Do heavy metals and metalloids influence the detoxification

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COST ACTION859•PHYTOREMEDIATION•RESEARCH ARTICLEDo heavy metals and metalloids influence the detoxification of organic xenobiotics in plants?Peter Schröder&Lyudmila Lyubenova&Christian HuberReceived:30January2009/Accepted:21April2009/Published online:22May2009#Springer-Verlag2009AbstractBackground,aim and scope Mixed pollution with trace elements and organic industrial compounds is character-istic for many spill areas and dumping sites.The danger for the environment and human health from such sites is large,and sustainable remediation strategies are urgently needed.Phytoremediation seems to be a cheap and environmentally sound option for the removal of un-wanted compounds,and the hyperaccumulation of trace elements and toxic metals is seemingly independent from the metabolism of organic xenobiotics.However,stress reactions,ROS formation and depletion of antioxidants will also cause alterations in xenobiotic detoxification. Here,we investigate the capability of plants to detoxify chlorophenols via glutathione conjugation in a mixed pollution situation.Materials and methods Typha latifolia and Phragmites australis plants for the present study were grown under greenhouse conditions in experimental ponds.A Picea abies L.suspension culture was grown in a growth chamber.Cadmium sulphate,sodium arsenate and lead chloride in concentrations from10to500µM were administered to plants.Enzymes of interest for the present study were:glutathione transferase(GST),glutathione reductase,ascorbate peroxidase and peroxidase.Measure-ments were performed according to published methods. GST spectrophotometric assays included the model sub-strates CDNB,DCNB,NBC,NBoC and the herbicide Fluorodifen.Results Heavy metals lead to visible stress symptoms in higher plants.Besides one long-term experiment of 72days duration,the present study shows time and concentration-dependent plant alterations already after24 and72h Cd incubation.P.abies spruce cell cultures react to CdSO4and Na2HAsO4with an oxidative burst,similar to that observed after pathogen attack or elicitor treatment. Cd application resulted in a reduction in GSH and GSSG contents.When a heavy metal mixture containing Na2HAsO4,CdSO4and PbCl2was applied to cultures, both GSH and GSSG levels declined.Incubation with80µM arsenic alone doubled GSSG values.Based on these results,further experiments were performed in whole plants of cattail and reed,using cadmium in Phragmites and cadmium and arsenic in Typha as inducers of stress.In Phragmites australis,GST activities for CDNB and DCNB were significantly reduced after short-term Cd exposure(24h).In the same samples,all antioxidant enzymes increased with rising heavy metal concentrations. Typha latifolia rhizome incubation with Cd and As leads to an increase in glutathione reductase and total peroxi-dase activity and to a decrease in ascorbate peroxidase activity.Measurements of the same enzymes in leaves of the same plants show increased GR activities,but no change in peroxidases.GST conjugation for CDNB was depressed in both cattail rhizomes and leaves treated with Cd.After As application increased,DCNB enzyme activities were detected.Discussion tifolia and P.australis are powerful species for phytoremediation because they penetrate a large volume of soil with their extensive root and rhizome systems. However,an effective remediation process will depend on active detoxifying enzymes,and also on the availability ofEnviron Sci Pollut Res(2009)16:795–804DOI10.1007/s11356-009-0168-7Responsible editors:Peter Schroeder and Jean-Paul Schwitzguébel P.Schröder(*):L.Lyubenova:C.HuberDepartment of Microbe–Plant Interactions,Helmholtz ZentrumMünchen,German Research Center for Environmental Health, Ingolstädter Landstrasse1,85764Neuherberg,Germanye-mail:peter.schroeder@helmholtz-muenchen.deconjugation partners, e.g.glutathione and its analogues. Species-specific differences seem to exist between the regulations of primary defence enzymes like SOD,catalase, peroxidases,whereas others prefer to induce the glutathione-dependent enzymes.As long as the pollutant mix encountered is simple and dominated by heavy metals, plant defence might be sufficient.When pollution plumes contain heavy metals and organic xenobiotics at the same time,this means that part of the detoxification capacity,at least of glutathione-conjugating reactions,is withdrawn from the heavy metal front to serve other purposes.In fact, glutathione S-transferases show strong reactions in stressed plants or in the presence of heavy metals.The spruce cell culture was a perfect model system to study short-term responses on heavy metal impact.Overall,and on the canopy level,this inhibitory effect might result in a lower detoxification capacity for organic pollutants and thus interfere with phytoremediation.Conclusions We present evidence that pollution with heavy metals will interfere with both the oxidative stress defence in plants,and with their ability to conjugate organic xenobiotics.Despite plant-species-dependent differences, the general reactions seem to include oxidative stress and an induction of antioxidative enzymes.Several processes seem to depend on direct binding of heavy metals to enzyme proteins,but effects on transcription are also observed.Induction of xenobiotic metabolism will be obtained at high heavy metal concentrations,when plant stress is elevated.Recommendations and perspectives Plants for phytoreme-diation of complex pollution mixtures have to be selected according to three major issues:uptake/accumulation capac-ity,antioxidative stress management,and detoxification/ binding properties for both the trace elements and the organic xenobiotics.By way of this,it might be possible to speed up the desired remediation process and/or to obtain the desired end products.And,amongst the end products,emphasis should be laid on industrial building materials,biomass for insulation or biogas production,but not for feed and fodder. Each of these attempts would increase the chances for publicly accepted use of phytoremediation and help to cure the environment.Keywords Detoxification.Heavy metals.Inducible metabolism.Multiple anic xenobiotics. Phytoremediation1Background,aim and scopeAnthropogenic and especially industrial activities have impacted severely in our environment during the last century.In many cases,environmental pollution by trace elements and especially by heavy metals is accompanied by pollution with organic foreign compounds and vice versa (Pambrun et al.2008).Surveys alone in Germany resulted in an inventory of more than80,000spill sites with potential for pollutant removal(Franzius1994).Phytor-emediation,i.e.the removal of pollutants with the help of plants,but also phytostabilization in such areas affected by multiple pollution is complicated,and only few plant species have been shown to survive under the adverse conditions of mixed contamination.Amongst trace ele-ments,predominantly arsenic,cadmium and lead have been investigated with respect to these effects(Schröder et al. 2002;Memon and Schröder2009).Arsenic,a metalloid,frequently included in the heavy metal group,is one of the oldest known inorganic toxins, therapeutics and intentional poisons.Disodium arsenate (Na2HAsO4)is a chemical analogue of phosphate and acts as an uncoupler of oxidative phosphorylation and catalase inhibitor.In the environment,arsenate is the predominant form(Peel et al.1991).Cadmium(Cd)is a true heavy metal.It evokes a whole array of toxic effects,due to its long biological half-life and storage in plant vacuoles,interacts with photosynthesis and nitrogen metabolism,and causes oxidative damage not only in mammals and plants(Dixit et al.2001),but also in microbes(Shen et al.2005).Lead is one of the most widely used metals in human history and very widespread in the environment(Tully et al.2000).It is classified as a possible carcinogen, whereby acute toxicity is revealed through its binding to enzyme prosthetic groups and inhibition of heme complex formation.Contamination of soil or water with these heavy metals is a serious issue with concentration levels exceeding the WHO drinking water guideline values in the lower µg/l regime in many regions of the world.A plethora of studies exists on heavy metals,metalloids and plants.Trace elements are well known for their ability to interfere with plant metabolism and to induce cellular damage resulting in the formation of reactive oxygen species(ROS),albeit through different reactions.The rise of reactive oxygen species in cell metabolism is a frequently observed phenomenon,and it has been shown that ROS may serve as signalling molecules for a number of defence reactions in plants,including alterations in primary or secondary metabolism.Tightly connected to the sulphur metabolism in plants is the predominant detoxification pathway of halogenated organic pollutants and herbicides,i.e.glutathione S-transferase(GST)dependent conjugation.GST catalyses the conjugation of reduced glutathione to electrophilic centres of such halogenated xenobiotics(Coleman et al. 1997;Schröder2001).They comprise a very heterogeneousfamily of enzymes with diverse tasks in plant metabolism and defence(Dixon et al.2001).More than50GST genes have been identified in model plants like Arabidopsis.To date, their functions are not known in too much detail,but it is clear that their expression and activity may be strongly modulated by oxidative stress and ROS.In order to obtain information on the physiological background of ROS formation under heavy metal stress and its influence on detoxification of organic pollutants, we have investigated reactions of plants used in phytor-emediation, e.g.Typha and Phragmites,but also in a weed,and a plant cell culture that has been used as a biomonitor of stress.The current compilation tries to elucidate detrimental effects of multiple pollution in pulling data together from different studies and exposure of different plant species.12Materials and methods2.1Plant materialExperiments were performed with three different species: P.australis,Typha latifolia,and a heterotrophic cell culture of Picea abies.Phragmites australis plants were grown hydroponically in2-L glass pots under greenhouse conditions in0.5L full-strength MS medium.CdSO4was used to set up final Cd concentrations of10and100µM in0.5L of growth medium.After24h of incubation,rhizome and leaf samples were taken;rhizomes were then washed thorough-ly with deionised water and frozen immediately in liquid nitrogen.Samples were kept at−80°C before analysis.Each sample consists of three single plants,respectively.For each treatment,three independent replicates were taken.Typha latifolia plants were grown in a greenhouse at 14h day,10h night and humidity of50%.After a growing period of72days,the plants were treated with three different metals and four different concentrations(10µM, 50µM and250µM)for72h.The control was grown at the same conditions,but not treated with heavy metals.Three plant replicates for each treatment were used.P.abies L.Karst.heterotrophic suspension-cultured cells have been described elsewhere(Messner and Berndt1990, Schröder2001).Media were changed after3days and the cultures allowed to grow for four additional days in the presence of heavy metals(Schröder2001).The cells were then sedimented,the medium discarded,50%of the cells removed and supplied with new medium,and then used as starting culture for the next experiments.The remaining cells received fresh medium and were further incubated. Upon termination of the incubation period,cells were harvested by removal of media and immediately immersed in liquid nitrogen.2.2IncubationsThe used metals are:cadmium(as cadmium sulphate), arsenic(as sodium arsenate)and lead(as lead chloride). Plants or freshly transferred cells were incubated with media containing CdSO4,Na2HAsO4or PbCl2(from50to 500µM).The concentration of particular metals was selected on the basis of previous experiments(Schröder et al.2002)and levels found in real water samples.To investigate the pathogenesis related oxidative burst in the cell culture,Rhizosphaera kalkhoffii was maintained on malt agar(30g l−1malt extract,10g l−1agar(type a, Sigma)).Preparation of the fungal cell wall fraction was performed as described previously(Schröder2001).2.3Protein extractionThe extraction procedure for soluble detoxification enzymes is carried out according to Schröder et al. (2005).Frozen plant material was powdered and extracted (0.1M Tris/HCl pH7.8,5mM EDTA,5mM dithioery-thritol DTE,1%Nonidet P40,1%insoluble polyvinylpyr-rolidone PVP K90),homogenised and centrifuged for 30min at20,000rpm.Proteins in this crude extract were precipitated by addition of ammonium sulphate in two steps of40%and80%saturation,respectively.Proteins were centrifuged after each step and the pellet finally resus-pended in2.5mL of25mM Tris/HCl buffer pH7.8.This step was followed by desalting with Sephadex PD-10 columns(Pharmacia,Germany).2.4GSH transferase assaySpectrophotometer assays for determination of GST activity using the standard substrates1-chloro-2,4-dinitrobenzene(CDNB,ε340nm=9.6mM−1cm−1),1,2-dichloro-4-nitrobenzene(DCNB,ε345nm=8.5mM−1cm−1), 4-nitrobenzyl chloride(NBC,ε310nm=1.8mM−1cm−1),p-nitrobenzoyl chloride(NBoC,ε310nm=1.9mM−1cm−1) followed the method of Schröder et al.(2002).Assays with the herbicide fluorodifen(ε400nm=3.1mM−1cm−1)followed Scalla and Roulet(2002).2.5Antioxidative enzymes assayGlutathione reductase(GR),peroxidase(POX)and ascor-bate peroxidase(APOX)were assayed following published1The present paper is based on an oral presentation delivered at the 4th European Bioremediation Conference,held in Chania,Greece, September3rd to6th,2008.methods.Peroxidase activity was measured according to Drotar et al.1985using Guajacol (2-Metoxy-phenol)as a substrate.Measurements were performed at pH 6.0and 420nm (ε420nm =26.6mM −1cm −1).The activity of ascorbate peroxidase and glutathione reductase were determined as described by Vanacker et al.1998.Ascor-bate peroxidase measurement is based on a decrease in extinction at 290nm through the oxidation of ascorbate (ε290nm =2.8mM −1cm −1)at pH 7.0.For glutathione reductase,the consumption of NADPH (ε340nm =6.2mM −1cm −1)can be followed spectrophotometrically at 340nm and pH 7.8according to Schickler and Caspi (1999).Protein content of the samples was measured by the method of Bradford (1976)with bovine serum albumin as a standard protein.3Results3.1Visual symptomsWhen whole plants were incubated with cadmium sulphate,they showed time and concentration-dependent visual symptoms and alterations in growth patterns,whereas the cell cultures treated with arsenate,cadmium sulphate and lead chloride,due to their short incubation time,remained void of visual symptoms.The strongest effects of heavy metal exposure were observed in the long-term Typha experiment,where incubation for 72days led to severe changes in biomass production and health (Fig.1).In order to obtain data forshort-term exposure under comparable experimental con-ditions,a set of Typha plants was exposed for 72h with the same concentrations.3.2Oxidative stressThe general response of plants toward heavy metals in concentrations ranging from 10to 250µM seems to be an increase in oxidative stress,directly coupled to an induction of enzymes like peroxidase,ascorbate peroxidase and glutathione reductase,i.e.of the Halliwell –Asada cycle,and connected to strong alterations in the glutathione pool.It had been previously shown that spruce cell cultures react to CdSO 4and Na 2HAsO 4with a pronounced oxidative burst,similar to that observed after pathogen attack or elicitor treatment (Messner and Schröder 1999;Schröder 2001).H 2O 2concentrations of up to 45µM were measured in spruce cells within 8h after the onset of the incubation.After addition of up to 150µM PbCl 2,the same cells developed only a weak oxidative burst at rates of 1to 2µM h −1H 2O 2.In the media of untreated spruce cells,H 2O 2levels never exceeded 1µmol h −1.However,and contrary to elicitor treatment,the H 2O 2extrusion was delayed between 4and 8h.This delay seems to indicate that the cellular signal leading to the HM mediated oxidative burst is different from the pathogen answer.Application of 150µM CdSO 4resulted in a reduction in GSH and GSSG of 75%and 96%,respectively.When a heavy metal mixture containing 80µM Na 2HAsO 4,150µM CdSO 4and 150µM PbCl 2was applied to the cultures,both GSH and GSSG levels declined by 60%and 99%,respectively.Incubation with 80µM arsenic alonedoubledFig.1Visual effects of Cd appli-cation in sublethal concentrations to Typha latifolia plants grown under greenhouse conditions after 72daysthe GSSG values.Effects of arsenic seem to be intercon-nected with the rise of reactive oxygen species (ROS)and active oxygen species (AOS)in the cell,leading to SOD or NADH-oxidase catalysed formation of H 2O 2.Incubating the cells for longer time periods (i.e.24h),led to similar effects on thiol contents (data not shown).Incubation with elicitor resulted in the doubling of GSH values within 16h to 933nmol g fwt −1,but GSSG concentrations remained unchanged.On the contrary,incubation with 117mM H 2O 2decreased GSH levels by 30%and doubled GSSG contents (Schröder 2001).Inter-estingly,arsenic appears to mimic oxidative stress processes as its effect is comparable to elicitor and H 2O 2addition to the cell cultures.From these results,it becomes clear that heavy metals influence thiol contents mainly by depleting the pool,e.g.by the larger demand for phytochelatins.3.3Induction of enzyme activitiesCdSO 4in concentrations up to 500µM significantly induced GST activities for the four substrates measured (p <0.05)in the treated cell culture.Except for EPNP,the inductive effect seemed to be pared to cadmium,arsenic was a strong inducer.Arsenic concentrations (up to 80µM)induced GST for all substrates,except EPNP,by a factor of 2(p <0.05),and the effect was strictly concentration-dependent for CDNB,DCNB and pNBC conjugation.It could be clearly shown that EPNP activity of the cells was lost proportionally with increasing metal concentrations.Cellular responses to PbCl 2contrasted to those of other metals tested,in that,with the exception of EPNP,no significant changes were observed for the conjugation of any of the substrates.EPNP conjugation was depressed by 56%of controls,similar to the treatments with the other heavy metals.In addition to experiments with pure heavy metals,mixtures of the mentioned heavy metals were,irrespective of concentrations applied,always inductive to GST activities of CDNB,DCNB and EPNP.A mixture of 150µM CdSO 4,150µM PbCl 2and 80µM NaHAsO 4led to a doubling of conjugation rates of the two former substrates,but tripled the activity towards the latter (72%,111%,231%above controls,p <0.01).Having recorded these effects in a cell culture,we focused on experiments in whole plants with cadmium as the lead heavy metal.We obtained very similar results in Typha and in reeds (Phragmites ).Some reports on Cd-dependent increases in CAT activity,but simultaneously high SOD increases,are available (Vitoria et al.2001).Current literature on the inducibility of detox enzymes indicates ROS and AOS as potent triggers of GST induction.Interestingly,and different from the general opinion,we found that even mild heavy metal stress might lead to the inhibition of detoxification reactions in reeds (Fig.2).Phragmites glutathione S -transferase activity for specific organic substrates (CDNB and DCNB)decreased within 24h of incubation with cadmium sulphate,whereas the antioxidative enzymes performed at increasingly higher rates.The decline in CDNB activity was around 20%,and 35%in DCNB.Similarly the increases in antioxidative enzymes were around 20%to 30%(Fig.3).This develop-ment depends strongly on the heavy metal to be considered and its concentration,as is reported below.Ongoing experiments with Typha show similar effects (Figs.4,5,6).In rhizomes,antioxidative enzymes like POX and GR are induced with higher HM concentration,but APOX decreases under Cd and As.In the leaves of the same plants,Cd influences only GR activities,whereas APOX and POX remain at more or less the same levels.The massive increase of GR activity points to the consumption of GSH for phytochelatin production.Arse-nate,contrary to this,leaves GR activities unchanged,but doubles APOX activities and leads to significant increases of POX.An interesting observation is connected to thefactFig.2Activity of glutathione S -transferase enzymes inPhragmites for chlorobenzene substrates A :CDNB(1-chloro-2,4-dinitrobenzene)and B :DCNB (1,2-dichloro-4-nitrobenzene)after 24h in the presence of CdSO 4(K control,a 10µM CdSO 4,b 100µM CdSO 4).Enzyme assays were performed for 5min,and con-jugate formation was recorded at the wavelengths given in M&M.All measurements were done in triplicate,and error bars represent standard deviationsthat low arsenate concentrations of 10µM will always lead to an induction of antioxidative enzymes in the leaves,whereas 50µM and above generally inhibit them.It becomes clear that rhizomes,receiving by far higher concentrations of heavy metals,will react more distinctly to this stress.In leaves of treated plants,additional signalling processes or absence of certain defence genes might modulate the stress response.The same is principally true for the glutathione S -transferases responsible for the detoxification of organic xenobiotics.GST conjugation is depressed for CDNB in both leaves and rhizomes after Cd treatments,whereas As treatment induces DCNB activity massively.Conjugation of fluoro-difen decreases in both treatments,albeit with low significance,except at low concentrations of CdSO 4.NBC and NBoC conjugation are both partly induced by cadmium,but generally inhibited strongly by arsenic.In our experiments with arsenate,conjugation rates under HM influence increased gradually,but did not reach control levels.Even more thrilling is the observa-tion that lead chloride (data not shown)and arsenic (see Fig.5C,H)both stimulate the conjugation of the herbicide fluorodifen at lower concentrations.This pro-vokes the question,whether a multiple pollution scenario,including organic pollutants,pesticides and heavy metals at the same time,would increase a given plant's capability for phytoremediation.In essence,cadmium seems to be inductive to some GST activities in Typha at high concentrations,but arsenate exerts inhibitory effects on all GST except the DCNB conjugating form.It is doubtful whether Cd is directly active as GST inducer,rather than depleting GSH and damaging cellular compartments,so that GST induction would occur.Experiments on the direct influence of heavy metals on isolated GST enzymes have shown that decreases in enzyme activity could also be observed when heavy metalsdirectly interfere with catalytic processes (Lyubenova et al.2007).We used a fraction of partially purified GST from Calystegia sepium ,common bindweed,because this plant had previously been described as very tolerant against organic pollutants and herbicides,although it produces an extensive rhizome that will allow for relatively high uptake rates of xenobiotics.When C.sepium GST was assayed for CDNB and NBOC conjugation in presence of CdSO 4,CDNB conjugation was inhibited by 50%at 250µM,whereas NBOC conjugation remained unaffected.Unexpect-edly,the activity of glutathione reductase,the enzyme responsible for the reduction of GSH,was inhibited by 90%already by the lowest HM concentration.Here,it might be speculated that consumption of GSH for heavy metal sequestration and for conjugation of xenobiotics could also lead to additional plant stress.4DiscussionPlants growing in serpentine soils,on pollution plumes or in phytoremediation facilities frequently encounter variable concentrations of heavy metals,amongst them cadmium and arsenate species.If these plants do not avoid metal uptake,they are forced to distribute dissolved heavy metals in their tissues and have to cope with the resulting stress.We have investigated several unrelated plant species,amongst them Typha ,Picea ,Phragmites and Calystegia for their response towards cadmium and arsenic,and found some similarities in their reactions.Being typical geophytic plants,the species chosen develop extensive root and rhizome systems that might penetrate a large volume of soil or sediment.Hence,these plants might be of some interest in soil remediation.In any case,the use of cell cultures as model organisms is of importance and will allow to obtain a large number of results under standardised conditions (Langebartels and Harms 1986).Fig.3Activity of antioxidative enzymes,A :ascorbate peroxidase (APOX ),B :guajacol peroxidase (POX )and C :glutathione reductase (GR )in Phragmites after 24h in the presence of CdSO 4(K control,a 10µM Cd,b 100µM Cd).All measurements were done in triplicate according to the conditions described in M&M,and error bars represent standard deviationsGenerally,all investigated species showed increases in most of the antioxidative enzymes of the Halliwell –Asada cycle,obviously after an oxidative burst signal initiating the response few hours after HM uptake.Species-specific differences seem to exist between the regulations of induction:some plants have stronger induction of the primary defence enzymes like SOD,catalase,peroxidases,whereas others prefer to induce glutathione-dependent enzymes that provide the cell with high antioxidant concentrations.Glutathione homeostasis may be decisive in this respect (Noctor et al.2002).As long as the pollutant mix encountered is simple and dominated by heavy metals only,plant defence might be sufficient.However,most pollution plumes contain both heavy metals and organic xenobiotics at the same time.For a given plant,this means that part of its predisposed detoxification capacity,at least of glutathione-conjugating reactions,is withdrawn from the heavy metal front to serve other purposes.In fact,glutathione S -transferases show strong reactions in stressed plants or in the presence of heavy metals.The spruce cell culture was a perfect model system to study short-term responses onheavyFig.4Influence of cadmium sulphate and sodium arsenate on the antioxidative enzymes ascorbate peroxidase (APOX ),guajacol peroxidase (POX )and glutathione reductase (GR )of Typha latifolia :left A –C (enzyme activities in therhizome);right D –F (enzyme activities in the leaves).All bars represent means±standard deviations of a minimum of three independent samples.K control,a 10µM Cd,b 50µM Cd,c 250µM Cd,d 10µM As,e 50µM As,f 250µM Asmetal impact.It was shown that glutathione pools were depleted,and that several GST isoforms were induced with Cd or Pb,but strongest with As.Pollution mixtures exerted even more complex reac-tions (Schröder 2001).An interesting lesson to learn wasthat the standard model substrate,CDNB,would not yield complete insight in the GST reactions in affected cells.This was also true for the other investigated plant species.Except for the conjugation of CDNB,where no significant changes in conjugation rates are observed,GSTactivitiesFig.6Sketch of heavy metal influence on plant detoxifica-tion,especially on theglutathione-dependent enzymes depicted in the triangle (from Schröder et al.2002,modified).Abbreviations:AOS activated oxygen species,APX ascorbate peroxidase,CAT catalase,Cd-PC cadmium phytochelatin complex,DAR dihydroascorbate reductase,GR glutathione reductase,GSH glutathione (red.),GSSG glutathione (ox.),GST glutathione S -transferase,HM heavy metals,MDA monodehydroascorbate,PC phytochelatins,ROS reactive oxygen species,SOD superoxidedismutaseFig.5Influence of heavy metals on GST conjugation of DCNB,CDNB,NBC (nitrobenzylchloride),NBoC (nitrobenz-O -yl-chloride)and the herbicide Fluorodifen in Typha plants treated with 10,50or 250µM of the respective HM.Graphs left A –E (enzyme activities inthe rhizome)right F –J (enzyme activities in the leaves);Treatments with CdSO 4and NaHAsO 4,respectively:(K control,a 10µM Cd,b 50µM Cd,c 250µM Cd,d 10µM As,e 50µM As,f 250µM As)。

6. Bioremediation of heavy metal-contaminated effluent using optimized activated sludge bacteria

ORIGINAL ARTICLEBioremediation of heavy metal-contaminated efﬂuent using optimized activated sludge bacteriaEbtesam El.Bestawy •Shacker Helmy •Hany Hussien •Mohamed Fahmy •Ranya AmerReceived:14October 2011/Accepted:3December 2012/Published online:16December 2012ÓThe Author(s)2012.This article is published with open access at Abstract Removal of heavy metals from contaminated domestic-industrial efﬂuent using eight resistant indigenous bacteria isolated from acclimatized activated sludge was investigated.Molecular identiﬁcation using 16S rDNA ampliﬁcation revealed that all strains were Gram-negative among which two were resistant to each of copper,cadmium and cobalt while one was resistant to each of chromium and the heavy metal mixture.They were identiﬁed as Entero-bacter sp.(Cu 1),Enterobacter sp.(Cu 2),Stenotrophomonas sp.(Cd 1),Providencia sp.(Cd 2),Chryseobacterium sp.(Co 1),Comamonas sp.(Co 2),Ochrobactrum sp.(Cr)and Delftia sp.(M 1)according to their resistance pattern.Strains Cu 1,Cd 1,Co 2and Cr were able to resist 275mg Cu/l,320mg Cd/l,140mg Co/l and 29mg Cr/l respectively.The four resistant strains were used as a mixture to remove heavy metals (elevated concentrations)and reduce the organic load of wastewater efﬂuent.Results revealed that using the pro-posed activated sludge with the resistant bacterial mixture was more efﬁcient for heavy metal removal compared to the activated sludge alone.It is therefore recommended that the proposed activated sludge system augmented with the acclimatized strains is the best choice to ensure high treatment efﬁciency and performance under metal stresses especially when industrial efﬂuents are involved.Keywords Acclimatization ÁActivated sludge ÁBioremediation ÁDomestic ÁHeavy metals ÁIndustrial wastewaterIntroductionHeavy metals are considered one of the most common and hazardous pollutants in industrial efﬂuents that might cause serious problems to the sewage network pipelines.The deleterious effects of heavy metals on biological processes are complex and generally related to species,solubility and concentration of the metal and the characteristics of the inﬂuent,such as pH,as well as presence and concentration of other cations and/or molecules and suspended solids (Gikas 2008).Metal toxicity results from alterations in the conformational structure of nucleic acids,proteins or by interference with oxidative phosphorylation and osmotic balance (Yaoa et al.2008).The most common mechanisms by which metals are eliminated from wastewater treatment processes depend on precipitation,adsorption to suspended solids during primary sedimentation or adsorption to extra-cellular polymers (Qodah 2006).Use of bio-adsorbentsE.El.Bestawy (&)ÁS.HelmyDepartment of Environmental Studies,Instituteof Graduate Studies and Research,Alexandria University,163Horria Ave.El-Shatby,P.O.Box 832,Alexandria,Egypt e-mail:ebtesamelbestawy@ S.Helmye-mail:shackerh@Present Address:E.El.BestawyDepartment of Life Sciences,Faculty of Science,King Abdul Aziz University,P.O.Box 42805,Jeddah 21551,Kingdom of Saudi ArabiaH.Hussien ÁM.Fahmy ÁR.AmerDepartment of Environmental Biotechnology,City for Scientiﬁc Research and Technology Application,P.O.Box 21934,Alexandria,Egypt e-mail:hhussein66@ M.Fahmye-mail:mohamedfahmy73@R.Amere-mail:ranyaamer@;r.amer@mucsat.sci.egAppl Water Sci (2013)3:181–192DOI 10.1007/s13201-012-0071-0such as bacteria,fungi,algae and some agricultural wastes that emerged as an eco-friendly,effective and low cost material option could offer potential inexpensive alterna-tives to the conventional adsorbents(Valls and Lorenzo 2002).Different species of Aspergillus,Pseudomonas, Sporophyticus,Bacillus,Phanerochaete,etc.,have been reported as efﬁcient chromium and nickel reducers.The response of microorganisms towards toxic heavy metals is very important for reclamation of polluted sites(Conge-evaram et al.2007).Adsorption of heavy metals on the sludge surface is usually attributed to the formation of complexes between metals and carboxyl,hydroxyl and phenolic surface func-tional groups of the extracellular polymeric substances (EPS)produced by many different species of bacteria isolated from activated sludge(Yuncu et al.2006).EPS are metabolic products of bacteria result from the organic matter of the efﬂuent and from microbial lysis or hydro-lysis.They serve as a protective barrier for cells against the harsh external environment and play a crucial role in sequestration and biosorption of metal ions as well as ions which constitute metabolic elements for bacteria.Metal biosorption performance depends on external factors,such as pH,other ions in bulk solutions(which may be in competition),organic material in bulk solution and tem-perature(Comte et al.2007).Identiﬁcation of sludge bacteria that are adapted to the new toxic metal environment provide efﬁcient potential candidates for heavy metal bioremoval from contaminated media.Because of its size(*1,500base pairs),the16S rRNA gene is the most widely used sequence in bacterial identiﬁcation(Weisburg et al.1991;Drancourt et al.2000). Segments from these genes can be easily ampliﬁed by PCR using universal primers and sequenced.Sequence com-parison of16S rRNA has been used as a powerful tool for establishing phylogenetic and evolutionary relationships among organisms(Amer2003).Alexandria is the second largest industrial city in Egypt where more than40%of the industrial activity localized.In Alexandria,industrial efﬂuents are mixed with domestic sewage and discharged into the main sewage network (Hussein1999).The aim of the present study was to isolate and identify heavy metal resistant bacteria from acclimatized activated sludge to be used for remediation of heavy metal contaminated wastewater at their highest expected levels. Materials and methodsSampling and pre-treatmentActivated sludge samples were obtained from the aeration tank of the activated sludge unit at Damanhore Wastewater Treatment Plant located in the North West side of Egypt. Prior to use,sludge was pre-treated by allowing the sludge to settle at room temperature followed by decanting the supernatant and replacement with synthetic wastewater with aeration to maintain dissolved oxygen above4mg/l. After24h,aeration was stopped and the mixture was allowed to settle,then the supernatant was decanted and fresh synthetic wastewater was added to provide the same original volume.The procedure of settling,decanting, addition of new efﬂuent and nutrients was repeated at24-h interval until the desired acclimation time(approximately 1month)had been achieved.Heavy metal determinationFour heavy metals(Cu,Cd,Cr and Co)in the wastewater and sludge were determined using Atomic Adsorption Spectrophotometry(Perkin Elmer)after digestion with microwaves digester following the manual instructions with the addition of nitric acid,hydroﬂuoric acid and hydrogen peroxide for sample digestion(Costley and Wallis2001;Borowski2004).Measurement of dehydrogenase activity(DHA) Dehydrogenase activity was determined using method described by Weddle and Jenkins(1971).Triphenyl tetra-zolium chloride(TCC)and sodium sulfate solutions(5%) were prepared in distilled water.One milliliter of TCC, 10ml liquor or cell suspension and3drops of NaSO3were placed in centrifuge tube and shaken at30°C for1h in dark.This was followed by centrifugation for5min;the color of the supernatant was determined using spectro-photometer at wavelength480nm.Inhibition of the bac-teria and consequently the activated sludge activity were determined according to the following equation: Inhibition%ðÞ¼I0ÀIðÞ=I0½ Â100Where I0represents intensity of the control without treat-ment and I represents intensity of sample after treatment with heavy metal(Weddle and Jenkins1971;Shim et al. 2003).Isolation of heavy metal-tolerant bacteriaTolerant bacterial isolates were collected fromﬁve differ-ent acclimatized activated sludge batch reactors treated by synthetic wastewater(Table1)with elevated levels of Cu, Cd,Co,Cr(prepared as10%stock solutions of CuSO4, CdCl2ÁH2O,K2Cr2O7,CoCl2Á6H2O salts)and metal mix-ture(El.Bestawy et al.2012)as described by O¨zbelge et al. (2005).Tolerant bacteria were isolated from theﬁnal stage of the metal enrichment experiment at all reactors.One-milliliter liquor from each reactor was used to inoc-ulate liquid Luria–Bertani(LB)medium containing the investigated heavy metal(sterilized byﬁltration)in that reactor.Heavy metal concentrations used during bacterial isolation were15,100,40and40mg/l for the individual Cu,Cr,Cd and Co,respectively.While a mixture of Cu, Cr,Cd and Co at2,10,10and10mg/l respectively was used for isolation of strains with multiple accumulation ability.After24h incubation at30°C,100l l of the grown culture was transferred into10-ml fresh LB medium con-taining the same metal concentration and left for another 24h under the same conditions.The highest metal con-centration reduced the activated sludge activity by50% was amended in Petri dish containing20ml sterile LB agar and mixed well.100l l of the grown culture was spread on the surface of the plate and incubated for24h at30°C. Large identical colonies from each plate were picked out and cultured for puriﬁcation and identiﬁcation. Molecular identiﬁcationTotal genomic DNA was extracted from5ml overnight LB culture of the puriﬁed isolates(Sambrook et al.1989).PCR was performed in a light cycler Eppendorf PCR machine. A1,300-bp fragment was obtained by PCR ampliﬁcation of the16S rDNA gene(Ausubel et al.1999)using the primers B341F(50-CCTACGGGA GGCAGC)and1392R(50-ACG GGCGGTG TGTRC-30).The PCR mixture was composed of100ng of genomic DNA,30pmol of each primer, 200l M of dNTPs,1U of Taq polymerase and10l l of10X PCR buffer,the reaction volume was adjusted to100l l in 0.5ml eppendorf tube.The PCR ampliﬁcation conditions were performed by an initial denaturation step at94°C for 10min followed by30denaturation cycles at94°C for 1min,annealing at60°C for1min and an extension at 72°C for1min followed by aﬁnal extension step at72°C for10min.Amplicons of16S rDNA were puriﬁed using PCR puriﬁcation kit(QIAGEN).Each of these puriﬁed products was sequenced by the chain terminator method (ABI3130XL system,DNA technology,Denmark)using the two corresponding PCR primers separately.The resulted DNA sequences were phylogenetically analyzed using the BLAST search program.Multiple sequence alignment and molecular phylogeny were performed using BioEdit software(Hall1999).Screening for heavy metal resistance patternEffect of the individual and mixed tested metals on the growth(measured as optical density,OD at600nm)and dehydrogenase activity of each of the resistant strains was investigated at elevated metal concentrations compared to their controls.An overnight culture of each strain was inoculated in20-ml LB medium supplemented with deﬁ-nite concentrations of the corresponding heavy metals and incubated for24h under the previously mentioned condi-tions.Concentrations of the tested metals(not shown)were calculated according to the IC50which is also included in the screening test.Concentrations of heavy metal mixtures were calculated by dividing the growth IC50of each heavy metal by4and two higher and two lower concentrations were applied for screening bioassay.Treatability studiesThe most efﬁcient strains for metal removal were selected based on the resistance screening bioassays and manipu-lated in activated sludge treatment of mixed domestic-industrial wastewater contaminated with heavy metals and organic matter.The acclimatized sludge was prepared by centrifugation to remove the supernatant which was replaced by wastewater.The resistant strain for each investigated metal was seeded individually in LB-medium and incubated at30°C and200rpm for24h.Cultures were centrifuged and the supernatants were replaced with wastewater and used as inocula for batch reactors for the treatability study with moderate concentrations of heavy metals.Three sets of500-ml reactors were used to compare the removal efﬁciencies of the sludge,bacterial strains and mixture of both.Each set contained four different con-centrations(not shown)of the heavy metals with initial COD of1,400mg/l as following:1.Theﬁrst set consists of200ml of sludge with differentconcentrations of the investigated heavy metals.2.The second set consists of200ml of bacterial cultureonly with different concentrations of the investigated heavy metals.Table1Composition of the concentrated synthetic wastewater Serial Constituents Concentration(g/l)1Tripton122.12NaCl40.73Na2SO4 4.464K2H2PO4 4.465MgCl2Á6H2O0.376CaCl2Á2H2O0.377MnSO40.578H2MoO40.319NaOH0.0810ZnSO40.4611CoSO40.4912CuSO40.7610ml of concentrated synthetic wastewater were added to each 1,000ml of deionized waster3.The third set consists of100ml of sludge and100mlof bacterial culture with different concentrations of the investigated heavy metals.Heavy metal removal per unit biomass was determined using the following equation:M uptake=g¼½MðInf:ÞÀMðEff:Þ =MðMLSS)where M=uptake/g:heavy metal removed per unit mass of biomass,M(Inf.)=the initial heavy metal concentration in the inﬂuent,M(Eff.)theﬁnal metal concentration after treatment, MLSS:Biomass concentration in gramResultsIsolation and molecular identiﬁcation of metal resistant-sludge bacteriaA total of eight strains representing morphologically different bacterial colonies were isolated and puriﬁed. Two showed resistance to copper(Cu1and Cu2),two for cadmium(Cd1and Cd2),two for cobalt(Co1and Co2), one for chromium(Cr)and the heavy metal mixture resistant strain(M1).PCR products of the chromosomal DNA extracts of the resistant isolates were puriﬁed, sequenced and aligned against the16S rDNA sequences of the ribosomal database project http://www.cme.msu. edu/RDP/html/index/html(Maidak et al.1994;Rainey et al.1996).Sequences were deposited in GenBank for obtaining accession numbers.The Phylogenetic relation-ship among the tested isolates and the closely related species(Fig.1)was analyzed using multi-sequence alignment program(BioEdit Sequence Alignment Editor). Accession numbers and similarity of the tested isolates to the related species are presented in Table2.It is well documented that both Gram-positive and Gram-negative bacteria are ubiquitous in nature with highly resistant anionic cell walls.This anionic cell wall canﬁx metal and provide sites for nucleation and growth of minerals, a property that has been the basis for many heavy metal removals using bacterial biomass(Kelly et al.2004). Heavy metal resistance and/or sensitivity of the sludge bacteriaResistance and/or sensitivity of the acclimatized sludge bacteria towards the investigated metals(individuals or mixture)were determined in terms of growth(OD)and dehydrogenase activity to select the most efﬁcient for treatability study.Effect of individual metalsComparing data of the individual effects of the four tested metals on the acclimatized strains(Fig.2)revealed the following points:1.IC50of all the tested metals that induced50%reduction in the growth of all the investigated strains was higher than that induced50%reduction in their DHA.This indicates that tested metal could inhibit the enzyme activity much higher than its inhibition to the growth because dehydrogenase activity is a direct reﬂection of microbial activity(Gong1997;Mathew and Obbard2001)and metal toxicity(Aoyama and Nagumo1997;Kelly and Tate1998;Nweke et al.2006).2.IC50of each metal depends mainly on the microbialspecies as well as acclimatization period,culturing or operation conditions,nutrient status and the presence of other inhibitors.Isolated strains Cu1,Cd1,Co2and Cr had the highest ability to grow individually in the presence of Cu,Cd,Co and Cr with IC50of275,320, 140and29mg/l,respectively.3.Although possessing multiple resistances,most of theacclimated strains exhibited their highest resistance with their corresponding metals.For example Enter-obacter sp.(Cu1and Cu2)showed the highest OD(275 and190,respectively)with copper that declined to190 and46,respectively with Cd followed by more toxicity with Co and Cr.Similarly,Stenotrophomonas sp.(Cd1)and Providencia sp.(Cd2)also showed high resistance to Cd(OD320and260,respectively;DHA 50and15,respectively)compared to their resistance pattern for other metals.4.Ochrobactrum sp.(Cr)isolated from activated sludgeamended with chromium was more sensitive to chromium(OD29)compared to copper and cadmium (OD75and150,respectively).On contrary Ochro-bactrum sp.strain CSCR-3isolated from chromium landﬁlls was able to tolerate and reduce Cr VI to Cr III in cultures containing800mg/l Cr(He et al.2009).This is mainly attributed to factors controlling resis-tance and/or toxicity of metals of which microbial species and the form of the metal are key factors (Gikas and Romanos2006).Results conﬁrmed that gradual increase of the applied metals produced generations with high metal resistance range that can be efﬁciently used in decontamination pur-poses.These results are in agreement and supported by other studies.For example a Cd-resistant Stenotropho-monas sp.showed resistance to Se(Antonioli et al.2007) and Cu(Zaki and Farag2010);a Cd-resistant Providencia sp.showed very high resistance(1,000–1,500mg/l)to Cd,Cr,Ni,Co and Zn(Thacker et al.2006)and Pseudomonas with multi resistance to Cr,Cu,Cd and Ni(Kelly et al. 2004).In addition,Comamonas sp.(Co2)that showed moderate growth and DHA activity(140and82,respec-tively)when cultivated with50mg/l cobalt was found containing plasmid encoding cobalt and nickel resistance (Siunova et al.2009).According to their average DHA(reﬂecting their resis-tance)against the individual metals,acclimatized bacteria can be arranged in the following order from the highly resistant to the highly sensitive Cd1[Co2[Cr[ Cu2[Cu1[M1[Cd2.Therefore,Stenotrophomonas sp. (Cd1)represents the highest DHA activity indicating the lowest toxic effects upon exposure to individual metals.Effect of mixed metalsTheﬁnal stage in the screening process was to investigate each of the acclimatized strain isolated against mixture of the tested heavy metals(Cu,Cd,Co and Cr)based on its IC50concentrations obtained in the previous stage.The concentration of each heavy metal for each strain was calculated by dividing its growth IC50by4,and then two higher and two lower concentrations were used for the screening process(i.e.,ﬁve mixtures with elevated metals concentration).Applying the investigated metals as mix-tures led to completely different responses from the investigated acclimatized strains(Fig.3).Results revealed the followingpoints:1.As with the individual metals,IC50of all testedmetals,those induced50%reduction in the growth,of all the investigated strains were higher than those induced50%reduction in their DHA,indicating that metal inhibits enzyme activity much higher than growth.2.As expected,tolerance of the investigated strains wasreduced dramatically towards the tested metal mixtures compared to their individual application.This was shown by the growth IC50recorded70,81.25,43.75 and12.5mg/l for strains Cu1,Cd2,Co2and Cr, respectively in the metal mixtures compared to275, 320,140and29mg/l,respectively when grown individually in their corresponding metals.3.IC50of DHA of the isolated strains Cu1,Cu2,Cd1,Cd2,Co2,Cr and M against the tested metals(Cu,Cd, Co and Cr)recorded35,20,2and2;37.5,18.75,11.25 and1.5;5,20,7.5and1;5,16.25,1.75and1.75;8,12.5,17.5and2;10,20,3.5and5;andﬁnally18,18,30and6mg/l respectively(Fig.3).4.According to their average DHA against the mixedmetals,acclimatized bacteria can be arranged in the following order from the highly resistant to the highly sensitive M[Cu2[Cu1[Co2[Cr[Cd.Delftia sp.exhibited the highest resistance to the mixture ofthe investigated metals due to the adaptation acquired during the acclimatization process when grown in the presence of heavy metal mixture.These results are consistent with those of Jackson et al. (2009)who reported a multiple heavy metal resistance of Delftia sp.when used within a consortium for bioremedi-ation of River Plankenburg water in South Africa con-taminated with aluminum,nickel,copper and manganese due to the presence of highly incidence plasmids(Zolg-harnein et al.2007).Treatability bioassayThree parallel strategies were used to evaluate the reme-diation capability of the proposed system towards heavy metal-and organic matter-contaminated wastewater. Treatment technologies included the proposed activated sludge unit amended with the acclimatized bacterial strains (ASBS),the plain activated sludge unit(AS)and the acclimatized bacterial cultures(BS)alone represented by Cu1,Cd1,Co2and Cr.The performance of the three pro-cedures was evaluated to determine the most efﬁcient application.Also a comparison was made between the removal efﬁciency of the contaminated metals andtheCFig.1continuedorganic matter load (as COD).Four different concentra-tions of each heavy metal were investigated in the three planned treatment procedures and the removal was deter-mined after 24h exposure.Treatment of wastewater efﬂuent under individual metal stressesWastewater collected from the Western Wastewater Treatment Plant (WWTP),Alexandria was subjected to 24h treatment using ASBS,AS and BS technologies after the addition of four elevated concentrations of individual heavy metals (20–200mg/l for each of Cu,Cd and Co and 20–300mg/l for Cr).Results achieved (Figs.4and 5)can be summarized asfollows:Table 2Resistant isolates’accession numbers and their similarity to the related species Isolate Accession no.Similar Strain Similarity (%)Cd 1FJ205387Stenotrophomonas sp.99Cd 2FJ205388Providencia sp.98Cu 1FJ205389Enterobacter sp.98Cu 2FJ205390Enterobacter sp.99Co 1FJ205391Chryseobacterium sp.98Co 2FJ205392Comamonas sp.97Cr FJ205394Ochrobactrum sp.98M 1FJ205393Delftia sp.9950100150200250300350Fig.2IC50for the growth (OD)and dehydrogenase activity (DHA)of the acclimatized strains after 24-h exposure to individual heavy metals:Cu copper,Cd cadmium,Co cobalt,Cr chromiumFig.3IC50for the growth (OD)and dehydrogenase activity (DHA)of the acclimatized strains after 24h exposure to the heavy metals in mixture:Cu copper,Cd cadmium,Co cobalt,Cr chromium1.As a general rule,removal of all the investigated metals was stimulated with increasing metal levels in the three investigated procedures reaching maximum efﬁciency at the highest applied concentration while COD removal decreased.Moreover,the removal of the investigated metals by the proposed technologies recorded the following order Co [Cd [Cr [Cu.2.The highest Cu removal achieved (at 200mg/l)by the ASBS,BS and AS technologies recorded 15.84,22.29and 31.37Cu/g biomass,respectively.Therefore,the magnitude of increasing Cu removal efﬁciency (RE %)is AS [BS [ASBS.On the contrary,COD removal under the highest Cu stress recorded 58.10,57.82and 38.16%using the ASBS,BS and AS technologies,respectively.Therefore,the magnitude of increasing COD RE %is ASBS [BS [AS.3.The proposed technologies ASBS,BS and AS could achieve Cd removal (at 200mg Cd/l)of 53.17,69.36and 56.25mg Cd/g sludge biomass,respectively.Therefore,Cd removal magnitude power order is BS [AS [ASBS technology.While COD removalwas recorded as 23.12,16.64and 50.99%using the ASBS,BS and AS technology,respectively.Therefore,the magnitude of increasing COD RE %is AS [ASBS [BS.4.Co removal achieved (at 200mg/l)by the ASBS,BS and AS technologies recorded 60.93,70.72and 67.12Co/g biomass,respectively indicating that Co removal efﬁciency by the three techniques is in the following order BS [AS [ASBS technology.COD removal under Co stress recorded 64.44,48.44and 55.16%using ASBS,BS and AS technology,respec-tively indicating magnitude of increasing COD RE %as ASBS [AS [BS.5.Cr removal (at 300mg/l)recorded as 55.17,47.89and 44.04mg Cr/g using ASBS,BS and AS technology,respectively indicating an order of ASBS [BS [AS technology in Cr removal efﬁciency.COD removal under the highest Cr stress recorded 67.51,10.51and 51.05%using the ASBS,BS and AS technology,respectively.Therefore,the magnitude of increasing COD RE %is ASBS [AS [BS.C DFig.4Comparison among the three bioremediation technologies AS,ASBS and BS in the removal efﬁciency of a Cu,b Cd,c Co and d Cr after 24-h exposureTreatability bioassays of the contaminated wastewater in the presence of the four tested metals applied as individuals achieved the following points:1.Native activated sludge (AS technology)exhibited the maximum efﬁciency in the removal of Cu.2.For Cd and Co removal,BS technology where acclimatized bacterial cultures were used,exhibited the highest efﬁciency followed by AS technology and ASBS technology with no signiﬁcant variations.3.Activated sludge amended with the acclimatized bacteria (ASBS technology)performed the highest efﬁciency for removing Cr.4.Activated sludge amended with the acclimatized bacteria (ASBS technology)performed almost the highest efﬁciency for removing COD from wastewater under the stress of all the investigated metals even under their highest levels.Metal biosorption by microbial biomass is a well-known advanced biotechnological tool used for efﬁcient removal of contaminant metals.Optimization tools of metalbiosorption included many factors among which pH is the most important one (Comte et al.2007)as well as the C/N ratio of activated sludge (Yuncu et al.2006)and temper-ature (Zouboulis et al.2004).Acclimatization is a very important process for acquiring new advanced features of microorganisms that can be efﬁciently applied in metal biosorption.During the present study,using strains previ-ously acclimated by exposing microbial biomass to metal concentration gradients (El.Bestawy et al.2012)proved high efﬁciency in the enhancement of activated sludge performance for metals and organic matter removal even at high metal stresses.For example,Enterobacter sp.(Cu 1)acclimated to survive under high levels of Cu stress (200mg/l)showed high efﬁciency for Cu (31.37Cu/g biomass)and organic matter (58.10%)removal compared to 2.93mg Cu/g achieved as the maximum removal by plain activated sludge (Principia et al.2006).Similar results were obtained by manipulating metal binding capacity of Nocardia amarae cells to optimize the overall Ni,Cu and Cd binding capacity of activated sludge (Kim et al.2002).As in the present study the pure cultureofC DFig.5Comparison among the three bioremediation technologies AS,ASBS and BS in the removal efﬁciency of COD in the presence of elevated levels of a Cu,b Cd,c Co and d Cr after 24-h exposureNocardia exhibited signiﬁcantly higher metal sorption capacity than the activated sludge biomass attributed to the fact that the Nocardia cells growing at stationary phase have substantially more speciﬁc surface area than that of activated sludge.The metal sorption capacity of activated sludge increased proportionally with the amount of Nocardia cells present in the mixed liquor.Sirianuntapi-boon and Ungkaprasatcha(2007)also reported efﬁcient removal of Pb and Ni as well as organic matter by com-paring acclimatized and un-acclimatized biosuldge.Reductions in the COD removal with increasing metal concentrations during the present study are attributed mainly to the toxic effects and inhibition of the biodegra-dative microbes in the proposed treatment systems induced by the investigated metals at their elevated levels(Jefferson et al.2001;Gikas2007).Treatment of wastewater efﬂuent under mixed heavy metal stressesTreatability study was also performed with mixtures of the four investigated metals with elevated concentrations after 24-h exposure.Three technologies were applied here included BS,where mixture(strains Cu1,Cd1,Co2and Cr) of the acclimatized bacteria was used,ASBS,where acti-vated sludge supplied with the acclimatized bacteria was used and plain activated sludge(AS).Heavy metal removalsAs general trends for all the investigated metals and the three proposed treatment technologies,increasing metal concentrations from5to75mg/l enhanced their removal which was in the following order Cu\Cr\Cd\Co. The highest achieved RE(s)%of Cu,Cd,Co and Cr recorded were6.61,16.58,27.58and19.83mg/g sludge, respectively using ASBS technology(Fig.6a);4.39,22.89, 30.38and26.52mg/g biomass,respectively using BStechnology(Fig.6b)andﬁnally7.09,15.02,21.08and 18.58mg/g biomass,respectively using AS technology (Fig.6c).The efﬁciency of using activated sludge under the stress of microbial inhibiting agents(i.e.heavy metals)is one of the issues with lots of environmental concern.Many factors affect the efﬁciency of activated sludge systems used for metal removal from contaminated media.pH considered as the most critical factor for microbial metal biosorption (Ozdemir et al.2003;Sheng-lian et al.2006;Congeevaram et al.2007)which is mainly attributed to organism-speciﬁc physiology(Congeevaram et al.2007).Other factors such as initial metal concentrations,activated sludge dosage, temperature,contact time(treatment duration),presence of other ions and organic materials in the bulk solutions (which may be in competition)could affect the efﬁciency of activated sludge for heavy metal removal.(Sheng-lian et al.2006;Comte et al.2007;Pamukoglu and Kargi 2007).Bieszkiewicz and Hoszowski(1978)reported that acti-vated sludge could tolerate up to0.8,1.15and20mg/l only of Cu,Cr III and Cr(VI),respectively after which the activated sludge experienced inferior puriﬁcation and reduced intensity of respiration of its microorganisms.In another study,removal efﬁciency%of Cu,Cd and Cr using activated sludge recorded only80,62and46%, respectively with initial concentrations of0.8,20.9and 0.63mg/l of the same metals(Petrasek and Kugelman 1983).Comparison with the present work indicated remarkable enhancement of the metal tolerance acquired 051015202530355101520253035510152025303502550750255075 0255075 ACBFig.6Comparison among the removal efﬁciency of the investigated metals mixture of Cu,Cd,Co and Cr using the proposed a ASBS, b BS and c AS technology for24-h exposure。

Characteristic of heavy metals in biochar derived from sewage sludge

ORIGINAL ARTICLECharacteristic of heavy metals in biochar derived from sewage sludgeTao Lu 1,2,3•Haoran Yuan 1,2•Yazhuo Wang 1,2•Hongyu Huang 1,2•Yong Chen 1,2Received:25September 2014/Accepted:4March 2015ÓSpringer Japan 2015Abstract This study investigates the characteristic of heavy metals (Pb,Zn,Cu,Cd,Cr,Ni and As)in biochar derived from sewage sludge at different pyrolysis tem-peratures (300,400,500,600and 700°C).The heavy metal concentrations,chemical speciation distribution,leaching toxicity,and bio-available contents were investigated using ICP-OES after microwave digestion,a sequential extraction procedure recommended by the Community Bureau of Reference (BCR),an improved nitric acid–sulphuric acid method,and diethylenetriamine pentaacetic acid (DTPA)extraction method,respectively.The results showed that a great percentage of the heavy metals remained in biochar,the concentrations of heavy metals in biochar (except Cd in B7)were higher than that in sludge,and the enrichment of the heavy metals in biochar enhanced with the pyrolysis temperature.Although the effect of pyrolysis temperature on the chemical speciation distribution,the leaching toxi-city and the bio-available contents of heavy metals in bio-char was inconsistent,the potential risk of biochar on soil and groundwater contamination was lower than sewage sludge.Keywords Sewage sludge ÁPyrolysis ÁBiochar ÁHeavy metal ÁToxicityIntroductionThe sewage sludge is a by-product generated in the sewage treatment process,containing rich organic material and nutrient elements such as nitrogen,phosphorus and potas-sium,and lots of toxic and harmful substances like germs,ova and heavy metals [1].With the greatly increasing de-mands for resources,the purpose of sewage sludge treat-ment was converted from volume and mass reductions to energy and resource utilization [2,3].Although farm ap-plication effectively re-utilized nutrient elements contained in sewage sludge,there are potential environment risks,germs,ova and heavy metals originally contained in sew-age sludge may result into soil and groundwater con-tamination,which greatly restricted large-scale farm application of sewage sludge as soil conditioner.Pyrolysis treatment,transferring the chemical energy from sewage sludge to syngas and bio-oil,is a relatively clean energy utilization method,but pyrolysis solid residual has long not been given enough attention to its further utilization [4].Recently,the pyrolysis solid residual,biochar,has at-tracted great attention because of its carbon sequestration and soil amendment beneﬁts [5,6].Because of its good biological and chemical stability,biochar can exist in the soil for more than a thousand years,thus reduce carbon emission effectively [7].In addition,biochar will help to improve the soil through raising the soil organic matter content,nutrient content,enhancing the water and nutrient holding capacity,aeration etc.[7,8].Compared to biomass feedstocks,sewage sludge is rich in nutrient elements (N,P and K)and minerals;therefore,sewage sludge biochar has inherent advantage of improving the soil fertility.How-ever,the majority of heavy metals (except the volatile elements Hg and Cd)originally contained in sewage sludge still remained in biochar [9–11],thus almost all the&Haoran Yuanyuanhr@1Guangzhou Institute of Energy Conversion,Chinese Academy of Sciences,Guangzhou 510640,China 2Key Laboratory of Renewable Energy,Chinese Academy of Sciences,Guangzhou 510640,China 3University of Chinese Academy of Sciences,Beijing 100049,ChinaJ Mater Cycles Waste Manag DOI 10.1007/s10163-015-0366-yresearches about sewage sludge biochar includes the in-vestigation on heavy metal migratory behaviors and its potential risk on soil and groundwater contamination.For example,Hossain et al.[12]reported the accumulation of heavy metal in biochar,and an obvious decline in available heavy metal content.Jin et al.[4]also found that all heavy metals in sewage sludge were kept in biochar except As, and their contents were enriched2.5–3.5times,whereas fast pyrolysis signiﬁcantly suppressed heavy metals leaching from biochar.Moreover,Agraﬁoti et al.[3]found pyrolysis suppressed heavy metal release in acetic acid extraction with pH of5.9and6.0.Additionally,the results from leaching experiments also showed that the leaching contents of heavy metals in biochar were signiﬁcantly lower than those in sewage sludge[13,14].He,Hwang and Kistler et al.[15–17]also concluded that heavy metals are immobile and stable in the biochar and that pyrolysis is able to reduce their potential release.Although extensive studies were conducted,the speciﬁc and comprehensive investigation on heavy metals in sew-age sludge biochar was still scarce.The main objective of the present work is to study the characteristic of heavy metals in biochar derived from sewage sludge.Hence, heavy metals contents in biochar produced at various temperatures were investigatedﬁrst,then their leaching contents were investigated to reﬂect their leaching toxicity. Extraction solvents with pH of5.0and6.0were used here to better simulate the actual acid soil environment.Finally, their chemical speciation distributions and bio-available contents were investigated to reﬂect their eco-toxicity and bioavailability,respectively.Materials and methodsExperimental materialsThe entire sewage sludge sample used in experiments was collected from Guangzhou Liede sewage treatment plant, with the treatment process of anaerobic–anoxic–aerobic. Dewatering of sewage sludge was conducted using a belt ﬁlter press and conditioning by cationic polymericﬂoccu-lants.The sewage sludge sample wasﬁrst separated from other impurities,such as glass fragments and plastic bags, and then dried in a convection laboratory oven at room temperature until it was qualiﬁed for pulverization.The sewage sludge sample was then ground to1-to2-mmﬁne particles,stirred and mixed in mortar to ensure that the collected sewage sludge sample was homogenous and representative for subsequent experiment.Finally,the sewage sludge sample was dried again overnight at tem-perature of105°C in a convection laboratory oven and was stored in sealed plastic bags until subsequent experiment.The ultimate analysis and proximate analysis of sewage sludge sample are shown in Table1.Experimental device and processThe experimental device(showed in Fig.1)for biochar preparation was horizontal quartz tube pyrolysis furnace, with a temperature range of room temperature and 1000°C,and a temperature precision of1°C.The quartz tube,with600mm length and50mm inner diameter,had two detachable rubber plugs on both sides,respectively, connected with inlet and outlet pipes.Puriﬁed nitrogen (99.999%)was provided by nitrogen cylinder to ensure that the whole pyrolysis process was under the oxygen-free atmosphere.Theﬂow meter1,connected with the inlet pipe,was used to measure the nitrogenﬂow to maintain the purgingﬂow at1000mL min-1.Theﬁrstﬂask,loaded with ice water mixture,was used to condense and collect the bio-oil and water vapor.The secondﬂask,loaded with sodium hydroxide solution,was used to absorb acidic gases.Theﬂowmeter2,connected with the outlet pipe,was used to measure theﬂow of nitrogen and syngas.Experimental process was described below.100±1g sewage sludge sample was loaded into the middle of the quartz tube,then the quartz tube was sealed with two rubber plugs,and the nitrogenﬂow was opened to purge the quartz tube for30min.Meanwhile,the pyrolysis fur-nace was heated with a heat rate of10°C per minute.The quartz tube was placed into the pyrolysis furnace when it reached the desired temperature(300,400,500,600and 700°C).During pyrolysis,the gasﬂow rate was measured byﬂowmeter2,which gradually increased and then de-creased,when it was consistent with that measured by ﬂowmeter1,indicating the pyrolysis process completed (no syngas emitting).Then,the pyrolysis furnace was turned off and the quartz tube was removed from the py-rolysis furnace quickly.When the quartz tube naturally was cooled to room temperature,the puriﬁed nitrogenﬂow was stopped,the biochar was recovered and kept in the sealed bags for further experimental analysis.In the following, B3,B4,B5,B6and B7represented the biochar produced at the temperatures of300,400,500,600and700°C, respectively.Analytical methodThe ultimate analysis and proximate analysis of sewage sludge sample were analyzed in triplicate according to Ultimate Analysis of Coal(GB/T476-2001)and Proximate Analysis of Coal(GB/T212-2001).Heavy metals were measured using ICP-AES(USA Thermo Jarrell Ash Cor-poration),after0.1g of the sewage sludge sample,biochar and bio-oil was dissolved in a mixture of HCI(2.25mL,J Mater Cycles Waste Manag5%),HNO3(0.75mL,5%)and HCIO4(2mL,5%), respectively(CJ221-2005).To better simulate the actual soil environment,an improved nitric acid–sulphuric acid method(HJ/T299-2007),the pH of extraction solvent was 5.0and 6.0,respectively,was used to investigate the leaching toxicity of heavy metals in sewage sludge and biochar.A sequential extraction procedure,recommended by BCR[18],was used to determine the heavy metals chemical speciation distribution,and DTPA method(NY/T 890-2004)was chosen to determine the bio-available heavy metals contents in sewage sludge sample and biochar.Statistic analysisANOVA was used to test ultimate analysis and proximate analysis results of sewage sludge,the heavy metals con-tents,bio-available heavy metals contents in sewage sludge and biochar,heavy metals concentrations in leachate of sewage sludge and biochar.The least signiﬁcant difference (p\0.05)test was applied to assess the differences among the means of three replications.Result and analysisHeavy metals contents in sewage sludge and biocharAs shown in Table2,the contents of heavy metals in sewage sludge varied greatly,the sequence was Pb[Zn[Cu[Cd[Cr[Ni[As,among which the content of Pb reached3740mg kg-1,while the content of As was only26mg kg-1.It was obvious that domestic wastewater generally contained low heavy metals contents, whereas the discharge of industrial wastewater into urban wastewater treatment plants would dramatically increase the content of speciﬁc heavy metal in sewage sludge. Although the contents of heavy metals in various biochar were varying,which were still higher than that in sewage sludge,additionally,the tendency was the contents of heavy metals increased with the pyrolysis temperature (except Cd in B7).Previous researches also reported heavy metals accumulation in the biochar with the increase of pyrolytic temperature[10,19].Although Zn,Cu,Cr,Ni, and As contained in biochar met the control standards for agricultural use(CJ/T309-2009),the excessive quantities of Pb and Cd limited the biochar application as soil amendment.The retention rate was deﬁned as the ratio of heavy metal quantities in biochar and sewage sludge,the result showed that90.4–98.3%of Pb,96.4–99.5%of Zn, 92.5–99.3%of Ni,85.5–92.5%of As,81.5–94.5%of Cu,and70.0–87.5%of Cr remained in biochar.Kistler et al.[17]reported the similar result,almost all the Cr,Ni, Cu and Zn remained in solid residues(biochar)over the temperature range of350–750°C.The heavy metals may exist as various mineral salts(carbonate,sulfate,chlorate, phosphate,etc.),sulﬁdes,hydroxide and oxide in sewage sludge,among which mineral salts and hydroxide areTable1Ultimate analysis and proximate analysis of sewage sludgeUltimate analysis(%)Proximate analysis(%)C H N S O a VM FC Ash12.15±0.23 2.74±0.08 1.57±0.040.48±0.0214.49±0.6928.34±0.22 3.09±0.5468.57±0.32The relative and absolute contents are on dry basisVM volatile matter,FCﬁxed carbona By differenceJ Mater Cycles Waste ManagT a b l e 2B i o c h a r y i e l d s ,h e a v y m e t a l s c o n t e n t s i n s e w a g e s l u d g e ,h e a v y m e t a l s c o n t e n t s a n d r e t e n t i o n r a t e i n b i o c h a rH e a v y m e t a l P a r a m e t e rS l u d g eB 3B 4B 5B 6B 7C J a C J bB i o c h a r y i e l d (%)83.374.069.2567.165.0P b C o n t e n t (m g k g -1)3740±27a4410±38b 4900±34c 5120±35d 5250±37e5200±46e 3001000R e t e n t i o n r a t e (%)98.397.095.194.290.4Z nC o n t e n t (m g k g -1)735±14.1a875±12.3b 986±17.5c 1040±13.4c d 1090±16.1d 1090±18.2d15003000R e t e n t i o n r a t e (%)99.299.398.399.596.4N i C o n t e n t (m g k g -1)72.4±2.8a86.3±2.9b 95.4±3.8c 97.7±3.5c 101±4.4c 103±4.6c100200R e t e n t i o n r a t e (%)99.397.593.893.692.5C dC o n t e n t (m g k g -1)169±4.3b197±4.6c 225±5.4d 235±5.6d 229±5.8d123±5.6a315R e t e n t i o n r a t e (%)97.198.596.690.985.8A sC o n t e n t (m g k g -1)26±1.3a27±1.4a 31±1.3a b 32±1.3a b 35±1.2b37±1.4b3075R e t e n t i o n r a t e (%)86.588.285.590.392.5C uC o n t e n t (m g k g -1)172±3.4a195±3.5b 213±3.5b 215±3.4b 209±3.4b227±3.5c5001500R e t e n t i o n r a t e (%)94.591.686.981.585.8C r C o n t e n t (m g k g -1)100±2.0a105±2.1a 118±2.2b116±2.1b106±2.0a103±2.1a5001000R e t e n t i o n r a t e (%)87.587.380.671.170.0B i o c h a r y i e l d (%)=(b i o c h a r m a s s /f e e d s t o c k m a s s )9100R e t e n t i o n r a t e (%)=(h e a v y m e t a l c o n t e n t i n b i o c h a r /h e a v y m e t a l c o n t e n t i n f e e d s t o c k )9b i o c h a r y i e l dD i f f e r e n t c h a r a c t e r s i n a s i n g l e r o w i n d i c a t e s i g n i ﬁc a n t d i f f e r e n c e b e t w e e n t h e t r e a t m e n t s a t p \0.05aD i s p o s a l o f s l u d g e f r o m m u n i c i p a l w a s t e w a t e r t r e a t m e n t p l a n t —A c o n t r o l s t a n d a r d s f o r a g r i c u l t u r a l u s e (C J /T 309-2009).S l u d g e m e t c o n t r o l s t a n d a r d s A i s p e r m i t t e d t o f e r t i l i z e v e g e t a b l e s ,f o o d c r o p s ,o i l c r o p s ,f r u i t t r e e s ,f e e d c r o p s ,a n d ﬁb e r c r o p sbD i s p o s a l o f s l u d g e f r o m m u n i c i p a l w a s t e w a t e r t r e a t m e n t p l a n t —B c o n t r o l s t a n d a r d s f o r a g r i c u l t u r a l u s e (C J /T 309-2009).S l u d g e m e t c o n t r o l s t a n d a r d s B i s p e r m i t t e d t o f e r t i l i z e o i l c r o p s ,f r u i t t r e e s ,f e e d c r o p s ,a n d ﬁb e r c r o p s ,a n d i s f o r b i d d e n t o f e r t i l i z e v e g e t a b l e s a n d f o o d c r o p sJ Mater Cycles Waste Managgenerally converted into oxide or sulﬁdes with better thermostability during reductive pyrolytic condition,thus a great proportion of the heavy metals remained in biochar. Correspondingly,0.5–8.6%of Pb,0–3.1%of Zn, 0–6.8%of Ni,6.1–12.4%of As,4.1–17.5%of Cu,and 11.1–28.2%of Cr were found in bio-oil.Similar contents of heavy metals were also detected in bio-oil from py-rolysis of sewage sludge in the previous research[20]. Thus,the recovery rates(the rate of the amount of each heavy metal detected in both of biochar and bio-oil,and that in sewage sludge)of Pb,Zn,Ni,As,Cu,and Cr was 98.8–99.0,99.0–99.5,99.0–99.3,97.9–98.6,98.6–99.0, and98.2–98.6%,respectively.Still a very small fraction of volatilized heavy metals was thought to remain in the cold surface of quartz tube and theﬁrstﬂask,therefore, syngas was regarded as heavy metals free.Chemical speciation of heavy metals in sewage sludge and biocharHeavy metals could be divided into exchangeable fraction (F1),reducible fraction(F2),oxidizable fraction(F3),and residual fraction(F4)according to BCR sequential extraction procedure.F1has good mobility,which has di-rect eco-toxicity and bioavailability to environment.F2 refers to Fe and Mn oxides and hydrous oxide,F3mainly refers to organic matter and sulphide,both of them have potentially eco-toxicity and bioavailability.F4refers to minerals as quartz,feldspars,etc.,which is stable in various environment,and has no eco-toxicity and bioavailability [18].As shown in Fig.2,for Cu,F3and F4fractions were the main forms,F1and F2fractions only accounted for small proportions,respectively,in sewage sludge.In B3,F1dis-appeared,F2and F4were almost the same as that in sewage sludge,while F3increased.In B4–B7,Cu existed as F3and F4,F1and F2disappeared.Yuan et al.[21]also found that Cu existed as F1and F2was reduced in biochar,the high percentages of Cu existed as F3was attributed the fact that the preference of Cu for organic matters.For Cr,The dis-tribution was similar to Cu in sewage sludge,while that was different in biochar.In B3–B7,F1disappeared,F3and F4 were the main forms,while F2still accounted for a very small fraction.Walter et al.[22]also showed over95%of Cr in sewage sludge existed in F3and F4,because most of Cr was in the organic and residual phases[23].Zn followedJ Mater Cycles Waste Managthe order of F2[F3[F1[F4in sewage sludge. Although pyrolysis leaded to decrease of F1fraction,still 1.4–11.2%of Zn existed as F1,thus Zn in biochar had a direct eco-toxicity and bioavailability.Additionally,F2 fraction decreased while F3fraction increased,therefore, Zn in biochar also had a potential eco-toxicity and bioavailability,especially in the oxidizing environment. Walter et al.also found high percentage of F3fraction owing to the afﬁnity of Zn for entering organic complexes [22].As followed the order of F1[F3[F4[F2in sewage sludge.With the increase of pyrolysis temperature, F1fraction in biochar gradually dropped off,thus the direct eco-toxicity and bioavailability of As declined,whereas As still had potential eco-toxicity and bioavailability,espe-cially in reducing environment,because F2fraction gradually rose,although F3fraction decreased with py-rolysis temperature.Ni existed mainly as F3form in sewage pared with sewage sludge,F2fraction dropped sharply,F3fraction increased,while F1and F4fractions changed little in biochar.According to these results,it may be concluded that Ni in biochar had higher potential eco-toxicity and bioavailability in oxidizing environment than in reducing environment.Almost all Pb and Cd existed as F4fraction in sewage sludge and biochar,which indicated that Pb and Cd were few eco-toxicity and bioavailability although Pb and Cd concentrations in sewage sludge and biochar exceeded Chinese sludge control standard for agricultural use(CJ/T309-2009).The above result showed that the chemical speciation distribution of heavy metals in biochar was largely determined by their distribution in sewage sludge.However,the eco-toxicity and bioavail-ability of heavy metal in biochar were relieved,as the loss of the mobile and easily available heavy metal,and the transformation of them into relatively stable forms during the pyrolysis process[21,24].Leaching toxicity of heavy metals in sewage sludge and biocharHeavy metals concentrations(Table3)in both leachate followed the order of Zn[Cu[Cr[Pb[Ni[As[ Cd,which was different from the order of heavy metals concentrations in sewage sludge and biochar,the result indicated that the leaching toxicity was inconsistent with the content of heavy metal.Moreover,the heavy metals concentrations in biochar leachate were lower thanJ Mater Cycles Waste Managthat of sewage sludge leachate,owing to the transform of some heavy metals from easy-to-leach forms to hard-to-dissolve forms through pyrolysis[21,24],and some loss of the mobile and easily available heavy metals during the pyrolysis process.The concentrations of heavy metals in leachate with pH of5and6were very low and similar, illustrating that heavy metals contained in biochar had similar immobility in acidic soil with pH of5and6.The reason might be theﬂourishing pore structure and high BET surface area of biochar enhanced its ability to im-mobilize heavy metals[3,15].It has also been reported that compounds and functional groups of biochar might sup-press the release of heavy metal through the formation of organometallic complex[4,13,16].Hence,the leaching toxicity of heavy metals in biochar was lower than that in sewage sludge,which corresponded to the reduction of the eco-toxicity and bioavailability of heavy metal in biochar resulted from BCR sequential extraction procedure.How-ever,the effect of pyrolysis temperature was limited,there was no signiﬁcant difference among the concentrations of heavy metals in leachate of biochar produced at different temperatures except at300°C,because the temperature was not high enough to stabilize heavy metals through melting like the high temperature incineration[25,26].The leaching rate,deﬁned as the ratio of the leachable heavy metal to the total content of each heavy metal,was employed to investigate the proportion of leachable heavy metal.The results are shown in Table4.Obviously,the leaching rates of heavy metals in sewage sludge were much lower than that of biochar,which indicates converting sewage sludge into biochar signiﬁcantly reduces heavy metals leaching toxicity.However,pyrolysis temperature exerted various effect on different heavy metal leaching rate.Speciﬁcally,For Pb,Ni,Cd and Cr,their respective leaching rates had no signiﬁcant difference during the range of300and700°C.For Zn,the leaching rate had no signiﬁcant difference during the range of400and700°C, however,it was lower than that of biochar produced at 300°C.For As and Cu,both their leaching rates decreased with the pyrolysis temperature rising,which may ascribed to more transformation of As and Cu from easy-to-leach forms to hard-to-dissolve forms,and more loss of the mobile and easily available As and Cu with the increasing pyrolysis temperature,as discussed above.Bio-available heavy metals in sewage sludgeand biocharBio-available heavy metal was the heavy metal fraction which could be directly absorbed by plants,the DTPA extractable fraction has been widely used to estimate bioavailability of heavy metals in sludge[12,13],due to its capacity to chelate a wide range of metallic elements.The bio-available heavy metal contents in sewage sludge and biochar are shown in Table5,the changes of bio-available heavy metals contents reﬂected the transformations of chemical speciation of heavy metals during the pyrolysis process.The contents of bio-available Pb,Cd,Ni and Cr in biochar produced at different temperatures were similar (p\0.05),respectively,the contents of bio-available Zn and As peaked in B3,and the content of bio-available Cu peaked in B6;hence,the pyrolysis temperature had in-consistent effect on the contents of bio-available heavy metals in biochar,which was similar to the result obtainedTable3Heavy metals concentrations in leachate of sewage sludge and biocharLeachate pH Concentration(mg L-1)Pb Zn Ni Cd As Cu CrSludge5 3.93±0.15b22.96±0.48c 2.94±0.09b0.11±0.01b 2.8±0.1c13.78±0..93c13.33±0.94b6 3.89±0.14b22.12±0.47c 2.96±0.09b0.12±0.01b 2.8±0.1c13.65±0.97c13.23±0.96b B35 3.52±0.12ab12.04±0.23b 1.83±0.06a0.03±0.01a 1.0±0.1b 5.23±0.33b 2.34±0.12a6 3.53±0.13ab11.89±0.20b 1.85±0.06a0.03±0.01a 1.0±0.1b 5.18±0.37b 2.28±0.11a B45 3.61±0.14ab 5.57±0.16a 2.31±0.07ab0.03±0.01a0.3±0.1a 2.71±0.15a 2.11±0.10a6 3.56±0.13ab 5.62±0.16a 2.23±0.07ab0.03±0.01a0.3±0.1a 2.65±0.16a 2.03±0.11a B55 3.65±0.14ab 5.34±0.15a 1.94±0.05a0.02±0.01a0.3±0.1a 2.20±0.15a 2.10±0.10a6 3.67±0.13ab 5.57±0.15a 1.89±0.05a0.03±0.01a0.3±0.1a 2.34±0.14a 2.08±0.11a B65 3.55±0.13ab 5.67±0.15a 2.06±0.06a0.03±0.01a0.3±0.1a 2.25±0.14a 2.24±0.10a6 3.62±0.13ab 5.68±0.15a 2.01±0.06a0.03±0.01a0.3±0.1a 2.30±0.13a 2.21±0.10a B75 3.38±0.14a 5.63±0.15a 2.15±0.06a0.02±0.01a0.2±0.1a 2.07±0.13a 2.13±0.10a6 3.33±0.14a 5.64±0.15a 2.10±0.06a0.03±0.01a0.3±0.1a 2.01±0.13a 2.18±0.10a Different characters in a single column indicate signiﬁcant difference between the treatments at p\0.05J Mater Cycles Waste Managby Hossain et al.[12].However,it was consistent and obvious that biochar had lower contents of bio-available heavy metals than sewage sludge,indicating that pyrolysis process could repress the release of heavy metal in DTPA extractant,which was similar to the decrease of the leaching concentrations of heavy metals in biochar dis-cussed above,hence the absorption ofﬂourishing pore structure,high BET surface area of biochar on heavy metals,and the formation of organometallic complex could be used to explain the low bio-available heavy metals contents in biochar[4,13,16].ConclusionBiochar was prepared with sewage sludge by pyrolysis at the temperature range of300and700°C.A great per-centage of heavy metals remained in biochar,the concentrations of heavy metals in biochar(except Cd in B7)were higher than that in sewage sludge,and the en-richment of the heavy metals in biochar enhanced with the pyrolysis temperature.The chemical speciation distribution of heavy metals indicated biochar had less eco-toxicity and bioavailability than sewage sludge,although it was largely determined by their chemical speciation distribution in sewage sludge.Although leaching toxicity of heavy metals in biochar was lower than that in sewage sludge,the effect of pyrolysis temperature was limited,the leaching toxicity of heavy metals in various biochar was similar to each other except in B3.Pyrolysis process also repressed the release of heavy metals in DTPA extractant,which indi-cated biochar had lower bio-available heavy metals con-tents than sewage sludge.Thus,biochar has low potential risk on soil and groundwater contamination when it is used as soil amendment,however,there are still some discrep-ancies between the experimental situation and the actualTable4Leaching rates of heavy metals in sewage sludge and biocharLeachate pH Leaching rate(%)aPb Zn Ni Cd As Cu CrSludge50.70±0.03b20.83±0.62c27.07±1.76b0.43±0.03b71.79±2.56d53.41±1.56d88.87±2.89c60.69±0.02b20.06±0.67c27.26±1.71b0.47±0.03b72.31±2.78d52.91±1.58d88.20±2.92c B350.53±0.02a9.17±0.35b14.13±1.45a0.10±0.01a25.19±1.32c17.88±0.83c14.86±0.67a60.53±0.02a9.06±0.32b14.29±1.43a0.10±0.01a25.68±1.12c17.71±0.88c14.48±0.66a B450.49±0.02a 3.78±0.21a16.14±1.49a0.09±0.01a 6.67±0.53b8.48±0.45b13.92±0.56a60.48±0.02a 3.80±0.22a15.58±1.48a0.09±0.01a 6.02±0.64b8.29±0.43b13.47±0.57a B550.48±0.02a 3.42±0.21a13.24±1.44a0.06±0.01a 6.88±0.51b 6.82±0.39b13.07±0.57a60.48±0.02a 3.57±0.20a12.90±1.43a0.09±0.01a 6.88±0.52b7.26±0.44b12.95±0.58a B650.45±0.02a 3.47±0.20a13.60±1.44a0.09±0.01a 4.76±0.36a7.18±0.45b14.09±0.62a60.46±0.02a 3.47±0.20a13.27±1.46a0.09±0.01a 5.33±0.46ab7.34±0.42b13.90±0.60a B750.43±0.02a 3.44±0.21a13.92±1.45a0.11±0.01a 4.14±0.35a 6.08±0.43a13.79±0.61a60.43±0.02a 3.45±0.20a13.59±1.43a0.11±0.01a 4.50±0.36a 5.90±0.42a14.11±0.59a Different characters in a single column indicate signiﬁcant difference between the treatments at p\0.05a Leaching rate=(heavy metal concentration9leachate volume)/(heavy metal concentration9raw material mass)Table5Bio-available heavy metals contents in sewage sludge and biochar(DTPA extractant)Sludge(mg kg-1)B3(mg kg-1)B4(mg kg-1)B5(mg kg-1)B6(mg kg-1)B7(mg kg-1)Pb74.3b47.8a52.3a49.6a51.1a51.4aZn105d84.2c73.6b70.4b39.0a37.5aNi 4.32b 2.12a 1.89a 2.05a 2.11a 2.13aCd8.25b 3.16a 3.23a 2.89a 3.21a 3.06aAs0.84c0.51b0.21a0.18a0.20a0.18aCu8.97c0.30a0.35a0.21a 1.41b0.41aCr 4.23b 1.67a 1.54a 1.47a 1.62a 1.53a Different characters in a single row indicate signiﬁcant difference between the treatments at p\0.05J Mater Cycles Waste Manag。

The remediation of heavy metals contaminated sediment

Journal of Hazardous Materials 161(2009)633–640Contents lists available at ScienceDirectJournal of HazardousMaterialsj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /j h a z m atReviewThe remediation of heavy metals contaminated sedimentJian-feng Peng,Yong-hui Song ∗,Peng Yuan,Xiao-yu Cui,Guang-lei QiuChinese Research Academy of Environmental Sciences,100012Beijing,Chinaa r t i c l e i n f o Article history:Received 23June 2007Received in revised form 19March 2008Accepted 21April 2008Available online 24April 2008Keywords:Heavy metal Sediment DistributionInﬂuence factorsIn situ and ex situ remediationa b s t r a c tHeavy metal contamination has become a worldwide problem through disturbing the normal functions of rivers and lakes.Sediment,as the largest storage and resources of heavy metal,plays a rather important role in metal transformations.This paper provides a review on the geochemical forms,affecting factors and remediation technologies of heavy metal in sediment.The in situ remediation of sediment aims at increasing the stabilization of some metals such as the mobile and the exchangeable fractions;whereas,the ex situ remediation mainly aims at removing those potentially mobile metals,such as the Mn-oxides and the organic matter (OM)fraction.The pH and OM can directly change metals distribution in sediment;however oxidation–reduction potential (ORP),mainly through changing the pH values,indirectly alters metals distribution.Mainly ascribed to their simple operation mode,low costs and fast remediation effects,in situ remediation technologies,especially being ﬁt for slight pollution sediment,are applied widely.However,for avoiding metal secondary pollution from sediment release,ex situ remediation should be the hot point in future research.©2008Elsevier B.V.All rights reserved.Contents 1.Introduction.........................................................................................................................................6342.Metals species in sediment .........................................................................................................................6343.Inﬂuence factors for heavy metal distributions ....................................................................................................6343.1.The inﬂuence of pH values...................................................................................................................6343.2.The inﬂuence of OM species.................................................................................................................6353.3.The inﬂuence of ORP ........................................................................................................................6353.4.The inﬂuence of some other factors.........................................................................................................6364.Remediation technology............................................................................................................................6364.1.In situ remediation technology..............................................................................................................6364.1.1.Amendments.......................................................................................................................6364.1.2.Sand cap............................................................................................................................6364.1.3.Phytoremediation..................................................................................................................6364.2.Ex situ remediation technology.............................................................................................................6374.2.1.Washing............................................................................................................................6374.2.2.Electrochemical remediation......................................................................................................6374.2.3.Flotation ...........................................................................................................................6384.2.4.Ultrasonic-assisted extraction.....................................................................................................6384.2.5.Immobilization ....................................................................................................................6385.Conclusions .........................................................................................................................................638Acknowledgement..................................................................................................................................639References ..........................................................................................................................................639∗Corresponding author.Tel.:+861084928381.E-mail address:pjf1995@ (J.-f.Peng).0304-3894/$–see front matter ©2008Elsevier B.V.All rights reserved.doi:10.1016/j.jhazmat.2008.04.061634J.-f.Peng et al./Journal of Hazardous Materials161(2009)633–6401.IntroductionNowadays,heavy metals originating from anthropogenic activities are frequently detected in sediments and water columns of river/lake,which cause a considerable number of the world’s rivers/lakes severely contaminated[1–4].Heavy metals pollution had gradually become a major concern world-wide.In aquatic environment,heavy metal is usually distributed as follows:water-soluble species,colloids,suspended forms and sedimentary phases.However,unlike organic pollutants,natural processes of decomposition do not remove heavy metals.On the contrary,they usually are enriched in sediment by organisms or some other compounds.In some conditions,more than99%of heavy metal entering into river can be stored in river sediments in various forms[5].However,heavy metals cannotﬁx in sediment forever.With the variation of the physical–chemical characteris-tics of water conditions,part of theseﬁxed metals will re-enter the overlying water and become available to living organisms.Thus, sediment often acts as both carriers and potential sources for metals in aquatic environment[1].Heavy metals usually possess signiﬁ-cant toxicity to aquatic organisms,and then affect human health through food chain.Therefore,investigating the transformation and distributions mechanisms of heavy metal in sediment becomes necessary.The main purpose of this paper is to provide a review on the geochemical forms,affecting factors and remediation technologies of heavy metal in sediment,depending on literatures and practi-cal working experiences.Only based on these,the optimization of remediation technologiesﬁtting for polluted sediment can be achieved.2.Metals species in sedimentIn sediments,metals can be bound to various compartments in different ways:occluded in amorphous materials;adsorbed on clay surfaces or iron/manganese oxyhydroxides;presenting in lattice of secondary minerals like carbonates,sulfates or oxides;complexed with organic matter(OM)or lattice of primary minerals such as silicates[6,7].Since each form has different remobilization poten-tial,and then affects its respective bioavailability and toxicity,the measurement of total metal may not be able to provide exact infor-mation about the characteristics of pollution[8].To clearly investigate the toxicity of heavy metal to aquatic biota, in the past decades,different sequential extraction procedures for partitioning the metals bound to various mineral components had been developed[9].Based on these,the community Bureau of Reference of the Commission of the European Union initiated a major effort to harmonize these extraction procedures.Their works produced the deﬁnition of an extraction protocol(the BCR protocol)and a purely operational deﬁnition of sequential frac-tionation.For enlarging the research scopes,depending on the different partition conditions among various metals,some assis-tant sequential extraction procedure were also established and popularized.Some primary characteristics of them are shown in Table1.In all of these sequential extraction methods,the four steps method mainly established by Tessier et al.won the most popu-lar application[6].This method partitions the heavy metals into ﬁve fractions:extractable and exchangeable,carbonate bound,iron and manganese oxides bound,organic matter bound and residual metal.Exchangeable fraction,usually extracted with magnesium chlo-ride solution or sodium acetate solution(1M)at pH8.2for1h,refers to the metals directly adsorbing on sediments[16].Through some typical sorption–adsorptions processes,these metals can be exchangeable and are in equilibrium with the ionic content in water. Generally,this fraction is usually used to represent the environmen-tally available components.Carbonates fraction,continuously extracted with NaOAc or HOAc solution(1M)at pH5.0for5h,mainly refers to the met-als that are precipitated or co-precipitated with carbonate.This fraction is sensitive to pH variations.Fe–Mn oxides fraction includes the soluble metal oxides/hydroxides under slightly acidic pH as well as the metal associated with reducible amorphous Fe–Mn oxyhydroxides, which was extracted with25%(v/v)acetic acid containing some NH2OH·HCl at96◦C for6h.This fraction can be dissolved with oxidation–reduction potential(ORP)varying.Organic fraction may be associated with various forms of organic material such as living organisms,detritus or coatings on mineral particles through complexation or bioaccumulation process.It is extracted mainly with0.02M nitric acid and30%hydrogen peroxide at pH2.0and85◦C.This kind of metals can exist in sediment for longer periods,and can also be released with OM decomposition.Residual fraction,namely the metals still remained in sediment after the above extraction procedures,usually presents as consoli-dated oxides,co-precipitates,and strongly held complexes,which keeps relatively stable and does not show signiﬁcant transforma-tion in various conditions.Normally,the summation of the mobile and the exchangeable fractions can be used to assess the environmentally available com-ponents.The fractions bound to Mn oxides and organic material are supposed to represent the potentially mobile component under changing conditions,which are reviewed as the most important components in sediments for metals binding.While the residual fraction represents the more stable metal forms associated with anthropogenic or geogenic components,the inﬂuence of which to ecological system is much less than the others in major condi-tions.3.Inﬂuence factors for heavy metal distributionsIt has been validated that each environmental factor presents unique inﬂuence on metals distribution in sediment.The inﬂuences of some factors,such as pH,ORP and OM are more crucial than the others.Only a slight variation of them,the distributions of metals would be producing some signiﬁcant variations.Correspondingly, some other factors(e.g.salinity,temperature)can only alter met-als distribution to a less extent.In this paper,only those relatively important factors are introduced.3.1.The inﬂuence of pH valuesThe pH is a key parameter controlling heavy metal transfer behavior in sediment.Normally,with pH decreasing in sediment, the competition between H+and the dissolved metals for ligands (e.g.OH−,CO32−,SO42−,Cl−,S2−and phosphates)becomes more and more signiﬁcant.It subsequently decreases the adsorption abilities and bioavailability of the metals,and then increases the mobility of heavy metal.Sometimes,only with a few lower of pH units,theﬁxation percentage of heavy metals on sediment parti-cles may range from almost a100%to none[17].In sediment,due to the OM degradation and the acid volatile sulﬁde oxidation,the pH of sediment usually decreases from the neutral in the initial to acid,sometimes even decreasing to pH1.2,which often results in some metals being released into water again even under stable water conditions[15,18].J.-f.Peng et al./Journal of Hazardous Materials161(2009)633–640635Table1Characteristic of different sequential extraction proceduresSequence number Total extraction steps Extracted metal species Reference1Three steps Acid soluble fraction;reducible fraction;oxidizablefraction;residual fraction[10,11]Exchangeable fraction;OM fraction;carbonate fraction;residual fraction[12]2Four steps Extractable/exchangeable fraction;carbonate fraction;ironand manganese oxides fraction;OM fraction;residualfraction[6]3Five steps Exchangeable Cations fraction;carbonates fraction;easilyreducible fraction(e.g.manganese oxides);moderatelyreducible fraction(e.g.amorphous iron oxyhydroxides);oxidizable fraction(organic phase+sulﬁdes);residualfraction[1,8]Exchangeable metals fraction;iron and manganese oxidesfraction;weakly bound to organic matter fraction;stronglybound to organic matter fraction;sulﬁde phase fraction;residual fraction.[13]4Six steps Mobile fraction;exchangeable fraction;Mn-oxide fraction;organic fraction;amorphous Fe oxides fraction;crystallineFe oxides fraction;residual fraction.[14]5Seven steps Mobile fraction;exchangeable fraction;Mn-oxidesfraction;OM fraction;Fe oxides(anthropogenic)fraction;Fe oxides(geogenic)fraction;sulﬁde fraction;residualfraction.[15]In sediment,there exists a limit pH controlling heavy metal mobility.And the trace metal would be released only as they reaching such pH value.For different metals,this limit pH is dif-ferent correspondingly,which can be found in Table2.Therefore, under similar pH value,the potential mobility of heavy metals in sediment is different signiﬁcantly.For example,when pH was con-trolled at4.0,the potential mobility of metal decrease as follows: Zn>Cd>Ni>As>Cu>Pb[19].3.2.The inﬂuence of OM speciesOrganic compounds in sediment,frequently existing in consid-erable amounts in particle form,play an important role in heavy metal transformation.For example,in the sediment of some rivers or lakes,the heavy metal bound to OM generally takes up the largest fraction.Additionally,in sediment,the solubility of organic matters usually directly determines the mobility of heavy metals.Normally, the complexation of metal ions with insoluble organic compounds can strongly lower their mobility,whereas the formation of sol-uble metal complexes with dissolved organic compounds would enhance their mobility[20].In natural rivers or lakes,OM is mainly composed of humic and fulvic substances.The complexation reaction between heavy metals and organic complexants is usually recognized as the most important reaction pathway,due to this reaction determining,to a large extent,the speciation and bioavailability of metal,and then inﬂuencing the mobility of trace metal in natural water environ-ment.However,in severely polluted river,due to the complexity of organic matter,the reaction types between organic complexes andTable2The limit pH values of different metals in sedimentMetal species pH limitZn 6.0–6.5 Cd 6.0Ni 5.0–6.0 As 5.5–6.0 Cu 4.5Pb 4.0Al 2.5Fe 2.5metals are difﬁcult to predict.In most conditions,precipitation,co-precipitation orﬂocculation usually plays the most important role in heavy metalﬁxation.3.3.The inﬂuence of ORPIt is generally accepted that sediment ORP is also a most impor-tant factors controlling heavy metal mobility[5].In anaerobic sediment,Acid volatile sulﬁde(AVS),a key component controlling the activities of some divalent cationic metals,usually present nat-urally[21].Initially,the majority of AVS contained in the anaerobic sediment is bound to iron as solid iron monosulﬁde(FeS),crys-talline mackinawite(FeS),pyrrhotite(FeS),greigite(Fe3S4),or exits as free sulﬁdes.However,if divalent metals,such as cadmium,cop-per,chromium,lead or zinc are present,the iron in iron-sulﬁde are displaced and one of these heavy metals rapidly bind to AVS with stronger afﬁnity[22].Finally,in those sediment contaminated,the metal bound with sulﬁde usually takes up a rather high propor-tion.When ORP in sediment increases,the oxidization rate of metal sulﬁdes and the degradation rate of organic compounds will increase correspondingly.Both can accelerate the release of the adsorbed/complexing heavy metal[23].The reaction can be expressed as:MS2+(15/4)O2+(7/2)H2O M(OH)3+2SO42−+4H+. The release of H+ions into porewater would decrease the pH of sediment and then cause a secondary release of heavy metals.Part of this released material will be re-adsorbed,especially onto the more labile binding fractions.For example,with ORP in sediment increase,the Cd bound to organic sulﬁde,a stable metal forms, would decrease from65%to30%and form a more mobile form [1,24].Accordingly,with the annual variations of ORP in sediment,the heavy metal presents seasonal release andﬁxation.These phe-nomena are very signiﬁcant in some seasonallyﬂooding rivers. For example,in Mulde reservoir,approximately18t of Zn can be released from sediment into water only due to the sediment being disturbed and oxidized inﬂooding,which led to a signiﬁcant increase of Zn concentration in water[1].Therefore,in the dredging process of river or lake,for decreasing the release of metal from sediment,oxidation of sediment should be avoided.636J.-f.Peng et al./Journal of Hazardous Materials161(2009)633–6403.4.The inﬂuence of some other factorsExcept pH,OM and ORP,some other factors,such as temper-ature,salinity,metal species and retention time,can also affect the distributions of heavy metal in sediment.For example,due to the differences of cation exchange capacity among different metal, their mobility capacity varies correspondingly,and usually ranges as follows:Cs>Zn>Cd>Fe>Ag>Co>Mn[25];with temperature increasing,the adsorption content of heavy metal on sediment often decreases gradually;with salinity in porewater increasing,the total adsorption content of heavy metal would decrease ascribed to the competition among heavy metal and some other cations [26].Additionally,long time kinetic adsorption–desorption exper-iments also show that the metal freshly associated with particles presents less stable and higher potential bioavailability than those associated for a long time.4.Remediation technologyJust like soil remediation,a two-tiered remediation strategy has also been adopted for remedying the sediment contaminated by heavy metal.Theﬁrst tier aimed at in situ increasing the stabiliza-tion of metals on sediment particles(e.g.immobilization)and the second tier aimed at ex situ extracting or separating metals from sediment(e.g.washing,ﬂotation)[4,27].Theﬁrst strategy focus on improving metal stabilization,in which mainly by enhancing metal sorption,precipitation and complexation capacity on sediment,the potential mobility or bioavailability of the toxic metals to environment are lowered.Due to these stabilization techniques usually being carried out“in situ”, their remediation cost is relatively low[28,29].Noteworthily,these techniques just improve the immobility of heavy metal on sedi-ment and their total content in sediment does not lower.Therefore, in some special conditions,part of these immobilized metals will be released into the water again.The other strategy is extraction,in which sediment polluted is dredged from the river bed and heavy metal is extracted from the sediment through a series of chemical,physical,biological methods in a specially designed reactor.This kind of remediation techniques is usually carried out“ex situ”,and can remove almost all mobile metal.However,it also implies the sediment structure deterioration and high costs,which limit their popularization on vast contami-nated areas[30,31].Compared with ex situ remediation techniques,the key advantage of in situ stabilization approach is that only need a simple mixture of amendments with sediment.And the major disadvantage of this approach is that theﬁnal product of reme-diation,although existing in inactive form,still remains in sediment.4.1.In situ remediation technologyDue to low cost and non-disruptive to natural hydrological con-ditions than conventional ex situ extraction technologies,in situ metal immobilization technologies developed rapidly in the past several decades.Several typical in situ remediation technologies are introduced as follows.4.1.1.AmendmentsAmendment,usually possessing high cation exchange capacity, can lower metal mobility and bioavailability in sediment by precip-itation or sorption,thereby decreasing their solubility.The in situ immobilization of metals,usually using inexpensive amendments such as minerals(e.g.apatite,zeolites,steel shot,or beringite),is considered as a promising alternative to the currently available remediation pared to the amendments used in soil, that used in sediment usually has higher sorption capacity,lower water solubility(K sp=10−40),higher stability under reducing and oxidizing conditions,and lower cost[28].Apatite is usually selected as an ideal amendment for sediment remediation.Generally,it is formulated in the form of Ca10–a−b Na a Mg b(PO4)6−x(CO3)x F2+0.4x with isomorphic substitution of carbon-ate for phosphate,F for hydroxy,and minor substitution of Ca2+ by Na+and Mg2+atoms.In sediment remediation process,metal ﬁrstly incorporates into the apatite lattice through ion exchange with Ca2+.This reaction can stimulate apatite dissolution and phos-phate is released correspondingly.Due to the lower solubility between metal ions and phosphate,a new metal-phosphate solid phase(such as Ca10−x Pb x(PO4)6(OH)2)would form[32].Through theseﬁxing process,apatite minerals can effectively immobilize almost all Pb,Mn,Co,Cu,Cd,Zn,Mg,Ba,U,and Th in sediment [28,33].4.1.2.Sand capNormally,decreasing the direct contact area between water and the contaminated sediment is a good choice for lowering the release content of heavy metal.Therefore,capping the contaminated sedi-ment with sandy materials,such as clean sediment,sand,or gravel becomes an effective remediation technique.Through physical iso-lation,chemical isolation or sediment stabilization,the mobile and the exchangeable metals are transformed from the contaminated sediment into the clean cap and combined with particles in more stable forms.When properly designed,the placement of a rela-tively coarse-grained cap does not disturb or mix with underlying very softﬁne-grained sediments.Some researchers also showed that a good cap thickness was approximately50cm;and through capping the sediment by sands materials,the heavy metal concen-tration in water could reduce to80%[1].Additionally,compared with other in situ remediation methods,this approach has a lower cost.However,this remediation technique can only reduce the trans-fer rate of metal in sediment,while their immobilization effect for heavy metal is small.Therefore,for enhancing their immobiliza-tion capacity,some amendments(such as apatite,rock phosphate, lime or zeolite)can also be added into the sand cap.These reac-tive materials would demobilize heavy metals from solution and enhance the cap quality.For example,Jacobs and F¨orstner found that through adding the natural zeolite in sand cap,theirﬁxa-tion capacity for heavy metals and organic contaminants increases sharply[34].4.1.3.PhytoremediationPhytoremediation is the use of plants to extract,sequester,or detoxify pollutants.This technology is widely viewed as an eco-logically responsible alternative to the environmentally destructive chemical remediation methods currently practiced[35].This tech-nology is popularly applied in soil remediation,and also shows some excellent remediation effects in some shallow rivers,lakes and wetlands.At present,this technology had presents good immo-bilization effects for Zn,Fe,Mn and Cd in sediment.Phytoremediation is comprised of two tiers,one by plants themselves and the other by the root colonizing microbes,which degrades the toxic compounds to further non-toxic metabolites. Normally,hydrophytes have the ability to uptake and accumu-late various heavy metals by the action of phytochelatins and metal lothioneins[36].However,mass balance experiments show that metal uptake by hydrophytes were not high enough for phy-toextraction.This indicates that in hydroremediation,the direct uptake of hydrophytes is small,and the indirect reactions,such as stimulation of microbial activity,redox reactions/formation andJ.-f.Peng et al./Journal of Hazardous Materials161(2009)633–640637precipitation of insoluble metal compounds in the rhizosphere, may play a relative important role[37].Therefore,the direct uptake capacity of phytoremediation for heavy metal can be achieved by most plant species able to grow in the contaminated sediment.Cor-respondingly,the choice of hydrophytes would not depend on their apparent uptake capacity,but on their practical immobility capacity for metal.4.2.Ex situ remediation technologyIn situ remediation techniques are usually applied for the sedi-ment slightly polluted by heavy metal.However,for the sediments heavily polluted,their remediation effects can be ignored to a large extent.Under such conditions,ex situ sediment remediation becomes theﬁrst choice[38].Most ex situ remediation technolo-gies for soil or mineral ores can be used for dredged sediment. However,due to higher workload and different environmental char-acteristics in sediment,some technologies have higher costs and more complex when used in sediment remediation.Only those promising alternative technologies are introduced as follows.4.2.1.WashingSediment washing is a relatively simple and useful ex situ reme-diation technology,which involves through adding washing water, heavy metal can be transferred from the dredged sediment to wash solution.To enhance the performance of sediment washing,various additives can be employed,such as acid washing(e.g.H2SO4and HNO3),chelating agents(e.g.EDTA,DTPA and EDDS)or surfactants (e.g.rhamnolipid).These additives can assist in the solubilization, dispersal and desorption of metal from dredged sediments.This technology is most appropriate for the weaker bound metals in the form of exchangeable,hydroxides,carbonates and reducible oxides fraction.Residual fractions,the most difﬁcult ones to remove,are not affected during the washing process[38,39].Additionally,ﬁne grain sediments are difﬁcult to decontaminate through washing solutions,therefore washing is most applicable to sands and grav-els.Acid leaching,as a typical washing method,refers to the reme-diation of sediment through extracting the metals with sulfuric acid.Considering the sources of leaching agent,acid leaching can be divided into abiotic leaching and microbial leaching[40,41].In abiotic solid-bed leaching,the H2SO4is supplied to the sediment by circulating water with a rate highly dependent on the solid-bed height and percolationﬂow.However,in microbial solid-bed leaching,elemental sulfur added to the sediment is oxidized to sul-furic acid within the package which,in turn,solubilize the heavy metals.Here,the percolationﬂow and the solid-bed height do not affect the rate of metal solubilization[42,43].Both methods present perfect removal efﬁciencies(>90%)for total extractable Ni,Zn,Cu and Cr.And the pH ranges are controlled depending on the heavy metal species.For example,Al is markedly solubilized at pH<4,and Fe at pH<2.4.However,due to a lesser requirement for acid and lime,which subsequently lower the operational difﬁculties of the microbial solid-bed leaching,the microbial process is80%cheaper than an abiotic solid-bed leaching[29].Additionally,when solid-bed leaching is performed on a larger scale,microbial leaching is much faster than abiotic leaching[40].Therefore,microbial solid-bed leaching presents great attraction in sediment remediation.Chelating agents used in sediment remediation usually possess higher chelating afﬁnity for metal.They can combine with aqueous metal to form chelate complex,which subsequently lower the metal concentration in water.Through the adsorption/release balance between sediment and water,the toxic and bioavailable metals adsorbed on sediment will be transferred to chelate complex grad-ually,andﬁnally removed with solution.This method is especially suitable for treating the dredged sediment contaminated concur-rently by organic pollutants and heavy metal.EDTA,as a chelating agent popularly applied,can effectively remove Cd,Cu,Pb and Zn with the removal efﬁciencies ranging between65and86%[44]. However,due to possible adverse health and environmental effects, EDTA is currently under scrutiny.Therefore,the optimization of chelating agents should be the research direction in future.Surfactants,especially biosurfactants,usually possess excellent surface active properties,anionic nature and low toxicity.Through adsorbing onto sediment surface,complexing with metal,detach-ing the metal from the sediment into the porewater and hence associating with surfactant micelles,surfactant can effectively remove the metals adsorbed on sediment particles.For example, when rhamnolipid,a glycolipid biosurfactant,without additives was applied,the removal of heavy metals from sediments was up to37%of Cu,13%of Zn,and27%of Ni[45].4.2.2.Electrochemical remediationElectrochemical remediation involves applying a low DC current or a low potential gradient to electrodes that are inserted into the sediment and encompass the contaminated zone[46].When DC electricﬁelds are applied to the contaminated sediment,migration of charged ions occurs.Positive ions are attracted to the negatively charged cathode,and negative ions move to the positively charged anode.For example,under an induced electric potential,the anionic Cr(VI)migrated towards the anode,while the cationic Cr(III),Ni(II) and Cd(II)migrated towards the cathode.Once the remediation process is over,the contaminants that are accumulated at the elec-trodes are eventually extracted by methods such as electroplating, precipitation/co-precipitation,pumping water near the electrodes, or complexing with ion-exchange resins[16].Because the electric conductivity is the highest in theﬁne particles of the sediment on which also most metals are adsorbed,and the electricﬁeld is the strongest where the metals are mainly found.This method is well suited forﬁne-grained dredged sediment.In electrochemical remediation,there are four mechanisms, namely electromigration,electro-osmosis,electrophoresis and dif-fusion,affecting the migration of metals in an imposed electricﬁeld. Electromigration is considered as the dominant transfer mecha-nism for metal due to the transfer rate from electromigration is one to two orders greater than the others[46].Using inert elec-trodes,the electrode reactions will produce H+at the anode and OH−at the cathode,which means that if pH is not controlled,the H+in the anode migrate through the sediment towards the cath-ode,whereas the OH−migrate towards the anode.Depending on the extent of migration of H+and OH−,pH will vary across the sed-iment.An increase in the OH−concentration causes an increase in the pH near the cathode.It has been proved that when heavy metals enter into basic conditions,they are likely to be adsorbed onto soil particles or form precipitate as hydroxides,oxyhydroxides,etc.,and in acidic conditions,those ions desorb,solubilize and migrate.The high pH region in the proximity to the cathode is the main obstacle to heavy metal removal.In order to solubilize the metal hydroxides and carbonates formed,or other species adsorbed onto sediment particles,as well as protonate organic functional groups,acidiﬁcation may be a very effective method.Generally,in electrochemical remediation pro-cess,the development of an acidic front is often couple with a successful remediation[47].However,this acid addition also has some evident drawbacks.Achieving these acidic conditions might be difﬁcult due to higher sediment buffering capacity;in addition, acidiﬁcation of dredged sediment may be not an environmen-tally acceptable method.Surfactants can increase the solubility and mobility of heavy metals during electrochemical remediation, depending on its function on decreasing the potential of sediment。

福州“PEP”2024年小学五年级上册K卷英语第1单元真题试卷[有答案]

福州“PEP”2024年小学五年级上册英语第1单元真题试卷[有答案]考试时间：90分钟（总分:100）B卷考试人：_________题号一二三四五总分得分一、综合题(共计100题共100分)1. 选择题：What is the main gas in the air we breathe?A. OxygenB. NitrogenC. Carbon dioxideD. Helium2. 听力题：A mineral's hardness can be tested using a simple ______.3. 听力题：She is _______ (taking) a picture of the sunset.4. 选择题：What do bees make?A. MilkB. HoneyC. ButterD. Cheese5. 听力题：The chemical properties of metals include being ______ and malleable.6. 选择题：What is the nearest galaxy to the Milky Way?A. TriangulumB. AndromedaC. WhirlpoolD. Sombrero7. 选择题：What is the largest ocean on Earth?B. IndianC. ArcticD. Pacific8. 选择题：Which insect makes honey?A. AntB. FlyC. BeeD. Butterfly答案:C9. 选择题：What is the capital of Hungary?A. BudapestB. PragueC. ViennaD. Bratislava答案:A10. 填空题：The __________ (新兴城市) is rapidly growing.11. 选择题：What is the past tense of "go"?A. GoesB. GoneC. WentD. Going12. 填空题：A plant’s life cycle includes germination, growth, and ______ (结果).13. 听力题：I want to _____ (play/learn) soccer.14. 填空题：The _______ (小果子狸) is nocturnal and adventurous.15. 选择题：What do you call the largest land animal?A. RhinoB. ElephantC. Hippo答案：B16. 填空题：The penguin is a flightless ______ (鸟) that swims well.17. 填空题：The _____ (植物研究) can lead to new medical discoveries.18. 填空题：The ______ (鲸鱼) is a gentle giant of the sea.19. 选择题：What is the name of the famous writer who created Sherlock Holmes?A. J.K. RowlingB. Agatha ChristieC. Arthur Conan DoyleD. Mark Twain20. 听力题：The study of how landscapes change is part of ______ science.21. 选择题：What color is the grass?A. BlueB. GreenC. RedD. Yellow22. 选择题：What color is the sky on a clear day?A. GreenB. BlueC. RedD. Yellow答案:B23. 听力题：The cat is ___ the tree. (in, on, under)24. 选择题：What do we call the process of taking care of animals?A. Animal husbandryB. Veterinary careC. Animal welfareD. All of the above答案: D. All of the above25. 填空题：I want to create a ________ with my toys.26. 选择题：What do you call a baby goat?A. KidB. LambC. CalfD. Foal27. 听力题：My mom is a ______. She takes care of our family.28. 填空题：The __________ (土壤的质量) is vital for growth.29. 填空题：The first man to fly at supersonic speed was _______. (查克·耶格)30. 听力题：The process of ______ can create sedimentary layers in rocks.31. 填空题：The _____ (香味) of flowers attracts bees.32. 填空题：The ________ jumps high in the air.33. 填空题：The parrot is very _________ (色彩斑斓).34. 选择题：What do we call a scientist who studies rocks?A. BiologistB. GeologistC. ChemistD. Physicist答案: B35. 选择题：Which of these is a cold drink?A. CoffeeB. JuiceC. SodaD. Both B and C答案:D36. 填空题：An iguana can change its ________________ (颜色) to blend in.37. 听力题：The chemical formula for calcium hydroxide is __________.38. 听力题：I can ___ (solve) puzzles quickly.39. 填空题：The newt can regenerate its ______ (尾巴) if lost.40. 填空题：中国的________ (history) 充满了起伏与变迁。

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福州“PEP”2024年小学五年级上册K卷英语第1单元真题试卷[有答案]

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