Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilize

International Biodeterioration &Biodegradation 57(2006)222–228

Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan ?akes

Alejandro R.Gentili a,?,Mar?a A.Cubitto a ,Marcela Ferrero b ,Mar?a S.Rodrigue

z c a

Departamento de Biolog?

′a,Bioqu?′mica y Farmacia,Universidad Nacional del Sur,San Juan 670,8000Bah?′a Blanca,Argentina b

Planta Piloto de Procesos Industriales Microbiolo

′gicos (CONICET),Av.Belgrano y Caseros,4000Tucuma ′n,Argentina c

Departamento de Qu?

′mica,Universidad Nacional del Sur,Alem 1253,(8000)Bah?′a Blanca,Argentina Available online 18April 2006

Abstract

In this laboratory-scale study,we examined the potential of chitin and chitosan ?akes obtained from shrimp wastes as carrier material

for a hydrocarbon-degrading bacterial strain.Flakes decontamination,immobilization conditions and the survival of the immobilized bacterial strain under different storage temperatures were evaluated.The potential of immobilized hydrocarbon-degrading bacterial strain for crude oil polluted seawater bioremediation was tested in seawater microcosms.In terms of removal percentage of crude oil after 15days,the microcosms treated with the immobilized inoculants proved to be the most successful.The inoculants formulated with chitin and chitosan as carrier materials improved the survival and the activity of the immobilized strain.It is important to emphasize that the inoculants formulated with chitin showed the best performance during storage and seawater bioremediation.r 2006Elsevier Ltd.All rights reserved.

Keywords:Bioaugmentation;Hydrocarbon-degrading bacterial strain;Chitin;Chitosan;Immobilization;Bioremediation

1.Introduction

Petroleum hydrocarbons are major pollutants of marine environments as a result of terrestrial and freshwater run-off,refuse from coastal oil re?neries,offshore oil produc-tion,shipping activities and accidental spills.Although evaporation and photo-oxidation play an important role in crude oil detoxi?cation,ultimate and complete degradation is accomplished mainly by microorganisms (Atlas,1981;Oudot,1984;Cerniglia,1984;Bartha,1986;Yakimov et al.,1998).

A popular option to favor the clean-up of hydrocarbons polluted environments has involved biostimulation.How-ever,for more recalcitrants compounds or if the biode-gradable pollutant is introduced to the environment at high concentrations (e.g.spills)and a rapid detoxi?cation of the chemical is required,it may not be appropriate to rely on the natural response of members of the native microbial community.For example,a slow biodegradation in an accidental oil spill in coastal seawater may result in the

movement of spilled crude oil to other coastal sites and probably its accumulation in the sediments,so the possibility of undesirable effects on the ecosystem is increased (Bartha,1986;Alexander,1999).Bioaugmenta-tion to enhance natural biodegradation is a useful alternative (Vogel,1996;Jansson et al.,2000;Cunningham et al.,2004).

Bioaugmentation has met with varying degrees of success (Crawford and Mohn,1985;Brodkorb and Legge,1992;Leavitt and Brown,1994;Vogel,1996;Atlas and Bartha,1992)and there has been a considerable debate over the ef?cacy of this methodology.The fact is that the use of selected bacterial strains,with broad substrate range and high metabolic rates,frequently has failed in natural environments.The selected microorganisms that have bene?cial traits for biodegradation must also be able to overcome biotic and abiotic stresses in the environment in which they are introduced (Alexander,1999).Macnaugh-ton et al.(1999)have demonstrated the early disappearance of the components of a microbial consortia introduced in a natural environment polluted with hydrocarbons.Hence,the maintenance of suf?cient activity of an inoculant population over a prolonged period after release,often

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0964-8305/$-see front matter r 2006Elsevier Ltd.All rights reserved.doi:10.1016/j.ibiod.2006.02.009

?Corresponding author.

E-mail address:agentili@https://www.sodocs.net/doc/d218322722.html,.ar (A.R.Gentili).

represents the main hurdle in the successful use of inoculants in bioremediation(Sanjeet et al.,2001).

The use of inoculant formulations involving carrier materials for the delivery of microbial cells to natural ecosystems,is an attractive option.Carrier materials are generally intended to provide protective niche to microbial inoculants,either physically,via the provision of a protective surface or pore space,or nutritionally,via the provision of a speci?c substrate.An optimal carrier should provide favorable conditions for survival as well as functioning of the inoculant cells,resulting in a suf?ciently long shelf life as well as improved survival and activity(van Veen et al.,1997).The carrier should,furthermore,be nontoxic,nonpolluting,have a constant quality and be locally available at low price(Ronchi and Ballatti,1996; Leenen et al.,1996).In the bioremediation of naturals environments the destruction of the carrier material once the introduced microorganisms have carried out their objective is desirable,so the materials should be biode-gradable(Pometto et al.,1998).A wide range of carriers prepared from natural materials,e.g.peat,clay and plant-derived compounds,have been tested and used specially in Rhizobium inoculants industry,but a little information has been reported about the development of inoculants with biodegradable carrier materials for seawater bioremedia-tion.

Chitin(1–4linked2-acetamido-2-deoxy-b-D-glucan)is the main component in the cuticles of crustaceans,insects, and mollusks and in the cell walls of fungi,is the second most abundant polysaccharide found on earth next to cellulose(Muzzarelli,1973).The exoskeletons of shrimps have long since attracted attention as a source of raw material for chitin production as the dry arthropod exoskeletons contain from20%to50%chitin(natural chelating polymers).This biopolymer is produced commer-cially from crab and shrimp exoeskeletons by treatment with dilute NaOH solution for deproteinization,followed by treatment with dilute HCL solution for demineraliza-tion.Chitosan,the deacetylated derivative of chitin,as a natural renewable resource,has numerous applications, which attract scienti?c and industrial interests(Li et al., 1997).

Bah?a Blanca is a port city located at south of Buenos Aires province(Argentina).It has an important crude oil re?nery and port facilities,destined to charge and discharge crude oil and its by-products.These activities generate hydrocarbon rich wastes and sometimes,acciden-tal oil spills in seawater.Several ports are located at the Estuary of Bah?a Blanca,which is a complex system of tidal channels,islands and extended tidal?ats.The last ones delimit the main channel of the estuary,which constitute the access to one of the most important harbor complexes of Argentina.

Since the exoskeletons of shrimps are an abundant residue of the local?shing industry,chitin and chitosan obtaining technology is available in our university,both materials are nontoxic,nonpolluting and biodegradable,the?akes of chitin and chitosan could be considered an alternative carrier material for immobilizing microorgan-isms for bioremediation purposes.Therefore,the?rst aim of the present study was to formulate inoculants with chitin and chitosan?akes as carrier of a hydrocarbon-degrading bacterial strain isolated from Bah?a Blanca coastal soils. The second aim was to evaluate the potential of the strain immobilized on chitin and chitosan?akes for the clean-up of crude oil-contaminated seawater.

2.Materials and methods

2.1.Obtaining of chitin and chitosan?akes

All reagents were of either analytical grade or the highest purity grade available.Chitin was obtained from shrimp(Pleoticus mu¨lleri)wastes at the Laboratorio de Investigaciones Ba sicas y Aplicadas en Quitina (LIBAQ).Raw material was homogenized and triturated in an industrial triturator(Westinghouse model DASO6).The product was rinsed with water at room temperature in order to remove organic materials.The cleaned residue was treated with9%(w/w)NaOH at651C for90min to remove proteins,then demineralized by treatment with10%(v/v)HCl at 201C for15min.Finally the obtained chitin was washed with water at room temperature,and then air-dried.Chitosan was obtained from chitin by heterogeneous deacetylation at1361C with50%(w/w)NaOH for1h. The characteristics of chitosan used in this study were:deacetylation degree:95,moisture:6.0%,ash content:0.55%.

2.2.Culture media

The culture media used in this study were:(a)SW:minimal salt medium of Winogradsky(Pochon,1962),amended with0.1%(w/v)NH4NO3and 0.1%(w/v)KH2PO4,pH:7.2;(b)ASW:SW with1.2%(w/v)ultra pure agar–agar;(c)Locke solution:1.5%(w/v)NaCl,0.04%(w/v)MgCl2, 0.01%(w/v)KCl,pH:7(Verna,1945).All the media and solutions were prepared with distillated water and autoclaved at1atm for15min.

2.3.Decontamination treatments

To determine a suitable method for reducing the microbial load of the carrier material,1g of chitin and chitosan?akes were put into respective glass Petri dishes and treated with wet heat in a Chamberland autoclave under different conditions:(a)?uent steam for15min,(b)?uent steam for 30min,(c)pressure saturated steam at1atm for15min and(d)pressure saturated steam at1atm for30min.The number of total viable heterotrophic aerobic bacteria was determined before and after each treatment.Samples of0.1g of?akes were taken out from each Petri dish. The?akes were suspended in9.9mL of sterile Locke solution.The?akes suspensions were disintegrated with a mixer at10,000rpm for1min. Successive decimal dilutions were prepared in Locke solution from the supernatant of the obtained suspension.From the dilutions,0.1mL portions were spread on Plate Count Agar(PCA).The plates were incubated at281C for5days.At the end of incubation the colonies were counted and the results expressed as CFU per gram of dry chitin or chitosan.

2.4.Dry weight

Viable counts were referred to the dry weight of?akes.From each sample a portion of chitin and chitosan?akes were dried at1051C overnight in aluminum boxes until constant weight.The?akes were weighed on a precision balance(AND ER-180A)and water content of the ?akes was calculated.

A.R.Gentili et al./International Biodeterioration&Biodegradation57(2006)222–228223

2.5.Microorganisms

The hydrocarbon-degrading bacterial strain(QBTo)used in this study was isolated in a previous research(results not shown),from coastal soils of Bah?a Blanca Estuary in?uenced by port and petrochemical industry activities.This strain has been characterized by16S rRNA gene sequence and proposed as Rhodococcus corynebacterioides(Barengo et al.,2002; NCBI GenBank accession number AY157677).

2.6.Production of inoculants

To immobilize the strain QBTo on the carrier material,the cells were cultured together with chitin and chitosan?akes in WS with kerosene as the sole source of carbon and energy.Previously,to determine the minimal suitable concentration of kerosene and the incubation time that could improve the strain QBTo attachment and the bio?lm formation on the ?akes,combination of three different kerosene concentration and three incubation times were tried.Eighteen250mL sterile erlenmeyers?asks containing100mL of SW were prepared.Nine?asks were added with 0.25g(w/v)of chitin?akes and nine with0.25g(w/v)of chitosan?akes. The?akes had been autoclaved at1atm for15min.0.5%(v/v)of kerosene were added to three?asks of each group,0.37%(v/v)to the other three and0.25%(v/v)to the last three ones.The kerosene had been autoclaved at 1.5atm for20min.All?asks were inoculated with1mL (108CFU mLà1)of72h strain QBTo culture in SW with crude oil as sole source of carbon and energy,incubated at281C.The growth conditions of the strain were established in a previous study(Gentili and Cubitto,2004).All?asks were incubated at281C and150rpm in a rotary shaker.At72,96and120h of incubation,samples of chitin and chitosan ?akes were taken from the?asks.Each sample was divided into three 0.01g portions,which were assigned to scanning electron microscopy (SEM),viable count and dry weight determinations,respectively.For SEM,the?akes were suspended in10mL of Locke solution and stirred at 500rpm during1min in order to wash the non-attached cells.Washed ?akes were taken with sterile forceps and placed in?xative(buffer phosphate,pH7.2and1%v/v glutaraldehyde)overnight.The?akes were prepared as was described by Lozano(1990)and?nally were observed in scanning electron microscope(JEOL35).

To assess the number of attached viable bacteria,0.01g of?akes sample were washed as was indicated above and then suspended in 9.99mL of Locke solution.The?akes in suspension were disintegrated with a mixer at10,000rpm for1min,successive decimal dilution were made from the supernatant and0.1mL from dilutions were spread on ASW.Filter papers saturated with crude oil were placed in the covers of Petri dishes to supply the carbon and energy source without affecting the isolation of the colonies(Robertson et al.,1973).The plates were incubated at281C for5days.

Once the more suitable conditions for the formation of an abundant bio?lm on the?akes were established,the inoculant was produced in a larger scale in a bioreactor with two glass tanks(New Brunswick Scienti?c Co.,USA)with a working volume of3L in each tank.SW medium with 0.25%(v/v)of kerosene as the sole source of carbon and energy was used in the bioreactor.A72h QBTo culture in SW with0.5%(v/v)crude oil was used as inoculum and7.5g of chitin or chitosan?akes were added to each tank.The conditions in the bioreactor were as follows:temperature, 281C;aeration,0.60volume of air/volume of medium/min;agitation, 150rpm;incubation time,5days.After this time,the?akes were?ltered aseptically through a sterile sieve,put on sterile metal trays and covered with sterile gauzes.The carrier-based culture was air dried at301C for24h and then distributed into sterile polyethylene bags in portions of2.5g in each bag.

2.7.Storage conditions and viability assessment

Polyethylene bags with2.5g of inoculants obtained as was described above,were stored at three different temperatures:(a)room temperature (251C72),(b)41C,and(c)201C.Each condition was tested by triplicate.Periodically,0.1g?akes samples were taken from each bag to establish the number of viable cells immobilized on each carrier material.Viable counts on ASW were carried out as was described in2.6and the counts were referred to the?akes dry weight.To monitor the staying of the strain QBTo hydrocarbon degrading activity along the storage time,0.01g of inoculants from each bag were put into tubes with5mL of SW amended with25m L of crude oil,the tubes were incubated at281C for96h.Tubes showing turbidity and visible changes in crude oil compared with controls prepared with sterile chitin and chitosan?akes were recorded as positive.

2.8.Seawater bioremediation

To evaluate the effectiveness of inoculants addition in a bioremediation process,seawater microcosms simulating a crude oil spill were prepared. Seawater was collected in sterile glass bottles from the Main Channel of Bah?a Blanca Estuary(381440–391270S;611450–621300W).The bottles were transported to the laboratory at101C and stored at41C for no more than24h.Twelve sterile250mL Erlenmeyer?asks were prepared with 150mL of seawater and0.75mL of autoclaved crude oil(type:‘‘Medanito’’;origin:Neuque n,Argentina;density:0.84),provided by a local re?nery.Small volumes of crude oil were autoclaved in10mL tubes at1.5atm for30min.Three?asks were inoculated with0.2g of the QBTo inoculant formulated with chitin,three with the QBTo inoculant formulated with chitosan and three with1mL of72h QBTo culture (108CFU mLà1)in seawater with crude oil as sole source of carbon and energy.The inoculants were produced as was indicated in2.7.The last three?asks were prepared without inoculum to assess the activity of the native microbial community alone(positive control).Three control?asks of the abiotic changes in the crude oil were prepared with autoclaved seawater and autoclaved crude oil(negative control).In order to obtain semi-continuous culture conditions,each?ask was supplemented with a glass device,designed to replace25mL of seawater in the?asks with fresh seawater without affecting the crude oil concentration(Cubitto and Cabezal?,2001).All microcosms were incubated for15days at251C on a rotary shaker set at100rpm.Every72h,25mL of seawater from the?asks were replaced by25mL of seawater recently collected.In the case of the negative controls,the seawater was previously autoclaved.

To monitor the survival and prevalence of the introduced bacterial strain,at the end of the experiment,1mL water samples were taken from all?asks and decimal successive dilutions were made in Locke solution. Viable counts were made on ASW plates with crude oil,prepared as was described in Section2.6.The plates were incubated at281C for5days. The number of the strain QBTo characteristics colonies(smooth,red-orange)was recorded.All colonies grown on agar plate were harvested, resuspended in TE buffer(10mM Tris HCl,1mM EDTA[pH8])and subjected to extraction of DNA for further DGGE analysis.DNA was extracted using BACTOZOL TM KIT Cat.No:BA154.The extracted genomic DNA was used as target in the PCR to amplify16S rRNA genes. Bacterial fragments suitable for subsequent denaturing gradient gel electrophoresis(DGGE)were ampli?ed with the primer combinations 341fGC-534r as was described by Ro lleke et al.(1996).We used6% polyacrylamide gel with empirically determined gradient of DNA-denaturant by mixing two stock solutions of acrylamide containing30% and70%denaturant(100%denaturant is de?ned as7M urea and40% deionized formamide).About800ng of PCR product was loaded for most of the samples and the gels were run at120V,601C for4.5h using 1?TAE buffer(40mM Tris base[pH7.4],20mM sodium acetate,1mM EDTA)in a CBS(Scientifc Co.,USA).The gels were stained with the nucleic acid stain SybrGold for45min,rinsed with TAE buffer,removed from the glassplate to a UV transparent gel scoop and visualized with UV radiation in a GelDoc2000Image Analyzer with the Quantity One software(Bio-Rad).

2.9.Hydrocarbon concentration

At the end of the incubation time,all microcosms were acidi?ed at pH2 with sulphuric acid.The residual hydrocarbons were recovered by?ve

A.R.Gentili et al./International Biodeterioration&Biodegradation57(2006)222–228 224

successive liquid–liquid extractions with5mL of n-hexane each time.The extraction was exhaustive including the devices and?akes.Solvent was evaporated at room temperature(251C72)during a normalized time of 72h to avoid variations in evaporations induced by differential volatiliza-tion of the naphtha(Oudot,1984).The amount of residual hydrocarbons recovered was determined gravimetrically in an analytical electronic balance(AND,model ER-180A).After gravimetric quanti?cation,1-eicoseno(Alltech Co.,USA,4174)was added as internal standard to the obtained extract.The extracts were suspended with5mL of hexane (Chromatographic quality)and resolved aliphatic hydrocarbons(RAH) were quanti?ed by gas chromatography.The chromatograph(Shimadzu GL-14A)was equipped with a?ame ionization detector(FID),split injection system and a capillary column(Shimadzu,S500.50CBP1). During analysis,the injector temperature was set at1701C,the detector temperature at3101C and the oven temperature was programmed to rise from1001C(3min)to3001C(10min)in31C minà1increments(Oudot, 1984,UNEP,1992).The RAH fraction was analyzed for individual n-alkanes,pristine,and phytane isoprenoids.

3.Results

The results obtained from the treatments to reduce the microbial load of chitin and chitosan?akes are shown in Table1.The?uent steam produced a99.9%reduction in the viable counts and the pressure saturated steam caused a reduction greater than the99.99%.No visible modi?cation in the?akes was observed after any treatments.

During the production of the inoculant,neither kerosene concentration nor incubation time produced signi?cant differences(p o0:05)in the viable counts obtained from the ?akes incubated with R.corynebacteriorides strain QBTo. Despite the viable counts results,SEM showed that?akes incubated with0.37%and0.50%(v/v)kerosene formed more compact bacterial aggregates than those incubated with0.25%during72h.The?akes incubated with0.25% (v/v)of kerosene did not show the compact cell aggregates until120h of incubation.

The images obtained from the culture with0.25%(v/v) kerosene after5days of incubation(Fig.1),decided us to choose these culture conditions that involved a lower concentration of kerosene and allowed a suitable coloniza-tion on the?akes surfaces.A low concentration of kerosene residues in the inoculant is desirable for bioremediation purposes.

Figs.2and3show the survival of strain QTBo cells immobilized on chitin and chitosan?akes during the storage at three different temperatures.All the inoculants maintained their initial viable counts for45days at the three temperatures tested.At90days,a decrease about2 orders of magnitude was detected in the inoculants stored at room temperature.The inoculants stored at41C and à201C showed a decrease only about one order of magnitude and yielded stable CFU counts(about 108CFU gà1of carrier material)for a period of135days. After6months of storage,chitosan?akes yielded counts about108CFU gà1only atà201C(Fig.5).Chitin?akes maintained the QBTo viable count about108CFU g–1at

41C and atà201C(Fig.2).The strain QBTo immobilized on the both materials preserved its crude oil—degrading capacity at the three storage temperatures tested.

In the seawater bioremediation trials,the highest biodegradation rates were obtained in the microcosms inoculated with R.corynebacteriorides QBTo immobilized Table1

Decontamination treatments applied to chitin and chitosan?akes Treatment Chitin?akes

(CFU gà1)

Chitosan?akes

(CFU gà1)

No treated 6.6?10 5.0?107

Fluent steam,15min 1.3?1037.0?102

Fluent steam,30min 2.5?102 1.0?102 Pressure-saturated steam1atm,

15min

5020

Pressure-saturated steam at

1atm,30min

o10a o10a

The viable counts are expressed as CFU per gram of chitin/chitosan dry weight.

a Below detection

level.

Fig.1.SEM showing the Rhodococcus corynebacterioides QBTo bio?lm on?akes incubated with0.25%kerosene(v/v)for120h:(A)chitin;(B) chitosan.Magni?cation3000?.Bars?10m m.

A.R.Gentili et al./International Biodeterioration&Biodegradation57(2006)222–228225

onto chitin and chitosan ?akes as carrier materials.In these microcosms,60%of hydrocarbons in the hexanic extract were removed compared with controls (Fig.4).In the seawater microcosms where the strain was inoculated without carrier,only a decrease about 30%of hexanic extract was obtained.The gas liquid chromatography of the hexanic extracts showed a higher degrading activity on RAH fraction in the microcosms where the inoculant formulated with the ?akes,were applied (Fig.5).The inoculants formulated with chitin showed the highest activity on this fraction.Changes in pristane and phytane isoprenoids concentrations were not observed in any microcosms during the experiment,probably because they are more resistant to bacterial degradation.

R.corynebacteriorides QBTo only was recovered from the microcosms seeded with the carrier-based inocula.DGGE analyses con?rmed the presence of the inoculated strain.

4.Discussion

The chitin and chitosan ?akes are natural products obtained from an abundant waste of the ?sh industry of Bah?a Blanca region.They are nontoxic,nonpollutant,have de?ned nature,and a constant quality can be obtained.

The ?akes can be ef?ciently decontaminated by ?uent steam and pressure saturated steam,which are common and available technologies in the industry.

The R.corynebacteriorides QBTo was cultured with the carrier materials in presence of kerosene as sole source of carbon and energy to favor the growth of the population on the ?akes surfaces and the subsequent formation of a bio?lm.We have previously observed that kerosene was rapidly adsorbed on the ?akes surfaces.This situation favored the growth of the population on the ?akes surfaces and inside its ‘‘pores’’and ‘‘crevices’’.It is well known that in these surface-attached population bacteria are protected from environmental stresses and predation.Bio?lm for-mation therefore emerges as an important process for microbial survival in the environment (Davey and O’Toole,2000).On the other hand,the presence of hydrocarbons in the production culture medium could help to maintain a selective pressure and the hydrocarbon-degrading activity of the cells to be applied in a bioremediation process.The differences observed between the viable counts and the SEM seem reasonable probably because many cells strongly attached could not be recovered from the super-natant of the ?akes suspension.

A high number of strain QBTo cells survived immobi-lized on both carrier materials for 6months.The storage at à201C was the most suitable storage temperature for the inoculants formulated with chitosan as carrier material.However,both 41C and à201C appeared as suitable for the inoculants formulated with chitin ?akes.

56789100

45

90135

180

Days

L o g 10 C F U g -1 d r y c h i t i n

Fig.2.Viable counts obtained from the inoculant formulated with chitin ?akes at three storage temperatures along time.Values are arithmetic means for three bags per temperature.Bars represent standard deviations.

5678910

Days

l o g 10 C F U g -1 d r y c h i t o s a n

Fig. 3.Rhodococcus corynebacterioides QBTo viable counts obtained from the inoculant formulated with chitosan at three storage temperatures along time.Values are arithmetic means for three bags per temperature.Bars represent standard deviations.

0.10.20.30.40.50.60.7Control -Control +

QTBo

QTBo-chitosan

QTBo-chitin g /100 m L s e w a t e r

Fig.4.Gravimetric determination of residual crude oil extracted with hexane from seawater microcosms after 15days of incubation.Contol à:autoclaved seawater;Control +:seawater without inoculant;QTBo:seawater inoculated with the strain QTBo without carrier material;QTBO-chitin:seawater inoculated with the inoculant formulated with chitin ?akes;QTBo-chitosan:seawater inoculated with the inoculant formulated with chitosan ?akes.Values are arithmetic means for three microcosms per treatment.Bars represent standard deviations.

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226

The bioremediation experience showed that the native population in the positive control microcosms could not produce any signi?cant reduction in the pollutants hydro-carbons concentrations.In the microcosms where the strain QBTo was inoculated without carrier,the low hydrocarbon removal would not justify the bioaugmentation.This result probably was due to the short survival of the strain in seawater,although it was originally isolated from Bah?a Blanca Estuary coastal environments and that at the beginning of the experience,it outnumbered the native microbial community.The bioaugmentation with the strain immobilized on chitin and chitosan ?akes enhanced signi?cantly the crude oil biodegradation.We consider that the principal reason for this result was the strain survival due to protective effect of the carrier material and the bio?lm structure that the cells have developed on it.From the results obtained in this laboratory scale study,we did not ?nd differences between chitin and chitosan ?akes about their decontamination,the inoculants produc-tion and the inoculant shelf-life.However,the inoculants formulated with chitin yielded the highest number of cell survival at 41C during 6months of storage and showed a higher degrading activity on RAH.One reason could be the crystalline structure of chitin that would provide more protective microhabitats to microbial inoculant.

Chitin production is cheaper than chitosan and since it is an abundant polysaccharide found in nature,there are several organisms that contain chitin-degrading enzymes in marine environments.These characteristics indicated the inoculant formulated with this carrier material as envir-onmentally friendly,avoiding the potential problems that

generate the synthetic supports materials (Leenen et al.,1996).

These results indicated that seawater represented a hostile environment to introduced microorganisms.The success of the application of a microbial inoculant depends to a large extent on how favorable to its survival the target environment is or can be made.In the present study a crude oil degrading bacterial strain was applied in form of carrier-based-inocula.The carrier material used,chitin and chitosan ?askes and the inoculant production method applied,allowed the development of a bio?lm,providing a protective niche to the bacterial strain and resulting in a long shelf life and in a high crude oil degrading activity in natural seawater.Acknowledgments

We thank the Science and Technology Secretary of the Universidad Nacional del Sur for funding of this research.The comments of the anonymous reviewers were appre-ciated.References

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0.10.20.30.40.50.60.70.80.911.110

1112

13

14

15

16

17

P

18

F

19

20

21

22

23

24

25

No of carbons atoms

% R A H

control -control +QTBo

QTBo-chitosan QTBo-Chitin

Fig.5.Percentage of resolved aliphatic hydrocarbons (RAH)after 15days of incubation.Contol à:autoclaved seawater;Control +:seawater without inoculant;QTBo:seawater inoculated with the strain QTBo without carrier material;QTBO-chitin:seawater with the inoculant formulated with chitin ?akes;QTBo-chitosan:seawater with the inoculant formulated with chitosan ?akes.Values are arithmetic means for three microcosms per treatment.Bars represent standard deviations.

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