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Bioremediation of polycyclic aromatic hydrocarbon-contaminated saline–alkaline soils of the former Lake Texcoco

Polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene, anthracene and Benzo[a]pyrene (BaP) are toxic for the environment. Removing these components from soil is difficult as they are resistant to degradation and more so in soils with high pH
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  Bioremediation of polycyclic aromatichydrocarbon-contaminated saline–alkalinesoils of the former Lake Texcoco L.A. Betancur-Galvis a,b , D. Alvarez-Bernal a ,A.C. Ramos-Valdivia a , L. Dendooven a,* a Laboratory of Soil Ecology, Department of Biotechnology and Bioengineering, Cinvestav, Me´xico D.F., C.P. 07360 Me´xico b Grupo Infeccio´ n y Ca´ ncer, Facultad de Medicina, Universidad de Antioquia, Medellı´n-Colombia Received 12 October 2004; received in revised form 14 April 2005; accepted 9 July 2005Available online 9 September 2005 Abstract Polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene, anthracene and Benzo[ a ]pyrene (BaP) are toxic forthe environment. Removing these components from soil is difficult as they are resistant to degradation and more so insoils with high pH and large salt concentrations as in soil of the former lake Texcoco, but stimulating soil micro-organ-isms growth by adding nutrients might accelerate soil restoration. Soil of Texcoco and an agricultural Acolman soil,which served as a control, were spiked with phenanthrene, anthracene and BaP, added with or without biosolid or inor-ganic fertilizer (N, P), and dynamics of PAHs, N and P were monitored in a 112-day incubation. Concentrations of phenanthrene did not change significantly in sterilized Acolman soil, but decreased 2-times in unsterilized soil and>25-times in soil amended with biosolid and NP. The concentration of phenanthrene in unsterilized soil of Texcocowas 1.3-times lower compared to the sterilized soil, 1.7-times in soil amended with NP and 2.9-times in soil amendedwith biosolid. In unsterilized Acolman soil, degradation of BaP was faster in soil amended with biosolid than in una-mended soil and soil amended with NP. In unsterilized soil of Texcoco, degradation of BaP was similar in soil amendedwith biosolid and NP but faster than in the unamended soil. It was found that application of biosolid and NP increaseddegradation of phenanthrene, anthracene and BaP, but to a different degree in alkaline–saline soil of Texcoco comparedto an agricultural Acolman soil. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Agricultural soil; Biosolid; Dynamics; Fertilizer; Microcosms assays; Sequestration 1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are a classof toxic environmental pollutants that have accumulatedin the environment principally due to anthropogenicactivities. Benzo[ a ]pyrene (BaP), a five-ring compound,has been classified by the US Environmental Protection 0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.chemosphere.2005.07.026 * Corresponding author. Tel.: +52 5 7477000; fax: +52 57477002. E-mail address: dendoove@cinvestav.mx(L. Dendooven).Chemosphere 62 (2006) 1749–1760www.elsevier.com/locate/chemosphere  Agency (USEPA, 1996, 1999) as a priority pollutant onthe basis of its known carcinogenicity, teratogenicityand acute toxicity (Juhasz and Naidu, 2000). Multipleanimal studies with many species have demonstratedthe carcinogenicity of BaP (IARC, 1983). Other PAHs,that are currently not considered as toxic and do not ap-pear in the Resource Conservation and Recovery Act(RCRA) hazardous waste lists, however do affect humanhealth. Phenanthrene and anthracene are skin and eyeirritants, whose effects increase in sunlight due to photo-sensitization (IARC, 1983).PAHs are persistent in terrestrial ecosystems andtheir carcinogenic properties have resulted in numerousstudies of contaminated soils with different approachesto bioremediate them (Breedveld and Sparrevik, 2000;Bogan et al., 2001; Atagana et al., 2003; Bengtssonand Zerhouni, 2003). Although a great diversity of organisms are capable of degrading low-molecular-weight PAHs, relatively few genera have been foundto degrade the high-molecular-weight ones (Cerniglia,1984, 1992; Walter et al., 1991; Juhasz et al., 1996;Schneider et al., 1996) and their activity towards high-molecular-weight PAHs is affected by site-specific envi-ronmental factors (Pignatello and Xing, 1996; Juhaszand Naidu, 2000).Alkaline saline soils are unique and extreme environ-ments (Ramirez-Fuentes et al., 2002; Luna-Guido et al.,2003; Vega-Jarquin et al., 2003) where processes such asnitrification and degradation of cellulose are inhibited(McClung and Frankenberger, 1987; Luna-Guidoet al., 2003; Vega-Jarquin et al., 2003). Little is knownabout degradation potential of PAHs in such an envi-ronment and factors controlling it, i.e. supply of macronutrients. An inhibitory effect of artificial salinityon mineralization of oil has been reported (Rhykerdet al., 1995).Mille et al. (1991)found an inhibitory effect of salinity above 2.4% w/v NaCl that was greater for thebiodegradation of aromatic and polar fractions than forthe saturated fraction of petroleum hydrocarbons. How-ever, different results have been obtained when investi-gating naturally salt-containing soils, since indigenousmicroorganisms in such environments are expected tobe salt-adapted (Bertrand et al., 1990, 1993; Geiselbrechtet al., 1998).Jackson and Pardue (1999)found low de- gradation rates (0–3.9% day À 1 ) for alkane components(C11–C44) in salt marshes, but high rates (8–16% day À 1 )for PAHs.Bertrand et al. (1990)observed thathalophilic microorganisms, i.e. archaea, degradedPAHs (acenaphthene, naphthalene and phenanthrene;500 mg l À 1 ) in a medium prepared with natural hypersa-line water from salt marsh.Jackson and Pardue (1999)demonstrated high degradation rates of naphthaleneand phenanthrene in microcosm laboratory studies.Mineralization rates of phenanthrene ranged from0.5% to 4.5% day À 1 in unfertilized soil, but increasedto 1.9–12.1% day À 1 in fertilized ones (Jackson andPardue, 1997, 1999). Other studies, however, haveshown that nutrients did not stimulate biodegradationrates of PAHs with more than two rings in salt marshes(Wright et al., 1997; Lin et al., 1996, 1999; Xu and Ob-bard, 2004). Not only addition of nutrients might affectdegradation of PAHs, butChen and Aitken (1999)havedemonstrated that an added co-substrate or carbonsource increased mineralization of PAHs and especiallyBaP.Salt-affected soils represent about 40% of the world Õ slands (Serrano and Gaxiola, 1994; Zahran, 1997) andPAHs are ubiquitous in terrestrial ecosystems. Contam-ination of soil with hydrocarbons and PAHs often oc-curs in Mexico, one of the most important oilproducing countries, and a lot is known about remediat-ing soil, but there is little or no information about bior-emediating alkaline–saline soils contaminated withPAHs. The soil at former lake Texcoco which lies some-what east of Mexico City is alkaline–saline. It wasdrained from the 17th century onwards to reduceinundation of the city, and an area of 5000 ha of alka-line–saline soil with a pH > 8 on the srcinal sedimentof volcanic ash was created with little or no vegetation.The large electrolytic conductivity (EC) and high pH of the soil inhibits microbial activity (Beltra´n-Herna´ndezet al., 1999; Ramirez-Fuentes et al., 2002; Luna-Guidoet al., 2003) and addition of inorganic N increaseddecomposition of glucose and maize (Conde-Barajaset al., 2005). Addition of nutrients and biosolid mightthus stimulate microbial activity and allow greater de-gradation of PAHs. In this study soil of former lake Tex-coco was used as a model to investigate the effect of mineral nutrients, N and P, and biosolid, which containslarge amounts of nutrients and organic material, in therestoration of hydrocarbon-contaminated alkaline–sal-ine soil. Anthracene, phenanthrene and BaP were addedunder laboratory conditions to soil of the former lakeTexcoco; dynamics of inorganic N (NH þ 4 and NO À 3 ),PO 3 À 4 and degradation of PAHs were then monitoredin an aerobic incubation at 22 ± 2 ° C for 112 days. Asoil under cultivation with maize and located near theformer lake was used as a control. 2. Materials and methods  2.1. Experimental site, collection and characteristicsof soil  Details of the first experimental site located in theformer lake of Texcoco in the valley of Mexico City(Mexico) (N.L. 19 ° 30 0 , W.L. 98 ° 53 0 ) at an altitude of 2250 m above sea level with a mean annual temperatureof 22–24 ° C and mean annual precipitation of 705 mm(http://www.inegi.gob.mx) can be found inLuna-Guido et al. (2000). Briefly, the soil is alkaline–saline, NaCl and 1750 L.A. Betancur-Galvis et al. / Chemosphere 62 (2006) 1749–1760  Na 2 CO 3 being dominant, with pH between 8.5 and 10.5,EC in saturation extracts ranging from 4 and 70 dS m À 1 and large exchangeable sodium percentage (60–80%).Soil was sampled at random by augering the 0–15 cmtop-layer of three plots of approximately 0.5 ha (Table1). The soil from each plot was pooled.The second experimental site, which served as a con-trol, is located near the ex-convent of Acolman in theState of Me´xico, Me´xico, (N.L. 19 ° 38 0 , W.L. 98 ° 55 0 )near the former lake Texcoco. Its average altitude is2250 m above sea level and characterized by a sub-humid temperate climate with a mean annual tempera-ture of 14.9 ° C and average annual precipitation of 624 mm mainly from June through August (http://www.inegi.gob.mx). Briefly, the soil is clayey with pH6.3 and EC 0.8 dS m À 1 , and that area is mainly culti-vated with maize and that for >20 years, receiving aminimum amount of inorganic fertilizer without beingirrigated (http://www.inegi.gob.mx)(Table 1). This soil was selected as contamination with petroleum had oc-curred in this area when an oil duct ruptured, but noton sampled fields. Soil was sampled at random by auger-ing the 0–15 cm top-layer of three plots of approxi-mately 0.5 ha (Table 1). The soil from each plot waspooled. In total six soil samples were obtained, threefrom Acolman and three from Texcoco.The soil was taken to the laboratory and treated asfollows. The soil from both sites and each plot waspassed separately through a five-mm sieve, adjusted to40% water holding capacity (WHC) by adding distilledwater (H 2 O) and conditioned at 22 ± 2 ° C for 10 daysin drums containing a beaker with 100 ml 1 M sodiumhydroxide (NaOH) to trap CO 2 evolved, and a beakerwith 1000 ml distilled H 2 O to avoid desiccation of thesoil.  2.2. Characteristics of the biosolid, PAHs Anthracene, phenanthrene, BaP, (NH 4 ) 2 SO 4 andKH 2 PO 4 were purchased from Sigma Chemical Com-pany (USA). The organic C content was 940 g C kg À 1 for phenanthrene, 940 g C kg À 1 for anthracene and950 g C kg À 1 for BaP.Details of where the biosolid was obtained from canbefoundinFranco-Herna´ndez etal.(2003). Briefly,Rec-iclagua (Sistema Ecolo´gico de Regeneracio´n de AguasResiduales Ind., S.A. de C.V.) in Lerma, State of Me´xico(Me´xico) treats waste water from different companies.Ninety percent of the waste water is of industrial srcin,mainly from textile industries, and the rest from house-holds. The waste water from each company has to com-ply with the following guidelines: biological oxygendemand <1000 mg l À 1 , lipids <150 mg l À 1 and phenol<1 mg l À 1 . The collected wastewater is digested aerobi-cally in a reactor. The biosolid obtained after the addi-tion of a flocculant is passed trough a belt filter toreduce water content. Twenty kg of aerobically digestedindustrialbiosolidwassampledasepticallyinplasticbagsafter it passed through the belt filter. Heavy metal con-centrations in the biosolid were low (Franco-Herna´ndezet al., 2003) making these biosolid of excellent quality(USEPA, 1994), as were concentrations of toxic organiccompounds (Reciclagua, Personal communication)(Table 2). The biosolid can be classified as a class ‘‘B’’biosolid (Franco-Herna´ndez et al., 2003) considering itspathogen content (USEPA, 1994).  2.3. Chemical and microbiological analyses Soil pH was measured in 1:2.5 soil–H 2 O suspen-sion using a glass electrode (Thomas, 1996). Large Table 1Some characteristics of soil of Texcoco and AcolmanSite pH WHC b Carbon TotalnitrogenEC a (dS m À 1 )Particle size distribution TexturalclassificationOrganic Inorganic Clay Silt Sand(g kg À 1 soil)Acolman 6.3 896 19.0 0.6 1.4 0.8 440 300 260 ClayeyTexcoco 10.0 600 38.8 8.0 2.9 12 54 87 859 Loamy sand a EC: electrolytic conductivity. b WHC: water holding capacity.Table 2Some characteristics of the biosolidpH WatercontentOrganic C Total N Total P Available P NH þ 4 Total heavy metalsPb Cu Cr Cd(g kg À 1 dry biosolid)6 660 499 41 5.1 0.400 3.071 0.019 0.029 0.298 0.019 L.A. Betancur-Galvis et al. / Chemosphere 62 (2006) 1749–1760 1751  concentrations of Cl À in soil of Texcoco might affect thedetermination of carbon (C) by wet oxidation (Nelsonand Sommers, 1996), so the total C in soil was deter-mined by oxidation with potassium dichromate andtrapping the evolved CO 2 in NaOH, followed by titra-tion with 0.1 M HCl (Amato, 1983). Inorganic C in soilwas determined by adding 20 ml 1 M HCl solution to 1 gair-dried soil and trap CO 2 evolved in 20 ml 1 M NaOHand then titrate. The organic C was defined as the differ-ence between total and inorganic C. Total N was mea-sured by the Kjeldahl method (Bremner, 1996), soilparticle size distribution by the hydrometer method asdescribed byGee and Bauder (1986), and cation ex-change capacity with the barium acetate method (Jack-son et al., 1986). Total P was measured by aqua regia digestion with sodium carbonate fusion (Crosland et al.,1995). The WHC was measured on soil samples water-saturated in a funnel and left to stand overnight. NH þ 4 and nitrate (NO À 3 ) in the K 2 SO 4 extracts were deter-mined colourimetrically (Mulvaney, 1996). Available Pwas determined as described byMurphy and Riley (1962).Concentrations of phenanthrene, anthracene andBaP in the soil were analyzed using an ultrasonic extrac-tion method developed bySong et al. (1995). The 1.5 gsub-sample of soil was mixed with 3 g of anhydrous so-dium sulphate to form a fine powder, and placed in aPyrex tube; 12 ml acetone was added. The tubes wereplaced in a sonicating bath at 35–40 ° C for 20 min,mechanically shaken on a vortex for 15 s, and sonicatedagain for 20 min. The extracts were separated from thesoil by centrifugation at 3500 · rpm for 15 min. Thisprocess was repeated three times. The extracts were com-bined, evaporated, and dissolved in 1 ml acetone. Fromeach tube, a 2.0 l l aliquot was immediately analyzed forPAHs on a Hewlett–Packard 4890-D GC (USA) fittedwith a flame ionization detector. A HP-5 column fromHewlett–Packard (USA) with length 15 m, inner diame-ter 0.53 mm and film thickness 1.5 l m was used to sep-arate the PAHs with carrier gas He flowing at a rateof 7 ml min À 1 . The oven temperature at 140 ° C was in-creased to 170 ° C at a rate of 2 ° C min À 1 , maintained at170 ° C for 5 min and increased to 280 ° C at 30 ° C min À 1 and maintained at 280 ° C for 10 min. The temperatureof the injector was 280 ° C and that of the detector300 ° C.The percentage recovery of the PAHs was tested byadding 1.5 g dry soil of each of the six soil samples (intriplicate) to a Pyrex tube and spiking them with280 mg BaP kg À 1 dry soil, 1200 mg phenanthrene kg À 1 dry soil, 520 mg anthracene kg À 1 dry soil. PAHs in thesoil were then extracted for the added PAHs as de-scribed above.Total metals in the biosolid were determined aftermicrowave digestion (Q Lab 6000, Questron) with0.5 g soil, 10 ml nitric acid (HNO 3 ) and 2 ml 10% hydro-gen peroxide (H 2 O 2 )(USEPA, 1998method 3051). Total Pb, Cu, Cr, and Cd were measured by flame atom-ic absorption spectrometry (Varian SpectrAA220 FastSequential). The plastic beakers used for analysis of met-als were new and treated with 2% HNO 3 for 24 h beforeuse.  2.4. Treatments and experimental set-up Sub-samples (105) of 30 g soil of each of the six soilsamples were added to 120 ml glass flask. Twenty-oneflasks were used for each of the five treatments. In a firsttreatment soil was spiked with 280 mg BaP kg À 1 dry soil,1200 mg phenanthrene kg À 1 dry soil and 520 mg anthra-cene kg À 1 dry soil. In a second treatment soil was spikedwith 280 mg BaP kg À 1 dry soil, 1200 mg phenanthrenekg À 1 dry soil and 520 mg anthracene kg À 1 dry soil andamended with 108 g dry biosolid kg À 1 dry soil (pH 6and water content of 660 g H 2 O kg À 1 ). In a third treat-ment soil was spiked with 280 mg BaP kg À 1 dry soil,1200 mg phenanthrene kg À 1 dry soil and 520 mg anthra-cene kg À 1 dry soil and amended with 300 mg(NH 4 ) 2 SO 4 -N kg À 1 dry soil plus 39 mg KH 2 PO 4 -Pkg À 1 dry soil. In a fourth treatment was spiked with280 mg BaP kg À 1 dry soil, 1200 mg phenanthrene kg À 1 dry soil and 520 mg anthracene kg À 1 dry soil andamended with 108 g dry biosolid kg À 1 dry soil and ster-ilized. In a fifth treatment unamended soil was usedwhich served as control. The amount of solution or bio-solid added was such that the water content of the soilwas adjusted to approximately 60% WHC. The amountof NH þ 4 -N and PO 3 À 4 -P contained in the biosolid addedwas the same added with the (NH 4 ) 2 SO 4 - KH 2 PO 4 solution, i.e. 300 mg N and 39 mg P. The amount of Cadded with the PAHs and biosolid was 1.89 g kg À 1 and 54 g kg À 1 respectively. The remaining 21 flasks wereadded with 1.95 g dry biosolid kg À 1 dry soil (108 g drybiosolid kg À 1 dry soil) and sterilized three times for30 min, with an interval of a day, with pressurized steamat 121 ° C supplied by an autoclave (Wolf and Skipper,1996). The sterilized soil was then spiked with 280 mgBaP kg À 1 , 1200 mg phenanthrene kg À 1 , and 520 mganthracene kg À 1 under sterile conditions to investigateabiotic factors that might affect dynamics of hydrocar-bons, NH þ 4 and available P. Three flasks were chosenat random from each treatment. Ten g soil was extractedfor inorganic-N with 100 ml 0.5 M K 2 SO 4 solution, sha-ken for 30 min, filtered through Whatman Ò no 42 filterpaper and analyzed, while 1.5 g soil was extracted forPAHs with acetone and analyzed on a GC. The remain-ing 18.5 g soil was extracted for soluble phosphorus with50 ml of 0.5 M NaHCO 3 . These provided zero-timesamples.The remaining flasks were placed in 945 ml glass jarscontaining 10 ml distilled H 2 O. The jars were sealed andstored in the dark for 112 days 22 ± ° C. After 7, 14, 28,56 and 112 days, three jars were selected at random from 1752 L.A. Betancur-Galvis et al. / Chemosphere 62 (2006) 1749–1760  each treatment and the soil was analyzed for inorganicN, P and PAHs as mentioned before. The remainingflasks were opened, except for the sterilized ones, andaired for 10 min to avoid anaerobicity, sealed and fur-ther incubated.  2.5. Statistical analyses Concentrations of inorganic N (NH þ 4 and NO À 3 ),available P and PAH Õ s were subjected to one-way ana-lysis of variance to test the significant differencesbetween the treatments. All analyses were performedusing SAS statistical analysis (SAS Institute, 1989). 3. Results 3.1. Dynamics of  NH  þ 4 Concentrations of NH þ 4 in the unamended Acolmansoil remained below 2 mg NH þ 4 -N kg À 1 soil for the entireincubation (Fig. 1a). In soil spiked with PAHs, the meanconcentration of NH þ 4 was 7 mg NH þ 4 -N kg À 1 soil andnot significantly different from the unamended soil. Inthe sterilized soil to which PAHs and biosolid wereadded, concentrations of NH þ 4 did not change signifi-cantly over time and were on average 240 mg NH þ 4 -Nkg À 1 soil. As such, approximately 60 mg NH þ 4 -N kg À 1 of the 300 mg NH þ 4 -N kg À 1 added with the biosolid,could not be accounted for due to abiotic factors within6 h, i.e. the time between application of biosolid andextraction with 0.5 M K 2 SO 4 . In the unsterilized soilamended with biosolid, a further 50 mg NH þ 4 -N kg À 1 could not be accounted for at the onset of the incuba-tion. Concentrations of NH þ 4 decreased further and after112 days <10 mg NH þ 4 -N kg À 1 was found. Concentra-tions of NH þ 4 in soil amended with inorganic fertilizerdecreased even more rapidly.Concentrations of NH þ 4 in the unamended soil of Texcoco and soil spiked with PAHs remained <4 mgNH þ 4 -N kg À 1 (Fig. 1b). In sterilized soil with both PAHsand biosolid, mean concentrations of N remained con-stant and was on average 120 mg NH þ 4 -N kg À 1 . As such,even larger amounts of NH þ 4 could not be accounted forin soil of Texcoco due to abiotic processes compared toAcolman soil. In the unsterilized soil of Texcocoamended with biosolid, a further 20 mg NH þ 4 -N kg À 1 could not be accounted for at the onset of the incuba-tion. Concentrations of NH þ 4 in soil amended with bio-solid decreased and after 112 days <20 mg NH þ 4 -N kg À 1 was found. Concentrations of NH þ 4 decreased even morerapidly in soil amended with inorganic fertilizer andeven less NH þ 4 -N was detected after 112 days. 3.2. Dynamics of  NO À 3 Concentrations of NO À 3 remained constant in thesterilized Acolman soil and increased by 39 mg NO À 3 -Nkg À 1 in unamended soil and 50 mg NO À 3 -N kg À 1 in soilspiked with PAHs (Fig. 2a). Increases in concentrationsof NO À 3 in soil amended with biosolid was 132 mgNO À 3 -N kg À 1 and 91 mg NO À 3 -N kg À 1 in soil amendedwith NP.Concentrations of NO À 3 remained also constant inthe sterilized soil of Texcoco and increased to 71 mgNO À 3 -N kg À 1 in unamended soil (Fig. 2b). The increasein concentrations of NO À 3 in soil amended with NPwas similar as in the unamended soil, but lower in soilspiked with PAHs and biosolid, although the concentra-tion of NO À 3 was larger at the onset of the incubation. 3.3. Concentrations of available phosphorus In Acolman soil, concentrations of available PO 3 À 4 re-mained constant in sterilized soil, unamended soil andsoil with PAHs. Concentrations of available PO 3 À 4 de-creased to % 15 mg PO 3 À 4 -P kg À 1 in soil spiked with NPand biosolid (Fig. 3a). In soil of Texcoco, concentrations Time (days)    C  o  n  c  e  n   t  r  a   t   i  o  n  o   f   N   H    4   +     (  m  g   N   k  g   -   1    s  o   i   l   ) (a) Acolman 0801602403204000 14 28 42 56 70 84 98 112 NPbio con hyd ster (b) Texcoco 0801602403204000 14 28 42 56 70 84 98 112 NP bio con hyd ste Fig. 1. Concentration of NH þ 4 (mg N kg À 1 dry soil) in (a)Acolman soil and (b) alkaline saline soil of Texcoco (con, s )amended with phenanthrene, anthracene and BaP (hyd, d ), NPfertilizer (NP, h ) or biosolid (bio, j ), and sterilized soil spikedwith phenanthrene, anthracene, BaP and biosolid (ster, Æ ),incubated aerobically at 22 ± 1 ° C for 112 days. Bars are ±minimum significant difference ( P  < 0.05). L.A. Betancur-Galvis et al. / Chemosphere 62 (2006) 1749–1760 1753
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