. 3.1. Isolation of pure culture The relative potentials for bioremediation of the major types of petroleum compounds decrease in the order, monoaromatics)straight chain alkanes)branched alkanes)naphthenes)polynuclear aromatics)polars (Huesemann, 1994b). Futhermore, microorganisms are selective and attack specific hydrocarbons rather than all the components of the oily waste (Brown, 1987). An appropriate mixture of different microbial species is needed to form a commensurable association that can degrade all of the components to the same extent (Gruiz and Kriston, 1995). To create such an artificial mixture is not easy, but a suitable adaptation technique may be useful. From the result of the soil microbiological enumeration study presented above, it may be inferred that artificial mixtures of microbial consortia will be necessary for biodegradation of petroleum compounds in the contaminated soil of Borhola oil fields.

B.K. Gogoi et al. / Advances in Environmental Research 7 (2003) 767782 Table 4 Properties of biosurfactanta produced in a n-hexadecane grown culture Strain (N) 10.5 12 CRL-4 7.2
a

773

Surface tension (dynesycm) 32 38 31 28

Functional characterisation Oil soluble, produce creamy oyw emulsion Oil soluble, produce creamy oyw emulsion Water soluble, produce stable oyw emulsion Water soluble, produce stable oyw emulsion

Partial chemical characterisation Glycolipid Similar to phospholipid Glycoprotein

Rhamnolipid isolated from Pseudomonas aurigenosa G6.

Pure cultures were isolated from the consortia of soil sample nos. 4 and 5 (which exhibited biodegradation capability for hexadecane and BTEX) as discussed below. Each consortium, developed in crude oil containing liquid medium (12 days old), was spread on agar plates after appropriate dilution; sterilised basal medium agar was used for preparing the agar plates. n-Hexane (0.2 ml) or phenanthrene (0.2 ml of 0.05% w in ether) was then spread on the agar surface. The plates were then incubated at 30 8C in an incubator for 4 days. The wellseparated bacterial colonies were transferred to agar slants containing hexadecane or phenanthrene, and were then allowed to grow for 4 days at 30 8C. Each culture was tested for its capacity to degrade hexadecane and phenanthrene in liquid medium. Pure cultures from these consortia having the capacity to degrade aliphatic hydrocarbons as well as phenanthrene were isolated. n-Hexadecane degrading pure culture was obtained from sample no 5, whereas phenanthrene degrading pure bacterial culture was obtained from consortia of sample no. 4. These pure cultures were maintained in the laboratory. 3.2. Hydrocarbon solubilisation and soil desorption testing As hydrocarbons are mostly insoluble in water, bacterial cultures producing biosurfactant will be useful in solubilising andyor emulsifying hydrocarbons leading to desorption and thereby enhancing biodegradation rate (Rogers et al., 1993). Biosurfactant production was only detected in the n-hexadecane grown culture; the detection method was based on measuring the surface tension of the medium using a Du-Nouy ring method (De Groot and Monson, 1931). The pertinent properties of the detected biosurfactant (from Pseudomonas sp. 7.2) are shown in Table 4. Due to surface tension of aqueous biosurfactant solution, oil solubilisation and consequent

desorption from the soil matrix may be expected to be higher. The effects of the isolated biosurfactant and synthetic surfactant on desorption and degradation were separately studied in a microslurry reactor operated for 48 h. For this study, 5 g of homogeneous, contaminated soil was stirred with 150 ml of aqueous medium in a flask and the content was mixed in a shaker bath. The extent of biodegradation was estimated from the total oil content of the soil (on dry basis), before and after the slurry treatment. The extent of desorption was estimated from the total oil content in contaminated soil before treatment and that present in the soil as well as in the liquid dispersion after the treatment. The oil content in the supernatant was determined by a standard gravimetric method (Franson, 1976a). Typical results of desorption and biodegradation are shown in Table 5 from which it is apparent that, although the biosurfactant effects are realised significantly in the desorption process, the enhancement of biodegradation can be considered to be only marginal
Table 5 Effect of biosurfactant on soil desorption and biodegradation Sample Treatment no. 1 2 3 4 5 6 Control (sterilised soil) Soilqbiosurfactant Soilqsynthetic surfactanta Soilqconsortia Soilqconsortiaq biosurfactant Soilqconsortiaq synthetic surfactant Desorption Biodegradation (% oil) (% oil) 13.27 71.05 77.27 21.41 29.24 28.98

a Tween 80: polyoxyethylene sorbitan monoaleate (nonionic).

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Table 6 Degradation of crude oil contaminated seashore sand in a batch reactor Sample consortia no. Control 4 5 Mix Time variation of oil content (gyg) 0 days 0.048 0.048 0.048 0.048 15 days 0.048 0.038 0.040 0.038 30 days 0.044 0.035 0.034 0.036 60 days 0.044 0.035 0.030 0.038

after Soxhlet extraction of the dried soil sample by the procedure described in Section 2.2. Results of degradation are presented in Table 6; what is provided are average values from analysis in triplicate and the reproducibility was within "5%. It is apparent that added bacteria degraded the hydrocarbon content of the crude oil, but the extent of degradation of individual hydrocarbons needs to be quantified. 4. Field pilot test A practical bioremediation technology should lend itself to design of a controlled and regulated system of optimum efficiency through optimal aeration (oxygen or another electron acceptor), optimal nutrient supply (N, P, etc., supplementation) and the best possible bacteria. In order to assess the optimal combination of these parameters, a field pilot study was carried out as discussed below. Six test cells of dimensions shown in Fig. 3 with an air sparger were used for test experiments. The cells designated as A, B, C, D, E and F were filled with approximately 500 kg each of crude oil contaminated soil prepared by mixing in a Banbury mixer. The soil pH was maintained between 6 and 8 by adding approximately 300 g of calcium carbonate during the mixing operation. The experimental conditions in each cell (Table 7) were different such that effects of aeration, nutrient supply and added bacterial consortia could be assessed. In test cells E and F, 25 kg each of oil contaminated soil collected from the top surface of the

under the ideal conditions of the slurry treatment. It may be possible, based upon the hypothesis that mass transfer limitations dominate contaminant fate in the soil matrix, to determine the treatment end-point of the hydrocarbons. Under the ideal conditions of the slurry reactor, desorption and solubilisation with cultured microorganisms are maximal which, in turn, maximises the extent of biodegradation. 3.3. Batch reactor microcosm test In general, laboratory pan microcosm andyor column tests are conducted to determine the kinetics of biodegradation, depending on whether an ex- or in situ approach is desired. Alternately, a microcosm test can be used to model essential characteristics of the environment to predict the consequences of bioremediation treatment. However, due to the constraints of conducting the demonstration within the budgetary and time limitations, the objectives of this test have been confined to seeing whether the inoculation of bacterial culture has any effect on the extent of biodegradation. The tests have been conducted using fresh sand contaminated with crude oil in an open batch reactor as described below. Sand (700 g) was mixed thoroughly with 35 g of Borhola crude oil. The sandoil mixture was distributed in seven open flasks (100 g of sand in each flask) where 10 ml each of inoculum was added. For the inoculum, a consortium was developed in basal medium containing 1% crude oil for 8 days at 30 8C and 200 revymin in a rotary shaker. The culture broth (50 ml each) was passed through glass wool to remove lumps of undegraded crude oil. The collected culture broth was then centrifuged, the cell mass collected and suspended in 20 ml of basal medium (used as inoculum). Inocula of pure cultures were also prepared using the respective hydrocarbons (either n-hexadecane or phenanthrene) in place of crude oil. A control experiment was also carried out with 10 ml of the basal medium. The flasks were kept in an incubator at 30 8C and a moisture level of 1015% was maintained with sterile diluted water. The residual oil content was determined

Fig. 3. Schematic diagram of field pilot study test cell.

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775

drainage channel at the waste pit were added as a source of naturally adapted hydrocarbon degrading microbial consortia. Aeration was provided for 1 h every day at a rate of 100 m3 yh. The moisture content in each cell was maintained between 50 and 65% during the entire period by adding water from time to time; the moisture level was monitored by weight loss (see Section 2.2 under oil and moisture content in soil samples) taking 1012 random samples from each cell. It may be noted that the moisture content of field samples was in the 12 15% range. In each of the cells E and D, consortia from a bacterial suspension obtained from sample no. 5 (discussed earlier) were added, at levels of 1.2 mlykg. During the course of investigation, the progress of bioremediation was monitored by estimating periodically the oil content, pH, moisture content, nitrogen and phosphorus levels along with the total microbial content of the soil samples. The samples were withdrawn at various depths, i.e. 0, 3, 6, 9 and 12 inches from the top surface and at these positions: one sample at the centre, and four at diagonally opposite positions, 9 inches away from the corners. Averaged values of the parameters from a sampling in triplicate were considered for data interpretation. The number of soil microbes was estimated by the most probable number (MPN) enumeration test (Franson, 1976b). 4.1. Results of the field pilot test The field pilot test was conducted for the duration of 1 year. The time variations of the oil content in the soils in the test cells, over this period, are shown in Fig. 4; this is a plot of time variations of normalised oil concentrations defined as the ratio of the oil concentration (C) in the test cell to that (Cc) in the control cell at any time. As evident from the figure, nutrient addition has an advantageous effect on the bioremediation process; also, there is an advantage in the addition of extraneous consortia. The advantageous effect of aeration seems to be marginally greater than nutrition if we go by the lower final concentration achieved by the soil in cell F as compared with that in cell D. The biodegradation capability of the indigenous microorganisms
Table 7 Experimental conditions in bench scale test cells Test cell A (control) B C D E F
a

Fig. 4. Variation of normalised oil concentrations with time in test cells; normalisation is done with respect to the concentration in the control cell. The errors in all cases are within 10%. The legends are defined in Table 7.

seems to be adequate for the process under conditions of added nutrients (which may also be further enhanced through aeration) since the performance of the soil in cell B is not very far behind those in C, D, E and F where extraneous consortia were employed. It is seen that under such ideal conditions, the extent of degradation of the crude oil was as high as 75% during this period. Fig. 5 shows the microbial growth pattern during the treatment period and it is apparent that the biodegradation process is associated with microbial growth especially under the influence of nutrients and aeration; the microbial growth in the control cell remained essentially constant during the test period. These results suggest that the conditions in the test cells with nutrients and aeration are conducive to increased microbial growth and metabolism of the petroleum hydrocarbon utilisers. 5. Mathematical model and computer simulation 5.1. Model Modeling and simulation offer promising means for assessing the migration and attenuation of the contami-

Soil (kg) 500 500 500 500 500 500

Crude (% wt.) 4.8 4.8 4.4 4.7 4.4 3.5

Nutrient (kg) w(NH)2HPO4x No 0.5 0.5 0.5 0.5 No

Consortia No No Yes Yes Yesa Yesa

Aeration No No Yes No Yes Yes

Naturally adapted.

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Fig. 5. Variation of microbial population with time in test cells; the legends are defined in Table 7. Errors are within 10% in all cases.

nants, being treated in situ, in the subsurface. Sorption, convective flow, and biological transformation are three major processes controlling the ultimate fate and transport of contaminants in the subsurface. A completely mixed pore model, accounting for bioremediation in the interstitial spaces among soil aggregates (macropores), has been combined with another model, which accounts for diffusion and biodegradation in the micropores among the soil particles of the aggregates, developed in a previous work (Dhawan et al. (1993); hereafter referred to as Dh93). This model has been employed by us in the present study and analysed further in light of the experimental results. A brief sketch of the model is provided here for convenience. The basic assumptions made in deriving the model are: 1. The bed is composed of spherical soil aggregates of a uniform radius, R. 2. The aggregates are saturated, homogeneous and isotropic, and are composed of soil particles of silt and clay. 3. The temperature is constant in the bed. 4. Transport in the aggregates is by diffusion only. 5. The mobile liquid in the macrovoids of the bed is completely mixed. Thus, the concentrations of oxygen, biomass and substrate leaving the solid bed are equal to those in the macropores of the bed. 6. The adsorption process is sufficiently rapid so that an equilibrium exists at the soil surface between the concentrations of components in the liquid and adsorbed phases. 7. A concentration equilibrium is maintained between the components in the liquid phase in the macro-

8. 9.

10.

11.

12.

13.

pores and those on the surface of the aggregates in the bed. The contaminants are not toxic and do not inhibit biodegradation. The contaminants are biodegraded to carbon dioxide and water. In general, the effects of by-products on bioremediation rates are considered negligible. The reaction rate follows the Monod kinetic model (see, e.g. Bailey and Ollis, 1986; see also Rashid and Kaluarachchi, 1999 for a comprehensive discussion on its role in biodegradation models) and depends only on the concentrations of three components: substrate (contaminants), oxygen, and biomass. The transport resistances of substrate and oxygen to and through the microcolonies attached to the surface of soil particles are negligible. Therefore, the microcolonies respond to the variations in the bulk concentrations in the pore liquid. Transport of microorganisms within the aggregates can be represented by a diffusion term in the equation for the biomass. The effect of biofilms in the macrovoids on the convective flow rate is neglected.

The justification for the above assumptions have been discussed in Dh93 (see also Dhawan et al., 1991, hereafter referred to as Dh91). The effects of sequestration and consequent non-bioavailability of contaminants for biodegradation are not considered herein; it may be remembered that the field samples are high in initial contaminant concentrations. The equations for the macropores (see Dh93 for a full derivation), with S, X

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Table 8 Parameter values for numerical simulation; in brackets are given for those few parameters, whose values have been changed from the ones used by Dh93, their Dh93 values for comparison b90s1.0=10y7 gycm3 X9is8.0=10y6 gycm3 Yss0.5 g yg Kdss45.0 cm3yg mms5.5=10y5 ys (2.78=10y5ys) Ds 9s2.5=10y6 cm2 ys (4.0=10y6 cm2 ys) kds1.0=10y7 ys (2.78=10y7ys) ts2.0 (1.4) Ds 9,sos0.0 Kxs1.0=10y8 gycm3 Y0s1.0 g yg Rs0.5 cm as0.45 (0.37) Qs4.5 cm3 ys (2.16=10y3 cm3 ys) Kss5.0=10y7 gycm3 (1.0=10y6 gycm3) Db 9 s1.0=10y6 cm2 ys Dx 9 s2.0=10y5 cm2 ys Kdbs30.0 cm3yg q0 s s43 500.0 mg y kg ls0.45 (0.37) Vs62 000.0 cm3 (30.48 cm3) rs2.5 gycm3 (1.72 gycm3)

and B indicating concentrations of substrate, oxygen and biomass, respectively, are given below; s, x and b indicate corresponding quantities in the micropores. The equations are being presented in the dimensionless form and the notations are listed in the Appendix A. B Rs E dS s Z FBhymDs Z sgSiyS.yf2 (1) 1C D Rb G du r rs1
Z Z

The value of the retardation factor, Rs, for the contaminants depends on the linear soilwater partition (or distribution) coefficient Kds ; it is large for compounds that adsorb strongly to soil particles. The concentration of the adsorbed component can be given, with the equilibrium relation for adsorptiondesorption described by a linear adsorption isotherm, as qssKdss9

BWE dX x Z FBhymDx Z sg1yX.yf2 1C D Rb G du r rs1

Z Z

(2)

where qs is the concentration in the solid phase. The initial conditions are as follows: At us0, Ss1.0, Xs0.05 and BsB0

dB b Z 2 sgBiyB.qf2 1Bhyf2BymDb du r Zrs1

Z Z

(3) For the micropores, the mass balance equations are as follows (detailed derivation in Dh91). s B Ds EB 2 s 2s E sC FC q Fyf2 1bh9 u D Rs GD r r r2 G
B 2 x 2x E x 2 sDxC q 2 Fyf1 Wbh9 D r r u r G

where the Monod reaction term, h, is given by hsC

B

S EB X E FC F D 1qbsS GD 1qbxX G

(4) (5) (6)

The correspondence with the physical (dimensional) quantities are as follows: B9Rb X9 S9 b9Rb x9 , Xs i , Ss 0 , bs , xs i , RsYsso X s RsYss0 X s9 mmts0Xi kdt t ss 0 , f1sR , f2sR , us , s KsKxDs 9 Ds 9 ur Xi s0 rKdb bxs , bss , Rbs1q , Kx Ks a Bs

b B Db EB 2 b 2b E 2 Fqf2 sC FC q 1bh9yf2b u D Rb GD r r r2 G where the Monod reaction term is h9sC

B

s EB x E FC F D 1qbss GD 1qbxx G

Rss1q Dss

rKds

, urs

R 2t s0RsYs , Ws i , Ds 9 X Yx

The initial and boundary conditions are: At us0, ss1.0, xs0.05, bsB0 At rs0, V s0, where Vss, x or b r for u)0 for 0FrF1 for uG0

Dx 9 Db 9 , Dxs , Dbs , Ds 9 yt Ds 9 Ds 9 3 1 y . l a lV r9 uls , ms , rs Q l R Further, we have introduced gsur yul.

Ds 9ytqDs,soRsy1.

At rs1, ssS, xsX, bsB

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5.2. Simulation Table 8 gives the parameter values which match the situation at Borhola oil fields, Assam wsee Regional Research Laboratory (1997) for discussions; this is referred to as RRL97 hereafterx; the parameter values in the studies reported hereafter will be these unless otherwise specifically mentioned. Many of these values are the same as in Dh93 where their choice was discussed and justified. We have had to alter only a few of them to suit the Borhola conditions. Where they have been altered, we have given the Dh93 values in brackets for easy comparison (except for q0 s , R and Kds, which were varied for sensitivity studies in Dh93). This model does not reproduce closely the experimental results using the laboratory-estimated parameters (Krishna Mohan, 1998). Nevertheless, the parameter values from the model that give output that compares favourably with experimental results are not outside realistic ranges of these parameters. The coupled set of ODEs is integrated by using IMSL (1991) subroutine DIVPAG. This employs Greens algorithm for stiff equations and is based on backward differentiation formulas. It requires an algebraic system of equations to be solved at each time step and resorts to the chord iteration method that calculates the Jacobian internally by finite differences. The coupled set of PDEs is integrated by using IMSL (1991) subroutine DMOLCH. This algorithm is based on the method of lines and employs a series of cubic Hermite polynomials to obtain the solution. This scheme requires a large number of mesh points for spatial accuracy and consequently large computation times. In our simulations, we have also employed a NAG (1993) routine equivalent to DIVPAG, viz. DO2NBF, for integrating the set of ODEs leaving DIVPAG for exclusive use by DMOLCH; this is called for because sharing of DIVPAG by both the ODEs and PDEs necessitates downloading of the routine, each time, from memory before making it available for the other which will make it expensive in terms of time. 5.2.1. Essential behaviour of the equations The simulation is terminated when the contaminant concentration drops below 1 ppb everywhere within the soil bed; we refer to the time taken to achieve this as the bioremediation time (Tb ). The results of a typical simulation are shown in Fig. 6. For illustration we have used a much reduced initial contaminant concentration, 3 q0 s s60 mg y kg and, also, Rs1.0 cm and Kdss150 cm y g. In Fig. 6a,b, the evolution of the concentrations of substrate, oxygen and biomass is shown for two points in the aggregate. It is seen that the degradation of substrate takes place much later at the centre (Fig. 6a), than at the halfway point to the centre (Fig. 6b). It is also seen that the start of degradation coincides with

Fig. 6. Evolution of concentrations inside the aggregate; (a) at rs0.0, and (b) at rs0.5.

the increase in oxygen and concomitant increase in biomass. Therefore, the delay in the oxygen front moving into the aggregate is what causes the delayed degradation of substrate at the centre of the aggregate. Furthermore, the degradation of substrate takes place very early in the macropores (in this particular case, we noted that by us0.2, substrate concentration, S is approx. 0) and subsequently, biomass and oxygen concentrations stay at high levels. In summary, it can be stated that the bioremediation process is a moving front that advances from the macropores into the micropores along with the oxygen front which enables the bacteria to start consuming the contaminants and multiply. It is to be expected then that the model itself can be made more efficient by converting it to a moving boundary problem. Such a strategy can be expected to reduce the computation time significantly (by orders of magnitude) since wasteful integration would be carried out during most times outside the zone of the oxygen front where the activity levels are practically zero. This strategy is being worked out by

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Fig. 7. Time evolution of substrate concentration, on a logarithmic scale, inside the aggregate at rs0.0.

Fig. 8. Laboratory results for biodegradation plotted on a logarithmic scale; variation of (suitably) averaged substrate concentration with time for a period of 360 days.

designing moving grid methods and will be reported elsewhere. Note that for the concentrations encountered at Borhola oil fields (higher by a couple of orders of magnitude than typically encountered in the literature; see Table 8), numerical integration is extremely costly even on a Convex C3820 computer; 1 years integration takes approx. 24 h real time and the concentrations have been reduced by just an order of magnitude (see below; reduction by seven orders is required for complete remediation to levels of 1 ppb or less). In Fig. 7, we have depicted the decay of the substrate concentration on a logarithmic scale at the centre of the aggregate (Fig. 6). What is emphasised here, is the exponential nature of the degradation once the oxygen front arrives. Notice that reduction by several orders of magnitude is needed to make the concentration levels acceptable, in spite of the low value of q0 s employed for these simulations. 5.2.2. Comparison with experimental results An important feature of the situation at Borhola oil fields is that the initial contaminant concentrations 4 (q0 s ;10 mg y kg, see Table 8) are higher by a couple of orders of magnitude than the values typically found 2 in the literature (q0 s ;10 mg y kg in Dh93). This implies that numerical integration is expensive (the equations are stiff) and it becomes impractical to run the model, in the present form, till the contaminant concentration drops below 1 ppb everywhere. However, the field pilot studies (see RRL97) were conducted only over a period of 1 year and, as such, the simulation results have also to be compared over the same length of time which is within the practical limits. Note that some of the assumptions made in the model cannot be expected to hold over the length of 1 year; for example, the temperature cannot be expected to be constant and there are bound to be associated variations in diffusion coef-

ficient. Nevertheless, the model can be expected to give some useful bounds on the bioremediation time. We will refer to the value of the contaminant concentration at the end of 360 days as the year-end value in the following. To see the effect of the laboratory parameters on Tb, it was decided to run some sample cases reported by Dh93 with the Borhola values of parameters as given 0 in Table 8. The Dh93 values of R, Kds and qs , for the specific cases we have tested (we retain the same case numbers as Dh93) are as follows: q0 s is 600 mg y kg for case 2, and 150 and 1500 mgykg for cases 9 and 10, respectively, while Kds is 15 cm3 yg and Rs1 cm for these selected cases. We found it convenient to compare the results using the exponents of exponential degradation (see Fig. 7); the results of the field pilot study plotted on a semi-log graph (Fig. 8) fit the exponential pattern well within the available decade and yield an exponent of y0.004. Table 9 gives the results of the comparison tests. It is seen that for the three cases investigated, the value of the degradation exponent with the laboratory parameters is lower than with the Dh93 parameters. At the same time, we also see that the percentage differences vary much from case to case. It has been demonstrated that the sensitivity to most
Table 9 Comparison of bioremediation times obtained with the Dh93 and Borhola parameter sets Dh93 case no. 2 9 10 Dh93 Tb (days) 22.6 8.1 41.3 Borhola Tb (days) 24.3 11.39 72.2 Dh93 exponent y0.2887 y1.2415 y0.1340 Borhola exponent y0.2811 y1.2139 y0.0850

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parameters is non-linear in nature (Krishna Mohan, 1998). Dh93 had studied the sensitivity of the model to Kds, R and q0 s and found that Tb is directly proportional to all of them. Our studies have revealed, however, that the dependence of Tb on Kds is non-linear in nature and their conclusion of linear proportionality is a result of the restricted range in Kds, that they had investigated (Krishna Mohan, 1998). We have observed a quadratic behaviour of Kds with Tb; a monotonous increase y decrease of Kds brings it to a turnaround value where Tb once again starts increasingydecreasing after the initial decreaseyincrease with change in Kds. It may be expected that the presenceyabsence of surfactants (bio or synthetic) will alter the force of adsorption of contaminants to the surfaces, thus affecting the speed of remediation and Kds should be capturing this effect. However, what should be noted here is that its role is limited and there is a turnaround value after which the amount of remediation decreases with further loweringy raising of Kds; also, the exponent value remains low regardless of the value of Kds for the Borhola case (Krishna Mohan, 1998). In general, the turnaround value will be different for different sets of parameters. We have also observed, as we have reported elsewhere (Krishna Mohan, 1998), that the model is not very sensitive to changes in the coefficients of either the input terms (first terms in the R.H.S. of Eqs. (1) (3)) or the biodegradation terms (second terms in the R.H.S. of Eqs. (1) (3)). Furthermore, the model is not sensitive to changes in the diffusion coefficients of biomass and oxygen (Db and Dx, respectively). The model is sensitive to changes in the diffusion coefficient of substrate. Our results indicate that a reduction of the whole coefficient of the third term of Eq. (1) by approximately 25%, to a value 0.78 times itself, results in the experimentally correct exponent of y0.004 being obtained. However, this change cannot be effected through alteration in Ds 9 because of the form of definition of Ds involving Ds,so and Rs; Ds is forced to be at 1.0 or greater in the present form (see Krishna Mohan, 1998, for a different form). The change can be effected by altering l and a (for l, as0.57, the same exponent is obtained). However, such a change affects other terms in the equations as well (though the results are not sensitive to changes in those terms as reported earlier) and also, the value of 0.57 for l, a does not agree well with the Borhola values. A reduction in the substrate diffusion coefficient (to obtain the correct exponent) implies that the leaching of substrate from the aggregates to the macropores is reduced. It has also been seen from the model output (Krishna Mohan, 1998) that the degradation had not yet reached the aggregates and that macropore activity was still going on after 1 year. This would imply that the reduced leaching prevents an extra load being put

into the macropore and that this enhances remediation; the extra load probably induces an inhibitory effect. However, since inhibition has not been explicitly modelled here, we see this as an artefact of the boundary conditions in the model. Instead of matching the variable concentrations at the surface of the aggregate with the corresponding macropore values, it seems that the inclusion of an exchange factor for the transfer would be more appropriate; this is being pursued further. Leaching of the contaminant into the macropores indicates that the desorption and diffusion mechanisms are operative, which should lead to more rapid bioremediation, in the absence of inhibitory terms, because of the increased availability of contaminant in the liquid medium. In the pilot study, the contaminant concentration was estimated from thoroughly homogenised samples extracted from the different parts of the soil column; only averaged (over the micropores and macropores) concentration values were available. We have assigned only 15% weightage to the macropores in the averaging and, with more weightage, the remediation activity will show an increase because all the activity is concentrated in the macropore in this 1 year; this is another one of the sensitive factors. Further studies are required to fine tune the model with experimental results. In the present form of the model, the optimal values (in the sense of least change in l and a) of Kds, Ds9 and (l, a), for attaining the degradation exponent of 0.004, are 60, 2.6=10y4 and (0.54, 0.54), respectively; the corresponding values estimated for the Borhola samples in the laboratory, were 45, 2.5=10y6 and (0.45, 0.45). 6. Conclusions The present report is essentially the result of the first phase of study on the design, construction and operation of above-ground biodegradation facilities; the complete study will include earthwork design and installation of piping, liners, drain etc., and finally, detailed cost estimation of the technology. The results and discussion presented above are expected to provide a meaningful insight into the biodegradability of the crude oil contaminated soil of Borhola oil fields. The experimental results of this study can be utilised to assess the role of various factors which control the success of bioremediation. These factors include the availability of microorganisms that can metabolise the contaminant utilising it as a carbon source through solubilisationydesorption under favourable conditions of temperature, pH, sufficient metabolic nutrients and moisture. The field pilot study showed that by applying nutrient and microbe enriched solution, the crude oil components were reduced under aerated conditions by 75% within a time span of 1 year. This observation is supplemented by a laboratory enumeration of adequate soil microbial

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and chemical properties, which can be exploited for designing a large-scale process system for field application. However, further studies may be required to tune the system suitably so as to achieve an even more accelerated process of biodegradation; this is necessary considering the high levels of contaminant concentration present at the site. In the above context, the role of computer simulation is of paramount importance. The laboratory experiments take periods of the order of 1 year, and it is difficult to tune the system efficiently through such a process. Computer simulation studies carried out alongside the laboratory work help in tuning the system satisfactorily. Our computer simulation study indicated, with the set of parameters measuredyemployed in the laboratory, that the degradation activity is restricted to the macropores during the initial period of 1 year. Mathematically, the necessity for an exchange factor in the matching of concentrations at the surface of the aggregates with the macropore values in the boundary conditions has been seen. The need for a better model, especially in the context of such high initial concentrations, is felt. It is expected that a moving boundary model will run efficiently; work in this direction is on going and will be reported elsewhere. We have also pointed out that contrary to what is suggested in Dhawan et al. (1993), bioremediation time is non-linearly related to several of the parameters in this model. Acknowledgments Financial support for this work from ERBC-ONGCL, Nazira, Assam, India, is hereby gratefully acknowledged. Appendix A: Notation
B9, b9 Dj9 Ds 9,so Kds Kj kd Q qs R Rj r9 S9ys9 Biomass concentration in macropores and inside aggregates, respectively, MyL 3 Diffusion coefficient of component j in pore liquid, where jss, x or b, L 2yT Diffusion coefficient of substrate in the solid phase, L 2 yT Linear partition coefficient for substrate, L 3 yM Saturation constant of component j, where jsS or X, M yL 3 Reaction rate constant for the decay of biomass, T y1 Volumetric flow rate in the bed, L 3 yT Concentrations of substrate in the solid phase, MyM Radius of the aggregate, L Retardation factor for component j, where jsS or B Radial position in the aggregate, L Substrate concentration in macropores and inside aggregates, respectively, MyL 3

S 0, X 0, B 0 Initial concentrations of substrate, oxygen and biomass, respectively S i, X i, B i Concentrations of substrate, oxygen and biomass, respectively, at the inlet Time taken for remediation of soil bed, T Tb t Time, T V Volume of the soil bed, L 3 W Oxygen supply factor X9, x9 Oxygen concentration in macropores and inside aggregates, respectively, MyL 3 Yj Yield factor of component j, where jsS or X, MyM

Appendix B: Greek letters

bs, bx

a l
mm r u h,h9 ul ur f2,f2

Saturation parameters of substrate and oxygen, respectively Volumetric fraction of liquid in the aggregate Volumetric fraction of liquid in the macrovoids of the bed Maximum specific growth rate of biomass, T y1 Bulk density of the aggregate particle, MyL 3 Dimensionless time Monod reaction term for macropores and aggregates, respectively Time to process one macrovoid volume of the bed, T Characteristic time for diffusion, T Thiele moduli for the growth and decay, respectively, of biomass (note that the corresponding quantity in Dh93 is lesser by a factor of three) Tortuosity of pores in the aggregate

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Binod Kumar Gogoi has an M.Sc. and Ph.D. in Chemistry with over 22 years of research experience in industrial and environmental biotechnology. Narendra Nath Dutta holds a Masters and Ph.D. (Tech) degree in Chemical Engineering from Bombay University. During his 25 years of service, he has been involved in R&D activities on chemical and biochemical processes, particularly in the field of petroleum technology. He has several papers and patents to his credit. Pranab Goswami has an M.Sc. and Ph.D. in Chemistry, and has worked for the last 15 years in the area of environmental biotechnology, particularly on the mechanism of microbial degradation of aliphatic hydrocarbons. T.R. Krishna Mohan is a Physicist in the area of nonlinear dynamical systems and chaos theory. At CMMACS, apart from his basic interest, he also works in the area of mathematical modelling applied to various natural and industrial systems: modelling the growth of Indian transport sector, biochemical oscillations (bifurcations and various dynamical patterns), bioremediation etc.