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Dibenzothiophene biodegradation by a Pseudomonas sp. in model solutions
Process
Biochemhy, Vol. 30, No. 8, pp. 721-728,1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. Al1 rights reserved 0032-9592/95 $9.50 + 0.00
0032-9592(94)00040-9
DibenzothiopheneBiodegradationbya Pseudomonas sp.inModelSolutions Leonardo Setti,” Gaetano Lanzarini & Pier Giorgio Pifferi Department of Industrial Chemistry and Materials, Interdepartmental Center of Biotechnological Research, Industrial Chemistry Section, We de1 Risorgimento, 4,40136 Bologna, Italy (Received 30 March 1994; revised manuscript received and accepted 30 July 1994)
The presence of a fany acid and an n-alkane may affect the biodegradation rate of aromatic sulphur compounds such as dibenzothiophene (DBTJ A fatty acid (hexadecanoic acid) may form micellar structures favouring DBT bioavailability. n-Alkanes, such as n-dodecane or n-hexadecane, form a film around the aromatic sulphur molecule as a consequence of solvation, thus increasing DBT bioavailability. The mass-transfer rate from the solid to the aqueous phase controls the DBT biodegradation rate when DBT is the only carbon source. [email protected] coassimilation and microbial hydrophobic fleets are rate-limiting steps in DBT biodegradation in the presence of aliphatic compounds. Di&sion depends on the DBTconcentration in n-allcane, while cometabolism is associated with different n-alkane biodegradation rates. Through the definition of biodesulphurization selectivity and biodesulphurization @ciency, our investigations have shown that a selective aerobic biodesulphurization process is possible by using an unselective biocatalyst, such as a Pseudomonas sp.
INTRODUCTION
All oil biodegradation processes are limited by the heterogeneous system, which comprises an oil fraction (organic phase) and a biocatalyst (aqueous phase). Hydrocarbon bioavailability is affected by the chemical-physical characteristics of the substrate, viz., solubility, viscosity, surface tension, and so on.’ Naphthalene, bibenzyP and phenathrene3 were found to be used by some bacteria only when dissolved. The biodegradation rates of palmitic acid4 and phenathrene5 are limited by their dissolution rates in water. Bacteria may release compounds capable of either increasing the aqueous solubihty of the substrate6T7 or the rate of substrate diffusion in the water phase.5 Hydrophobic microorganisms may, however, use an insoluble compound through direct contact. In fact, microorganism adsorption
Biological and chemical-physical factors affect heavy oil biodegradation and/or biotransformation processes such as biodesulphurization. Biological factors depend on the type of microorganism employed and on its capability to assimilate hydrocarbons after adaptation to the substrate or after mutagenesis. Chemical-physical factors, however, such as, agitation, aeration for aerobic processes or the presence of hydrogen for anaerobic processes, as well as medium conditions and substrate characteristics, play a very important role in hydrocarbon assimilation by microorganisms. *To whom correspondence should be addressed. 721
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Leonardo Setti, Gaetano Lanzarini, Pier Giorgio Pifferi
in the oil phase is the most likely mechanism for explaining n-alkane biodegradation.8,9 Substrate uptake presumably occurs through diffusion or active transport at the point of contact. As is well known, most aerobic microorganisms adhere to the n-alkanes (below n-C16) which are in a liquid form at room temperature.l’ The microbial utilization of insoluble substrate in the case of direct of the microorganism on solid adsorption hydrocarbon has been reported in only a few papers.” The carrier effect of liquid n-alkanes on the degradation of solid hydrocarbons has been reported for paraffins by Cameotra et ~1.‘~ and Miller & Johnson,‘” for asphaltenes by Rontani et &.I4 and for sulphur aromatic compounds by Setti et a1.‘5~‘h Moreover, bacteria capable of degrading substrates partitioned into a non-biodegradable hydrocarbon solvent are known-l7 A Pseudomonas sp. may use diphenylmethane in the liquid, but not in the solid, form at the aqueous-organic interface, while another strain of Pseudomonas utilizes naphthalene at the water-heptamethylnonane interface but not at the surface of naphthalene crystals.ls Knowledge of how microorganisms assimilate solid hydrocarbon in the presence of a liquid solvent is very important for developing industrial oil biotransformation processes such as biodesulphurization. In fact, heavy oil is a complex mixture of different hydrocarbons which may affect the degradation of the sulphur aromatic compounds. A study was conducted to assess the role of aliphatic hydrocarbons in determining the means by which bacteria degrade sulphur aromatic compounds. Since dibenzothiophene (DBT) derivatives are the most frequently encountered sulphur organic compounds, DBT is generally employed as a model molecule in microbiological studies of heavy oil desulphurization. As toxic waste sites, industrial effluents and polluted areas around hydrocarbon reservoirs contain a non-aqueous phase, these investigations may provide useful ecological and toxicological information for reducing environmental pollution.
MATERIALS
AND METHODS
Medium The buffered inorganic salt solution used for the experiments contained:
at pH 7.0 3.52 g of
KH,PO,, 12.5 g of Na,HPO,.12H,O, NH&l and 0.04 g of MgSO,*7H,O deionized water.
0.8 g of per litre of
Chemicals Hexadecanoic acid (aC16) was obtained from Fluka AG. Buchs SG, n-dodecane (C12) and IZhexadecane (Cl 6) were purchased from Aldrich Chemical Co., Gillingham, England, and dibenzothiophene (DBT ) was obtained from Merck. Other chemicals used were reagent grade and were supplied by Carlo Erba, Milan. Microorganisms The enrichment culture was obtained from fuelcontaminated soil material of AGIP-Petroli refineries in Gela. The pure strain capable of degrading DBT was isolated by enrichment culture and identified according to the procedure described in a previous paper.15 To prepare inocula, the culture was grown at 28°C on a rotary shaker operating at 200 rpm in the 0.02% aC16salt medium for 4 days. For all experiments, the inoculum size was equal to 10% of the medium volume. Fermentation conditions DBT degradation was performed by adding 0.2% (v/v) DBT solution to 25 ml culture medium in a 250-ml volumetric flask fitted with breakwater. The following DBT concentration and solvent were employed as the case required: 54 ells in dimethylformamide (DMFA), 54-272 mu in ndodecane (C12) or n-hexadecane (C16), while the hexadecanoic acid (aC16) concentration was 19 or 190 mM. All solutions were autoclaved and added to the sterilized salt medium. All experiments were followed by control trials without inoculum. Culture hydrophobic assay The assay was made according to the procedure described by Rosenberg et a1.l9 Bacteria were grown in SALT medium containing a carbon source, viz. 0.02% (w/v) aC16, O-2% (v/v) Cl2 and 0.2% (v/v) C16. After 4 days growth, 5 ml of broth were diluted with distilled water to a turbidity, at 600 nm, close to O-3 and added in a tube ( 1 cm diameter) containing 5 ml of Cl2 or C16. The mixtures were agitated uniformly for 5 min and allowed to settle for 10 min to permit the hydrocarbon phase to rise completely. The aqueous phase was carefully removed with a
Biodegradation
723
of Pseudomonas sp.
Pasteur pipette and transferred to a 1.5ml cuvette. Light absorbance of the aqueous solution was determined at 600 nm before and after agitation using a Uvikon 860 spectrophotometer (Kontron Instr.). The absorbance values were directly proportional to the amount of cells in the aqueous solution, Analysis The residues of DBT microbial degradation were recovered from the culture medium by liquid-toliquid extraction using n-hexane after acidification of the aqueous phase to pH 2*0. Controls were carried out before each trial without inoculum. The amount of DBT was determined by spectrophotometric analysis using a Uvikon 860 spectrophotometer on the basis of the two relative maximum values of 3 12 and 325.5 nm, which are typical of this compound. The amount of Cl2 and Cl6 was determined by gas-chromatographic analysis using a Hewlett Packard 5890 CG fitted with a Flame Ionization Detector and a methylsilicone capillary column (0.25 mm X 25 m). Hydrogen was employed as a carrier gas (4 ml/min). The temperatures at which trials were carried out were 270°C for the injector, 270°C for the detector and a programmed temperature of 40°C for 5 min, 50°C/min up to 90°C and 4O”C/min up to 270°C. One microlitre of sample was injected into the capillary column. Solubility of DBT in the medium at 28°C The solubility of the aromatic compound was determined in duplicate by adding 0.2% (v/v) DBT solution in DMFA (54 mu) to 25 ml culture medium in a 250-ml volumetric flask. The bottle was incubated at 28°C for 24 h on a rotary shaker operating at 200 rpm. After centrifugation at 2600 g for 10 min, samples (10-O ml) containing no visible particles were withdrawn for solvent extraction and spectrophotometric analysis according to the procedure described above.
the solid to the aqueous phase may be a ratelimiting step.*O Setti et aZ.*l have shown that the biodegradation of DBT, as the only carbon source, by a Pseudomonas sp. is a typical example of the theoretical model suggested for PAH biodegradation. At the beginning of fermentation (Fig. l), the DBT degradation rate was controlled by bacterial exponential growth. After about 6 days, the dissolved DBT concentration (420 ng/ ml) was close to zero so that further degradation was controlled by the mass-transfer rate of the sulphur compound from the solid to the aqueous phase, 42 ng/ml/h, determined by the slope from 6 to 14 days fermentation. Consequently, the DBT degradation rate was independent of the amount of solid DBT in the medium. The presence of the fatty acid, hexadecanoic acid (aC16), during fermentation affects DBT degradation as reported in a recent note (Fig. l).*l When aC16 (19 mu) and DBT (54 mu) were added simultaneously to the medium from dimethylformamide solution, a different DBT biodegradation pattern was observed as compared to free dibenzothiophene. At the beginning of fermentation, no exponential biodegradation was seen to occur, which suggests a diffusion-limiting factor at work. When the amount of aC16 was close to zero, after five days of fermentation, as shown by the stationary phase of the growth trend on aC16 (Fig. 2), the DBT degradation rate became constant and was equal
T
0.1 0 0
RESULTS
AND DISCUSSION
The effect of the fatty acid on DBT biodegradation Wodzinski and Coyle3 have suggested that polycyclic aromatic hydrocarbons (PAHs) can be converted by microorganisms only in the dissolved state. This means that PAH mass transfer from
2
4
6
8
10
12
14
16
18
20
Fermentation time (days)
DBT removed as a function of fermentation time in Fig. 1. the presence of aC16: (0), DBT (54 mu) and aC16 (19 mM) added from the same initial solution in DMFA; (V), DBT (540 mu) and aC16 (190 ITLM)added from the same initial solution in DMFA; (a), DBT (54 mM) and aC16 (19 mM) added from different initial solution in DMFA; ( +), DBT (54 ~llhl) in DMFA solution as the only carbon source. All experiments were performed adding 02% (v/v) DMFA solution to the medium.
724
Leonardo
Setti, Gaetano
Lamarini,
Pier Giorgio Pifferi
;.
I
0
2
4
6
6
10
12
14
Fermentation time (days) 0
1 F.rm.mal0”
a
9
tm.
4
m4y-a)
Fig. 2. Batch growth of a strain of Pseudomonas aC16 (19 mM) dispersed in water.
sp. on
to that calculated for free dibenzothiophene, as it was controlled by mass transfer. The amount of DBT removed increased by proportionally increasing DBT (540 mu) and aC16 (190 mu) in the medium. The reverse was observed when aC16 ( 19 mu) and DBT (54 mu) from different dimethylformamide solutions were added to the medium. In fact, at the beginning of fermentation, the DBT removal rate was lower than that reported for free dibenzothiophene dibenzothiophene. The degradation rate increased exponentially with fermentation time and was higher when hexadecanoic acid degradation was nearly complete. After 10 days of fermentation, this rate became constant and was equal to that calculated for free dibenzothiophene, as degradation was controlled by mass transfer. These different behaviours and the higher dissolution rate of hexadecanoic acid, 70 ng/ml/ h,4 suggest that hexadecanoic acid favours dibenzothiophene assimilation, forming a kind of micelle when the fatty acid and the sulphur compound are added simultaneously to the medium. A similar model was suggested by Velankar et al** for hydrocarbon uptake by microorganisms. According to this model, the presence of micelles of surface-active agents is essential for the growth of microbial cells on hydrocarbon substrates. These micelles act as ‘transport packets’ for hydrocarbons. When the micelles are in close proximity to the cells, hydrocarbon is transferred more quickly to the cell surface as diffusion through the aqueous phase is eliminated.
Fig. 3. Amount of DBT removed as a funtion of fermentation time on 02% (v/v) n-dodecane at different DBT concentrations: (o), 54 mu; (A), 163 mu; (L), 243 mu; (O), 272 mu; (---) DBT as the only carbon source (data from Fig. 1).
0.6
,
0.5 o---------O
0
2
4
6 Fermentation
6
10
12
14
time (days)
DBT removed as a function of fermentation time on Fig. 4. 0.2% (v/v) n-hexadecane at different DBT concentrations: (o), 54 rnq (A), 163 mu; (*), 243 mu; (0), 272 mu; (---), DBT as the only carbon source (data from Fig. 1).
On the other hand, when hexadecanoic acid and dibenzothiophene were added from different solutions, the fatty acid did not necessarily form micelles containing the sulphur molecule, so that a competitive degradation involving the highly biodegradable fatty acid and the DBT dissolved in the medium was established. The effect of n -alkane on DBT biodegradation To evaluate this effect, two n-alkanes, Cl2 and C16, were chosen as representatives of two different degradation subgroups of the aliphatic fraction which makes up a heavy ~il.*~ When DBT was dissolved in II-alkane, the sulphur aromatic compound biodegradation trend was similar to that observed for the fatty acid micellar system (Figs 3 and 4). The DBT
725
Biodegradation of Pseudomonas sp.
degradation rate increased as the sulphur concentration compound in the n-alkane increased, this effect being much more significant when DBT was dissolved in n-hexadecane. At the beginning of fermentation, DBT degradation was higher in C 12 than in C 16 while these differences were minimal after 13 days of fermentation. At high DBT concentrations, close to saturation in n-alkane, biodegradation of the aromatic sulphur compound was always higher that that reported for free dibenzothiophene, which suggests that the n-alkane has a carrier effect on DBT degradation. When the sulphur compound was dissolved in ndodecane, a small carrier effect was always observed, while in n-hexadecane, at a low dibenzothiophene concentration, DBT degradation was lower than that observed for free dibenzothiophene. In this case, n-alkane was seen to have a barrier effect. The dependence of DBT degradation rate DBT concentration in n-alkane may be explained by diffusion. DBT assimilation in n-alkane is affected by two main concurrent factors: a coassimilation factor due to the simultaneous assimilation of the aliphatic and sulphur compound by the microorganisms, and, a hydrophobic factor characterized by the capability of the bacteria to adhere to Cl 2 or to C16. n-Dodecane was degraded better than n-hexadecane (Fig. 5) and complete degradation was reached after 6-8 days of fermentation compared to more than 20 days of fermentation for C16. These different degradation rates have been reported by Goma et aZ.,24 who studied the growth of Candida lipolyvica on a mixture of several hydrocarbons. These authors
0
2
4
6
8
10
12
14
16
18
20
Fermentation time (days)
n-Alkane degradation percentage as a function of Fig. 5. fermentation time on O-2% (v/v) of n-alkane added to the medium.
found that the short-chain hydrocarbons were used up faster than the longer chain hydrocarbons. This is due to the fact that the shorter chain hydrocarbon, Cl 2, is solubilized faster than the long-chain one, C16, probably moving across the oil-cell boundary faster. This means that the aliphatic degradation behaviour in heavy oil is a function of the single n-alkane surface tension.’ In fact, surface tension affects mass-transfer rate across the cell membrane. The n-alkane biodegradation rate, however, is also affected by the capability of the microorganisms to adhere to the aliphatic phase. The hydrophobic character of a microorganism is an index of this capability. The hydrophobicity of the strain of Pseudomonas sp. employed depends on the substrate in which the bacteria were grown. In fact, when palmitic acid was used as a substrate, the microorganisms did not exhibit hydrophobic characteristics, as testified by the absence of the biomass in the organic phase (Fig. 6). Bacteria grown on Cl 2 or Cl6 exhibited a good hydrophobic behaviour. Microbial pa&ion in the organic phase was higher in n-dodecane than in n-hexadecane, regardless of the kind of n-alkane used as a growth substrate. Diffusion, coassimilation and microbial hydrophobicity may explain why DBT degradation was found to be highest when the sulphur compound was dissolved in rt-dodecane at the beginning of
B
C
~ Cl2
Cl8
Microorganisms partitioned in n-alkane,Cl2 and Fig. 6. C16, as a function of the difEerent carbon source for the growth of the cells: A, palmitic acid; B, n-dodecane; C, nhexadecane.
726
Leonardo Seni, Gaetano Lanzarini, Pier Giorgio Pi#eti
fermentation. After 13 days of fermentation, Cl2 degradation was nearly complete so that n-alkane no longer exerted its carrier effect for the biodegradation of residual dibenzothiophene. The DBT biodegradation rate was thereafter conditioned by mass transfer from the solid to the aqueous phase, as reported for free dibenzothiophene. Conversely, n-hexadecane was not completely degraded so that the n-alkane may still have been able to exert its carrier effect. It may be reasonably assumed that the diffusional rate of DBT from the organic phase to the adsorbed microorganism is higher than the mass-transfer rate of free DBT.22 This may explain the similar DBT degradation values observed in Cl2 and Cl6 after 13 days fermentation at DBT concentrations above 100 mu. At lower concentrations, the diffusional rate in n-hexadecane was very low when compared to the coassimilation effect exhibited in n-dodecane. This suggests a simple model similar to that reported for the fatty acid. n-Alkane forms a film around the aromatic sulphur compound and as this fihn is easily attacked by aerobic microorganisms, the bioavailability of the sulphur compound increases. Biodesulphurization selectivity The biodesulphurization process selectivity, Ds, is represented by the ratio between the initial and final DBT concentrations in n-alkane. Es = (n-alkane recovered)/(initial n-alkane) X (DBT removed)/(initial DBT ) = Co/ Cf where Co and Cf are the initial and the final DBT concentrations in n -alkane, respectively. A microbial treatment is considered to be a selective biodesulphurization process when Ds is above 1. In fact, in this case the DBT biodegradation rate is higher than the n-alkane biodegradation rate and, consequently, the concentration of the sulphur aromatic compound decreases in n-alkane after the treatment. Ideally, desulphurization should be capable of removing all sulphur without destroying useful products. Figure 7 shows that Ds depends on the percentage amount of n-alkane removed and is independent of the type of n-alkane employed. The value of selectivity was above 1 when the percentage of n-alkane biodegraded was very low, i.e. 510%. The fact that Ds is apparently dependent on the type of n-alkane as a function of fermentation time, as shown in Fig. 8, can be ascribed to the higher biodegradation rate of Cl2
1.2
1
1 0.3 g 0.6 0.4 0.2 0
Fig. 7. Biodesulphurization selectivity, Ds, as a functionof the n-alkane degradation percentage in both Cl2 and in C16.
10
0123456780
11
12
13
Fermentation time (days)
Biodesulphurization selectivity, Ds, as a function of Fig. 8. fermentation time in Cl2 and in Cl6 at different DBT con;Fdtra,“,o;: (0) 5:’ 11~1, (A) 163 mu, (0) 272 mu. Cl2 (---)
compared to that of C16. The value of selectivity broke down very quickly and was close to zero after five days fermentation as a consequence of a high Cl 2 biodegradation rate, while this decrease is slower in n-hexadecane. Selectivity is affected by the DBT concentration in n-alkane. In fact, it increases as the DBT concentration increases, reaching its peak in n-hexadecane after the first three days of fermentation. At the beginning of fermentation, high DBT concentration and slow n-alkane biodegradation rates favour the DBT diffusional rate from the organic phase to the surface of the microorganisms adsorbed on the aliphatic compound. The value of Ds above 1 observed in nhexadecane after three days of fermentation suggests that a selective biodesulphurization process in aerobic conditions is possible by using an
Biodegradationof Pseudomonas sp.
unselective biocatalyst. A selective biodesulphurization process is already known for some anaerobic microbial treatments.25 Biodesulphurization selectivity was not observed in Cl2 due to the early n-alkane biodegradation which occurred at the beginning of fermentation. Biodesulphurization efficiency Biodesulphurization efficiency is an index biocatalyst efficiency for the removal of DBT the presence of n -alkane, and is given as: Es = ( n -alkane recovered)/( initial n-alkane) (DBT removed)/( initial DBT )
of in x
Biodesulphurization selectivity is not sufficient to characterize a biodesulphurization process, as it does not take into account the amount of DBT removed. Biodesulphurization efficiency compares the amount of recovered n-alkane with the amount of recovered DBT and then, when Es is equal to 1, biodesulphurization is an ideal process in which all sulphur is removed selectively without destroying n-alkane. A higher efficiency was observed when DBT was dissolved in n-hexadecane (Fig. 9). Efficiency reached its peak after one day of fermentation in n-dodecane and after 3-6 days of fermentation in n-hexadecane. The low efficiency observed in n-dodecane is due to the high biodegradation rate of this alkane, even though it is effective as a carrier for DBT biodegradation. In n-hexadecane, efficiency increased as DBT concentration in n-alkane increased, this behaviour being more significant at the beginning of fermentation. In n-dodecane, efficiency was
012345676
9
10
11
12
13
Fermentation time (days)
efficiencv, Es. as a function of Fie. 9. Biodesulohurization feromentation time-in Cl2 and in Cl6 Lt different DBT concentration: (V) 54 mM, (0) 163 mu, (A) 243 mu, (0) 272 ) with filled symbols. mu. Cl2 (---) and Cl6 (-
727
minimal at a DBT concentration of 163 mu. These results may be due to the two concurrent effects previously mentioned: coassimilation on one hand and diffusion on the other. The latter is a rate-limiting step in n-hexadecane, as efficiency is proportional to DBT concentration, while in IZdodecane the high value of Es at a low DBT concentration is due to the coassimilation limiting effect. In fact, at a low DBT concentration, the diffusional rate of the sulphur molecule from the bulk of the organic phase to the microorganism adsorbed on the aliphatic compound, is slower. IIAlkanes work as carriers for DBT biodegradation, although the different capabilities of the hydrocarbon microbial uptake affect biodesulphurization selectivity and efficiency. In fact, the model system in which dibenzothiophene is dissolved in n-hexadecane exhibits the best selectivity and efficiency as a consequence of the worst biodegradability of Cl6 with respect to C12.
CONCLUSIONS The study of the biological and chemical-physical parameters affecting biodegradation processes may be interesting for developing selective biodesulphurization treatments in heavy oil as well as biodegradation processes in complex organic solvents. The present study shows that a selective aerobic biodesulphurization process is possible by using an unselective biocatalyst. Further progress needs to be made in order to improve the selectivity of aerobic heavy oil biodesulphurization treatments. In fact, the biocatalytic activity of aerobic microorganisms is higher than that of anerobic ones, even though the latter hold considerable interest for industrial applications.2” Biodesulphurization selectivity and efficiency have been examined as the two important indexes characterizing the process, as they suggest that diffusion and cometabolic effects are responsible for the rate-limiting-step in DBT degradation. Diffusion depends on the microbial affinity between the microorganism and the organic phase in which the substrate is dissolved and on the substrate concentration in the same organic phase, while coassimilation depends on the organic phase biodegradation rate. These two effects may affect the removal of the sulphur aromatic compounds in heavy oil.
728
Leonardo Setti, Gaetano Lanzarini, Pier Giorgio Pifferi
ACKNOWLEDGEMENTS
microorganisms. II: uptake of solid n-alkanes by yeast and bacterial soecies. Biotechnol. Bioena.. _. 25 (1983)
Special thanks to Prof. D. Matteuzzi and Dr. M. Rossi from the Department of Pharmaceutical Science (University of Bologna, Italy) who supplied us with the culture of Pseudomonas sp. The research was supported by funds provided by the Enichem-ANIC S.p.A. (Milan, Italy).
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Edizioni
M.A.F.
Biochemhy, Vol. 30, No. 8, pp. 721-728,1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. Al1 rights reserved 0032-9592/95 $9.50 + 0.00
0032-9592(94)00040-9
DibenzothiopheneBiodegradationbya Pseudomonas sp.inModelSolutions Leonardo Setti,” Gaetano Lanzarini & Pier Giorgio Pifferi Department of Industrial Chemistry and Materials, Interdepartmental Center of Biotechnological Research, Industrial Chemistry Section, We de1 Risorgimento, 4,40136 Bologna, Italy (Received 30 March 1994; revised manuscript received and accepted 30 July 1994)
The presence of a fany acid and an n-alkane may affect the biodegradation rate of aromatic sulphur compounds such as dibenzothiophene (DBTJ A fatty acid (hexadecanoic acid) may form micellar structures favouring DBT bioavailability. n-Alkanes, such as n-dodecane or n-hexadecane, form a film around the aromatic sulphur molecule as a consequence of solvation, thus increasing DBT bioavailability. The mass-transfer rate from the solid to the aqueous phase controls the DBT biodegradation rate when DBT is the only carbon source. [email protected] coassimilation and microbial hydrophobic fleets are rate-limiting steps in DBT biodegradation in the presence of aliphatic compounds. Di&sion depends on the DBTconcentration in n-allcane, while cometabolism is associated with different n-alkane biodegradation rates. Through the definition of biodesulphurization selectivity and biodesulphurization @ciency, our investigations have shown that a selective aerobic biodesulphurization process is possible by using an unselective biocatalyst, such as a Pseudomonas sp.
INTRODUCTION
All oil biodegradation processes are limited by the heterogeneous system, which comprises an oil fraction (organic phase) and a biocatalyst (aqueous phase). Hydrocarbon bioavailability is affected by the chemical-physical characteristics of the substrate, viz., solubility, viscosity, surface tension, and so on.’ Naphthalene, bibenzyP and phenathrene3 were found to be used by some bacteria only when dissolved. The biodegradation rates of palmitic acid4 and phenathrene5 are limited by their dissolution rates in water. Bacteria may release compounds capable of either increasing the aqueous solubihty of the substrate6T7 or the rate of substrate diffusion in the water phase.5 Hydrophobic microorganisms may, however, use an insoluble compound through direct contact. In fact, microorganism adsorption
Biological and chemical-physical factors affect heavy oil biodegradation and/or biotransformation processes such as biodesulphurization. Biological factors depend on the type of microorganism employed and on its capability to assimilate hydrocarbons after adaptation to the substrate or after mutagenesis. Chemical-physical factors, however, such as, agitation, aeration for aerobic processes or the presence of hydrogen for anaerobic processes, as well as medium conditions and substrate characteristics, play a very important role in hydrocarbon assimilation by microorganisms. *To whom correspondence should be addressed. 721
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Leonardo Setti, Gaetano Lanzarini, Pier Giorgio Pifferi
in the oil phase is the most likely mechanism for explaining n-alkane biodegradation.8,9 Substrate uptake presumably occurs through diffusion or active transport at the point of contact. As is well known, most aerobic microorganisms adhere to the n-alkanes (below n-C16) which are in a liquid form at room temperature.l’ The microbial utilization of insoluble substrate in the case of direct of the microorganism on solid adsorption hydrocarbon has been reported in only a few papers.” The carrier effect of liquid n-alkanes on the degradation of solid hydrocarbons has been reported for paraffins by Cameotra et ~1.‘~ and Miller & Johnson,‘” for asphaltenes by Rontani et &.I4 and for sulphur aromatic compounds by Setti et a1.‘5~‘h Moreover, bacteria capable of degrading substrates partitioned into a non-biodegradable hydrocarbon solvent are known-l7 A Pseudomonas sp. may use diphenylmethane in the liquid, but not in the solid, form at the aqueous-organic interface, while another strain of Pseudomonas utilizes naphthalene at the water-heptamethylnonane interface but not at the surface of naphthalene crystals.ls Knowledge of how microorganisms assimilate solid hydrocarbon in the presence of a liquid solvent is very important for developing industrial oil biotransformation processes such as biodesulphurization. In fact, heavy oil is a complex mixture of different hydrocarbons which may affect the degradation of the sulphur aromatic compounds. A study was conducted to assess the role of aliphatic hydrocarbons in determining the means by which bacteria degrade sulphur aromatic compounds. Since dibenzothiophene (DBT) derivatives are the most frequently encountered sulphur organic compounds, DBT is generally employed as a model molecule in microbiological studies of heavy oil desulphurization. As toxic waste sites, industrial effluents and polluted areas around hydrocarbon reservoirs contain a non-aqueous phase, these investigations may provide useful ecological and toxicological information for reducing environmental pollution.
MATERIALS
AND METHODS
Medium The buffered inorganic salt solution used for the experiments contained:
at pH 7.0 3.52 g of
KH,PO,, 12.5 g of Na,HPO,.12H,O, NH&l and 0.04 g of MgSO,*7H,O deionized water.
0.8 g of per litre of
Chemicals Hexadecanoic acid (aC16) was obtained from Fluka AG. Buchs SG, n-dodecane (C12) and IZhexadecane (Cl 6) were purchased from Aldrich Chemical Co., Gillingham, England, and dibenzothiophene (DBT ) was obtained from Merck. Other chemicals used were reagent grade and were supplied by Carlo Erba, Milan. Microorganisms The enrichment culture was obtained from fuelcontaminated soil material of AGIP-Petroli refineries in Gela. The pure strain capable of degrading DBT was isolated by enrichment culture and identified according to the procedure described in a previous paper.15 To prepare inocula, the culture was grown at 28°C on a rotary shaker operating at 200 rpm in the 0.02% aC16salt medium for 4 days. For all experiments, the inoculum size was equal to 10% of the medium volume. Fermentation conditions DBT degradation was performed by adding 0.2% (v/v) DBT solution to 25 ml culture medium in a 250-ml volumetric flask fitted with breakwater. The following DBT concentration and solvent were employed as the case required: 54 ells in dimethylformamide (DMFA), 54-272 mu in ndodecane (C12) or n-hexadecane (C16), while the hexadecanoic acid (aC16) concentration was 19 or 190 mM. All solutions were autoclaved and added to the sterilized salt medium. All experiments were followed by control trials without inoculum. Culture hydrophobic assay The assay was made according to the procedure described by Rosenberg et a1.l9 Bacteria were grown in SALT medium containing a carbon source, viz. 0.02% (w/v) aC16, O-2% (v/v) Cl2 and 0.2% (v/v) C16. After 4 days growth, 5 ml of broth were diluted with distilled water to a turbidity, at 600 nm, close to O-3 and added in a tube ( 1 cm diameter) containing 5 ml of Cl2 or C16. The mixtures were agitated uniformly for 5 min and allowed to settle for 10 min to permit the hydrocarbon phase to rise completely. The aqueous phase was carefully removed with a
Biodegradation
723
of Pseudomonas sp.
Pasteur pipette and transferred to a 1.5ml cuvette. Light absorbance of the aqueous solution was determined at 600 nm before and after agitation using a Uvikon 860 spectrophotometer (Kontron Instr.). The absorbance values were directly proportional to the amount of cells in the aqueous solution, Analysis The residues of DBT microbial degradation were recovered from the culture medium by liquid-toliquid extraction using n-hexane after acidification of the aqueous phase to pH 2*0. Controls were carried out before each trial without inoculum. The amount of DBT was determined by spectrophotometric analysis using a Uvikon 860 spectrophotometer on the basis of the two relative maximum values of 3 12 and 325.5 nm, which are typical of this compound. The amount of Cl2 and Cl6 was determined by gas-chromatographic analysis using a Hewlett Packard 5890 CG fitted with a Flame Ionization Detector and a methylsilicone capillary column (0.25 mm X 25 m). Hydrogen was employed as a carrier gas (4 ml/min). The temperatures at which trials were carried out were 270°C for the injector, 270°C for the detector and a programmed temperature of 40°C for 5 min, 50°C/min up to 90°C and 4O”C/min up to 270°C. One microlitre of sample was injected into the capillary column. Solubility of DBT in the medium at 28°C The solubility of the aromatic compound was determined in duplicate by adding 0.2% (v/v) DBT solution in DMFA (54 mu) to 25 ml culture medium in a 250-ml volumetric flask. The bottle was incubated at 28°C for 24 h on a rotary shaker operating at 200 rpm. After centrifugation at 2600 g for 10 min, samples (10-O ml) containing no visible particles were withdrawn for solvent extraction and spectrophotometric analysis according to the procedure described above.
the solid to the aqueous phase may be a ratelimiting step.*O Setti et aZ.*l have shown that the biodegradation of DBT, as the only carbon source, by a Pseudomonas sp. is a typical example of the theoretical model suggested for PAH biodegradation. At the beginning of fermentation (Fig. l), the DBT degradation rate was controlled by bacterial exponential growth. After about 6 days, the dissolved DBT concentration (420 ng/ ml) was close to zero so that further degradation was controlled by the mass-transfer rate of the sulphur compound from the solid to the aqueous phase, 42 ng/ml/h, determined by the slope from 6 to 14 days fermentation. Consequently, the DBT degradation rate was independent of the amount of solid DBT in the medium. The presence of the fatty acid, hexadecanoic acid (aC16), during fermentation affects DBT degradation as reported in a recent note (Fig. l).*l When aC16 (19 mu) and DBT (54 mu) were added simultaneously to the medium from dimethylformamide solution, a different DBT biodegradation pattern was observed as compared to free dibenzothiophene. At the beginning of fermentation, no exponential biodegradation was seen to occur, which suggests a diffusion-limiting factor at work. When the amount of aC16 was close to zero, after five days of fermentation, as shown by the stationary phase of the growth trend on aC16 (Fig. 2), the DBT degradation rate became constant and was equal
T
0.1 0 0
RESULTS
AND DISCUSSION
The effect of the fatty acid on DBT biodegradation Wodzinski and Coyle3 have suggested that polycyclic aromatic hydrocarbons (PAHs) can be converted by microorganisms only in the dissolved state. This means that PAH mass transfer from
2
4
6
8
10
12
14
16
18
20
Fermentation time (days)
DBT removed as a function of fermentation time in Fig. 1. the presence of aC16: (0), DBT (54 mu) and aC16 (19 mM) added from the same initial solution in DMFA; (V), DBT (540 mu) and aC16 (190 ITLM)added from the same initial solution in DMFA; (a), DBT (54 mM) and aC16 (19 mM) added from different initial solution in DMFA; ( +), DBT (54 ~llhl) in DMFA solution as the only carbon source. All experiments were performed adding 02% (v/v) DMFA solution to the medium.
724
Leonardo
Setti, Gaetano
Lamarini,
Pier Giorgio Pifferi
;.
I
0
2
4
6
6
10
12
14
Fermentation time (days) 0
1 F.rm.mal0”
a
9
tm.
4
m4y-a)
Fig. 2. Batch growth of a strain of Pseudomonas aC16 (19 mM) dispersed in water.
sp. on
to that calculated for free dibenzothiophene, as it was controlled by mass transfer. The amount of DBT removed increased by proportionally increasing DBT (540 mu) and aC16 (190 mu) in the medium. The reverse was observed when aC16 ( 19 mu) and DBT (54 mu) from different dimethylformamide solutions were added to the medium. In fact, at the beginning of fermentation, the DBT removal rate was lower than that reported for free dibenzothiophene dibenzothiophene. The degradation rate increased exponentially with fermentation time and was higher when hexadecanoic acid degradation was nearly complete. After 10 days of fermentation, this rate became constant and was equal to that calculated for free dibenzothiophene, as degradation was controlled by mass transfer. These different behaviours and the higher dissolution rate of hexadecanoic acid, 70 ng/ml/ h,4 suggest that hexadecanoic acid favours dibenzothiophene assimilation, forming a kind of micelle when the fatty acid and the sulphur compound are added simultaneously to the medium. A similar model was suggested by Velankar et al** for hydrocarbon uptake by microorganisms. According to this model, the presence of micelles of surface-active agents is essential for the growth of microbial cells on hydrocarbon substrates. These micelles act as ‘transport packets’ for hydrocarbons. When the micelles are in close proximity to the cells, hydrocarbon is transferred more quickly to the cell surface as diffusion through the aqueous phase is eliminated.
Fig. 3. Amount of DBT removed as a funtion of fermentation time on 02% (v/v) n-dodecane at different DBT concentrations: (o), 54 mu; (A), 163 mu; (L), 243 mu; (O), 272 mu; (---) DBT as the only carbon source (data from Fig. 1).
0.6
,
0.5 o---------O
0
2
4
6 Fermentation
6
10
12
14
time (days)
DBT removed as a function of fermentation time on Fig. 4. 0.2% (v/v) n-hexadecane at different DBT concentrations: (o), 54 rnq (A), 163 mu; (*), 243 mu; (0), 272 mu; (---), DBT as the only carbon source (data from Fig. 1).
On the other hand, when hexadecanoic acid and dibenzothiophene were added from different solutions, the fatty acid did not necessarily form micelles containing the sulphur molecule, so that a competitive degradation involving the highly biodegradable fatty acid and the DBT dissolved in the medium was established. The effect of n -alkane on DBT biodegradation To evaluate this effect, two n-alkanes, Cl2 and C16, were chosen as representatives of two different degradation subgroups of the aliphatic fraction which makes up a heavy ~il.*~ When DBT was dissolved in II-alkane, the sulphur aromatic compound biodegradation trend was similar to that observed for the fatty acid micellar system (Figs 3 and 4). The DBT
725
Biodegradation of Pseudomonas sp.
degradation rate increased as the sulphur concentration compound in the n-alkane increased, this effect being much more significant when DBT was dissolved in n-hexadecane. At the beginning of fermentation, DBT degradation was higher in C 12 than in C 16 while these differences were minimal after 13 days of fermentation. At high DBT concentrations, close to saturation in n-alkane, biodegradation of the aromatic sulphur compound was always higher that that reported for free dibenzothiophene, which suggests that the n-alkane has a carrier effect on DBT degradation. When the sulphur compound was dissolved in ndodecane, a small carrier effect was always observed, while in n-hexadecane, at a low dibenzothiophene concentration, DBT degradation was lower than that observed for free dibenzothiophene. In this case, n-alkane was seen to have a barrier effect. The dependence of DBT degradation rate DBT concentration in n-alkane may be explained by diffusion. DBT assimilation in n-alkane is affected by two main concurrent factors: a coassimilation factor due to the simultaneous assimilation of the aliphatic and sulphur compound by the microorganisms, and, a hydrophobic factor characterized by the capability of the bacteria to adhere to Cl 2 or to C16. n-Dodecane was degraded better than n-hexadecane (Fig. 5) and complete degradation was reached after 6-8 days of fermentation compared to more than 20 days of fermentation for C16. These different degradation rates have been reported by Goma et aZ.,24 who studied the growth of Candida lipolyvica on a mixture of several hydrocarbons. These authors
0
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16
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Fermentation time (days)
n-Alkane degradation percentage as a function of Fig. 5. fermentation time on O-2% (v/v) of n-alkane added to the medium.
found that the short-chain hydrocarbons were used up faster than the longer chain hydrocarbons. This is due to the fact that the shorter chain hydrocarbon, Cl 2, is solubilized faster than the long-chain one, C16, probably moving across the oil-cell boundary faster. This means that the aliphatic degradation behaviour in heavy oil is a function of the single n-alkane surface tension.’ In fact, surface tension affects mass-transfer rate across the cell membrane. The n-alkane biodegradation rate, however, is also affected by the capability of the microorganisms to adhere to the aliphatic phase. The hydrophobic character of a microorganism is an index of this capability. The hydrophobicity of the strain of Pseudomonas sp. employed depends on the substrate in which the bacteria were grown. In fact, when palmitic acid was used as a substrate, the microorganisms did not exhibit hydrophobic characteristics, as testified by the absence of the biomass in the organic phase (Fig. 6). Bacteria grown on Cl 2 or Cl6 exhibited a good hydrophobic behaviour. Microbial pa&ion in the organic phase was higher in n-dodecane than in n-hexadecane, regardless of the kind of n-alkane used as a growth substrate. Diffusion, coassimilation and microbial hydrophobicity may explain why DBT degradation was found to be highest when the sulphur compound was dissolved in rt-dodecane at the beginning of
B
C
~ Cl2
Cl8
Microorganisms partitioned in n-alkane,Cl2 and Fig. 6. C16, as a function of the difEerent carbon source for the growth of the cells: A, palmitic acid; B, n-dodecane; C, nhexadecane.
726
Leonardo Seni, Gaetano Lanzarini, Pier Giorgio Pi#eti
fermentation. After 13 days of fermentation, Cl2 degradation was nearly complete so that n-alkane no longer exerted its carrier effect for the biodegradation of residual dibenzothiophene. The DBT biodegradation rate was thereafter conditioned by mass transfer from the solid to the aqueous phase, as reported for free dibenzothiophene. Conversely, n-hexadecane was not completely degraded so that the n-alkane may still have been able to exert its carrier effect. It may be reasonably assumed that the diffusional rate of DBT from the organic phase to the adsorbed microorganism is higher than the mass-transfer rate of free DBT.22 This may explain the similar DBT degradation values observed in Cl2 and Cl6 after 13 days fermentation at DBT concentrations above 100 mu. At lower concentrations, the diffusional rate in n-hexadecane was very low when compared to the coassimilation effect exhibited in n-dodecane. This suggests a simple model similar to that reported for the fatty acid. n-Alkane forms a film around the aromatic sulphur compound and as this fihn is easily attacked by aerobic microorganisms, the bioavailability of the sulphur compound increases. Biodesulphurization selectivity The biodesulphurization process selectivity, Ds, is represented by the ratio between the initial and final DBT concentrations in n-alkane. Es = (n-alkane recovered)/(initial n-alkane) X (DBT removed)/(initial DBT ) = Co/ Cf where Co and Cf are the initial and the final DBT concentrations in n -alkane, respectively. A microbial treatment is considered to be a selective biodesulphurization process when Ds is above 1. In fact, in this case the DBT biodegradation rate is higher than the n-alkane biodegradation rate and, consequently, the concentration of the sulphur aromatic compound decreases in n-alkane after the treatment. Ideally, desulphurization should be capable of removing all sulphur without destroying useful products. Figure 7 shows that Ds depends on the percentage amount of n-alkane removed and is independent of the type of n-alkane employed. The value of selectivity was above 1 when the percentage of n-alkane biodegraded was very low, i.e. 510%. The fact that Ds is apparently dependent on the type of n-alkane as a function of fermentation time, as shown in Fig. 8, can be ascribed to the higher biodegradation rate of Cl2
1.2
1
1 0.3 g 0.6 0.4 0.2 0
Fig. 7. Biodesulphurization selectivity, Ds, as a functionof the n-alkane degradation percentage in both Cl2 and in C16.
10
0123456780
11
12
13
Fermentation time (days)
Biodesulphurization selectivity, Ds, as a function of Fig. 8. fermentation time in Cl2 and in Cl6 at different DBT con;Fdtra,“,o;: (0) 5:’ 11~1, (A) 163 mu, (0) 272 mu. Cl2 (---)
compared to that of C16. The value of selectivity broke down very quickly and was close to zero after five days fermentation as a consequence of a high Cl 2 biodegradation rate, while this decrease is slower in n-hexadecane. Selectivity is affected by the DBT concentration in n-alkane. In fact, it increases as the DBT concentration increases, reaching its peak in n-hexadecane after the first three days of fermentation. At the beginning of fermentation, high DBT concentration and slow n-alkane biodegradation rates favour the DBT diffusional rate from the organic phase to the surface of the microorganisms adsorbed on the aliphatic compound. The value of Ds above 1 observed in nhexadecane after three days of fermentation suggests that a selective biodesulphurization process in aerobic conditions is possible by using an
Biodegradationof Pseudomonas sp.
unselective biocatalyst. A selective biodesulphurization process is already known for some anaerobic microbial treatments.25 Biodesulphurization selectivity was not observed in Cl2 due to the early n-alkane biodegradation which occurred at the beginning of fermentation. Biodesulphurization efficiency Biodesulphurization efficiency is an index biocatalyst efficiency for the removal of DBT the presence of n -alkane, and is given as: Es = ( n -alkane recovered)/( initial n-alkane) (DBT removed)/( initial DBT )
of in x
Biodesulphurization selectivity is not sufficient to characterize a biodesulphurization process, as it does not take into account the amount of DBT removed. Biodesulphurization efficiency compares the amount of recovered n-alkane with the amount of recovered DBT and then, when Es is equal to 1, biodesulphurization is an ideal process in which all sulphur is removed selectively without destroying n-alkane. A higher efficiency was observed when DBT was dissolved in n-hexadecane (Fig. 9). Efficiency reached its peak after one day of fermentation in n-dodecane and after 3-6 days of fermentation in n-hexadecane. The low efficiency observed in n-dodecane is due to the high biodegradation rate of this alkane, even though it is effective as a carrier for DBT biodegradation. In n-hexadecane, efficiency increased as DBT concentration in n-alkane increased, this behaviour being more significant at the beginning of fermentation. In n-dodecane, efficiency was
012345676
9
10
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13
Fermentation time (days)
efficiencv, Es. as a function of Fie. 9. Biodesulohurization feromentation time-in Cl2 and in Cl6 Lt different DBT concentration: (V) 54 mM, (0) 163 mu, (A) 243 mu, (0) 272 ) with filled symbols. mu. Cl2 (---) and Cl6 (-
727
minimal at a DBT concentration of 163 mu. These results may be due to the two concurrent effects previously mentioned: coassimilation on one hand and diffusion on the other. The latter is a rate-limiting step in n-hexadecane, as efficiency is proportional to DBT concentration, while in IZdodecane the high value of Es at a low DBT concentration is due to the coassimilation limiting effect. In fact, at a low DBT concentration, the diffusional rate of the sulphur molecule from the bulk of the organic phase to the microorganism adsorbed on the aliphatic compound, is slower. IIAlkanes work as carriers for DBT biodegradation, although the different capabilities of the hydrocarbon microbial uptake affect biodesulphurization selectivity and efficiency. In fact, the model system in which dibenzothiophene is dissolved in n-hexadecane exhibits the best selectivity and efficiency as a consequence of the worst biodegradability of Cl6 with respect to C12.
CONCLUSIONS The study of the biological and chemical-physical parameters affecting biodegradation processes may be interesting for developing selective biodesulphurization treatments in heavy oil as well as biodegradation processes in complex organic solvents. The present study shows that a selective aerobic biodesulphurization process is possible by using an unselective biocatalyst. Further progress needs to be made in order to improve the selectivity of aerobic heavy oil biodesulphurization treatments. In fact, the biocatalytic activity of aerobic microorganisms is higher than that of anerobic ones, even though the latter hold considerable interest for industrial applications.2” Biodesulphurization selectivity and efficiency have been examined as the two important indexes characterizing the process, as they suggest that diffusion and cometabolic effects are responsible for the rate-limiting-step in DBT degradation. Diffusion depends on the microbial affinity between the microorganism and the organic phase in which the substrate is dissolved and on the substrate concentration in the same organic phase, while coassimilation depends on the organic phase biodegradation rate. These two effects may affect the removal of the sulphur aromatic compounds in heavy oil.
728
Leonardo Setti, Gaetano Lanzarini, Pier Giorgio Pifferi
ACKNOWLEDGEMENTS
microorganisms. II: uptake of solid n-alkanes by yeast and bacterial soecies. Biotechnol. Bioena.. _. 25 (1983)
Special thanks to Prof. D. Matteuzzi and Dr. M. Rossi from the Department of Pharmaceutical Science (University of Bologna, Italy) who supplied us with the culture of Pseudomonas sp. The research was supported by funds provided by the Enichem-ANIC S.p.A. (Milan, Italy).
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Edizioni
M.A.F.