Rhamnolipids enhance marine oil spill bioremediation in laboratory system
Qingguo Chen a ,b ,Mutai Bao b ,⇑,Xiaoning Fan b ,Shengkang Liang b ,Peiyan Sun c
a
College of Marine Science and Technology,Zhejiang Ocean University,Zhoushan 316004,China
b
Key Laboratory of Marine Chemistry Theory and Technology,Ministry of Education,Ocean University of China,Qingdao 266100,China c
North China Sea Environmental Monitoring Center of State Oceanic Administration,Qingdao 266033,China
a r t i c l e i n f o Keywords:
Bioremediation Rhamnolipids Normal alkanes
Polyaromatic hydrocarbons Biomakersrais
a b s t r a c t
This paper prents a simulated marine oil spill bioremediation experiment using a bacterial consortium amended with rhamnolipids.The role of rhamnolipids in enhancing hydrocarbon biodegradation was evaluated via GC–FID and GC–MS analysis.Rhamnolipids enhanced total oil biodegradation efficiency by 5.63%,with variation in normal alkanes,polyaromatic hydrocarbons (PAHs)and biomakers biodegra-dation.The hydrocarbons biodegradation by bacteria consortium overall follows a decreasing order of PAHs >n -alkanes >biomarkers,while in different order of PAHs
>biomarkers >n -alkanes when rhamn-olipids was ud,and the improvement in the removal efficiency by rhamnolipids follows another order of biomarkers >n -alkanes >PAHs.Rhamnolipids played a negative role in degradation of tho hydrocar-bons with relatively volatile property,such as n -alkanes with short chains,PAHs and squiterpenes with simple structure.As to the long chain normal alkanes and PAHs and biomakers with complex structure,the biosurfactant played a positive role in the hydrocarbons biodegradation.
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1.Introduction
The demand of petroleum as an energy source increas with the increasing worldwide industrialization.Oil spill occurs fre-quently around the world in the petroleum exploitation and trans-portation (Brito et al.,2009;Joo et al.,2008).Large amount oil was relead into the a in oil spills and it has certainly done devastat-ing effects on the marine environment.Physico-chemical methods are not completely effective in oil spill removal (Prince et al.,2003).Bioremediation,with the advantages such as low cost and environ-mentally-friendliness,bioremediation could be an alternative to solve oil pollution problem and is currently receiving favorite pub-licity (Rahman et al.,2
002).Some studies have been reported to successfully u bioremediation for spilled oil (Atlas,1995;Green-wood et al.,2009;Hii et al.,2009;Mearns,1997;Radwan et al.,2005).
The bioavailability of petroleum hydrocarbons is limited since they are mostly insoluble in water.Surfactant amendment may en-hance the oil mobility and thereby improve the biodegradation of petroleum (Laha et al.,1995).Microbial biosurfactants,as the nat-urally occurring surfactants,can also exert some influence on interfaces in both aqueous solutions and hydrocarbon mixtures (Bordoloi and Konwar,2009).This will increa insolubility of the hydrocarbons and emulsify hydrocarbon–water mixtures (Banat,1995),resulting in a growth of oil-degrading bacteria and an improvement in bacterial ability to utilize hydrocarbons (Ron and Ronberg,2002;Bao et al.,2012).
Rhamnolipid biosurfactants have been widely reported to en-hance petroleum hydrocarbons biodegradation (Mulligan et al.,2001).They were ud to improve the bioavailability of crude oil,diel,or PAHs (Bordoloi and Konwar,2008;Providenti et al.,1995;Whang et al.,2009)in different places such as wastewater,contaminated soil and marine environments (Nikolopoulou and Kalogerakis,2008;Sponza and Gök,2009;Whang et al.,2008).The studies mainly focud on the improvement of degrading efficiency on total n -alkanes or total PAHs by rhamnolipids,and none of them reported the function of rhamnolipids in specific n -alkane,PAH or biomarker biodegradation in
oil samples.
This work prents a simulated experiment using rhamnolipids to enhance oil bioremediation with a bacterial consortium.The function of rhamnolipids in the main petroleum hydrocarbon com-ponents biodegradation was explored by gas chromatography.2.Materials and methods 2.1.Materials
2.1.1.Oil sample for biodegradation
The crude oil was obtained from Shengli Oilfield of China.This light crude oil has a viscosity of 22.2m Pa s (determined at 50°C in 50r min À1)and a density of 0.855g cm À3.It was pretreated fol-lowing the steps before ud:(1)10g crude oil was dissolved by 200ml petroleum ether (boiling point of 30–60°C,after aromatics
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Corresponding author.Tel.:+8653266782509;fax:+8653266782540.
E-mail address:mtbao@ouc.edu (M.Bao).
removal treatment)and wasfiltered by qualitativefilter paper in order to remove insoluble material,(2)thefiltrate was dried by so-dium sulfite for12h andfiltered again to remove sodium sulfite hydrates,(3)thefiltrate was heated in water bath(temperature of65±5°C)to distill most of the petroleum ether,and(4)the trea-ted oil sample was placed in vacuum desiccation chamber at65°C to remove the remaining petroleum ether.
2.1.2.Seawater sample
The awater ud for oil spill bioremediation experiment was collected from the Bathing Beach of Qingdao,Shandong Province, China.It has a pH of8.1and a salinity of32.1‰.
2.1.
3.Rhamnolipids
Rhamnolipids were produced by Pudomonas sp.0-2-2,which was previously isolated in our laboratory,the purification of the rhamnolipids ud in this study has been described previously (Wang et al.,2005).Rhamnolipids solutions(0.1g LÀ1)was pre-pared by dissolving0.1g of purified Rhamnolipids into1L of dis-tilled water.
2.1.4.Bacterial consortium ud for bioremediation
The bacterial consortium ud in this study was isolated from awater near Qingdao port of China,which consists of four strains including N1,N2,N3and N4.N1is affiliated to Ochrobactrum sp., while N2,N3and N4are affiliated to Brevibacillus sp.The nucleotide quences of16S rDNA of N1,N2,N3,and N4were stored in the GenBank databa with an accession number of HQ231209, HQ231210,HQ231211and HQ231212.The optimum growth condi-tions for the four bacteria are pH of7.5–8.5and temperature of15–30°C.They show high crude oil degradation efficiency at salinity of 20–35‰.The consortium of the four strains has a strong ability to utilize the crude oil,and the oil removal efficiency was up to74.8%.
The bacterial cells were obtained by centrifugation at6150times gravitational acceleration centrifugal force after the bacterial con-sortium incubated in LB culture broth for3days.The obtained cells were washed by distilled water and centrifuged for3times to re-move the residual nutrition in the culture broth.The purified cells was mixed with phosphate buffer to get the bacterial suspension, which was1.5of absorbance adjusted via absorbance at600nm (Bao et al.,2010),then the bacterial suspension was ready for u.
2.1.5.Media
The medium for biodegradation is1L of awater sample spiked with2g of pre-treated oil sample.They were sterilized in an autoclave at120°C before u.Three media ud in oil spill bio-remediation include:(1)1L of the described medium for control (MC),(2)1L of MC with10mL bacterial consortium suspension (prepared in Section2.1.4)for bacterial control(BC),and(3)1L of BC with10mL rhamnolipids solutions prepared in Section2.1.3 for bacterial control added with rhamnolipids(BCR).The oil in the media on the surface of the awater formed an oil slick.
2.2.Oil spill bioremediation simulated experiment
The simulation of marine oil spill bioremediation was carried out in three aquariums.Two of the aquariums werefilled with 1L MC and the other wasfilled with1L BCR.The aquariums were put in clean bench(YT-CJ-IND,Beijing Yatai-Kelong instrument technology Co.,LTD.,China)to prevent contamination at ambient temperature for30d.The temperature was allowed tofluctuate in the range of16–27°C in order to make sure the temperature condition clo to real marine environment.During this experi-ment,distilled water was added at regular intervals as the supple-ment of the evaporated water in the aquariums.No aeration equipment or agitating device was ud in this work.2.3.Oil sample processing and gas chromatography analysis
Oil samples in the biodegraded samples and the control were extracted by chloroform and concentrated by a rotary evaporator. The oil samples were vacuum dried at65°C to remove the rest of chloroform.The oil samples were accurately weighed and dis-solved in hexane,and then centrifuged at3000r minÀ1speed.
0.2mL of above oil solution was quantitatively loaded onto a3g silica gel microcolumn(200mmÂ10.5mm I.D.)which was pre-conditioned with20mL of hexane.The saturated hydrocarbons were eluted with12mL of hexane.The eluent was concentrated with a stream of nitrogen and then adjusted to accurate1.0mL. This fraction was analyzed by chromatography–flame ionization detector(GC–FID)analys and gas chromatography–mass spectra (GC–MS)for the aliphatic compounds(Sun et al.,2006,2009).多拉a梦全集
The analys of individual n-alkanes,pristane and phytane were performed on a Shimadzu GC-2010(Kyoto,Japan)with a FID detector.Analys for polycyclic aromatic hydrocarbons(PAHs) and biomarkers were performed on a Shimadzu GC–MS-QP2010.
A HP-5capillary chromatographic column(30mÂ0.32mm I.D.) and a DB-5MS capillary column(60mÂ0.25mm I.D.)were ud, respectively for the GC–FID and GC–MS.The biomarkers of s
esqui-terpenes,tricyclic and pentacyclic terpanes,steranes were deter-mined at m/z123,m/z191and m/z217,respectively.pottery
Quantitation of the n-alkanes,unsubstituted PAHs and their alkylated homologues,and biomarker compounds were accom-plished using internal standards.C24D50,D14-trichlorodiphenyl, 5-a-Androstane and17b(H),21b(H)-hopane were ud as the internal standards for the analysis of the n-alkanes,PAHs and bio-marker compounds,respectively.System control and data acquisi-tion was achieved with a GC solution and GC–MS solution software,respectively.Chromatographic conditions and quality control refer to Sun et al.(2009).
2.4.Calculation of hydrocarbon compounds removal efficiency
The removal efficiency of the hydrocarbon compounds was cal-culated by the difference of the concentration between the control and biodegraded sample divided by the concentration of the control.
3.Results and discussion
As en in Fig.1,the oil slick in BC and BCR was significantly al-tered after30d’s bioremediation,com
pared with it in MC.Espe-cially for BCR,the oil slick was completely destroyed and disperd in the medium,which mainly due to the emulsification by rhamnolipids.Tho three samples were procesd as described in Section2.2.The recovered oil from MC,BC and BCR was1.67g, 1.22g and1.13g,respectively.After bioremediation,the removal efficiency of oil by the bacterial consortia alone was26.72%,and the oil removal efficiency through combined effect of the bacterial consortia and the amendment of rhamnolipids was up to32.3%. The rhamnolipids existed in BCR enhanced the oil biodegradation efficiency by5.63%.
In order to better understand the specific role of rhamnolipids in the degradation of target hydrocarbon compounds by the bacte-rial consortium,n-alkanes,PAHs and biomakers in the three oil samples were detected by gas chromatograph.
3.1.n-Alkanes,pristane and phytane
It was obrved that only n-C13to n-C30were detected in the three oil samples.The GC chromatograms of oil samples in MC,BC and BCR were shown in Fig.2a,and the comparison of n-alkanes of
270Q.Chen et al./Marine Pollution Bulletin71(2013)269–275
three samples prented in Fig.2b.The loss of normal alkanes n-C13is attributed to be attributable tobe attributable evaporation in the experiment,and the abnce of n-alkanes -C30was due to the natural of the oil sample,becau sample is one kind of light crude oil,it doesn’t contain n-alkanes after n-C30.The total removal efficiency of
by the bacterial consortium and rhamnolipids was compared with41.7%by bacterial consortium alone.It is clear
the bacterial consortium is able to completely degrade n-alkanes in the oil samples but rhamnolipids enhanced the process of biodeg-
radation.It is interesting to note that rhamnolipids played a nega-tive role to the biodegradation of short-chain n-alkanes(C13–C15). This could be explained by volatile properties of the alkanes (Rahman et al.,2002)and emulsification of rhamnolipids.Rhamn-olipids in BCR not only enhanced the dissolubility and stability of short-chain n-alkanes in the culture broth,but also reduced volatil-ization of the alkanes.This is called‘n-alkanefixation’function. This‘n-alkanefixation’predominated over the promoting effect of rhamnolipids on biodegradation.As a result,rhamnolipids dem-onstrate a negative effect on short-chain n-alkanes biodegradation. The volatilization of alkanes decrea with i
ncrea of the length of alkyl chain.The enhancement of biodegradation by rhamnolipids was obrved for>n-C15.Rhamnolipids obviously showed a posi-tive effect on n-alkanes biodegradation from C16to C25.Rhamn-olipids had less noticeable on the biodegradation of long chain n-alkanes(C26to C30).Long chain n-alkanes were more difficult to be consumed by bacteria consortium than short chain alkanes. Although rhamnolipids could improve the solubility of alkanes in aqueous pha,it likely had no obvious effect on the metaboliza-tion of the alkanes.
Several studies reported that rhamnolipids improved n-alkanes biodegradation,however,they mainly focud on the improved ef-fects on total n-alkanes(Hua et al.,2003;Nikolopoulou and Kalo-gerakis,2008;Rahman et al.,2003),or some individual n-alkanes
The experiment photos before and after bioremediation.(a)Before
mediation and(b)after bioremediation.
Fig.2.The changes of n-alkanes,pristane and phytane distribution in MC,BC and
BCR after bioremediation detected by GC–FID.(a)The GC–FID spectrum of MC,BC
and BCR and(b)the comparison of n-alkanes in the three samples.
(hexane,decane,
studies on the
rhamnolipids in crude
Fig.2a and b also
tane and phytane.Thetragedy是什么意思
was13.4%andrfi
Their removal
39.5%,respectively,
that rhamnolipids
ciency of pristane
(Section3.3).
3.2.PAHs
Five target
were detected by
total PAHs by
of PAHs was
bacterial consortium
moval efficiency of
than that of n
prefer to biodegrade
alloys
showed positive role
biodegrading
kanes biodegradation.
Rhamnolipids
thalene,
their alkylated ries.
英语关联词大全tuted PAHs by the
total removal
tium and
uted to the volatile
alkanes
argue的名词The degradation of
that of unsubstituted
ated PAHs is61.5%,
the bacterial
played a negative
(even dimethyl and
hanced
played a positive role
alkylated compounds.
role to the
homologues.
the compounds
ber of the PAHs
Though there are
the removal of PAHs,
tion by rhamnolipid
Sponza and Gök,
olipid on alkylated
江户英语3.3.Biomarkers
The biomarkers
GC–FID(Fig.4a–c).
as squiterpenes(Fig.
of
terpanes(Fig.4f),and
the main components
ples,accounts for61.8%,64.0%and64.3%of the total biomarkers in
MC,BC and BCR samples,respectively.
Biomarkers were often ud to evaluate oil biodegradation and identify the source of spilled oil(Wang and Fingas,1997;Zakaria et al.,2001).There are few studies on the function of rhamnolipids in biomarkers biodegradation.The removal efficiency of total bio-markers by the bacterial consortium is36.3%,significantly lower than that of n-alkanes or PAHs.Rhamnolipids could dramatically enhance the degradation of biomarkers.The total biomarkers re-moval efficiency by the bacterial consortium and rhamnolipids in-creas to58.3%,which is22.0%higher than that by the bacterial consortium alone.However,rhamnolipids played a negative role in terpanes biodegradation with simple structure such as Fig.3.The changes of PAHs distribution in MC,BC and BCR after bioremediation.(a) The PAHs GC–MS spectrum of MC,BC and BCR and(b)the comparison of PAHs in the three samples.
272
The biomarkers GC–MS spectra of MC,BC and BCR after bioremediation.(a)m/z123,(b)m/z191,(c)m/z217,(d)the comparison of squiterpenes in (e)the comparison of tricyclic terpanes in the three samples,(f)the comparison of pentacyclic terpanes in the three samples and(g)the comparison of
samples.
