天然菌群、生物刺激和生物强化修复烃污染南极土壤的有效性研究-

1、 International Biodeterioration &Biodegradation 52(2003115125E ectivenessofthe natural bacterial ora,biostimulation and bioaugmentation on the bioremediation ofa hydrocarboncontaminated Antarctic soilLucas Ruberto a , Susana C. Vazquez a , Walter P. Mac Cormack b ; a Facultad de Farmacia y Bioqu

2、Ã mica, Universidad de Buenos Aires, Argentina b InstitutoAntÃa rtico Argentino, Cerrito 1248Buenos Aires (1010,ArgentinaReceived 14June 2002; received in revised form 6January 2003; accepted 8January 2003AbstractMicrocosms systems (250g soil in 1l askswere performed in Jubany Station (Kin

3、gGeorge Island, South Shetland Islands to analysebiodegradation ofgas-oil in Antarctic soils under natural conditions. Abiotic loss ofhydrocarbons, biodegradation activity ofindigenous micro oraand biostimulation with N and P were studied. In addition, biaugmentation with a previously isolated psych

4、rotolerant strain (B-2-2was analysed. Hydrocarbon concentration, heterotrophic and hydrocarbon-degrading bacterial counts and predominant bacterial groups were evaluated during 51days. A signiÿcantloss ofhydrocarbons was observed in abiotic control. Indigenous micro orashowed increased heterotr

5、ophic counts and hydrocarbon-degrading/heterotrophicratio. This fact was associated with a signiÿcantdegrading activity (35%higher than the control. Bioaugmentation with B-2-2strain improved the bioremediation e ciency(75%ofthe hydrocarbon was removed. High levels ofN and P produced an initial

6、inhibition ofbacterial growth. bacterial diversity was reduced in contaminated soil. Our results showed that bacterial orafrom Antarctic soils is able to degrade an important fraction of the gas-oil and that bioaugmentation represents a valuable alternative tool to improve bioremediation. ? 2003Else

7、vier Ltd. All rights reserved.Keywords:Bioremediation; Antarctic soils; Hydrocarbon-degrading bacteria1. IntroductionDuring the last two decades many investigations were performed to determine the persistence of hydrocarbons in di erentnatural environments and the possible role ofthe in-digenous mic

8、ro oraon the degradation rate ofthese contam-inants (K a stner et al., 1994; Trzesicka-Mlynarz and Ward, 1996; Solano-Serena et al., 2000. Working with many di erentsystems (soil,freshwater, seawater these investi-gations showed that a small fraction of all natural bacterial communities grew on hydr

9、ocarbons. The size ofthis community depends on many di erentbiotic and abiotic factors but principally on the adaptive capacity of the microorganisms and previous exposure to the kind ofhy-drocarbon present in their habitats (Liu and Su ita,1993. This adaptation makes the chronically contaminated ar

10、easCorresponding author. Fax:+54-011-4508-3645.the choice sites for the screening of bacterial strains po-tentially useful in the design of bioremediation processes (Ashok et al., 1995; Aitken et al., 1998. However, the capacity ofthe isolated and inoculated bacteria to survive and carry out the bio

11、degradation activity under conditions prevailing in the polluted site undergoing the bioremedi-ation process is critical (Shannon and Unterman, 1993; Vogel, 1996. This is even more crucial under conditions ofextreme environment. Cold environments represent the most widespread situations. An importan

12、t fraction of the petroleum hydrocarbon produced worldwide are extracted and processed in cold areas. In the Antarctic continent, although petroleum exploitation is not permitted, the im-portant scientiÿcand logistic activities represent high risk ofpollution in an environment where temperature

13、 and other climate factors, strongly limit bacterial growth and activity. In fact, contaminated areas near the fuel tanks in the stations, as well as more signiÿcantspills in Antarctic lands and coastal waters have been reported (Cripps and0964-8305/03/$-see front matter ? 2003Elsevier Ltd. All

14、 rights reserved. doi:10.1016/S0964-8305(0300048-9116L. Ruberto et al. /International Biodeterioration &Biodegradation 52(2003115125Priddle, 1991; Green and Nichols, 1995; Aislabie, 1997. In Antarctica, bioremediation could only be carried out by psychrophilic and psychrotolerant microorganisms

15、as was indicated by several previous studies (Wardell, 1995; Aislabie et al., 1998, 2001. The objective ofour study was to analyze the response ofthe indigenous soil bacterial orato the presence of gas-oil (thefuel most often used in Argentine scientiÿcstations. For this purpose micro-cosms sys

16、tems with Antarctic soil supplemented with N and P (biostimulationwere used. In addition, the e ectofthe presence ofa previously described psychrotolerant hydrocarbon-degrading bacteria (B2-2strain on the hydro-carbon biodegradation rate (bioaugmentationwas studied. 2. Materials and methods2.1. Stud

17、y area and chemical analysis of soilStudies were carried out in summer (JanuaryFebruary1996 in Jubany scientiÿcstation (6214 S ; 5840 W lo-cated on Potter Cove, King George Island, South Shetland Islands. Jubany station is at high risk ofpollution, prin-cipally by jet fuel and gas-oil (Mac Corm

18、ack and Fraile, 1997.Soil used in this study was collected in Potter Peninsula, far from station facilities to minimise the presence of anthro-pogenic hydrocarbons. Soil was sieved (2mm-mesh screen and this fraction was analysed for texture (Gee and Bauder, 1986, pH (Seppelt, 1992, water content, to

19、tal and soluble carbon (Richter and von Wistinghausen, 1981, total nitro-gen (Bremner and Mulvaney, 1982 and phosphorus (Bray and Kurtz, 1945. Hydrocarbon traces from human activity as well as the presence ofnatural levels ofthese compounds in Antarctic soils (Cripps and Priddle, 1991, were deter-mi

20、ned by extraction in CCl 4and quantiÿcationby FT-IR spectrometry following EPA method 418-1(USEPA, 1983. Total hydrocarbon concentration (THCin soil during the study was determined by triplicates (10g ofsoil each f rom microcosms using the same methodology. Results are ex-pressed per gram ofdry

21、 soil. Samples f rom microcosms were placed in acid washed sterile vials and stored at 20C until analysis.Table 1Characteristics of the di erentmicrocosms performed for the hydrocarbon degradation test with Antarctic soilCondition HgCl 2Gas-oil pH controlN added P added B-2-2a (g(ml(mgkg 1(mgkg 1(ml

22、B A 15.215.225.2+35.2+180050045.2+3.055.2+18005003.0a Celldensity ofB-2-2suspension used to inoculate the soil was 1:1×109CFU ml 1.2.2. Microcosms designIn Table 1we summarise all the conditions performed in the microcosms systems. Six one-litre sterile asks(11cm internal diameter were ÿll

23、edwith 250g (2:5cm height ofthe sieved soil contaminated with 1.5%w/wgas-oil (5:2ml, =0:73g cm 3. An additional control askwas maintained uncontaminated (conditionB. Microcosms were exposed to the Antarctic climate conditions (withouttemperature and water content control during 51days. Activity ofth

24、e indigenous bacterial orawas observed in condition 2 ask.Nitrogen (1800mg kg 1 as NaNO 3and phosphorous (500mg kg 1 as Na 2HPO 4were added to two asks(biostimulatedsoil in order to evaluate the in- uenceofthese nutrients on the growth ofthe indigenous micro oraand the inoculated strain (conditions3

25、and 5, respectively. Biostimulation was performed due to the low levels ofN and P showed by this Antarctic soil (Mac Cormack, 1999, information that was conÿrmedby the chemical analysis ofsoil (seeresults section. N an P were added to reach a C:N:Pratio of100:12:3in the contami-nated soil. This

26、 fact resulted in higher initial concentration ofN and P than those used by several authors in previous re-ports (Wrenn et al., 1994; Aislabie et al., 2001; Mohn et al., 2001. The pH ofthe soil used in this study was 7.1and this value was maintained (inthe systems with pH control by addition ofsteri

27、le 0.1M NaOH solution. One ofthe askswas not pH controlled (condition1. An abiotic control ask(conditionA was prepared by addition to the contam-inated soil of1g ofHgCl 2as biocide (Trzesicka-Mlynarz and Ward, 1996; Solano-Serena et al., 2000. Bioaugmen-tation was studied (conditions4and 5 by inocul

28、ation of soil with B-2-2, a psychrotolerant hydrocarbon-degrading Acinetobacter strain previously isolated from a chronically polluted river (Espeche et al., 1994. This strain showed high degradation activity, mainly on gas-oil and other aliphatic-rich fuels that represent the main pollution sources

29、 near the stations. Inoculum was prepared from cultures of B2-2in saline basal medium (Espeche et al., 1994 with gas-oil (1%w/vat 15C. After 40h (endof exponential growth phase cells were centrifuged (10000×g ; 10min and the pellet was resuspended in saline solution (NaCl0.9%to a cell density o

30、f1:1×109CFU ml 1. ThisL. Ruberto et al. /International Biodeterioration &Biodegradation 52(2003115125117Table 2Main climatic parameters at which soil and microorganisms were exposed during the experimental period in Jubany StationJanuaryFebruary(untilday 19th Temperature (CAverage +2:10+2:8

31、8Maximum +10:10+8:40Minimum 1:402:00Relative humidity (%0.000.00suspension ofB-2-2strain was added to soil in microcosms under bioaugmentation in order to reach a cell density of 1:3×107CFU g 1ofdry soil.Since climate conditions given during the experiment was one ofthe major f actors that dete

32、rmined some ofthe re-sults observed, we considered it relevant to show the values observed for the main climatic parameters in January and the part ofFebruary during which the assay was developed. These data are shown in Table 2and were provided by the synoptic meteorological station located in Juba

33、ny Base. 2.3. Microbiological analysisSoil samples (1g were suspended in 10ml ofsterile saline solution (0.9%NaCl and vigorously shaken for 5min. Serial dilutions (102106 were prepared in saline solution for microbiological analysis.Heterotrophic aerobic bacteria (HABin soil samples were determined

34、(fourreplicates on caseinpeptonestarch(CPSagar, as was suggested by Wynn-Williams (1992for Antarctic soils analysis. Hydrocarbon-degrading bacteria (HDBwere enumerated on solidiÿedsaline basal medium (SBMsupplemented with 2%gas-oil as sole carbon source. SBM composition was previously reported

35、(Espeche et al., 1994. For both HAB and HDB counts, plates were incu-bated 30days at 4C and results expressed as colony forming units per gram ofdry weight (CFUg 1. Bacterial counts data from the di erentmicrocosms were analysed by re-peated measured ANOVA and Tukeysmultiple comparison test.From CPS

36、 and SBM-gas-oil agar plates at 0, 14and 51days, 30colonies were picked at random and puriÿedby re-streaking. These isolates were tested for morphology, mo-bility, Gram reaction, catalase production, oxidase reaction, spore formation and glucose breakdown under aerobic andanaerobic conditions.

37、Subsequently, isolates were charac-terised using the API system kits (Biomerieux.The objec-tive ofthis part ofthe work was not to make an exhaustive taxonomic analysis ofall isolates strains but to group them into dominant clusters present in the analysed samples. 2.4. Ampliÿcation,cloning and

38、sequencing of 16S rRNA geneStrain B2-2was isolated from a chronically polluted environment and its growth capability under di erentphysicochemical conditions was previously analysed (Espeche et al., 1994. The 16S rRNA gene was am-pliÿedfrom B-2-2strain genomic DNA by PCR using 5 -CCGAATTCGTCGAC

39、AACAGAGTTTGATCCTGGC-TCA-3 as forward primer and 5 -CCCGGGATCCAAGCT-TAAGGAGGTGATCCAGCC-3 as the reverse one. PCR was performed in 100 l reaction mixture consisted of 1 l DNA template solution (1mg m 1, 16 l ofeach primer solution (5pmol l 1, 10 l (eachdeoxynucle-oside triphosphates (2:5mM, 2:5U Taq p

40、olymerase in 10 l 10×reaction bu erwithout MgCl 2(Promegaand 10 l 25mM MgCl 2. Each ofthe 25ampliÿcationcycles included denaturation at 94C for 1min, annealing at 47C for 2min and extension at 72C for 2min. The ÿnalex-tension was performed at 72C for 8min. PCR products were puriÿ

41、edwith the Gen-Clean II kit and cloned into the SalI/BamHI-digestedplasmid pUC-18. Plasmid was used to transform competent Escherichia coli DH5 using standard techniques (Sambrook et al., 1989. The 5 and 3 ends of16S rRNA gene (491and 437base pairs, respec-tively were sequenced with an ABI Prism 377

42、automatic equipment. Previously published 16S rRNA gene sequences were obtained from EMBL/GenBank/DDBJdatabase. Both, B-2-2and reference strains 16S rRNA gene sequences were aligned with the CLUSTAL W program (Thompson et al., 1994 and phylogenetic inferences were made with SEQ-BOOT (forbootstrap an

43、alysis, NEIGHBOR (forneighbour joining analysis and DNADIST (formaximum likelihood estimates ofdistance programs f rom PHYLIP package (Felsestein, 1993.2.5. Nucleotide sequence accession numbersSequences corresponding to the 5 and 3 ends ofthe 16S rRNA gene from B-2-2strain used to the phylogenetic

44、inferences have been deposited in the GenBank database un-der accession numbers AY083506and AY083507, respec-tively. Accession numbers ofthe strains used to construct the phylogenetic tree are the folowings:U10874(A anitra-tus , X81661(A calcoaceticus , X81662(A haemolyticus , Z93437(A junii , AF188

45、302, (A lwo , U37348(Acineto-bacter sp., AF188300(A johnsonii , AB018487(Bacillus subtilis ATCC 21331.118L. Ruberto et al. /International Biodeterioration &Biodegradation 52(2003115125100Bacillus subtilisAcinetobacter anitratusAcinetobacter calcoaceticusAcinetobacter haemolyticusAcinetobacter ju

46、niiAcinetobacter lwoffiB-2-2Acinetobacter sp.Acinetobacter johnsonii88473899910009398711000Fig. 1. Neighbour-joining tree based on partial 16S rRNA gene sequences showing relationships between B-2-2and the most closely related species. The numbers at the nodes indicate the levels ofbootstrap support

47、 based on neighbour-joining analyses of1000resampled data sets. Bar indicates 0.01nucleotide substitution per position. Bacillus subtilis ATCC 21331was used as outgroup strain.3. Results3.1. Characterization of B-2-2strainPhysiological and molecular analyses showed that hydrocarbon-degrading strain

48、B-2-2belongs to the genus Acinetobacter. Partial sequencing ofthe 16S rRNA gene showed maximal similarity with Acinetobacter sp. and A. johnsonii (Fig.1. 3.2. Analysis of soilCharacterisation ofsoil showed the f ollowing results:pH, 7.10; water content, 10%;total C, 0.51%;total N, 0.054%;P, 14.6ppm;

49、 sand, 93.3%;silt, 4.00%;clay, 2.7%.3.3. Hydrocarbon concentration in microcosmsChanges in total hydrocarbon concentration are shown in Fig. 2. An important decrease in the hydrocarbon con-centration was observed in all systems, including the abi-otic control, during the ÿrst10days ofstudy (not

50、shown in Fig. 2. This fact decreased the initial hydrocarbon concen-tration in the soil from 14380ppm to the values observed inFig. 2at day 10, determining a percentage loss ranging between 54%and 61%depending on the treatment. This sig-niÿcantloss could be related to the volatilisation and str

51、ip-ping rates ofthe most volatile compounds ofthe gas-oil. No di erenceswere found among treatments in hydrocar-bon concentration values at this time (day10. Evidently the high rate ofabiotic elimination in all systems minimised the e ectcaused by the microbial metabolism during this initial period.

52、 Hydrocarbon concentration in the abiotic con-trol decreased until day 20. From this time to the end ofthe study THC in the control showed no signiÿcantchanges, 30%ofthe initial concentration ofhydrocarbons remaining. All biotic conditions showed a decrease in the hydrocarbon content. Fertilisa

53、tion with N and P determined higher values ofresidual THC whenever these nutrients were added com-pared with the corresponding non supplemented condition. At the end ofthe assay, these di erences(betweenf ertilised and non-fertilised systems were signiÿcant(p ¡0:05 in the case ofthe indige

54、nous micro ora(conditions2and 3 but showed no di erencesbetween the systems where B-2-2was used for bioaugmentation (conditions4and 5. Com-pared with the abiotic control, activity ofthe indigenous mi-cro orareduced 35%ofthe THC whereas bioaugmentation with B-2-2strain caused a decrease of65%.Includi

55、ng the abiotic elimination and the basal value ofthe non-pollutedL. Ruberto et al. /International Biodeterioration &Biodegradation 52(2003115125119 0100020003000400050006000700061218243036424854T o t a l h y d r o c a r b o n s (µg /g Fig. 2. Total hydrocarbons concentration in the di erent

56、microcosms performed to analyse bioremediation of gas-oil contaminated soil at Jubany Station. Initial concentration in all microcosms at day 1was 14380 g g dryweight1(notshown. Bar represents SD oftriplicates.soil, the fraction of the contaminant that was eliminated from the soil at the end ofthe s

57、tudy was 75%.It is important to remark that one day after the start of the study, bacterial concentration in the askwhere B-2-2strain was inoculated was 7:4×106CFU ml 1indicating that an important fraction of inoculum was not recovered. 3.4. Bacteriological countsHAB counts value ofthe soil use

58、d in this study was 2:2×106CFU g 1. Changes in the HAB counts in the di erentassayed conditions are shown in Fig. 3. After the addition ofHgCl 2to the abiotic control, no counts were de-tected all along the study (datanot shown. In Fig. 3, the ÿrstpoint represents the counts obtained in da

59、y 1, 24-h after ad-dition ofthe di erentcomponents ofeach assayed condition. HAB counts in the uncontaminated soil remained nearly constant throughout the assay. The presence ofgas-oil in the soil determined an increase in the bacterial counts (com-pared with the uncontaminated control that was signiÿcant(p ¡0:05 at day 28. At this time, 9:8×106CFU g 1were observed in contaminated soil and 2:0×106CFU g 1in the control. Addition ofN and P produced a decrease in HAB counts in all b