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Chowdhury R, Pedernera E & Reimert R, Trickle-bed reactor model for desulfurization and dearomatization of diesel, J. AIChE, 48 (2002) 126-135. 2. Barton L L ...
Indian Journal of Biotechnology Vol 6, January 2007, pp 107-113

Degradation of polyaromatic hydrocarbons by mixed culture isolated from oil contaminated soil—A bioprocess engineering study Ruma Roy, Raja Ray1, Ranjana Chowdhury, Pinaki Bhattacharya* Chemical Engineering Department, Jadavpur University, Kolkata 700 032, India Department of Microbiology, Institute of Post Graduate Medical Education and Research, Kolkata 700 020, India

1

Received 28 March 2005; revised 9 January 2006; accepted 27 March 2006 In the present investigation, decomposition of polyaromatic hydrocarbons was studied using mixed culture. Mixed strains were isolated from soil of petrol stations of different Indian cities and the best performing strains from these sources were used for subsequent bioprocess study using simulated mixture of anthracene and naphthalene as carbon source in methanol solution. The cell growth curve and substrate depletion time history curve obtained from batch fermentative process show that the reaction engineering behaviour of the systems under study can well be represented by classical substrate uninhibited Monod’s model. In a separate attempt the intrinsic kinetic parameters µmax and KS were evaluated following differential analysis of experimental data. Keywords: biodegradation, bioprocess engineering, isolation of bacterial strains; oil contaminated soil, polyaromatic hydrocarbons IPC Code: Int. Cl.8 B09 C1/10

Introduction Polyaromatic hydrocarbons (PAHs) have been identified as hazardous chemicals by different State and Central Pollution Control Boards, because of their toxic, carcinogenic and tetragenic effects on living body. At present, hydrocarbon fuels (mainly diesel) contain an excessive quantity of PAHs, causing abundant distribution of the same in the ecosphere. In order to protect environment from such PAH emission from diesel oil, a stringent EURO III standard has recently been enforced. This specifies that the maximum allowable concentration of PAH in diesel oil to be used as automobile fuel should be 11% by weight. Conventional hydro-treatment of diesel (using CoMo/Ni-Mo catalysts) to reduce the PAH content below this permissible limit failed. Even under high pressure (80 kPa) and temperature (633K) conversion of aromatics to naphthenes has so far been achieved only in order of 40%1. Investigation on the biodegradation on PAH is being carried out for a fairly long time and despite of the fact, as observed by some of the investigators, that these compounds may resist degradation by microbial enzymes2, many _______________ *Author for correspondence: Tel: 91-33-24146666; Fax: 91-33-24146378 E-mail: [email protected]

papers are appearing in literatures describing the success of biodegradation process of PAH3-12. This relatively new technology demands proper coordination between classical microbiological work and bioprocess engineering, followed by bioseparation for its successful use in industry. In the present investigation a systematic and programmed bioprocess study of a mixed culture system capable of degrading PAH from a simulated mixture has been reported. In order to initiate the bioprocess study, isolation of mixed culture from native source, viz. soil of petrol pump of three major Indian cities, Delhi, Kolkata and Hyderabad, has been carried out. Using microbiological and biochemical tests, different strains were identified. These mixed cultures constituted the living biocatalysts for degradation of the substrates under study. Based on the performance of mixed cultures collected from different cities, it was observed that sample collected from Delhi city worked more efficiently compared to the samples of the other two cities. Thus, for the subsequent bioprocess study, the mixed culture obtained from the soil of Delhi city was exclusively used. Study on the removal of PAH by the strains so isolated has been carried out using simulated mixture of either anthracene or naphthalene in methanol

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solution as substrate. Since such biodegradation reaction follows a complex path, the intention of this investigation was to establish a systematic reaction program under controlled condition and to evaluate intrinsic kinetic parameters essential for bioreactor design. By judiciously performing simulation work and comparing the experimental data with available model equations, it is observed that the reaction engineering behaviour of both naphthalene and anthracene degradation system can successfully be described by Monod’s substrate uninhibited model equation. In a subsequent attempt, the complete rate equation relating cell growth and substrate depletion has been computed by evaluating the intrinsic kinetic parameters, viz., µmax and KS, present in the Monod equation using differential analysis of the experimental data. It is needless to state that these kinetic parameters are of essential requirements for bioreactor design. Materials and Methods Materials Chemicals Used

KH2PO4 (E Merck, India), Na2HPO4 (E Merck, India), (NH4)2SO4 (Ranbaxy, India), MgSO4.7H2O (S D Fine-Chem Pvt. Ltd, India), CaCl2.6H2O (E Merck, India), FeSO4.7H2O (E Merck, India), naphthalene (S D Fine-Chem Pvt. Ltd, India), benzene (E Merck, India), methanol (E Merck, India), anthracene (E Merck, India). All chemicals13-16 used for the microbiological and biochemical tests were purchased from Himedia, India. Source of Soils

Soils were collected from petrol/diesel stations of three Indian cities, viz., Delhi, Kolkata and Hyderabad. Methods Microbiological Methods

Soil sample (1 gm) from each site was suspended separately in 20 mL of sterile (0.2 MPa, T 121oC, 15 min) selective medium17 in a 50 mL Erlenmeyer flasks containing (per dm3): KH2PO4 (1 g), Na2HPO4 (1.25 g), (NH4)2SO4 (1 g), MgSO4.7H2O (0.5 g), CaCl2.6H2O (0.05 g) and FeSO4.7H2O (0.005 g). Each soil-medium suspension was supplemented with 100 µL of 1% (w/w) solution of naphthalene/anthracene in methanol, sterilized through a millipore membrane filter under positive pressure since normal autoclaving process of such hydrocarbon solution cannot be carried out, which was verified by subculture on to

nutritive bacterial culture medium. These flasks were then kept in an incubator shaker (28oC, 100 rpm) for 5 d, so that the bacteria can adapt the new laboratory environment. Growth of microorganisms was indicated by visible turbidity of the solution. Enriched culture was obtained by repeated inoculation of preceding bacterial culture into fresh selective medium along with polyaromatic hydrocarbon compounds dissolved in methanol. The nature of culture so prepared, from the soil of three cities mentioned earlier, was mixed culture, containing different strains of bacteria capable of degrading naphthalene/anthracene, which was later confirmed by experimental results using Gas Chromatograph. In the present investigation, strains isolated and purified from the soil sample collected from Delhi city have been identified using standard microbiological and biochemical tests13-16. The results of such study is given in Table 1. It is evident that Pseudomonas putida (JUPHB1), P. alcaligenes (JUPHB2), Alcaligenes sp. (JUPHB3) and Acinetobacter sp. (JUPHB4) were present in the mixed culture as major constituents since they individually respond positively to the confirmatory standard microbiological and biochemical tests as shown in Table 1. Among these four strains JUPHB1 and JUPHB2 degraded naphthalene while JUPHB3 and JUPHB4 degraded anthracene efficiently. Analytical Methods

Assay of cell mass18—The concentration of biomass in the reaction broth was determined by dry weight method. In this method the broth was centrifuged at the rate of 10,000 rpm for 15 min and the bacterial mass was transferred to a pre-weighed aluminum cup and was dried at 80°C for 24 h. The exact weight of the bacterial mass was determined by subtracting the weight of dry cup from that of the cup containing dry bacterial mass. GC analysis for the determination of aromatics— GC (Chemito-7610) equipped with 5% phenyl polysiloxane packed capillary column (BP-5) of 25 m length, 220 µm inner diam and 0.25 µm film thickness was used in FID mode for the determination of aromatics both in feed and in the reaction broth. In both the cases the medium was centrifuged at 10,000 rpm for 15 min and the supernatant was treated with benzene for the extraction of aromatics. The nonaqueous phase containing benzene and the aromatics was analyzed by the gas chromatograph for the

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Table 1—Microbiological and biochemical tests of microorganisms isolated from soil of Delhi city No.

1

Pseudomonas putida (JUPHB1)

Pseudomonas alcaligenes (JUPHB2)

Media used for inoculation

4 5

Selective medium Selective medium + naphthalene + naphthalene Colony character on selective Round, creamish, slightly Round, creamish, medium convex convex Colony character on nutrient agar Fluorescent and Yellow-orange, nondiffusible pigment diffusible pigment Gram-stain character Gram -ve rods Gram-ve rods Motility Motile Motile

6

Biochemical tests*:

2 3

a. b. c. d. e.

f. g. h. i. j. k. l. m. n. o. p. q. r. s. t. u.

Oxidase reaction Nitrate reduction Decarboxylases (Arginine Dihydrolase) Catalase test Oxidative- Fermentative test

Citrate Gelatin liquefaction tests Indole test TSI Methyl Red test Malonate test Phenylalanine deaminase test Voges-Proskauer Test Dnase test MacConkey Sugar tests (Xylose, Sucrose, Mannitol, Mannose) Growth in blood agar Esculin hydrolysis Lysine Urease Antibiotic sensitivity

Alcaligenes sp. (JUPHB3)

Acinetobacter sp. (JUPHB4)

Selective medium + anthracene Round, creamish, slightly convex Non-pigmented colonies Gram -ve rods Motile

Selective medium + anthracene Round, red, smooth, shiny, slightly convex Smooth, opaque, round, creamy Gram -ve rods Twitching motility

+ +

+ + +

+ +

+

+ Oxidatively utilizes sugars like glucose and xylose

+ Non-utiliser for all sugars

+ Non-utiliser for all sugars except isovalerate & valerate (utilizes oxidatively)

+ K/K Green NLF -

+ K/K Blue-green NLF -

K/K Dark green NLF -

+ Utilizes fermentatively the following four sugars- glucose xylose, arabinose, ribose. + K/K Green NLF -

+ P, GM, OF

+ + + OF, PI, CA, CS, GM, OF, PI, CA, CS, GM, OF, PI, CA, CS, GM, P TC TC v. Antibiotic resistance TC, CA, CS TC Nil Nil + = positive; - = negative; K/K= alkali/alkali; NLF = non-lactose fermenting; OF = ofloxacin; CA = carbenecillin; CS = ceftazidime; PI = piperacillin; TC = ticarcillin; GM = gentamicin; P = polymixin. *Biochemical tests were performed following standard methods13-16.

determination of concentration of aromatics. Nitrogen was used as the carrier gas. Flow rates of N2 and H2 were maintained at 30 mL/min each, while the airflow rate was kept at 7 mL/min. The temperatures of capillary injector port, detector and oven were kept at 240ºC, 260°C and 140°C, respectively for anthracene, and 220°C, 260°C and 100°C, respectively for naphthalene.

Experimental

Degradation of naphthalene and anthracene—The study on the degradation of naphthalene and anthracene was carried out in batch mode of operation. Fourteen 50 mL Erlenmeyer flasks were used for this purpose. 20 mL of selective medium along with 100 µL of methanol solution of the specific carbon source (either naphthalene or

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anthracene) were added in each flask. These flasks were then inoculated with 5% (v/v) innoculum previously prepared and the whole system was kept in an incubator maintained at 28ºC. The content of each flask represented sample under study at a specific time. First two samples (i.e. two separate flasks) were taken out of the incubator at zero time and after 30 min, respectively. Since then samples were drawn (i.e. a single flask was taken out) after regular interval of 4 h and continued for 48 h. The content of each flask was centrifuged, and the bacterial mass and the aromatic content were determined using dry weight method and GC analysis, respectively. Initial concentration of naphthalene was varied in the range of 1000-6000 mg/dm3 and that of anthracene was kept in the range of 500-800 mg/dm3, respectively. For each initial concentration of a specific aromatic compound, the same sampling protocol was followed. All the experiments were repeated for three times and the average value has been reported in the present investigation. Results and Discussion As mentioned earlier, the present system involved biodegradation of PAH through a competing route. Initially microorganism would utilize the specific substrates (either naphthalene or anthracene) for its own growth and later when an appreciable quantity of biomass was formed, biodegradation of PAH would take place through specific biocatalysis. Schematically, this may be represented as follows:Substrate X → nX , n >1

… (1)

Biomass ( nX ) Substrate  → product

… (2)

In order to compare relative effectiveness of different mixed cultures collected from different sites, it was felt that fractional conversion of the specific substrate might be important. Thus, in the present investigation, fractional conversion of the specific substrate (either anthracene or naphthalene) was computed and compared for mixed cultures collected from different sources (Figs 1 & 2). It was observed that microorganisms isolated from New Delhi city was the most efficient in decomposing both the substrates, while the microorganisms isolated from Kolkata and Hyderabad cities followed suit. It was also observed that for a fixed time of exposure the fractional conversion increased with increase in the initial concentration of the substrate for both the cases.

Fig. 1—Fractional conversion vs. naphthalene concentration for mixed culture system

Fig. 2—Fractional conversion vs. anthracene concentration for mixed culture system

In the present investigation, an attempt was made to study growth kinetics of microorganisms and the related substrate depletion kinetics. Fig. 3 shows a plot of cell mass concentration data against time with initial substrate concentration (naphthalene or anthracene) as parameter. It is evident from the figure that the growth of biomass increased with the increase in the initial substrate concentration studied (1000 – 6000 mg/dm3 in case of naphthalene and 500 –800 mg/dm3 in case of anthracene). No substrate inhibition was observed. The figure also shows a distinct exponential growth phase, followed by a stationary phase, although no lag phase was identified. This type of growth behaviour of the bacterial strains indicated that they follow classical substrate uninhibited Monod’s model19. The main aim of the present investigation was to study the reaction behaviour of the bacterial strains

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S

KS

Fig. 3—Biomass growth curve using naphthalene and anthracene as substrates [500mg/dm3: (•); 700mg/dm3: (+); 800mg/dm3 :(─); 1000mg/dm3 :(♦); 2000mg/dm3: (■); 3000mg/dm3: (▲); 4500mg/dm3: (x); 6000mg/dm3: (□)]

(JUPHB1, JUPHB2, JUPHB3, and JUPHB4) in decomposing naphthalene and anthracene present in different concentrations. Fig. 4 shows both the anthracene and naphthalene concentration time history curve as a function of initial substrate concentration. Here, anthracene was decomposed by mixed culture containing strains Acinetobacter sp. and Alcaligenes sp., while naphthalene was degraded by a consortium having bacteria like P. alcaligenes and P. putida. It is evident from the figure that for all the initial substrate concentrations studied; the depletion of substrates took place exponentially. Evidently, this is the definite indication that the system follows Monod’s substrate uninhibited model equation. Evaluation of Kinetic Parameters

The Monod’s substrate uninhibited model equation for the present system may be written as:Biomass:

dC B µ max C S C B = dt K S + CS

… (3)

Substrate:

dC S 1 µ max C S C B =− dt YB / S K S + C S

… (4)

The Monod’s rate equation may be rearranged in the following double reciprocal form (Lineweaver and Burk form).

Fig. 4—Substrates depletion time history curves [500mg/dm3: (•); 700mg/dm3: (+); 800mg/dm3: (─); 1000mg/dm3: (♦); 2000mg/dm3: (■); 3000mg/dm3: (▲); 4500mg/dm3: (x); 6000mg/dm3: (□)]

1

µ

=

KS 1 1 + × µ max µ max CS

where, µ =

…. (5)

1 dCB × CB dt

Thus, a double reciprocal plot of the specific growth rate and the substrate should be a straight line having slope KS / µ max and an intercept on the ordinate

1

µ max

.

In the present system, it has already been realized that a balanced growth prevails throughout the growth cycle. Moreover, since all the experimental data was collected from the exponential growth phase only, the yield co-efficient YB / S may be, therefore, considered constant over the entire growth period. The specific growth rate (µ) was calculated by dividing the biomass formation rate by the average cell concentration within the specific time span. It may be mentioned that cell growth curves for naphthalene and anthracene degradation was utilized (Fig. 1) for evaluation of biomass formation rate for the specific case. Fig. 5 shows that double reciprocal plot of specific growth rate of P. putida, P. alcaligenes and the substrate, namely naphthalene. On the same figure similar plot was made for the growth rate of Acinetobacter sp. and Alcaligenes sp. and anthracene. In above two cases, reasonably good straight lines were obtained, which clearly indicate that both the

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Table 2—Kinetic parameters for microbial growth Name of city

Anthracene Ks (mg/dm3)

Delhi Hyderabad Kolkata

139 165 175

µmax (h-1) 0.0188 0.0271 0.0293

Naphthalene YB/S

Ks (mg/dm3)

0.450 0.541 0.602

2938 4500 5200

Fig. 5—Double reciprocal plot of specific growth rate and initial substrate concentration

systems using separate mixed cultures as mentioned above follow Monod type substrate uninhibited model equation. The kinetic parameters (KS and µ max ) evaluated following the procedure outlined earlier are shown in Table 2. In each case the constant yield coefficient ( YB / S ), was also evaluated using substrate depletion time history curves (Fig. 4). These values are given in Table 2. The magnitude of KS (2938 mg/dm3) for degradation of naphthalene (1000-6000 mg/dm3) is evidently an indication that the growth kinetics was a shifting order reaction, i.e., zero order at high substrate concentration and shifting to first order at lower substrate concentration. This is verified by plotting specific growth rate (µ) against substrate concentration (figure not shown). In case of anthracene degradation, similar trend was observed. However, on comparing the kinetic parameter KS for anthracene and naphthalene system, it is evident that anthracene was more readily biodegradable (low value of KS) than that of naphthalene (high value of KS). This is also indicated in the magnitudes of µ max for the above two substrates. Comparison of anthracene lower µ max value to that of naphthalene shows that biodegradation of anthracene was much faster than that of naphthalene. Conclusion It may apparently appear that the treatment presented for kinetic analysis is rather oversimplified,

µmax (h-1) 0.107 0.182 0.195

YB/S 0.185 0.312 0.373

since at higher substrate concentration effect of substrate inhibition may play an important role in the transient dynamics of specific cell growth. However, in the present study, no such substrate inhibition is observed within the substrate concentration range studied (1000-6000 mg/dm3 in case of naphthalene and 500-800 mg/dm3 in case of anthracene). It may, therefore, be concluded that, within the substrate concentration studied, biodegradation of polyaromatic hydrocarbons may be successfully described by Monod’s substrate uninhibited model equation in the present range of substrate concentration. The magnitude of kinetic parameters (KS and µ max ) evaluated following the differential method of analysis indicate that the microbial removal process of polyaromatic hydrocarbons under the concentration range of present study is a shifting order reaction. It may be mentioned here that further work with higher initial substrate concentration involving substrate inhibition effect on the specific cell growth rate is in progress. Nomenclature C KS

YB / S

µ max

Concentration, (mg)(dm3)-1 Saturation constant, (mg)(dm3)-1 Yield coefficient =Mass of biomass produced/mass of substrate consumed Maximum specific growth rate, h-1

Subscript Anth Anthracene B Biomass Naph Naphthalene S Substrate B, Naph Biomass concerned with naphthalene system B, Anth Biomass concerned with anthracene system Acknowledgement Authors gratefully acknowledge the financial support rendered by University Grants Commission of India, New Delhi to carry out this research work.

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