Preliminary bioleaching of heavy metals from contaminated soil employing indigenous Penicillium

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Bioleaching of heavy metals from red mud using Aspergillus niger

Bioleaching of heavy metals from red mud using Aspergillus nigerYang Qu a ,b ,Bin Lian a ,c ,⁎,Binbin Mo a ,Congqiang Liu aa State Key Laboratory of Environmental Geochemistry,Institute of Geochemistry,University of Chinese Academy of Sciences,Guiyang,550002,Chinab Graduate University of Chinese Academy of Sciences,Beijing,100039,ChinacJiangsu Key Laboratory for Microbes and Functional Genomics,College of Life Sciences,Nanjing Normal University,Nanjing,210046,Chinaa b s t r a c ta r t i c l e i n f o Article history:Received 20October 2012Received in revised form 5March 2013Accepted 24March 2013Available online 28March 2013Keywords:Red mud Bioleaching Heavy metals Aspergillus nigerRed mud (bauxite residue)is the main waste product of the alkaline extraction of alumina from bauxite with high amounts of metals.In this study,bioleaching of heavy metals from red mud by using the fungus Aspergillus niger was investigated.Bioleaching experiments were examined in batch cultures with the red mud at various pulp densities (1–5%,w/v)under various bioleaching conditions (one-step,two-step and spent medium bioleaching).It was shown that the main lixiviant excreted by A.niger was citric acid.The highest leaching ratios of most various heavy metals were achieved under spent medium leaching at 1%pulp density.The increase in red mud pulp densities resulted in a general decrease in leaching ratios under all bioleaching conditions.However,in the case of the spent medium leaching the decrease in leaching ratios was lowest.The Toxicity Characteristic Leaching Procedure (TCLP)tests showed that the leaching toxicity of the bioleaching residue was far below the levels of relevant regulations.The micromorphology of the red mud particles were changed by the fungal activity during bioleaching process.©2013Elsevier B.V.All rights reserved.1.IntroductionThe production of alumina results in the generation of bauxite re-ﬁnery residue known as red mud,a highly saline and alkaline waste material,which represents the main disposal problem in the alumina industry (Clark et al.,2011).Producing one ton of alumina will con-sume approximately four metric tons of raw bauxite and simulta-neously generate above two metric tons of red mud (Ghosh et al.,2011).The storage volume of red mud in the whole world is estimat-ed to be over 2.7billion tons (Power et al.,2011).Furthermore,it is increasing with an annual rate of 120million tons according to the latest reports (Klauber et al.,2011).The management in the future of this kind of waste residue is of increasing environmental concern.The raw red mud or after processed could potentially be used in various environmental or industrial ﬁelds (Klauber et al.,2011).The main attempts for the effective application of red mud are:soil amendment to prevent nutrient loss and reduce heavy metal avail-ability (Alva et al.,2002);adsorbents for removal of heavy metal ions and metalloid ions (Cengeloglu et al.,2006;Zhang et al.,2008);absorbents for hydrogen sul ﬁde and sulfur dioxide in gas puri ﬁers (Fois et al.,2007);building materials as bricks and cements additive (Somlai et al.,2008);pigments and paints (Liu et al.,2009);catalysts (Liu et al.,2009;Wang et al.,2008).Though red mud could be potentially applied in many ﬁelds,the environmental risk of heavy metals leaching from red mud has never been thoroughly evaluated (Ghosh et al.,2011;Milacic et al.,2012).It is believed that the concentration of heavy metals (e.g.,V,Cr,Ni,Cu,Zn and As)are elevated in red mud and are approximately 20folds comparing with the surrounding soil (Kutle et al.,2004).The concentration of various heavy metals generally accounts for 0.01%to 1%respectively of the total weight according to the relevant studies (the concentration of Fe is even over 10%)(Akinci and Artir,2008;Ghosh et al.,2011;Kutle et al.,2004).It is also reported that the concentration of heavy metals such as Cd,Cu,Ni and Zn in the red mud are 3orders of magnitude more than the related regulation of Sediment Quality Guidelines Developed for the National Status and Trends Program enacted by National Oceanographic and Atmospheric Administration (NOAA)(Ghosh et al.,2011).Maybe the leaching tox-icity of heavy metals is low for the raw red mud due to the high alka-line characteristic itself.However,the leaching toxicity would be likely to increase after the change in surrounding environment or any process for various application purpose.It will have a harmful effect on plants,animals,aquatic life and humans once the heavy metals leach from red mud.Therefore,it is important to decrease the heavy metals contents in red mud before storage or application for the environmental safety.Biohydrometallurgical approaches (bioleaching)are generally considered as a ‘green technology ’with low-cost and low-energy requirement (Wu and Ting,2006).It can complete two aims simulta-neously in one process:(1)recover some valuable heavy metals and (2)reduce the leaching toxicity of heavy metals from waste ma-terials (Klauber et al.,2011).Some species of heterotrophic fungus (e.g.,Aspergillus and Penicillium )have shown potential for metal bioleaching of various waste materials,such as ﬂy ash (Bosshard et al.,1996;Wu and Ting,2006),spent catalysts (Amiri et al.,2011;Hydrometallurgy 136(2013)71–77⁎Corresponding author.Tel./fax:+868515895148.E-mail address:bin2368@ (B.Lian).0304-386X/$–see front matter ©2013Elsevier B.V.All rights reserved./10.1016/j.hydromet.2013.03.006Contents lists available at SciVerse ScienceDirectHydrometallurgyj o ur 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 /h y d r o me tSanthiya and Ting,2005)and electrical waste(Brandl et al.,2001). The most important mechanism of metal leaching by heterotrophic fungus is an indirect process with microbial production of metabolites, such as amino acids and organic acids(Burgstaller and Schinner,1993). Due to the strong adaptability,high metabolic activity and high produc-tion of organic acids,Aspergillus niger becomes the one of the most widely used fungi in bioleaching(Aung and Ting,2005;Ren et al., 2009).Although the process of metal bioleaching using A.niger seems promising,only few studies have been performed(Ghorbani et al., 2008;Vachon et al.,1994).Therefore,the focal point of this study was chosen to the bioleaching of heavy metals from red mud by using A.niger.The chemical characteristic of the red mud and the growth characteristics of the fungus(biomass dry weight,pH and ex-creted organic acids)in the pure culture were determined before bioleaching.Thereafter,the pH change and heavy metal leaching efﬁ-ciency under various bioleaching conditions(one-step,two-step and spent medium bioleaching)and pulp densities(1–5%,w/v)were ana-lyzed.Finally the Toxicity Characteristic Leaching Procedure(TCLP) tests,which are designed to determine the leaching toxicity of heavy metals from solid wastes,were conducted and the results were com-pared with the relevant regulations.2.Materials2.1.Leaching fungal strainA.niger(GenBank accession number is JF909353)was provided by Research Center For Bio-Resource&Technology,Institute of Geochemistry,Chinese Academy of Sciences.2.2.Red mudThe red mud samples were collected from the storage area of bauxite residue(26°41′N,106°35′E)which belongs to Chinalco in Guiyang.The semi-arid samples were collected by sterile steel con-tainers.When transported to the library,the red mud samples were dried to constant weight in the oven at80°C,and ground by usinga porcelain pestle and mortar and screened through74μm sieves.2.3.Chemical reagentsThe chemical reagents used in our experiments were all analytical reagent(AR).All the aqueous solutions were prepared using deion-ized distilled water.3.Methods3.1.Characterization of red mudFor analyzing the chemical composition of red mud,the process according to US EPA SW846Method3050B was used to totally digest the samples(Wu and Ting,2006).The metal ions containing in the digestive supernatant were analyzed using a Quadrupole Inductively Coupled Plasma Mass Spectrometry(Q-ICP-MS,PerkinElmer,ELAN DRC-e)and an Inductively Coupled Plasma Optical Emission Spectrom-eter(ICP-OES,VistaMPX).The pH value of red mud samples were deter-mined by using a digital pH meter(PHS-3C).The electrical conductivity (EC)was determined by EC meter(DDSJ-308A).The acid neutralizing capacity(ANC)was determined by a standard procedure of titration (endpoint of pH was4.5).3.2.TCLP testsThe TCLP tests were conducted based on U.S.Environmental Pro-tection Agency(EPA)SW846method1311(Aung and Ting,2005).The extraction supernatant was determined by using Q-ICP-MS and ICP-OES afterﬁltering through a0.45μm glassﬁbreﬁlter.3.3.Bioleaching of red mud by A.nigerA.niger was cultivated on potato dextrose agar plates at30°C for 7days in an incubator.The mature spores were harvested with a ster-ile solution of physiologic saline.The spores suspension was diluted and the number of spores was counted by using a haemocytometer and standardized to approximately1×107spores/mL.In order to observe the characteristics of fungal growth in the absence of red mud,two milliliter of spore suspension was inoculated into100ml of sucrose medium(autoclaved at121°C for15min)in250ml Er-lenmeyerﬂasks and cultivated in an orbital shaking incubator at 30°C and120rpm.The composition of sucrose medium is shown in Table1.Bioleaching studies were carried out using250mL Erlenmeyer ﬂasks in100mL of sucrose medium(autoclaved at121°C for 15min)with the sterilized red mud at various pulp densities.Three different bioleaching conditions were investigated.In one-step bioleaching,the fungus was incubated together with the sucrose me-dium and red mud.In two-step bioleaching,the fungus wasﬁrst incubated in sucrose medium in the absence of red mud for3days, after which the sterilized red mud was added.In spent medium bioleaching,the fungus wasﬁrst incubated in sucrose medium for 10days.Then the sterilized red mud was added into the cell-free spent medium which was obtained by centrifugation(3000rpm) and membraneﬁltration(0.2μm,Whatman)of the fungal culture. The cultures were incubated in an orbital shaking incubator at30°C and120rpm.Control experiments were carried out using fresh su-crose medium and deionized distilled water.All the experiments were conducted in triplicate.Two milliliters of samples were with-drawn at regular intervals for analyzing the sugar concentration, organic acids concentration,pH value and heavy metals concentra-tion.The biomass dry weight was also examined.3.4.Analytical methodsThe concentration of sugars and organic acids were determined using High Performance Liquid Chromatography(HPLC,Agilent 1200)with a refractive index detector(RID)for analyzing the sugars, and the variable wave-length detector(VWD)for the organic acids. The pH value and heavy metals concentration in the leachate was de-termined as described in Section3.1above.The percentage of metal extraction ratio was calculated through the concentration in theﬁlter liquor divided by the total concentration in red mud.The residue (biomass with bioleached red mud)obtained from theﬁlter paper was dried at80°C for24h,followed by ashing at500°C for4h to determine the biomass dry weight(Aung and Ting,2005).All exper-iments were performed in triplicate.The micromorphology of the fungi and red mud was observed using a Scanning Electron Microscopy(SEM,Shimadzu-SS550, 25kV,0.25nA).The samples were prepared by membraneﬁltration to remove redundant water.Then washed for1h with2%glutaralde-hyde solution in order to protect the intact appearance of microbial cell.After that a series of washings with mixtures of water andTable1Composition of bioleaching medium.Ingredients Concentration(g/L)Sucrose100KNO30.5KH2PO40.50.5Yeast extract 2.0Peptone 2.072Y.Qu et al./Hydrometallurgy136(2013)71–77ethanol were conducted for the purpose of cell dehydration.The sam-ples were coated with gold and submitted for SEM and EDS analysis.4.Results and discussion4.1.Characteristic of red mudThe pH of the red mud samples was12.9.Electrical conductivity was 21.8mS/cm.The ANC of red mud was3.53mmol H+/g.The weight percent concentration of major elements containing in the red mud(wt.%):Al(3.27);Ca(11.85);K(0.95);Mg(0.37);Na(5.30);Si (4.53).The weight concentration of heavy metals in red mud(unit: ppm):As(125);Ba(590);Cr(848);Cu(182);Fe(84200);Ni(169); Pb(332);Zn(670);Zr(2070).The heavy metals which weight percent concentrations were below0.01%were not shown.The high pH and ANC value is owing to abundant alkaline anions (OH−,CO32−/HCO3−,Al(OH)4−/Al(OH)3)dissolved from red mud (Gräfe et al.,2011).The high EC value is due to high ion strength in the red mud leachate(Gräfe et al.,2011).The high pH,ANC and EC value are indicative of the extreme alkalinity and salinity of the red mud.Furthermore,the high concentration of heavy metals makes the characteristic of red mud severely unfavorable for microorgan-isms to live in(Krishna et al.,2008).The extremely scarce of organic carbon,nitrogen and micronutrients will also limit the microbes to growth(Gräfe and Klauber,2011;Hamdy and Williams,2001; Thiyagarajan et al.,2009).Therefore,we chose the heterotrophic fun-gus A.niger which has a prominent tolerance of unfavorable condition (Brandl et al.,2001;Krishna et al.,2005;Ren et al.,2009)and an abil-ity of producing high volume of organic acids as the leaching strain for the further bioleaching research.4.2.Characteristic investigation of A.niger growth in pure cultureBefore the bioleaching,the pure culture experiments(in the absence of red mud)of A.niger were conducted until it reached the stationary phase in order to determine the optimum time for addition of red mud into fungal culture in two-step bioleaching,as well as to determine the optimum time to obtain the cell-free medium in spent medium bioleaching.Fig.1a shows the variation of sugars and biomass concentration during40days incubation.The concentration of sucrose drastically decreased to32.4mg/L within3day,andﬁnally completely hydro-lyzed to glucose and fructose through the invertase action after 10days incubation.With a decrease in sucrose,the concentration of biomass increased to the maximum of28.6mg/L along with the glycometabolism at the tenth day,then had a slight decrease during the rest time of incubation,which is probably due to the toxicity of secondary metabolism accumulating in the medium(Amiri et al., 2011).Fig.1b shows the change in pH value and organic acids concentra-tion.The concentration of citric acid drastically increased to the maxi-mum of82.3mmol/L within10days of incubation.The concentration of gluconic acid and oxalic acid also increased to the maximum of16.9 and18.3mmol/L during20and10days incubation respectively.The increase in organic acids was accompanied by a decrease in pH value. The pH value decreased to the minimum of1.8at the tenth day. Thereafter,with the decrease in organic acids,the pH marginally in-creased after the tenth day.It showed a high correlation(r=0.995, p b0.001)between an increase in biomass dry weight and a decrease in pH value,which indicates that the activity of the A.niger is the upper-most factor affecting the pH in the bioleaching culture.There are several mechanisms involved in the bioleaching of metals when use heterotrophic microbes as the leaching strains. However the most important mechanism is the acidolysis.The surface of metal compound covering by the oxygen atoms are proton-ated rapidly,thus the metal and water combine with the protons and oxygen is separated from the surface of metal compound(Burgstaller and Schinner,1993).The most important substances involved in the acidolysis secreted by fungus are the organic acids(Burgstaller and Schinner,1993).Through Fig.1b we can clearly see that the citric acid is the main leaching agent(the highest production among all the organic acids)for bioleaching of red mud by the fungi.The related reactions between the different organic acids and metal ions are listed as below(M n+represents the metal ions with certain valence):Gluconic acid:C6H12O7→C6H11OÀ7þHþðPk a¼3:86Þð1Þn½C6H11O−7 þM nþ→M½C6H11O7 nðGluconic metallic complexÞð2ÞOxalic acid:C2H2O4→C2HO−4þHþðPk a1¼1:25Þð3ÞC2HO−4→C2O2−4þHþðPk a2¼4:14Þð4Þn½C2HO−4 þM nþ→M½C2HO4 nðOxalic metallic complexÞð5Þ1008060402080604020abtime (days)time (days)Sugarconcentration(g/L)Organicacidsconcentration(mmol/L)Biomassdryweight(g/L)203010642pHFig.1.Variation of(a)sugars and biomass concentration and(b)organic acids concen-tration and pH as a function of time in the pure culture of A.niger during40days incu-bation.The experiments were performed in triplicate.73Y.Qu et al./Hydrometallurgy136(2013)71–77n½C2O2−4 þ2M nþ→M2½C2O4 nðOxalicmetalliccomplexÞð6ÞCitricacid:C6H8O7→C6H7O−7þHþðPk a1¼3:09Þð7ÞC6H7O−7→C6H6O2−7þHþðPk a2¼4:75Þð8ÞC6H6O2−7→C6H5O3−7þHþðPk a3¼6:40Þð9Þn½C6H7O−7 þM nþ→M½C6H7O7 nðCitricmetalliccomplexÞð10Þn½C6H6O2−7 þ2M nþ→M2½C6H6O7 nðCitricmetalliccomplexÞð11Þn½C6H5O3−7 þ3M nþ→M3½C6H5O7 nðCitricmetalliccomplexÞð12ÞThe nearly complete hydrolysis of sucrose and the maximum pro-duction of glucose and fructose at the third day indicate that the A.niger is in the active growth phase.Therefore after3days incuba-tion,the red mud added into the fungal culture under two-step bioleaching.The maximum biomass and minimum pH value were reached at the10th day of incubation.Therefore the cell-free medium was obtained throughﬁltering the culture after10days incubation under spent medium bioleaching.4.3.The change in pH value during various bioleaching conditionsThe pH value is a very important parameter in determining the bioleaching efﬁciency.Therefore,the pH change at different pulp den-sities under one-step,two-step and spent medium bioleaching were examined(Fig.2).In the one-step bioleaching with1%(w/v)red mud pulp density (Fig.2a),the initial pH(the value was approximately9.1)of the suspension gradually decreased to the lowest value of approximate 2.0after15days incubation,and then remain constant for up to 40days.In the two-step bioleaching containing1%(w/v)red mud, the initial pH was5.1when the red mud added into the three days culture.The pH drastically decreased to the lowest value of1.9after 6days incubation,and then remained constant in the rest time.The change in pH value during spent-medium process was much smaller than other bioleaching process,which is because there is no obvious metabolism activity in the leaching medium.The pH gently increased from2.6to3.2during40days incubation after the red mud(1%) added into the cell-free culture.The slowly increase of pH value in spent medium bioleaching is due to the continuous release of alkaline anions from red mud(Khaitan et al.,2009).With an increase in red mud pulp densities from1%to5%(w/v),the pH value during all the three bioleaching conditions increased. However,the increase extent in each bioleaching process was differ-ent.The minimum pH value in one-step bioleaching increased from 2.0at1%pulp density to5.0at2%pulp density.The increase extent of pH in two-step bioleaching was lower than that in one-step bioleaching.The minimum pH value increased from1.9at1%pulp density to3.9at5%pulp density.Furthermore,the time to reach the lowest value of pH in two-step bioleaching was also shorter than that in one-step bioleaching at all red mud pulp densities.These phenomena indicate that the production activity of organic acids by leaching fungus was obviously inhibited in one-step bioleaching,especially at high red mud concentration.The higher organic acids production in two-step bioleaching is probably because that the pre-culture of fungus in the absence of red mud is conducive for the production of organic acids and fungal growth(Amiri et al.,2011; Bosshard et al.,1996;Yang et al.,2008).The increase extent of pH value in spent medium bioleaching was the lowest among all the three bioleaching process.The minimum pH value increased from 2.6at1%pulp density to only3.5at5%pulp density(though the pH value increased in some degree with the leaching time prolonging). And in the presence of5%(w/v)red mud,theﬁnal pH value was lower than that in one-stepbioleaching.121086421210864212108642pHpHpHtime (days)time (days)time (days)abcFig.2.Variation of pH as a function of time during various bioleaching conditionsat different pulp densities of red mud:(a)1%(w/v),(b)2%(w/v)and(c)5%(w/v).The experiments were performed in triplicate.74Y.Qu et al./Hydrometallurgy136(2013)71–77The previous studies considered that the red mud of2%concentra-tion will exert severe toxicity to organisms(Pagano et al.,2002).Our results showed that the fungus had a favorable growth condition and organic acids production in the presence of5%(w/v)pulp density.There-fore,A.niger has a potential application for bioleaching of red mud.4.4.Leaching efﬁciency of heavy metals from red mud under different bioleaching conditionsThe leaching ratios of heavy metals under different bioleaching conditions and different red mud pulp densities are shown in Fig.3. The optimum pulp density in one-step,two-step and spent medium bioleaching were all1%.The highest leaching ratios of most heavy metals were achieved under spent medium bioleaching at1%pulp density,with leaching ratios at over80%of Pb and Zn,67%of Cu, 50%of Ni,44%of As,31%of Ba,26%of Cr and about11%of Fe and Zr.The leaching ratios data of fresh sucrose medium and deionized distilled water were not shown due to the negligible extraction.Different bioleaching conditions have different leaching efﬁciency orders of heavy metals.The leaching efﬁciency in descending order for the one-step,two-step and spent medium bioleaching can be roughly arranged as below,respectively,with slightly different.Zn≈Ni≈Pb>Cu≈As>Ba>Fe>Zr≈Crð13ÞZn>Pb≈Ni>Cu≈As>Fe≈Ba>Zr≈Crð14ÞPb>Zn≈Cu>Ni>As>Ba>Cr>Fe≈Zrð15ÞThe different leaching ratios between different heavy metals under various leaching conditions are due to:(1)the physical and chemical properties of heavy metals themselves;(2)the solubility of the com-plexes formed by organic acids and metal ions(Burgstaller and Schinner,1993);(3)the biosorption and bioaccumulation of heavy metals by the leaching fungus during bioleaching process(Yang et al.,2009);(4)the precipitation of heavy metals to the surface of leaching materials(Wu and Ting,2006).From Fig.3it can be found that the leaching efﬁciencies of Pb,Cu and Cr in the spent medium bioleaching were obviously higher than that in one-and two-step bioleaching.This is possibly because the adsorption capacities for the fungus of these heavy metals are com-paratively higher than other heavy metals(Volesky and Holan, 1995).They will be highly adsorbed by the fungus after leaching from the red mud.However,the biosorption will not occur frequently in spent medium bioleaching due to the scarce biomass in the leaching medium.Maybe this is an important mechanism that the spent medium bioleaching had better leaching efﬁciency of heavy metals than other bioleaching conditions.This is inconsistent with Wu's study(Santhiya and Ting,2005)that the leaching efﬁciency of spent medium bioleaching is lower when bioleaching ofﬂy ash,but is consistent with Amiri's study(Amiri et al.,2011)when usingspent hydrocracking catalyst as the leaching materials.The increase in red mud pulp densities result in a general decrease in leaching efﬁciency under each bioleaching process.This is probably due to two reasons:(i)the high concentration of red mud results in high pH value in leaching solution;(ii)the fungal growth is inhibited by the toxicity from red mud under one-and two-step bioleaching, which results in a decrease of metabolites(e.g.,organic acids)pro-duced from leaching strains(Aung and Ting,2005).Precisely because the second reason,with an increase in pulp densities,the decrease extents of leaching efﬁciency between the three bioleaching condi-tions were different.The highest decrease extent was occurred in one-step bioleaching,next was the two-step bioleaching.The de-crease extent of spent medium bioleaching was the lowest.According to our results,the leaching efﬁciency of spent medium bioleaching was the highest among all the bioleaching conditions, regardless of low or high pulp densities of red mud.Furthermore, the spent medium bioleaching also has other advantages,such as short processing time,easy handling and convenient optimization (Aung and Ting,2005).Therefore,it can be concluded that the spent medium bioleaching probably is the best choice for leaching heavy metals from red mud when using A.niger as the leaching fungus.4.5.TCLP tests of the bioleaching residueTable2shows a comparison of the TCLP test results of the red mud before and after bioleaching process against the identiﬁcationstandard 80604020Extractionratio(%)Extractionratio(%)Extractionratio(%)As Ba Cr Cu Fe Ni Pb Zn ZrAs Ba Cr Cu Fe Ni Pb Zn ZrAs Ba Cr Cu Fe Ni Pb Zn Zr abcFig.3.Extraction efﬁciencies of heavy metals at various pulp densities of red mud under(a)one-step bioleaching,(b)two-step bioleaching and(c)spent medium bioleaching.The experiments were performed in triplicate.75Y.Qu et al./Hydrometallurgy136(2013)71–77for hazardous wastes enacted by National Environmental Agency of China,recommended acceptance criteria for suitability of industrial wastes for landﬁll disposal enacted by National Environment Agency of Singapore,and TCLP regulatory level enacted by U.S.EPA,respectively.The concentration of As in the extract of raw red mud by TCLP tests was found to exceed the regulatory level.In contrast,the con-centration of heavy metals in the extract of the bioleaching residue by TCLP tests was reduced to well below the regulatory level.This in-dicates that the bioleaching process has a prominent effect on reduc-ing the leaching toxicity of heavy metals from red mud.Therefore,the red mud after bioleaching process can probably be disposed of safety or reused in other applicationﬁelds(e.g.,construction materials). However,considering the certain amount of heavy metals leached from raw red mud,it is urgency to develop relevant regulations on the storage and disposal of red mud in order to assess the environ-ment risk as well as to guarantee human health.4.6.Micromorphology analysis of red mud particles and A.nigerThe micromorphology of red mud particles and mycelium is shown in Fig.4.The size of individual raw red mud particles is largely different,which is ranging from nano-scale to micron-scale(Fig.4a). The amorphous and poorly crystalline structure is predominant in raw red mud particle which appear asﬂuffy aggregates.However,the morphology of the bioleaching residue particles is much different from the raw particles.Moreﬁne grained particles ap-pear,and crystalline structures also occur(Fig.4b).This is due to a comprehensive effect induced by fungal activity(Lian et al.,2008; Xiao et al.,2012).First,the hyphae will penetrate red mud particles through physical destruction force after the spores germinate and the hyphae elongate.Secondly,the organic acids and amino acids secreted by the hyphae will slowly erode red mud particles through chemical corrosive action.Thirdly,the CO2produced by respiration during metabolism activity will form carbonic acid when it reacts with water molecule.The carbonic acid can also have corrosive effect on red mud particles like other mineral weathering(Xiao et al.,2012). Theﬁnal result is the large particles will be split and destroyed to moreﬁne-grained particles through these comprehensive effects of physical destruction and chemical erosion.After bioleaching,the crystalline structures occur,while the non-crystalline structures dis-appear to some extent in red mud particles.Therefore it is speculated that the non-crystalline structures of the red mud tend to be eroded or damaged easily by the fungal activity,but the crystalline structures are difﬁcult to erode.When bioleaching was carried out at1%pulp density(Fig.4c), mostﬁne red mud particles adhere to the surface zone of mycelium.But if the red mud particle is large,the mycelium can penetrate through the whole particle(marked by the white arrow).This is ben-eﬁcial for the leaching of metals since it can expand the contacting area between fungus and red mud particles.Furthermore,the large particles have a greater tendency to be broken and turn into small ones by the physical destruction of mycelium,which is also conducive for the bioleaching.When bioleaching was carried out at5%pulp density(Fig.4d),the red mud particles cover almost the whole mycelial surface,which possibly form a thick barrier to impede the fungal metabolic product (e.g.,organic acids)escaping to the external space of the fungus,as well as reduce the speed rate to reach the chemical equilibrium and homogeneous state in solution.Therefore,the pH value inside this barrier will possibly decrease faster comparing to the outside when the fungal begins to secrete organic acids.Finally,due to the lower pH in the microenvironment,the actual concentration of heavy metal ions around the spores or the mycelium is probably higher than the measured value of that in solution.The fungal metabolic ac-tivities will beﬁercely restrained by high concentration of heavy metals inside this red mud barrier during bioleaching process.That is possibly one of the important reason that the fungi can't grow well at high concentration of red mud.However,enhancing the mass transfer rate is possibly a good way to alleviate this negative effect.5.ConclusionThis work has shown that heavy metals from red mud can be mo-bilized by leaching with A.niger.The main lixiviant excreted by the fungi was the citric acid.The highest leaching ratios were achieved under spent medium bioleaching at1%pulp density.According to our results,the spent medium bioleaching probably was the best choice for leaching heavy metals from red mud when used A.niger as the leaching fungus.The TCLP tests showed that through the bioleaching process,the leaching toxicity of red mud decreased obvi-ously.The micromorphology analysis indicates that the appearance of red mud particles is changed by the fungus activity during bioleaching process.AcknowledgmentThis work was jointly supported by the National Science Fund for Creative Research Groups(grant no.41021062)and the Guiyang Science and Technology Project([2012103]87).Table2TCLP test results of red mud(before and after bioleaching)in comparison with the various regulatory levels.Heavy metals Metal concentration in extractionﬂuid(mg/L)Raw red mud Bioleaching residue a Regulatory levels(China)b Regulatory levels(Singapore)c Regulatory levels(U.S.A.)dAs8.60±0.220.84±0.03555Ba 4.83±0.170.38±0.0410*******Cr 5.19±0.090.10±0.01155nsCu nd nd100100nsFe 2.42±0.210.31±0.02ns100nsNi 3.43±0.100.09±0.0155nsPb0.61±0.020.16±0.01555Zn15.00±0.85 1.30±0.11100100nsZr0.96±0.07nd ns ns nsnd:not detected;ns:not stated in regulation.a Bioleached residue under one-step bioleaching at1%pulp densities after40days.b Identiﬁcation standard for hazardous wastes—identiﬁcation for extraction procedure toxicity,National Environmental Agency,China(GB5085.3-2007).c Recommended acceptance criteria for suitability of industrial wastes for landﬁll disposal,National Environment Agency,Singapore.d“Identiﬁcation and listing of hazardous waste”U.S.Code of Federal Regulations(CFR),title40,Chapter1,Part261,U.S.Environmental Protection Agency.76Y.Qu et al./Hydrometallurgy136(2013)71–77。

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。

超富集植物吸收富集重金属的生理和分子生物学机制

超富集植物吸收富集重金属的生理和分子生物学机制3李文学　陈同斌33(中国科学院地理科学与资源研究所环境修复室,北京100101)【摘要】　与普通植物相比,超富集植物在地上部富集大量重金属离子的情况下可以正常生长,其富集重金属的机理已经成为当前植物逆境生理研究的热点领域.尤其是近两年,随着分子生物学等现代技术手段的引入,关于重金属离子富集机理的研究取得了一定进展.通过与酵母突变株功能互补克隆到了多条编码微量元素转运蛋白的全长cDNA ;也从分子水平上研究了谷胱甘肽、植物螯合素、金属硫蛋白、有机酸或氨基酸等含巯基物质与重金属富集之间的可能关系.本文从植物生理和分子生物学角度简要评述超富集植物对重金属元素的吸收、富集、螯合及区室化的机制.关键词　超富集植物　重金属　生理学机制　分子生物学机制文章编号　1001-9332(2003)04-0627-05　中图分类号　X171.5　文献标识码　APhysiological and molecular biological mechanisms of heavy metal absorption and accumulation in hyperaccu 2mu altors.L I Wenxue ,CHEN Tongbin (L aboratory of Environmental Remediation ,Institute of Geographical Sciences and N atural Resources Research ,Chinese Academy of Sciences ,Beijing 100101,China ).2Chin.J.A p 2pl.Ecol .,2003,14(4):627～631.In comparison with normal plants ,hyperaccumulators have the ability to accumulate heavy metals in their shoots far exceeding those observed in soil ,without suffering from detrimental effects.With the help of molecular tech 2nologies ,the research on the mechanisms of heavy metal accumulation in hyperaccumulators has been made a great progress.A number of trace element trans porters have been cloned by functional complementation with yeast mutants defective in metal absorption.The relations between glutathione ,phytochelatins metallothioneins ,organic acids and heavy metals have been studied by molecular technologies.This review concentrated on the physiological and molecular mechanisms of heavy metal absorption and sequestration in hyperaccumulators.K ey w ords Hyperaccumulator ,Heavy metal ,Physiological mechanisms ,Molecular biological mechanisms.3国家自然科学基金项目(40071075)、中国科学院知识创新工程重点方向项目(K Z CX 22401202)和王宽诚博士后工作奖励基金资助.33通讯联系人.E 2mail :chentb @ 2002-07-05收稿,2002-11-28接受.1　引言土壤重金属污染是一个重要的环境问题,传统的治理主要采用物理或化学方法,费用高,对大面积的污染效果差;与传统措施相比,植物修复技术以成本低、操作简单等优点而倍受青睐.广义上的植物修复是指利用植物去除土壤、水体或空气中重金属、有机污染物等污染物的技术,包含植物萃取(Phytoextraction )、根际过滤(Rhizofiltration )、植物挥发(Phytovolatilization )、植物固定(Phytostabilization )等技术,现在通常提到的植物修复主要是指植物萃取[32].超富集植物(Hyperaccumulator )是植物修复的基础,国际上已发现400多种超富集植物,国内对于超富集植物的研究相对较晚,研究较为系统的当属As 、Zn 等重金属的超富集植物[2,3,33].与普通植物相比,重金属离子进入超富集植物体内同样经过吸收/转运、富集/转化/矿化等生理生化过程,而且许多重金属离子进入植物体内的离子通道与必需营养元素相同,这就决定了超富集植物必然具有独特的生理代谢过程.关于这些过程的研究已经成为新的研究热点.本文对有关超富集植物吸收和富集重金属离子的生理及分子机制研究进行评述.2　重金属离子吸收的分子生物学机制遏蓝菜属(Thlaspi L.)植物具有非常强的富集Zn 的能力,能够在地上部富集高达3%(干重)的Zn ,同时植物正常生长,没有表现出任何中毒症状,它已经成为研究重金属富集机理的模式植物之一.但无论是超富集植物或是普通植物,金属离子进入植物体内的第一步是根系吸收,也就是说吸收过程很可能是超富集植物富集重金属离子的第一个限速步骤.T.caerulescens 与T.arvense 同属于遏蓝菜属,T.caerulescens 能够富集Zn 而T.arvense 则不具此能力,通过比较它们对Zn 2+的吸收动力学发现:两者Km 值差异不大,但T.caerulescens 的Vmax 要比T.arvense 高3.5倍[21],表明T.caerulescens 富集Zn 2+的能力并非是与Zn 2+有更高的亲和力,而很可能是因为锌离子的流入量加大所致,也就是说在T.caerulescens 根系细胞膜上分布有更多的锌离子转应用生态学报　2003年4月　第14卷　第4期 CHIN ESE JOURNAL OF APPL IED ECOLO GY ,Apr.2003,14(4)∶627～631运蛋白.近年来随着分子生物学等现代技术手段的引入,人们对金属离子如何进入细胞有了新的认识.通过对酵母突变株进行功能互补克隆到了多条编码微量元素转运蛋白的全长cDNA,其中研究最多的是ZIP基因家族(ZRT,IRT-like Protein).ZIP基因家族分布非常广泛,在真菌、动物、植物等真核细胞中均发现了ZIP基因家族成员.ZIP基因编码的蛋白一般具有8个跨膜区,C2端和N2端的氨基酸均位于细胞膜外.此家族包含至少25个成员,z rt1、z rt2(zinc2regulated transporter)和irt1(iron2regulated transporter)是最早克隆到的ZIP基因.z rt1、z rt2均由酵母中获得,与Zn的吸收密切相关[36,37];irt1编码的蛋白主要位于拟南芥的根系,体内缺Fe时可诱导irt1表达[8].另一类与金属离子吸收有关的蛋白是Nramp基因家族(Natural resistance associated macrophage proteins).Nramp基因家族编码的蛋白一般具有12个跨膜区,这与ZIP基因家族明显不同.Nramp最初在哺乳动物中发现,植物中的研究主要集中于水稻(Oryz a sativa)和拟南芥(A rabidopsis).O2 ryz a sativa和A rabidopsis的Nramp基因家族分为2类,Os2 Nramp1、OsNramp3和AtNramp5属于一类,OsNramp2、At2 Nramp1、AtNramp2、AtNramp3与AtNramp4属于另一类. Nramp基因家族在植物中的功能现在仍不清楚,AtNramp3和AtNramp4能够维持A rabidopsis体内铁离子的平衡[29].此外,AtNramp3很可能与Ca2+的吸收有关,破坏AtNramp3基因可增加植物对Cd的耐性,过量表达则导致植物对Ca2+的超敏感性.对于超富集植物而言,Zn的吸收过程研究相对较清楚.通过与酵母突变株进行功能互补,Pence等[24]在具有富Zn 能力的T.caerulescen中克隆到z nt1.z nt1编码Zn2+转运蛋白,属ZIP基因家族,缺Zn和Zn供应充足条件下均可以在根系和叶片中高量表达,表明其可能是组成型表达;对于不具有富Zn能力的T.arvense而言,z nt1主要在缺Zn件下表达,供Zn时,表达明显受到抑制.这种表达方式的不同很可能是造成Thlaspi富Zn能力差异的主要原因之一.Assun2 cao等[1]的研究结果也表明Zn转运蛋白基因T.caerulescen 的表达量要远高于T.arvense.从Pence等[24]、Assuncao等[1]与Lasat等[21]的实验结果可以发现根系Zn转运蛋白基因的表达量与Thlaspi富集Zn的能力正相关,初步验证了吸收过程是超富集植物富集重金属离子的首个限速步骤的假设.但是目前还不能肯定转运蛋白是否在超富集植物吸收重金属方面起到决定性作用.譬如说,尽管z nt1、z nt2在T. caerulescen的表达量要远高于T.arvense,但是它们在具有不同富集能力T.caerulescen中的表达量几乎相同[1],即T.caerulescen富集能力的差异与吸收并无太大的相关性.造成此现象的原因很可能在于:(1)一般来说,转运蛋白由一个基因家族控制,而现在得到的克隆还不足以代表整个家族,许多未知的基因可能起到更为重要的作用,如在T. caerulescen就又克隆到z at基因,它与Zn2+的区室化(Se2questration)密切相关,但是此基因与ZIP基因家族明显不同,仅含有6个跨膜区[34];(2)对已知转运蛋白的性质研究还不清楚,金属离子转运蛋白对底物专一性不强,造成多种吸收途径同时对一种金属离子发挥作用,所以在进行具体的分子生物学研究时,必须清楚那些转运蛋白对该金属离子起作用;(3)现在转运蛋白的研究主要集中于根系,叶片中转运蛋白的研究相对较少,但是对超富集植物而言,重金属离子在地上部的含量要远远高于根系,即叶片中的转运蛋白很可能起到更为主要的作用.3　木质部运输在木质部存在大量的有机酸和氨基酸,它们能够与金属离子结合,这种复合物是重金属离子在木质部中运输的主要形式.譬如在木质部,Fe主要是以柠檬酸铁的形式存在,Zn 主要是与柠檬酸或苹果酸结合,而Cu随着植物不同可与天冬酰胺酸、谷氨酸、组氨酸或烟碱结合,当然也有许多是以离子形态存在的,如Ca、Mg、Mn.在超富集植物中研究较多的为组氨酸.Kramer等[19]发现,组氨酸与A lyssum montanum 富集Ni的能力密切相关,当植物地上部Ni含量高时,木质部中组氨酸含量也较高,外源组氨酸的加入也能显著促进Ni装载入木质部,从而提高Ni向地上部的运输.然而,最近的研究表明,组氨酸反应很可能并不是Ni超富集植物的普遍机理.Persans等[25]在研究Ni的超累积植物Thlaspi geosingense时并没有发现His反应,同时他们克隆了控制His 合成的关键酶基因thg1、thb1、thd1,其表达量并没有随着Ni用量的增加而升高. 重金属由根系进入木质部至少需要3个过程:进入根细胞,由根细胞运输到中柱,装载到木质部.在内皮层由于凯氏带的存在,使得共质体运输在重金属进入木质部的过程中起到主导作用.在共质体运输中起关键作用的是膜转运蛋白,然而直到现在还没有在木质部中克隆到与重金属离子运输相关的基因,这方面的研究,尤其是在研究超富集植物时应该引起充分的重视.与普通植物相比,超富集植物能够高效、迅速地把重金属离子由根系运输到地上部,而通过凯氏带是重金属离子进入木质部主要屏障之一,探明此过程,将有利于提高植物修复的效果.4　对金属离子的解毒机制411　谷胱甘肽(GSH) 许多金属离子是植物必需的微量养分,它们参与植物体内众多的生理代谢过程.但如果含量过高,尤其是具有氧化还原活性的金属,会对植物产生毒害作用,这种毒害作用很可能是由于自由基的形成造成的.GSH含巯基,具有很强的氧化还原特性,可有效地清除活性氧等自由基,因此GSH在植物抗逆境胁迫中起重要作用.GSH为三肽,结构通式为γ2 G lu2Cys2G ly,合成主要通过两步依赖于A TP的反应完成,γ2 EC合成酶和GSH合成酶是其中的关键酶.γ2EC合成酶由gsh1编码,GSH合成酶由gsh2编码,gsh1与gsh2在拟南芥826应　用　生　态　学　报 14卷基因组中均以单拷贝的形式存在. 正常条件下,GSH的合成依赖于半胱氨酸的活性,同时存在明显的反馈抑制现象,表明由γ2EC合成酶催化的反应是整个合成的限速步骤.重金属胁迫条件下,重金属离子激活植物螯合素的合成,消除了GSH的反馈抑制作用,由GSH 合成酶催化的反应也成为限速步骤,此时如果加强gsh2的表达,则既可增加植物螯合素的合成又能避免GSH的耗竭,从而缓解重金属胁迫.Zhu等[38,39]的实验结果验证了此假设.他把大肠杆菌的gsh1与gsh2分别转入到印度芥菜(B rassica juncea),发现印度芥菜对Cd2+的耐性与富集能力均有明显增加,且耐性和富集能力还与gsh2的表达正相关.然而,Foyer等[10]把gsh2转入白杨树(Populus)后,白杨树抗氧化胁迫的能力(光抑制)并没有增加;G oldsbrough等[13]的结果也表明gsh2转入野生型的拟南芥后并不能增加其对Cd的抗性.由此可见,如何通过基因工程改造GSH,以增加植物对重金属的耐性和富集能力还有待于进一步研究.412　植物螯合素(PCs) 植物螯合素(PCs,=cadystins in S.pombe)由植物体内一系列低分子量、能够结合金属离子的多肽组成,其结构通式为(γ2G lu2Cys)n2G ly(图1),一般来讲,n为2～5,最高可达11[5].现已发现多种PC的同功异构体,主要是C端的G ly 被β2Ala、Ser取代形成.原来认为植物螯合素仅存在于植物中,但是随着研究的深入,陆续在线虫、蚯蚓等克隆到PC合成酶的类似基因. PCs不能由基因直接编码,必须在PCs合成酶的催化下完成[14].PC合成酶为四聚体,分子量95000道尔顿,等电点在p H4.8附近,最适反应温度和p H分别为35o℃、7.9[14].然而,由克隆到的编码PCs的全长cDNA推测的结果与此不符,推测结果表明PCs不是多聚体,分子量为42000～70000道尔顿,这种偏差很可能由于在Grill等提纯的酶中PCs并不是主要成分造成的.不同重金属离子诱导PCs合成的能力有很大差别[15],一般为Cd2+>Pb2+>Zn2+>Sb3+>Ag+> Hg2+>As5+>Cu+>Sn2+>Au3+>Bi3+;不同重金属离子诱导PC合成酶活性的能力与诱导PCs合成的能力稍有不同[35]:Cd2+>Ag+>Pb2+>Cu+>Hg2+>Zn2+>Sn2+> Au3+>As5->In3+>Tl3+>G e4+>Bi3+>G a3+.关于PCs 功能研究得相对清楚的是PCs与Cd之间的关系(图2).现图1　植物螯合素的化学结构示意图Fig.1Chemical structure of phytochelatin.已明确PCs在植物解Cd毒中起到重要作用,PCs2Cd复合物是Cd由细胞质进入液泡的主要形式.正是由于PCs在重金属离子区室化中所起的重要作用,近年来PCs已成为植物抗重金属胁迫的研究热点之一. 目前PCs的分子生物学研究基本集中于普通植物或耐性植物,而有关超富集植物的研究相对较少.Schmoger等[28]在用As处理过的蛇根木(Rauvolf ia serpentina)悬浮细胞及拟南芥幼苗中发现了PCs,Hartley2Whitaker等[17]在绒毛草(Holcus lanatus)上也证实了上述现象.但这些植物多属于耐性植物.Ebbs等[7]的实验表明,无论是否具有富集能力, Thlaspi用Cd处理后都会有大量PCs的合成,但是T.ar2 vense中PCs的总量要高于T.caerulescens,说明PCs与植物富Cd能力之间并无太大的关系.由于PCs在超富集植物中的研究还很少,所以PCs在超富集植物是否起到重要作用还有待于深入研究. Cobbett、Rea和等3个研究小组于1999年分别在拟南芥、小麦、酵母中克隆到了编码PC合成酶的全长cDNA.其中,通过对拟南芥cad1突变株(含有与野生型相似的GSH含量,但不含PC)定位克隆获得At PCS1[16],小麦耐Cd基因At PCS1与TaPCS1主要是通过与酵母突变株功能互补得到[4,30].对PC合成酶相应的全长cDNA对齐比较发现其保守区位于N端,同一性高达40%.长时间Cd2+处理cad1突变株也没有发现PCs的合成,表明PCs的合成可能是由单基因控制[18].但随着拟南芥基因组测序的完成,发现了与At PCS1高度同源的At PCS2基因[16],其功能尚不清楚,但与At PCS1相比,其表达量非常低.但植物在长期的进化历程中把At PCS2作为功能基因保留下来,尽管其在正常条件下表达量很低,可以想象在某些器官或环境下,At PCS2基因的表达肯定会起到重要作用.图2　以Cd为例说明谷胱甘肽、植物螯合素在抗重金属胁迫中的作用(+表示增加基因表达或酶活性,-表示减少基因表达或酶活性, HM T1表示位于液泡膜上的PC2Cd转运蛋白),参见Cobbert[5]并作修改Fig.2Function of GSH and PC in the metal tolerance of plants under metal stress(+and2indicate positive and negative regulation of enzyme activities or gene expression,respectively;HM T1is a vacuolar meme2 brane transporter of PC2Cd complex;revised from the figure of Cob2 bert[5]).413　金属硫蛋白(M T) 金属硫蛋白(Metallothioneins)是自然界中普遍存在的一种低分子量、富含半胱氨酸的蛋白质.它与PCs的本质区别在于M T由基因直接编码,而PCs在PCs合成酶的催化下完成.与PCs一样,金属硫蛋白能够通过巯基与金属离子结合,从而降低重金属离子的毒性,它对于Zn2+和Cu2+的解毒效9264期李文学等:超富集植物吸收富集重金属的生理和分子生物学机制果尤为明显[23]. 植物中首先鉴定的M T是Ec蛋白,它由小麦成熟胚芽中分离得到.在植物中已发现大约50种M T,根据半胱氨酸残基的排列方式,可以将其分为Ⅰ型、Ⅱ型、Ⅲ型和V型,大多属于Ⅰ型和Ⅱ型.Ⅰ型中的半胱氨酸残基仅有Cys2Xaa2 Cys一种排列方式;Ⅱ型中的半胱氨酸残基有两种排列方式,分别为Cys2Cys、Cys2Xaa2Xaa2Cys.编码I型M T的cDNA 在根系的表达水平较高,编码Ⅱ型M T的cDNA主要在叶片表达. 金属硫蛋白极易水解,尤其植物中的金属硫蛋白氨基酸链比较长,极易在半胱氨酸区水解,同时金属硫蛋白在有氧的条件下非常不稳定,所以难以获得相应蛋白质的资料,目前仅对小麦Ec蛋白及拟南芥M T1、M T2编码的蛋白进行了纯化,这就限制了对M T类似基因功能的研究.Murphy 等[22]证实Cu2+诱导拟南芥M T2表达,而且表达强度与不同基因型抗Cu胁迫的能力密切相关;Nathalie等[13]的研究结果也证实Cu的耐性植物Silene v ulgaris耐Cu胁迫的特性与M T2b的表达紧密联系.王剑虹等[31]在重金属耐性植物紫羊茅草(Festuca rebra)中克隆到mc M T1的全长cD2 NA,此基因编码70个氨基酸,含有12个Cys残基,在N端和C端分别含有3个Cys2Xaa2Cys结构,将此基因转入到酵母M T基因缺失突变株中发现,mc M T1的表达增加了酵母细胞对Cu、Cd和Pb的抗性.在拟南芥和蚕豆中,M T主要在毛状体中表达[9,12],而Cd等许多有毒重金属离子也在毛状体中累积[27],暗示M T和重金属累积有某种联系.414　细胞壁的固持与区室化作用植物细胞壁残基对阳离子有高亲和力,可以影响重金属离子向细胞内扩散速率,从而影响金属离子的吸收.比较黄花茅(A nthox anthum odoratum)悬浮细胞和原生质体固Pb 能力发现,Pb浓度对从耐Pb细胞克隆分离的悬浮细胞无太大影响,而原生质体的死亡率上升,相应地,从Pb敏感细胞克隆分离的悬浮细胞和原生质体对Pb极其敏感,表明细胞壁在A nthox anthum odoratum抗Pb胁迫中起到重要作用[26].需要明确的是,细胞壁对金属的固定作用不是一个普遍的抗金属毒害的机制,例如抗Zn毒和Zn敏感型菜豆的细胞壁物质表现出相似的亲和力,同时细胞壁有一定的金属容量,而超富集植物能够在地上部富集大量的重金属离子,暗示细胞壁不可能在超富集植物中起到重要作用.最近的研究表明,区室化作用与超富集植物富集重金属离子的能力密切相关.就Thlaspi而言,具有富集能力的T.geosingense液泡中Ni的含量要比不具有富集能力的T.arvense高1倍[20]; Frey等[11]也证实Zn在T.caerulescens中主要分布于表皮细胞液泡中.但区室化作用是否为超富集植物富集重金属离子的一个普遍机理还需对新发现的超富集植物进一步研究才能确定.5　研究展望关于超富集植物富集重金属离子的研究虽然取得了一定进展,但至今对其分子和生理机制仍不是很清楚,研究人员的看法也存在明显的分歧.在把超富集植物用于实践的过程中,首先要研究清楚对超富集植物富集的生理基础,譬如重金属离子如何进入根细胞,在木质部如何被运输,在叶片中如何分布;其次要注意不同生理过程的联系,就吸收而言,它其实是根系吸收与体内再分配的有机结合,所以在利用基因工程方法增加重金属离子吸收量时,不仅要考虑到增加根系的吸收位点,提高转运蛋白底物的专一性,同时要注意细胞器,尤其是液泡膜上与重金属离子区室化相关膜蛋白的表达,只有这样,才会达到比较好的效果;最后要强调的是学科交叉与渗透,Dhankher等[6]将细菌中的砷酸盐还原酶ArsC 基因和γ2谷氨酰半胱氨酸合成酶(γ2ECS)在拟南芥的叶子中表达,这样运输到地上部的砷酸盐在砷酸盐还原酶的作用下转化成亚砷酸盐,γ2ECS表达可增加一些连接重金属(如亚砷酸盐)并解除其毒性的化合物,将这些复合物限制在叶子中,从而使植物能够积累并忍耐不断增加的As含量.参考文献1　Assuncao A G L,Martins PDC,Polter SD,et al.2001.Elevated expression of metal transporter genes in three accessions of the met2 al hyperaccumulator Thlaspi caerulescens Plant Cell Envi ron,24: 217～2262　Chen T2B,Wei C2Y,Huang Z2C,et al.2002a.Arsenic hyperaccu2 mulator Pteris vittata L.and its arsenic accumulation.Chi n SciB ull,47:902～9053　Chen T2B,Fan Z2L,Lei M,et al.2002b.Arsenic uptake of hy2 peraccumulating fern Pteris vittata L.:Effect of phosphorus and its significance,Chi n Sci B ull,47:1876～18794　Clemens S,K im E J,Neumann D,et al.1999.Tolerance to toxic metals by a gene family of photochelatin synthase genes from plants and yeast.EMBO J,18:3325～33335Cobbett CS.2000.Phytochelatin biosynthesis and function in heavy metal detoxification.Curr Opi n Plant Biol,3:211～2166　Dhankher OP,Li Y,Rosen BP,et al.2002.Engineering toler2 ance and hyperaccumulation of arsenic in plants by combining arsen2 ate reductase and(2glutamylcysteine synthetase expression.N at ure Biotech,20:1140～11457　Ebbs S,Lau I,Ahner B,et al.2002.Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator Thlaspi caerulescen.Planta,214(4):635～6408　Eide D,Broderius M,Fett J,et al.1996.A novel iron2regulated metal transporter from plants identified by functional expression in yeast.Proc N atl Acad Sci,93:5624～56289　Foley RC,Singh K B.1994.Isolation of a V icia f aba metalloth2 ioneins2like gene:expression in foliar trichomes.Plant Mol Biol, 26:435～44410　Foyer CH,Souriau N,Perret S,et al.1995.Overexpression of glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in Polar trees.Plant Physiol,109: 1047～105711　Frey B,Keller K,Z ierold K,et al.2000.Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens.Plant Physiol,23:675～68712　G arcia2Hernandez M,Murphy A,Taiz L.1998.Metallothioneins 1and2have distinct but overlapping expression patterns in Ara2 bidopsis.Plant Physiol,118:387～39713　G oldsbrough PB.1998.Metal tolerance in plants:the role of phy2 tochelatins and metallothioneins.In:Terry N,Banuelos GS eds.Phytoremediation of trace elements.Michigan:Ann Arbor Press. 14　Grill E,Loffler S,Winnacker EL,et al.1989.Phytochelatins, the heavy metal binding peptides of plants,are synthesized from glutathione by a specificγ2glutamylcysteine dipeptidyl transpepti2 dase(phytochelatin synthase).Proc N atl Acad Sci,86:6838～684215　Grill E,Winnacker EL,Zenk MH.1987.Phytochelatins,a class of heavy metal binding peptides from plants,are functionally analo2036应　用　生　态　学　报 14卷gous to metallothioneins.Proc N atl Acad Sci,84:439～44316　Ha SB,Smith AP,Howden R,et al.1999.Photochelatin syn2 thase genes from A rabi dopsis thaliana and the yeast,Schizosac2 charomyses pombe.Plant Cell,11:1153～116417　Hartley2Whitaker J,Ainsworth G,Vooijs R,et al.2001.Pho2 tochelatins are involved in differential arsenate tolerance in Holcus lanat us.Plant Physiol,126:299～30618　Howden R,G oldsbrough PB,Andersen CR,et al.1995.Cadmi2 um2sensitive,cad1,mutants of A rabi dopsis thaliana are pho2 tochelatin deficient.Plant Physiol,107:1059～106619　Kramer U,Cotter2Howells JD,Charnock J M,et al.1996.Free histidine as a metal chelator in plants that accumulate nickel.N a2 t ure,379:635～63620　Kramer U,Pickering I J,Prince RC,et al.2000.Subcellular lo2 calization and speciation of nickel in hyperaccumulator and non2ac2 cumulator Thlaspi species.Plant Physiol,122:1343～135321　Lasat MM,Pence SN,G arvin DF,et al.2000.Molecular physi2 ology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens.J Ex p Bot,51(342):71～7922　Murphy A,Zhou J,G oldsbrough PB,et al.1997.Purification and immunological identification of metallothioneins1and2fromA rabi dopsis thaliana.Plant Physiol,113:1293～130123　Nathalie ALM,Hassinen VH,Hakvoort HW J,et al.2001.En2 hanced copper tolerance in Silene v ulgaris(Moench)garcke popu2 lations from copper mines is associated with increased transcript lev2 els of a2b2type metallothionein gene.Plant Physiol,126:1519～152624　Pence NS,Larsen PB,Ebbs SD,et al.2000.The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumu2 lator.Proc N atl Acad Sci,97:4956～496025　Persans MW,Yan XG,Patnoe J MML,et al.1999.Molecular dissection of the role of histidine in nickel hyperaccumulation in Thlaspi geosi ngense.Plant Physiol,121:1117～112626　Poulter A,Collin HA,Thurman DA,et al.1985.The role of the cell wall in the mechanism of lead and zinc tolerance in A nthoxan2 thum odorat um L.Plant Sci,42:61～6627　Salt DE,Prince RC,Pickering I J,et al.1995.Mechanisms of cadmium mobility and accumulation in Indian mustard.Plant Phys2 iol,109:1427～143328　Schmoger MEV,Oven M,Grill E.2000.Detoxification of arsenic by phytochelatins in plants.Plant Physiol,122:793～80129　Thomine S,Wang R,Ward J M,et al.2000.Cadmium and irontransport by members of a plant metal transporter family in A ra2 bi dopsis with homology to Nramp genes.Proc N atl Acad Sci,97: 4991～499630　Vatamaniuk O K,Mari S,Lu YP,et al.1999.AtpCS1,a pho2 tochelatin synthase genes from A rabi dopsis thaliana:isolation andi n vit ro reconstitution.Proc N atl Acad Sci,96:7110～711531　Wang J2H(王剑虹),Ma M(麻　密).2000.Biological mecha2 nisms of phytoremediation.Chi n B ull Bot(植物学通报),17(6): 504～510(in Chinese)32　Wei C2Y(韦朝阳),Chen T2B(陈同斌).2001.Hyperaccumu2 lators and phytoremediation of heavy metal contaminated soil:a re2 view of studies in China and abroad.Acta Ecol Si n(生态学报),21(7):1196～1203(in Chinese)33　Wei C2Y(韦朝阳),Chen T2B(陈同斌).2002.The ecological and chemical character of plants in arsenic abnormal areas.Acta Phytoecol Si n(植物生态学报),26:695～700(in Chinese)34　Zaal BJ VD,Neuteboom L W,Pinas J E,et al.1999.Overexpres2 sion of a novel Arabidopsis gene related to putative zinc2transporter genes from animals can lead to enhanced zinc resistance and accu2 mulation.Plant Physiol,119:1047～105535　Zenk MH.1996.Heavy metal detoxification in higher plants—a review.Gene,179:21～3036　Zhao H,Eide D.1996a.The yeast ZRT1gene encodes the zinc transporter protein of a high affinity uptake system induced by zinc limitation.Proc N atl Acad Sci,93:2454～245837　Zhao H,Eide D.1996b.The ZRT2gene encodes the low affinity zinc transporter in S accaromyces cerevisiae.J Biol Chem,271: 23203～2321038　Zhu Y L,Pilon2Smits EAH,Jouanin L.1999.Overexpression of glutathione synthetase in Indian mustard enhances cadmium accu2 mulation and tolerance.Plant Physiol,119:73～7939　Zhu Y L,Pilon2Smits EAH,Tarun AS,et al.1999.Cadmium tol2 erance and accumulation in Indian mustard is enhanced by overex2 pressingγ2glutamylcysteine synthetase.Plant Physiol,121:1169～1177作者简介　李文学,男,1973年生,博士后.主要从事植物营养遗传与重金属污染生态学研究,在国内外发表论文8篇. E2mail:liwx@1364期李文学等:超富集植物吸收富集重金属的生理和分子生物学机制。

金属硫蛋白作为重金属污染生物标志物的研究进展

农业环境科学学报2009,28(3):425-432Journal of Agro-Environment Science摘要：金属硫蛋白（MT ）是一类普遍存在于生物体内的低分子量、富含半胱氨酸、热稳定性、可诱导型非酶蛋白，具有维持生物体内金属含量动态平衡和重金属解毒作用双重机制。

大量研究表明，MT 可作为重金属污染的生物标志物，用来预测生物体受重金属暴露的状况和重金属污染的压力，且对制定预防性的管理措施、及时监控环境污染皆具有重要的现实意义。

本文根据国内外文献资料查阅及综合分析，主要阐述了金属硫蛋白的基本结构、多态性、生理功能及其作为生物标志物在环境污染检测与诊断方面的研究与应用，并展望其今后研究方向。

关键词：金属硫蛋白；重金属；动态平衡；解毒；生物标志物中图分类号：X835文献标志码：A 文章编号：1672-2043(2009)03-0425-08收稿日期：2008-06-13基金项目：高等学校科技创新工程重大培育资金项目（707011）作者简介：陈春（1980—），男，安徽宿县人，博士，主要从事分子生态毒理学研究。

E-mail ：chenchun@ 通讯作者：周启星E-mail ：zhouqx@ 金属硫蛋白作为重金属污染生物标志物的研究进展陈春1，周启星1，2（1.南开大学环境科学与工程学院环境污染过程与基准教育部重点实验室，天津300071；2.中国科学院沈阳应用生态研究所陆地生态过程重点实验室，辽宁沈阳110016）Researching Advance in Metallothionein and Its Biomarker of Heavy Metal ContaminationCHEN Chun 1,ZHOU Qi-xing 1,2（1.Key Laboratory of Environmental Pollution Process and Criteria,Ministry of Education,College of Environmental Science and Engineer －ing,Nankai University,Tianjin 300071,China;2.Key Laboratory of Terrestrial Ecological Process,Institute of Applied Ecology,Chinese A －cademy of Sciences,Shenyang 110016,China ）Abstract ：Metallothioneins （MT ）are ubiquitous,non-enzymatic proteins characterized by low molecular weight,cyst-eine-rich,heat stabili －ty,metal-induced.MT plays the double biologically mechanisms,mainly implicated in essential metals homeostasis and toxic trace metal detoxification.In recent years,it was researched to indicate that MT was used as a suitable biomarker of metal toxicity or exposure,it could e －valuate the quality of heavy metal exposure and pollution stress.Furthermore,the application of MT biomarker would enable environmental a －gencies or legislators to take precautionary measures and real-time monitor the environment pollution.In this review,the structure,isoforms and biological role of MT would be mentioned.Moreover,a brief overview of the use of MT as a biomarker was included the soil ecotoxicology,aquatic ecotoxicology and environmental health.Finally,the prospect and future of the development of MT biomarker were discussed.Keywords ：metallothioneins;heavy metal;homeostasis;detoxification;biomarker金属硫蛋白（metallothionein ，MT ）的化学名称为金属硫组氨酸三甲基内盐，是一类普遍存在于生物体内的低分子量（6~7kDa ）、富含半胱氨酸、热稳定性、可诱导型非酶蛋白[1]。

重金属盐沉淀蛋白质的实验探究

1　问题的提出在有关蛋白质性质的教学中，教师往往是通过直观的变性蛋白质沉淀的现象，帮助学生认识到重金属盐会使蛋白质变性。

然而，一些教师在教学实验中发现，硫酸铜使蛋白质变性产生的沉淀，会溶解在过量的硫酸铜溶液中，教师对此现象难以自圆其说，陷入了非常尴尬的窘境。

本研究尝试运用多种重金属盐进行变性蛋白质沉淀实验时，发现往鸡蛋清溶液中加入过量的硫酸锌溶液或硫酸铜溶液，会使蛋白质沉淀溶解。

过量的重金属盐溶液为什么会使蛋白质沉淀溶解？对于该问题的解释，已有的文献包含理论探讨与实验研究两类：1）理论探讨的文献指出，Cu2+、Zn2+造成的蛋白质沉淀源于盐析作用而不是变性[1]；2）实验探究的文献指出，Cu2+引起蛋白质沉淀溶解的主要原因是体系pH变化影响了蛋白质的溶解度[2]。

鉴于此，本研究设计了一系列的化学实验，旨在解决以下3个问题：1）Cu2+、Zn2+的蛋白质沉淀是由于盐析还是变性?2）除Cu2+外，体系pH的变化是否会导致Zn2+、Ag+的蛋白质沉淀溶解？3）如何防止过量的重金属盐溶液使变性蛋白质沉淀溶解？2　实验方案2.1　实验仪器台秤、烧杯、玻璃棒、100mL容量瓶、试管、吸量管、力辰科技PH-100型pH测试笔。

2.2　实验试剂鸡蛋清溶液：用台秤称取5.0g鸡蛋清，加水至100.0g，搅拌，用4层纱布过滤，得到澄清透明鸡蛋清溶液（pH =9.38）。

其他试剂：浓硫酸、浓硝酸；分析纯的硫酸铜、硝酸银、硫酸锌。

2.3　正式试验实验1：探究Cu2+、Zn2+的蛋白质沉淀是由于盐析还是变性。

①取两支试管编号1、2号管，分别加入2mL 鸡蛋清溶液，1号试管中滴入3~4滴5%硫酸铜溶液，2号试管中滴入3~4滴5%硫酸锌溶液，观察到两支试管中均出现白色沉淀。

②往1、2号试管中分别加入4mL水，观察到两支试管内的沉淀均不溶解。

实验2：探究pH的变化是否会导致Cu2+、Zn2+、Ag+的蛋白质沉淀溶解。

①取三个烧杯，编号为A1、B1、C1，分别量取20mL的蛋清溶液，按表1分别加入重金属盐溶液，观察现象并测量pH。

土壤重金属固化英语

土壤重金属固化英语Soil contamination by heavy metals is a big concern, especially for farmers and environmentalists. To tacklethis issue, one effective method is soil heavy metal stabilization. Basically, it's about locking those harmful metals in place so they don't spread or get absorbed by plants.The process involves using special materials that bind to the heavy metals, kind of like a magnet attracts metal filings. It's pretty amazing how science can come up with solutions like this. With stabilization, we can make the soil safer for agriculture and reduce the risk of these metals getting into our food chain.One thing I find fascinating is the variety of stabilization techniques. Some use natural materials like clays or organic amendments, while others rely on synthetic chemicals. Each has its pros and cons, but the goal is the same: keep those metals from moving around.For farmers, it's crucial to understand the options and choose the best stabilization method for their specific situation. After all, soil health is essential for growing healthy crops. And with soil heavy metal stabilization,we're taking a step towards ensuring a safer and more sustainable agricultural future.I've heard stories from farmers who've implemented stabilization techniques and seen.。

Assessment of the Heavy Metal Pollution

ASSESSMENT OF THE HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION IN THE BAIX LLOBREGAT(CATALONIA,NE SPAIN)DANUTA ZIMAKOWSKA-GNOI´NSKA1,JAUME BECH2and FRANCISCO J.TOBIAS21International Centre of Ecology PAS,Dziekanów Le´s ny n.Warsaw,05-092Łomianki,Poland 2Catedra de Pedologia,Facultat de Biologia,Universitat de Barcelona,Spain(Received21July1998;accepted13January1999)Abstract.A main goal of investigations is to determine could a soil respiration be an indicator of the soil pollution.In this case a measured level of the soil oxygen of its pollution.It also means that the pollution reduces biological processes soil samples were taken from polluted and non-polluted places in the Baix(Catalonia, NE Spain).Soil samples were taken from the top of soil(0–5cm)without a litter.Soil analysis were done,determining percentage shares of coarse fragments,coarse sand,ﬁne sand,coarse silt,ﬁne silt, clay,CaCO3,organic matter as well as water pH and conductivity CE(1:5[mS cm−1]).Also were determined(in mg kg−1)quantities of heavy metals,as Fe,Al,Mn,Zn,Cr,Ni,V,Cu,Cd,Pb.The soil respiration was investigated in temperatures15and30◦C and with controlled humidity.The respiration in30◦C is number of times greater then in15◦C both for polluted and non-polluted soils.Particularly high coefﬁcients of correlation between the soil respiration and soil pollution in polluted soils were obtained for Pb:r=0.75in15◦C and r=0.98in30◦C;for Ba:0.90and0.57;for V:0.99and0.81.In non-polluted soils highest correlation coefﬁcients are for Pb:r=0.70in15◦C; Fe:0.60and0.72;Al:0.68and0.64;Mn:0.51and0.66;Ba:0.63and0.61;Cr:0.94and0.70;Ni: 0.64and0.65;Cu:0.69and0.48;as well as V:0.62in15◦C;and Cd:0.69in15◦C.This way the soil respiration could be a good indicator of the soil pollution.Keywords:biological activity of soil,constant-pressure volumetric respirometer,heavy metals,respirom-etry methods,soil,soil degradation1.IntroductionThe aim of this study is to try correlating the effect of metal pollution and the respiration of soil in two areas–one polluted and a neighbour non-polluted area.An unfavourable inﬂuence of heavy metals on soil environment is well known. Many autors indicate their particularly destructive inﬂuence on soil microﬂora (Strojan,1975;Freedman and Hutchinson,1980).An activity of soil microbial population is also inﬂuenced by heavy metals as well as an intensity of biological processes in edaphon(Nordgren et al.,1983,1985,1986).Scientists are looking for relatively sensitive indicator of degradating processesin soil environment.An intensity of biological processes is generally estimated by quantity of ejected carbon dioxide(Witkamp,1969;Edwards,1974;Elmholt,1992;Environmental Monitoring and Assessment61:301–313,2000.©2000Kluwer Academic Publishers.Printed in the Netherlands.302 D.ZIMAKOWSKA-GNOI´NSKA ET AL.Gildon et al.,1993;Nakadai et al.,1993).This indicator covers both metabolisms –aerobic and anaerobic.Investigation of oxygen respiration could give more sen-sitive and explicit indication.Investigations of Fischer(1996)show that measured changes of the carbon dioxide ejection are less sensitive to changes of temperature and humidity of the soil as well as to changes of the soil pollution.Probably,the carbon dioxide ejection is a result of two simultanous processes–aerobic and anaerobic.The oxygen intake is connected only with aerobic process.In presented paper a respirometry method was applied to oxygen respiration measurements of polluted and non-polluted soils from the Barcelona area.Results have to show an inﬂuence of pollution on biological activity of soils. These results are also inﬂuenced by other physical and biological characteristics of investigated soil sample.The main objective of said study is to prove that res-piration of soil depends of soil pollution and moreover indicates the extent of the pollution harmfulness on soil biological processes.2.Area of Studies,Climatic Conditions and VegetationStudied areas are located in the Baix Llobregat,about20km SW of Barcelona and inside of the metropolitan area of the city.For purposes of this paper two areas were taken into account.Theﬁrst one,named‘non-polluted area’(Figure1),belongs to mountainous side of Gavá,Viladecans and Torelles de Llobregat.This area can be considered as non-polluted area because it is forest zone with relatively low anthropogenic impact.The second one,named‘polluted area’(Figure2),is located SW of Gaváand Viladecans and represents degradated part of metropolitan area.The climate of area is typically Mediterranean with mean annual temperature of 16◦C and mean annual rainfall(mostly in spring and in autum)of650mm.Spring and autumn precipitation are135and240mm,respectively;summer precipitation is only105mm.The summer is hot and dry with mean temperature over20◦C from June to September.The mean January temperature is9±10◦C.According to the Koppen climatic classiﬁcation,it is‘Csa’;pedoclimic regimes are xeric(SMR) and thermic(STR).A main vegetation in the non-polluted area are low‘garriga’scrubs,Quercus coccifera,Pistacia lentiscus,Olea europea,Chamaerops humilis,Rhamnus ly-cioides,Rosmarinus ofﬁcinalis,Erica multiﬂora,Ampelodesma mauritanica,Cer-atonia siliqua,Fumana laevipes,frequently accompanied by Pinus halepensis.In degradated area are herbes such as Hyparrhenia hirta,Sonchus tenerrimus, Brachypodium ramosum,Brachypodium phoenicoides,Foeniculum vulgare,Pso-ralea bituminosa,Inula viscosa etc.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION303Figure1.The sampling points of the non polluted area are indicated.Number2shows the location of the polluted area.304 D.ZIMAKOWSKA-GNOI´NSKA ET AL.Figure2.Sampling points in the polluted area:enlarged view(A:composting plant,B:used cars dump).3.Material and Methods3.1.M ATERIALWere studied22topsoil samples(0–5cm,without litter).Eleven samples were taken from non-polluted area(Figure1)and other eleven from polluted area(Fig-ure2).Those from polluted area were taken from three transects–two near a composting plant and one near an used car dump.3.2.A NALYTICAL METHODSSoil samples were air-dried and passed through a2mm sieve to obtain a‘ﬁne earth’(fraction less then2mm).Sieving has also to eliminate plant roots and other plant parts.In this‘ﬁne earth’shares of coarse fractions(>0.2mm)andﬁne fractions (<0.2mm)were determined for sands and for silts.In the‘ﬁne earth’the organic carbon content was determined using the Walkley-Black dichromate acid oxidation method.Soil pH values were determined for soil solution suspensions in distilled water and in the1N KCl.This determination was done potentiometrically with a glass electrode.Calcium carbonate contents were determined using the acid neutralization method.Particle-size distribution was determined using the pipette method and by sieving sand fractions.Conduc-HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION305 timetry was determined in1:5soil to water extract.Total nitrogen contents were determined using the Carlo Erba1500elemental analyzer.The‘ﬁne earth’was grounded in a tungsten carbide mill,dried at105◦C and then used to determine metal contents.Soil samples(3g)were extracted with28ml of aqua regia(HNO3to HCl as1:3)during two hours at130◦C after a predigestion of16hr at a room temperature.Metal concentrations were measured inﬁltered extract,using the Polyscan61E spectrometer.3.3.S OIL RESPIRATIONSoil respirations were determined in laboratory because in this case experimental conditions are more stabile than in the case ofﬁeld measurements.Soil samples were taken from the top of soil(0–5cm)without a litter,trans-ported to laboratory and stored there in a cooler in temperature3◦C.Soil samples were sieved to eliminate plant roots,as recommends Anderson(1982).Soil respirations were measured using constant pressure volumetric respirime-ters,modiﬁed by Klekowski(1975).Respirometers were placed in water ther-mostate with temperature controlled with±0.1◦C precision.Experiments were done in two temperatures:15and30◦C.In respirometric vessels were placed30g samples of soil.Samples were moist-ened up to60%of humidity adding sufﬁcient quantity of boiled cold water.Moist-ened samples were adapted during12hr to the temperature of experiments.Mea-surements of oxygen consumption were done for the next3hr.After respiration and after drying in temperature55◦C a dry weight of sample was weighted.An humidity of sample was determined from the ratio of dry weight to wet weight of sample.A share of organic matter in the soil sample was determined by combustion in a mufﬂe oven in temperature450◦C.Results obtained when using this method of determination are more applicable to biological processes in the investigated soil.The oxygen consumption for a sample30g of the wet soil is expressed in mm3O2·h−1and per gram of the dry soil or per gram of the organic matter in mm3O2·h−1·g−1.Mean values and standard deviations were calculated for all experiments.The aim of this preliminary study is toﬁnd simple correlations between heavy metal pollution and soil respiration in two areas:polluted and non-polluted.4.Results and DiscussionThe increase of antropogenic heavy metals in soils is signiﬁcant on outskirts of industrial zones like the Metropolitan Area of Barcelona.In Table I are compared physico-chemical characteristics of sampled soils.In Table II are shown shares of heavy metals in the same soil samples.In polluted area in the Llobregat delta plane306 D.ZIMAKOWSKA-GNOI´NSKA ET AL.TABLE IPhysico-chemical data of soil samplesNo.Coarse Coarse Fine Coarse Fine Clay Organic pH N CaCO3CE1:5 fragments sand sand silt silt matter H2O(%)(%)(%)(%)(%)(%)(%)(%)(%)(mS cm−1)1 3.6254.7122.53 4.407.5010.85 3.827.290.1025.580.23029.8922.0220.0215.6622.4819.83 1.887.340.0923.400.5843 3.5735.6010.0234.430.0019.97 3.597.120.2417.850.764419.6142.8611.719.8225.4410.17 2.907.230.1116.650.804565.1635.8815.677.0721.3120.078.037.260.7218.82 2.368619.7144.8812.267.1317.4018.32 4.167.540.2115.200.240722.2430.8216.2012.6319.8520.51 6.097.530.3118.820.303833.5746.4212.287.8919.2514.169.177.450.1529.680.347937.3634.7014.5412.8719.6218.27 4.277.270.1623.890.27710 6.3318.4110.758.1739.3823.29 6.557.300.2831.130.551 1119.8217.988.817.5640.3925.279.637.210.4329.440.653 1232.1617.55 6.5612.9338.7724.19 3.707.260.197.850.517 1352.0615.00 6.3214.1342.1122.44 3.137.360.17 2.500.103 1465.5522.52 6.3911.0339.5920.4714.537.200.60 3.450.213 1511.1340.3441.50 4.44 6.277.45 6.097.060.23 2.610.104 1646.5816.07 4.63 5.3137.6936.307.58 6.470.49 4.040.135 1715.7014.128.7210.7931.7334.64 4.967.180.1847.740.157 1813.667.8169.649.18 5.787.59 3.13 6.920.09 1.550.089 1913.527.4238.6516.4918.8118.62 6.557.370.2411.430.204 2019.6115.0245.407.9013.5918.09 6.217.480.1811.540.231 2140.2826.0916.2610.1124.5023.0412.827.070.38 5.590.319 2250.5514.94 5.447.4931.4340.718.157.300.22 6.880.183main sources of pollution are composting plant and the used car dump.The non-polluted area is nearby hilly forest area.Point16placed in this area is an anomaly –its soil contains natural high share of metals.These tables conﬁrm that the polluted area is really a polluted one and concen-tration of heavy metals grows when approaching to the plant and car dump.In paper Bech et al.(1994)were presented correlations between physico-chemical characteristics and concentrations of heavy metals.For example,conductivity(CE 1:5)positively correlates with concentration of heavy metals in the contaminated area,but not in noncontaminated area.Mean conductivity in polluted area is0.65mS cm−1.In extremely polluted point No.5conductivity is even2.37mS cm−1.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION307TABLE IIShares of heavy metals in soil samplesNo.%Fe%Al Mn Zn Cr Ni V Cu Cd Pb(ppm)1 1.56 1.35445.86197.0120.6616.1297.5871.930.71166.762206 2.24455.38122.1327.1920.69100.2833.040.4462.723 2.88 2.57575.72355.8050.0938.01128.49131.18 1.84241.304 3.12 2.34497.67898.4344.5628.79114.91196.33 4.46761.21513.64 2.961395.843770.36705.91130.17128.121093.7411.911970.656 1.98 1.78 4.38166.6324.0821.0298.9035.390.7465.067 3.08 2.24549.01537.1039.9335.21102.25112.85 1.30205.738 3.54 1.94610.201573.7155.3463.7383.16216.29 1.88458.669 2.32 2.36501.02391.9932.2024.13117.3858.310.76149.1710 2.22 2.48512.89199.0627.7622.89104.3745.200.4963.2911 2.15 2.48488.32247.5227.8922.96111.1629.140.9648.7212 4.11 3.10813.0877.6432.8437.7593.1025.200.3739.0813 4.60 3.68705.9484.1942.0544.21106.0327.270.3041.9714 3.47 2.79978.56214.3343.0638.25100.4033.090.5885.50 150.710.72103.4347.4511.608.7765.98 4.720.3028.04 1610.77 4.3313584.39111.4556.72111.15169.70129.82 1.2974.45 17 1.80 2.77426.8292.7127.9319.30117.8411.480.4740.94 180.870.98522.3323.2019.438.8879.07 3.980.1220.2619 1.79 2.20805.0652.0323.2519.9587.4015.550.3027.0020 1.68 1.851153.5742.6521.5915.1498.0310.020.3026.8621 3.64 1.74786.78128.3721.5036.3890.3358.600.6977.7122 4.31 4.16256.9178.3341.0442.74116.7225.17 1.0850.99In polluted area is observed considerable concentration of Fe,Mn,Zn,Be,Cr, Ni,V,Cu,Cd and Pb.In non-polluted area concentration of heavy metals is much lower and mean conductivity is only0.20mS cm−1.Share of organic matter decreases with increased concentration of heavy metals and increased salinization of soils.In the non-polluted area,natural Fe,Al,Zn,Cr,Ni,V,Cd and Pb show correla-tions with size fractions(negative correlations withﬁne sand and positive withﬁne silt and clay).In both areas metal contents generally correlate with each other:–in the non-polluted area these correlations are due to similar pedogonic processes and also to similar parent materials.308 D.ZIMAKOWSKA-GNOI´NSKA ET AL.Figure3.Oxygen consumptions of soil samples from contaminated areas:=15◦C, =30◦C.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION309Figure4.Oxygen consumptions of soil samples from contaminated areas:=15◦C, =30◦C.310 D.ZIMAKOWSKA-GNOI´NSKA ET AL.–in the polluted area these correlations are due to contaminant processes which lead to a proportional increase of metal concentrations.In Figure3are presented oxygen consumptions of soil samples from contaminated areas and in Figure4oxygen consumptions of soil samples from noncontaminated areas.In both cases results are given for two temperatures of experiment:15and 30◦C.Was observed generally lower oxygen consumption in soil samples from con-taminated areas in comparison with oxygen consumption in soil samples from noncontaminated areas.In the extremely contaminated point No.5oxygen con-sumption has the lowest value.In this point concentration of heavy metals is con-siderably higher then in other points and also there is an extremal salinization (conductivity2.37mS cm−1compared with mean values:0.65in contaminated area and0.20in noncontaminated).In contaminated area a share of the organic matter is signiﬁcantly lower,a mean value for all points in this area is only9.18%,when in noncontaminated area a mean value is12.77%.It could be a result of unfavourable inﬂuence of heavy metals and their salts on biological processes in edaphon,mostly on soil microfauna and soil microbial populations.In Table III are presented correlations between measured soil respirations and components of soil samples from contaminated area.In Table IV are presented correlations between measured soil respirations and components of soil samples from noncontaminated area.Particularly high coefﬁcients of correlation between the soil respiration and soil pollution in polluted soils were obtained for Pb:r=0.75in15◦and r=0.98in 30◦C,for Ba:0.90and0.57;for V:0.99and0.81.In non-polluted soils highest correlation coefﬁcients are for Pb:r=0.70in15◦C;Fe:0.60and0.72;Al:0.68 and0.64;Mn:0.51and0.66;Ba:0.63and0.61;Cr:0.94and0.70;Ni:0.64and 0.65;Cu:0.69and0.48;V:0.62in15◦C;and Cd:0.69in15◦C.Obtained results attest that measurements of soil respirations using volumetric respirometry could be used for estimations and comparations of soil ecological conditions as well as biological activity of soils.5.Conclusions1.Obtained results of experiments and analysis conﬁrm that some investigatedsoils in the Baix Llobregat near Barcelona are contaminated by heavy metals.Concentration of pollutants increases in vinicity of a composting plant and a car dump.2.In contaminated soils conductivities are three times higher than in noncontam-inated soils.Soil conductivity depends in high degree on salinization of soils by dissociated heavy metal salts.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION311TABLE IIICorrelations between measured soil respirationand components of this soil from contaminatedarea15◦C30◦CSoil humidity–0.326∗–0.471∗0.328∗∗0.144∗∗Organic matter–0.543∗–0.385∗0.084∗∗0.243∗∗CaCO3–0.544∗–0.334∗0.083∗∗0.315∗∗CE–0.493∗–0.528∗0.123∗∗0.095∗∗Fe–0.445∗–0.382∗0.170∗∗0.247∗∗Al–0.432∗–0.341∗0.185∗∗0.305∗∗Mn–0.532∗–0.442∗0.092∗∗0.173∗∗Sr–0.546∗–0.446∗0.082∗∗0.169∗∗Zn–0.476∗–0.304∗0.139∗∗0.364∗∗Ba0.044∗0.193∗0.897∗∗0.570∗∗Cr–0.418∗–0.399∗0.201∗∗0.224∗∗Ni–0.517∗–0.368∗0.104∗∗0.266∗∗V–0.003∗–0.084∗0.992∗∗0.806∗∗Cu–0.431∗–0.359∗0.186∗∗0.278∗∗Cd–0.352∗–0.295∗0.288∗∗0.379∗∗Pb–0.109∗0.010∗0.749∗∗0.976∗∗∗:r=Correlation Coefﬁcient.∗∗:Signiﬁcance level p<0.05.312 D.ZIMAKOWSKA-GNOI´NSKA ET AL.TABLE IVCorrelations between measured soil respirationand components of this soil from noncontami-nated area15◦C30◦CSoil humidity–0.325∗–0.569∗0.330∗∗0.068∗∗Organic matter–0.515∗–0.708∗0.105∗∗0.015∗∗CaCO3–0.655∗–0.515∗0.029∗∗0.105∗∗CE0.313∗–0.192∗0.349∗∗0.572∗∗Fe0.179∗–0.122∗0.599∗∗0.721∗∗Al–0.141∗–0.161∗0.679∗∗0.637∗∗Mn0.221∗–0.152∗0.513∗∗0.657∗∗Sr–0.595∗–0.606∗0.053∗∗0.048∗∗Zn–0.138∗–0.542∗0.685∗∗0.085∗∗Ba0.162∗–0.175∗0.633∗∗0.607∗∗Cr0.025∗–0.132∗0.941∗∗0.699∗∗Ni0.159∗–0.153∗0.641∗∗0.653∗∗V–0.170∗–0.337∗0.617∗∗0.311∗∗Cu0.135∗–0.238∗0.692∗∗0.482∗∗Cd–0.137∗–0.474∗0.690∗∗0.141∗∗Pb–0.133∗0.492∗0.697∗∗0.124∗∗∗:r=Correlation Coefﬁcient.∗∗:Signiﬁcance level p<0.05.HEA VY METAL POLLUTION EFFECTS ON THE SOIL RESPIRATION313 3.In contaminated area a share of the organic matter is signiﬁcantly lower(9.18%)then in noncontaminated area(12.77%).It could be a result of unfavourable inﬂuence of heavy metals and their salts on biological processes in edaphon, mostly on soil microfauna and soil microbial populations.4.Oxygen consumptions are considerably lower in soil samples from contami-nated areas in comparation with noncontaminated areas.5.V olumetric respirometry could be used for estimations and comparations ofecological condition and biological activity of soils.ReferencesAnderson,J.P.E.:1982,‘Soil Respiration’,in:Page,A.L.,Miller,R.H.and Keeney,D.R.(eds.),Methods of Soil Analysis:2Chemical and Microbiological Properties,Am.Soc.Agron., Madison,Wisconsin,pp.467–476.Bech,J.,Tobias,F.J.and Zimakowska-Gnoi´n ska,D.:1994,‘Comparison of Heavy Metals in pol-luted and unpolluted soils at the NE edge of the Garraf Massif(Catalonia,Spain)’,Abstracts of the15th International Congress of Soil Science,Acapulco,Mexico,152–153.Edwards,N.T.:1974,‘A moving chamber design for measuring soil respiration rates’,OIKOS25, 97–101.Elmholt,S.:1992,‘Effect of Propiconazole on Substrate Amended Soil Respiration Following Laboratory and Field Respiration’,Pestic.Sci.34,139–146.Fischer,Z.:1996,‘Ecological equilibrium disturbances in the soil exposed to simulated acid rain.Part VI’,Environ.Monit.Assess.41,61–65.Freedman,B.and Hutchinson,T.C.:1980,‘Effect of smelter pollutants on forest litter decomposition near a nickel-copper smelter at Sudbury,Ontario’,Can.J.Bot.58,1722–1736.Freedman,B.and Hutchinson,T.C.:1980,‘Pollutant inputs from the atmosphere and accumulation in soils and vegetation near a nickel-copper smelter at Sudbury,Ontario’,Can.J.Bot.58,108–132.Gildon,A.and Rimmer,D.L.:1993,‘Soil respiration on reclaimed coal-mine spoil’,Biol.Fertile Soils16,41–44.Klekowski,R.Z.:1975,‘Constant Pressure V olumetric Microrespirometer for Terrestrial Inver-tebrates’in W.Grodzi´n ski,R.Z.Klekowski and A.Duncan(eds.),Methods for Ecological Bioenergetics,IBP Handbook Blackwell Sci.Publ.,Oxford-London-Edinburgh-Melbourne,24, pp.212–225.Nakadai,T.,Koizumi,H.,Usami,Y.,Satoh,M.and Oikawa,T.:1993,‘Examination of the method for measuring soil respiration in cultivated land:Effect of carbon dioxide concentration on soil respiration’,Ecological Research8,65–71.Nordgren,A.,Baath,E.and Soderstrom,B.:1983,‘Microfungi and microbial activity along a heavy metal gradient’,Appl.Environ.Microbiol.46,1829–1837.Nordgren,A.,Baath,E.and Soderstrom,B.:1985,‘Soil microfungi in an area polluted by heavy metals’,Can.J.Bot.63,448–455.Nordgren,A.,Kauri,T.,Baath,E.and Soderstrom,B.:1983,‘Soil microbial activity,mycelial lengths and physiological groups of bacteria in a heavy metal polluted area’,Environ.Poll.(Serie A)41, 89–100.Strojan,C.L.:1978,‘Forest litter decomposition in the vicinity of a zinc smelter’,Oecologia(Berl.) 32,203–212.Witkamp,M.:1969,‘Cycles of temperature and carbon dioxide evolution from litter and soil’, Ecology50,922–924.。

Adsorption of heavy metal ion from aqueous single metal solution by chemically

Adsorption of heavy metal ion from aqueous single metal solutionby chemically modiﬁed sugarcane bagasseOsvaldo Karnitz Jr.a ,Leandro Vinicius Alves Gurgel a ,Ju´lio Ce ´sar Perin de Melo a ,Vagner Roberto Botaro a ,Taˆnia Ma ´rcia Sacramento Melo a ,Rossimiriam Pereira de Freitas Gil b ,Laurent Fre´de ´ric Gil a,*aDepartamento de Quı´mica,Instituto de Cie ˆncias Exatas e Biolo ´gicas,Universidade Federal de Ouro Preto,35400-000Ouro Preto,Minas Gerais,BrazilbDepartamento de Quı´mica,Instituto de Cie ˆncias Exatas,Universidade Federal de Minas Gerais,31270-901Belo Horizonte,Minas Gerais,BrazilReceived 22November 2005;received in revised form 28April 2006;accepted 2May 2006Available online 14July 2006AbstractThis work describes the preparation of new chelating materials derived from sugarcane bagasse for adsorption of heavy metal ions in aqueous solution.The ﬁrst part of this report deals with the chemical modiﬁcation of sugarcane bagasse with succinic anhydride.The carboxylic acid functions introduced into the material were used to anchor polyamines,which resulted in two yet unpublished modiﬁed sugarcane bagasse materials.The obtained materials were characterized by elemental analysis and infrared spectroscopy (IR).The sec-ond part of this reports features the comparative evaluation of the adsorption capacity of the modiﬁed sugarcane bagasse materials for Cu 2+,Cd 2+,and Pb 2+ions in aqueous single metal solution by classical titration.Adsorption isotherms were studied by the Freundlich and Langmuir models.Ó2006Elsevier Ltd.All rights reserved.Keywords:Adsorption;Modiﬁed sugarcane bagasse;Polyamines;Isotherm;Heavy metals1.IntroductionWater pollution is a major environmental problem faced by modern society (Baird,1995)that leads to eco-logical disequilibrium and health hazards (Kelter et al.,1997).Heavy metal ions such as copper,cadmium,lead,nickel,and chromium,often found in industrial waste-water,present acute toxicity to aquatic and terrestrial life,including humans.Thus,the discharge of eﬄuents into the environment is a chief concern.The methods commonly used to remove toxic heavy metal from municipal and industrial wastewater are based on the adsorption of ions onto insoluble compounds and the separation of the sed-iments formed.Many eﬀorts have been made recently toﬁnd cheaper pollution control methods and materials(Panday et al.,1985;Ali and Bishtawi,1997;Acemiog˘lu and Alma,2001).The new material world trends point to the importance of using industrial and agricultural residues as production starting materials.Reusing and recycling these residues can minimize the environmental problems associated with their build-up and reduce the use of noble starting materi-als.This trend has contributed to the reconsideration of the use of traditional biomaterials such as natural lignocellu-losic ﬁbers to substitute synthetic polymers,for example,since in many cases they have a better performance.Brazil is the world leading producer of sugarcane for both the alcohol and the sugar industries.These industries produce a large amount of sugarcane bagasse and although it is burned to produce energy for sugar mills,leftovers are still signiﬁcant.Thus,on account of the importance of0960-8524/$-see front matter Ó2006Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2006.05.013*Corresponding author.Tel.:+553135591717;fax:+55315511707.E-mail address:laurent@iceb.ufop.br (L.F.Gil).Bioresource Technology 98(2007)1291–1297bagasse sugar as an industrial waste,there is a great interest in developing chemical methods for recycling it.Sugarcane bagasse has around50%cellulose,27%polyoses,and23% lignin(Caraschi et al.,1996).These three biological poly-mers have many hydroxyl and/or phenolic functions that can be chemically reacted to produce materials with new properties(Xiao et al.,2001;Navarro et al.,1996).Despite the many studies of the chemical modiﬁcation of cellulose published around the world in this area(Gurnani et al.,2003;Gellerested and Gatenholm,1999),only a few have investigated the modiﬁcation of bagasse sugar(Krish-nan and Anirudhan,2002;Orlando et al.,2002).This work describes the preparation and the evaluation of new chelating materials from sugarcane bagasse to adsorb heavy metal ions in aqueous solution.In a prelimin-ary study,it has been chosen to study the adsorption of Cu2+,Cd2+,and Pb2+.Theﬁrst part of this work describes the modiﬁcation of sugarcane bagasse with succinic an-hydride to introduce carboxylic functions to sugarcane bagasse and the chemical introduction of commercial linear polyamine via the formation of amide functions.It is well known that polyamines have powerful chelating properties, mainly towards ions such as Cu2+,Zn2+,and Pb2+(Bian-chi et al.,1991;Martell and Hancock,1996).The second part of this work evaluates the adsorption of Cu2+,Cd2+,and Pb2+onto three modiﬁed sugarcane bag-asses(MSBs)from aqueous single metal ion solutions by classical titration.The results were analyzed by the Lang-muir and Freundlich models(Ho et al.,2005).2.Methods2.1.MaterialsPolyamines ethylenediamine3and triethylenetetramine 4were used in this work.Succinic anhydride,1,3-diiso-propylcarbodiimide(DIC),and triethylenetetramine,from Aldrich,were used without puriﬁcation.Ethylenediamine and dimethylformamide were distilled before use.Pyridine was reﬂuxed with NaOH and distilled.2.2.Sugarcane bagasse preparationSugarcane bagasse was dried at100°C in an oven for approximately24h and nextﬁber size was reduced to pow-der by milling with tungsten ring.The resulting material was sieved with a4-sieve system(10,30,45,and60mesh). Then,the material was washed with distilled water under stirring at65°C for1h and dried at100°C.Finally,it was washed anew in a sohxlet system with n-hexane/ ethanol(1:1)as solvent for4h.2.3.Synthesis of MSBs1and2Washed and dried sugarcane bagasse(5.02g)was trea-ted with succinic anhydride(12.56g)under pyridine reﬂux (120mL)for18h.The solid material wasﬁltered,washed in sequence with1M solution of acetic acid in CH2Cl2, 0.1M solution of HCl,ethanol95%,distilled water,and ﬁnally with ethanol95%.After drying at100°C in an oven for30min and in a desiccator overnight,MSB1(7.699g) was obtained with a mass gain of53.4%.MSB2was obtained by treatment of1with saturated NaHCO3solu-tion for30min and afterwards byﬁltering using sintered ﬁlter and washing with distilled water and ethanol.2.4.Synthesis of MSBs5and6The process used to introduce amine functions was the same as that used to prepare MSB5and6.MSB1was trea-ted with5equiv of1,3-diisopropylcarbodiimide(DIC)and 6equiv of polyamine in anhydrous DMF at room tempera-ture for22h under stirring.Afterﬁltration,the materials were washed with DMF,a saturated solution of NaHCO3, distilled water,andﬁnally with ethanol.Next,they were dried at80°C in an oven for30min and in a desiccator overnight.2.5.Kinetic study of metal ion adsorption of MSBs2,5,and6Experiments with each material and metal ion were per-formed to determine the adsorption equilibrium time from 10to90min in10min intervals.The amount of100mg MSB was placed in a250-mL Erlenmeyer with100.0mL metal ion solution with concentration of200mg/L under stirring.The experiments were done at pHs5.8for Cu2+, 7.0for Cd2+,and6.2for Pb2+,optimal values to obtain the best adsorption.To adjust pH values,was added NaOH solution(0.01mol/L)into metal solutions with MSB.Afterﬁltration,metal ion concentration was deter-mined by EDTA titration.2.6.pH study of metal ion adsorption of MSBs2,5,and6Experiments with each material and metal ion were per-formed to determine the eﬀect of pH on ion adsorption.An amount of100mg MSB was placed into a250-mL Erlen-meyer with100.0mL of metal ion solution200mg/L under stirring.pH was calibrated with HCl or NaOH solutions (0.1–1.0mol/L).The reaction times used were30min (MSB2)or40min(MSB5and6)for Cu2+and Cd2+, and40min(MSB2)or50min(MSB5and6)for Pb2+. Metal ion concentration was determined afterﬁltration by EDTA titration.No signiﬁcative variation of pH was observed at the end of each experiment.2.7.Adsorption isotherms of MSBs2,5,and6Experiments were performed for each material and metal ion to determine adsorption isotherms.In each experiment,100mg of MSB was placed into a250-mL Erlenmeyer with100.0mL of metal ion solution in speciﬁc concentrations(between200mg/L and400mg/L)under stirring.Each experiment was performed at the pH of1292O.Karnitz Jr.et al./Bioresource Technology98(2007)1291–1297larger ion adsorption during the time necessary for equilib-rium (Tables 3and 4).After ﬁltration,the metal ion con-centration was determined by EDTA titration.2.8.Characterization of the new obtained materials MSB 1,2,5,and 6were characterized by IR spectro-scopy in a Nicolet Impact 410equipment with KBr.Elemental analyses were accomplished in Analyzer 2400CHNS/O Perkin Elemer Series II.3.Results and discussion3.1.Synthesis of MSBs 1,2,5,and 6The synthesis route used to prepare MSBs 1,2,5,and 6are presented in Scheme 1.Prewashed sugarcane bagasse was succinylated for various periods of time.The degree of succinylation of the bagasse ﬁbers was determined by measuring the quantity of acid function.The results are shown in Fig.1.The concentration of carboxylic functions per mg of bagasse was determined by retro titration.For this,MSB 1was initially treated with an excess solution of NaOH (0.01mol/L)for 30min.Soon afterwards the material was ﬁltered and the obtained solution was titrated with an HCl solution (0.01mol/L).The highest degree of succinylation was reached after 18-h ing this reaction time,sugarcane bagasse was succinylated to pro-duce MSB 1,which presented a weight gain of 54%and a concentration of carboxylic acid function per mg of 3.83·10À6mol.Next,MSB 1was treated with a saturated NaHCO 3solution to produce MSB 2.Starting from MSB 1,two polyamines were introduced:ethylenediamine 3and triethylenetetramine 4.The method-ology used to introduce the polyamines was the same for the two MSBs 5and 6,as shown in Scheme 1.Concentra-tions of 2.4·10À6mol (5)and 2.6·10À6mol (6)of amine function per mg of material were determined by back titra-tion with excess HCl solution.The introduction of the amine functions was also veriﬁed by IR spectroscopy (Table 1)and elemental analysis (Table 2).3.2.Characterization of MSBs 1,5,and 6Characterization of carboxylated MSB 1was accom-plished by IR spectroscopy.The spectrum of unmodiﬁed sugarcane bagasse and MSB 1are presented in Fig.2.The spectrum of MSB 1displayed two strong bands at 1740and 1726cm À1in relation to that of unmodiﬁed sug-arcane bagasse.This demonstrated the presence of two types of carbonyl functions,one relative to carboxylic acid and another relative to the ester.The acid and ester IR bands indicate that succinic anhydride acylated theO.Karnitz Jr.et al./Bioresource Technology 98(2007)1291–12971293hydroxy group of bagasse to generate an ester bond with consequent release of a carboxylic acid functional group.The spectra of MSBs5and6(Figs.3and4,respectively) showed three new strong bands at1550–1650cmÀ1(see data in Table1)corresponding to the presence of amide and amine functions,and one band at1060cmÀ1 corresponding to C–N stretch.The bands at1635and 1650cmÀ1(Fig.3)correspond to the axial deformation of the carbonyl of the amide function and the angular deformation of the N–H bond of the amine function.The band at1575cmÀ1corresponds to the angular deformation of the N–H bond of the amide function.The band at 1159cmÀ1(Fig.4)corresponds to the asymmetric stretch of C–N–C bond.The main bands observed in all MSBs are presented in Table1.MSB elemental analysis data presented in Table2show a modiﬁcation in the carbon and hydrogen composition of MSB1and a larger proportion of nitrogen as the number of amine functions in the used polyamine increases.3.3.Study of adsorption of Cu2+,Cd2+and Pb2+on MSBs2,5,and6The study of the MSB adsorption properties was accom-plished for each material and metal ion.A kinetic study and an adsorption study as a function of pH wereﬁrst carried out.3.3.1.Eﬀect of contact timeThe kinetic study of MSB2with Cu2+,Cd2+,and Pb2+ ions in aqueous solution is presented in Fig.5.Adsorption equilibrium was reached after20min for Cd2+ions.A time of30min was chosen for all studies of MSB2with Cd2+. The adsorption equilibrium times chosen for pH and con-centration dependent experiments are presented in Table3.Similar studies were accomplished for MSBs5and6for Cu2+,Cd2+,and Pb2+.The results are presented in Table3.3.3.2.pH EﬀectThe removal of metal ions from aqueous solutions by adsorption is dependent on solution pH as it aﬀects adsor-Table1Main IR spectrum bands observed in MSBs1,5,and6MSB Main bands observed(cmÀ1)11740,172651745,1650,1635,1575,1423,1060 61738,1651,1635,1560,1400,1159,1060 Table2Elemental analysis of MSBs1,2,5,and6C(%)H(%)N(%) Sugarcane bagasse43.98 6.020.13MSB145.41 5.620.10MSB238.04 5.140.01MSB544.01 6.51 2.21MSB646.88 6.65 3.431294O.Karnitz Jr.et al./Bioresource Technology98(2007)1291–1297bent surface charge,the degree of ionization,and the species of adsorbates.The study of adsorption of Cd 2+,Cd 2+,and Pb 2+on MSB 2as a function of pH was accom-plished with the reaction times given in Table 3;the results are presented in Fig.6.The adsorption of the three metal ions increases with the increase in pH.Maximum removal of Cd 2+was observed above pH 6and in the case of Pb 2+and Cu 2,above pH 5and 5.5.Similar studies were accomplished for MSBs 5and 6and Cu 2+,Cd 2+and Pb 2+with similar results,as shown in Table 4.3.3.3.Adsorption isothermsThe Langmuir (Ho et al.,2005)(Eq.(1))and Freundlich (Eq.(2))isotherms were evaluated by adsorption experi-ments as a function of the initial metal ion concentrations in aqueous solution under equilibrium time and pH condi-tions given in Tables 3and 4.The results of each material and metal ion are presented in Fig.7(Langmuir)and Fig.8(Freundlich)and Table 5.c q ¼1Q max Âb þc Q maxð1Þln q ¼ln k þ1nln cð2ÞTable 3Adsorption equilibrium times of MSBs 2,5and 6MSB Equilibrium time (min)Cu 2+Cd 2+Pb 2+230304054040506404050Table 4pH of largest adsorption of MSBs 2,5and 6MSB pH of largest adsorption Cu 2+Cd 2+Pb 2+2 5.5–6.0 6.5–7.5 5.0–6.05 5.5–6.0 6.5–7.5 5.0–6.065.5–6.06.5–7.55.0–6.0O.Karnitz Jr.et al./Bioresource Technology 98(2007)1291–12971295where q(mg/g)is the concentration of adsorbed metal ions per gram of adsorbent,c(mg/L)is the concentration of metal ion in aqueous solution at equilibrium,Q max and b are the Langmuir equation parameters and k and n are the Freundlich equation parameters.High correlation coeﬃcients of linearized Langmuir and Freundlich equations indicate that these models can explain metal ion adsorption by the materials satisfactorily. Therefore,both models explained metal ion adsorption by MSBs2,5,and6as can be observed in Table5,with the exception of the Freundlich model for Pb2+adsorption by MSB2.The Langmuir isotherm parameter Q max indicates the maximum adsorption capacity of the material,in other words,the adsorption of metal ions at high concentrations. It can be observed in Table5that MSB5presents the larg-est Cu2+adsorption capacity while MSB6adsorbs Cd2+ and Pb2+the ngmuir parameter b indicates the bond energy of the complexation reaction of the material with the metal ion.It can be observed that MSB2presents the largest bond energy for Cu2+and Cd2+,while three materials do not diﬀer signiﬁcantly for Pb2.The Freundlich isotherm parameter k indicates the adsorption capacity when the concentration of the metal ion in equilibrium is unitary,in our case1mg/L.This parameter is useful in the evaluation of the adsorption capacity of metal ions in dilute solutions,a case closer to the characteristics of industrial eﬄuents.The values of k of MSB2and5are much similar for Cu2+and Cd2+ and much higher than that for MSB6.This shows the superiority of both materials in the adsorption of these metal ions in low concentrations.MSB5has a higher k value for Pb2+when compared to those of the other materials.These results were compared with those of Vaughan et al.(2001)for a commercial macroreticular chelating resin with thiol functional groups,Duolite GT-73.The Q max of Duolite GT-73for Cu2+,Cd2+,and Pb2+were 62mg/g,106mg/g,and122mg/g,respectively.Duolite GT-73exhibited Q max lower than those of MSBs(Table5).4.ConclusionsThrough a fast,eﬀective,and cheap methodology,it was possible to devise a strategy to introduce chelating func-tions(carboxylic acid and amine)to sugarcane bagasse. Modiﬁed sugarcane bagasses presented a good adsorption capacity for Cu2+,Cd2+,and Pb2+ions with maximum adsorption capacity observed for MSB6.It has been dem-onstrated that metal ion adsorption eﬃciency is propor-tional to the number of amine functions introduced into the material.MSB2,which contained only carboxylate functions,showed an eﬃciency similar to that of MSB5, a material of much more complex synthesis. AcknowledgementsWe thank FAPEMIG forﬁnancial support,CAPES and UFOP.Table5The Langmuir and Freundlich parameters for Cu2+,Cd2+and Pb2+ adsorptionMetalion MSB Langmuir FreundlichQ max (mg/g)b(L/mg)r2k(mg/g)n r2Cu2+21140.431191.623.90.919351390.1730.999898.315.80.906161330.0140.992722.8 3.640.9635Cd2+21960.1030.993459.4 4.160.977351640.0680.995762.8 5.490.983463130.0040.9528 5.15 1.630.9856Pb2+21890.1100.994566.0 4.660.757951890.1250.999914724.510.98163130.1210.9994121 5.210.8771296O.Karnitz Jr.et al./Bioresource Technology98(2007)1291–1297ReferencesAcemiog˘lu,B.,Alma,M.H.,2001.Equilibrium studies on adsorption of Cu(II)from aqueous solution onto cellulose.Journal of Colloid and Interface Science243,81–83.Ali,A.A.,Bishtawi,R.,1997.Removal of lead and nickel ions using zeolite tuﬀ.Journal of Chemical Technology and Biotechnology69, 27–34.Baird,C.,1995.Environmental Chemistry.W.H.Freeman and Company, New York.Bianchi,A.,Micheloni,M.,Paoletti,P.,1991.Thermodynamic aspects of the polyazacycloalkane complexes with cations and anions.Coordi-nation Chemistry Reviews110,17–113.Caraschi,J.C.,Campana,S.P.,Curvelo, A.A.S.,1996.Preparac¸a˜o e Caracterizac¸a˜o de Polpas Obtidas a Partir de Bagac¸o de Cana de Ac¸u´car.Polı´meros:Cieˆncia e Tecnologia3,24–29.Gellerested,F.,Gatenholm,P.,1999.Surface properties of lignocellulosic ﬁbers bearing carboxylic groups.Cellulose6,103–121.Gurnani,V.,Singh,A.K.,Venkataramani,B.,2003.2,3-Dihydroxypyri-dine-loaded cellulose:a new macromolecular chelator for metal enrichment prior to their determination by atomic absorption spectrometry.Analytical and Bioanalytical Chemistry377,1079–1086. Ho,Y.S.,Chiu,W.T.,Wang,C.C.,2005.Regression analysis for the sorption isotherms of basic dyes on sugarcane dust.Bioresource Technology96,1285–1291.Kelter,P.B.,Grundman,J.,Hage,D.S.,Carr,J.D.,Castro-Acun˜a,C.M., 1997.A discussion of water pollution in the United States and Mexico;with High School Laboratory Activities for the analysis of lead, atrazine,and nitrate.Journal of Chemical Education74,1413–1421. Krishnan,K.A.,Anirudhan,T.S.,2002.Removal of mercury(II)from aqueous solutions and chlor-alkali industry eﬄuent by steam activated and sulphurised activated carbons prepared from bagasse pith:kinetics and equilibrium studies.Journal of Hazardous Materials92,161–183. Martell, A.E.,Hancock,R.D.,1996.Metal complexes in aqueous solutions.Plenum,New York.Navarro,R.R.,Sumi,K.,Fujii,N.,Matsumura,M.,1996.Mercury removal from wastewater using porous cellulose carrier modiﬁed with polyethyleneimine.Water Research30,2488–2494.Orlando,U.S.,Baes,A.U.,Nishijima,W.,Okada,M.,2002.Preparation of chelating agents from sugarcane bagasse by microwave radiation as an alternative ecologically benign procedure.Green Chemistry4,555–557.Panday,K.K.,Gur,P.,Singh,V.N.,1985.Copper(II)removal from aqueous solutions byﬂy ash.Water Research19,869–873. Vaughan,T.,Seo,C.W.,Marshall,W.E.,2001.Removal of selected metal ions from aqueous solution using modiﬁed corncobs.Bioresource Technology78,133–139.Xiao,B.,Sun,X.F.,Sun,R.,2001.The chemical modiﬁcation of lignins with succinic anhydride in aqueous systems.Polymer Degradation and Stability71,223–231.O.Karnitz Jr.et al./Bioresource Technology98(2007)1291–12971297。

铁基材料修复重金属污染农田土壤的研究进展

• 116 •有色金属（冶炼部分）（h t t p://ysyl.bgri2021年第3期doi： 10. 3969/j. issn. 1007-7545. 2021. 03. 018铁基材料修复重金属污染农田土壤的研究进展黄剑、陈涛1>2,程胜1，蒋少军1，晏波1_2(1.华南师范大学环境研究院，广东省化学污染与环境安全重点实验室，广州510006;2.华南师范大学环境学院，广州510006)摘要：铁基材料可通过降低重金属的有效态比例.降低t壤重金属的生物可利用性，控制重金属毒性危害，具有来源广、生产成本低、稳固效果优良等优势。

参阅国内外相关文献.对铁基材料在农田土壤修复过程中存在的作用机理如吸附沉淀、还原、氧化等，以及影响因素如土壤水分、p H、有机质含量和离子竞争等进行了阐述。

对铁基材料在土壤重金属污染的修S潜力以及未来研究方向进行了相关展望。

关键词：铁基材料；土壤重金属修复；作用机理；影响因素中图分类号：X53文献标志码：A文章编号：1007-7545(2021)03-0116-06Research Progress of Iron-based Materials Remediationof Heavy Metal Contaminated Farmland SoilHUANG Jian' ,CHEN Tao K2,CHENG Sheng1 ,JIANG Shao-jun1,YAN Bo1-2(1.G u a n g d o n g P r o v i n c i a l K e y L a b o r a t o r y o f C h e m i c a l P o l l u t i o n a n d E n v i r o n m e n t a l S a f e t y.E n v i r o n m e n t a l R e s e a r c h I n s t i t u t e.S o u t h C h i n a N o r m a l U n i v e r s i t y,G u a n g z h o u510006,C h i n a；2.S c h o o l o f E n v i r o n m e n t,S o u t h C'h i n a N o r m a l U n i v e r s i t y.G u a n g z h o u510006，C h i n a)Abstract ：Iron-based materials can reduce effective state heavy metals proportion and heavy metals bioavailability in soil,and control heavy metals toxicity.There are advantages of wide source,low production cost and good stable effect.Operation mechanism of iron-based materials during soil heavy metals remediation such as adsorption,precipitation,reduction,oxidation,as well as influencing factors such as soil moisture,pH value，organic content and ion competition were summarized and discussed though referring to domestic and overseas literatures.Potentiality and future research direction of iron-based materials remediation in heavy metal contaminated soil were expected.Key w ords：iron-based materials；soil heavy metals remediation；action mechanisms；influence factors工业化和城市化，特别是采矿及冶炼生产导致大量重金属迁移至土壤中，造成了严重的土壤重金属污染。

英文文献-以农业为基础的废弃物的重金属吸收

Environmental Biotechnology Biotechnology Industry Electronic Journal of Biotechnology ISSN: 0717-3458Vol. 10 No. 3, Issue of July 15, 2007© 2007 by Pontificia Universidad Católica de Valparaíso -- Chile Received September 29, 2006 / Accepted March 8, 2007 DOI: 10.2225/vol10-issue3-fulltext-12How to reference this articleHeavy metal uptake by agro based waste materialsSuleman Qaiser*Department of Chemical Engineering University of Engineering and Technology Post code 54890 Lahore, Pakistan Tel: 92 42 6829488 Fax: 92 42 6822566 E-mail: engrsqaiser@Anwar R. Saleemi Department of Chemical Engineering University of Engineering and Technology Post code 54890 Lahore, Pakistan Tel: 92 42 6829288 Fax: 92 42 6822566 E-mail: darsaleemi@Muhammad Mahmood Ahmad Department of Chemical Engineering University of Engineering and Technology Post code 54890 Lahore, Pakistan Tel: 92 42 6829488 Fax: 92 42 6822566E-mail: chemengguet@*Corresponding authorFinancial support: Merit scholarship for PhD to Suleman Qaiser by Higher Education Commission Pakistan.Keywords: biosorption, ficus religiosa leaves, hexavalent chromium, kinetics, lead, wastewater.Abstract Reprint (PDF)Presence of heavy metals in the aquatic systems has become a serious problem. As a result, there has been a great deal of attention given to new technologies for removal of heavy metal ions from contaminated waters. Biosorption is one such emergingtechnology which utilized naturally occurring waste materials to sequester heavymetals from industrial wastewater. The aim of the present study was to utilize thelocally available agricultural waste materials for heavy metal removal from industrialwastewater. The wastewater containing lead and hexavalent chromium was treated with biomass prepared from ficus religiosa leaves. It was fund that a time of one hr wassufficient for sorption to attain equilibrium. The equilibrium sorption capacity after one hr was 16.95 ± 0.75 mg g-1 and 5.66 ± 0.43 mg g-1 for lead and chromium respectively.The optimum pH was 4 for lead and 1 for chromium. Temperature has strong influence on biosorption process. The removal of lead decreased with increase in temperature.On the other hand chromium removal increased with increase in temperature up to 40ºC and then started decreasing. Ion exchange was the major removal mechanism alongwith physical sorption and precipitation. The biosorption data was well fitted toLangmuir adsorption model. The kinetics of biosorption process was well described bythe pseudo 2nd order kinetics model. It was concluded that adsorbent prepared from ficus religiosa leaves can be utilized for the treatment of heavy metals in wastewater.ArticleThe application of biosorption in environmental treatment has become a significant research area in the past ten years. Heavy metal ions are reported as priority pollutants, due to their mobility in natural water ecosystems and due to their toxicity (Volesky and Holan, 1995). The discharge of heavy metals into surface waters has become a matter of concern in Pakistan over the last two decades. These contaminants are introduced into surface waters through various industrial operations. The pollutants of concern include lead, chromium, zinc, and copper. Heavy metals such as zinc, lead and chromium have number of applications in basic engineering works, paper and pulp industries, leather tanning, petrochemicals fertilizers, etc. The hexavalent and trivalent chromium is often present in electroplating wastewater (Kratochvil et al. 1998). Other sources of chromium pollution are leather tanning, textile, metal processing, paint and pigments, dyeing and steel fabrication. Lead is used as industrial raw material in the manufacture of storage batteries, pigments, leaded glass, fuels, photographic materials, matches and explosives (Raji and Anirudhan, 1997).Lead and chromium are toxic metal contaminants in water. According to Pakistan standards the maximum discharge limits for lead and chromium in wastewater are respectively 0.5 mg l-1 and 1.0 mg l-1. Maximum limit in drinking water is 0.05 mg l-1 for both metals. In fact there is no safe level of these metals in drinking water and even a very dilute content can cause adverse health effects. Lead is toxic to living organisms and if released into the environment can bio accumulate and enter the food chain. Lead is known to cause mental retardation, reduces haemoglobin production necessary for oxygen transport and it interferes with normal cellular metabolism. Lead has damaging effects on body nervous system. It reduces I.Q level in children. Strong exposure of hexavalent chromium causes cancer in the digestive tract and lungs and may cause gastric pain, nausea, vomiting, severe diarrhoea, and haemorrhage (Mohanty et al. 2005).The conventional methods for treatment of lead and chromium wastes include: lime and soda ash precipitation, adsorption with activated carbon, ion exchange, oxidation and reduction, fixation or cementation. These methods are economically unfavourable or technically complicated, and are used only in special cases of wastewater treatment (Kratochvil et al. 1998; Sharma, 2003).Biosorption of heavy metals from aqueous solutions is a relatively new technology for the treatment of industrial wastewater. Adsorbent materials derived from low cost agricultural wastes can be used for the effective removal and recovery of heavy metal ions from wastewater streams. The major advantages of biosorption technology are its effectiveness in reducing the concentration of heavy metal ions to very low levels and the use of inexpensive biosorbent materials (Holan and Volesky, 1994; Kratochvil and Volesky, 1998).Removing metals from wastewater requires development of new sorbents. A wide range of commercial sorbents including chelating resins and activated carbon are available for metal sorption, but they are relatively expensive. In recent years, numerous low cost natural materials have been proposed as potential biosorbents. These include moss peat, algae, leaf mould, sea weeds, coconut husk, sago waste, peanut hull, hazelnut, bagasse, rice hull, sugar beet pulp, plants biomass and bituminous coal. (Lee and Volesky, 1997; Singh and Rawat, 1997; Gupta et al. 1998; Quek et al. 1998; Brown et al. 2000; Chong and Volesky, 2000; Dakiky et al. 2002; Johnson et al. 2002; Reddad et al. 2002; Babel and Kurniawan, 2003; Pagnanelli et al. 2003; Sekhar et al. 2003). In this research adsorbent prepared from ficus religiosa leaves was used for treatment of lead and chromium wastes. Effect of operating conditions like temperature, pH and initial metal concentration, on lead and chromium biosorption were investigated.Composition of leavesLeaves of different trees are very versatile natured chemical species as these contain a variety of organic and inorganic compounds. Cellulose, hemicellulose, pectins and lignin present in the cell wall are the most important sorption sites (Volesky, 2003). Leaves have chlorophyll, carotene, anthocyanin and tannin which contribute to metal biosorption. The important feature of these compounds is that they contain hydroxyl, carboxylic, carbonyl, amino and nitro groups which are important sites for metal sorption (Volesky, 2003). Fourier transform infrared (FTIR) spectra of ficus religiosa leaves also indicated the presence of these functional groups (Figure 2).Cr(VI) is present in solution as CrO4-2 and Cr2O7-2 at normal pH values but when pH values are reduced below 3 then Chromium exists in the form of HCrO4- (Cimino et al. 2000; Demirbas et al. 2004; Park et al. 2006b). When adsorbent developed from ficus religiosa leaves is intimately mixed with chromium solution at low pH values then OH- group present in biomass are replaced by chromate ions in the solution. At pH values close to five the adsorbent surfaces are negatively charged due to release of H+ ions, therefore these attract lead cation (Pb+2). Leaves also have Ca, Mg, Na ions. These are present in the structure of complex organic compounds in leaves and exchange with Pb+2 cations during sorption process (Kratochvil et al. 1998). Leaves have considerable amounts of CaO, MgO, Na2O, K2O etc. When leaf powder is mixed in water these oxides are converted into hydroxides. These hydroxides precipitate the metal cations (Schneider et al. 2001).Materials and MethodsChemicals and instrumentsAll chemicals used were of analytical reagent grade. Lead solution of 1000 mg l-1concentration was prepared by dissolving 1.6 g lead nitrate in one litre distilled water. Hexavalent chromium solution of 1000 mg l-1concentration was prepared by dissolving 2.827 g potassium dichromate in one litre distilled water. These solutions were further diluted to get solutions of various known concentrations of lead and chromium. A variable speed shaker 20-500 rotations perminute (rpm) was used for batch experimentation. It has the capacity of holding eight Erlenmeyer flasks simultaneously. It was equipped with heating water bath. A vacuum filter assembly having Pyrex filter funnel of porosity grade 4 was used for separating adsorbent from solution. The unknown quantities of lead and chromium were determined by Shimadzu 6800 atomic absorption spectrophotometer using an air-acetylene flame. The adsorbent was analyzed by JASCO FTIR 4000 spectrometer.Biomass preparationFicus religiosa leaves were collected from local environment of University of Engineering and Technology Lahore, Pakistan. These leaves were washed with tap water and dried in shadow. Dried leaves were ground and sieved to 50 mesh sizes. This powder was soaked in 0.1 molarity (M) HNO3 for 24 hrs (50 g leaves powder was soaked per litre). It was filtered and washed with distilled water to remove acid contents. The washing was continued till the pH of the filtrate became near neutral. It was first dried at room temperature and then in an oven at 105ºC to remove moisture. This biomass was stored in air tight glass bottles to protect it from humidity.Fourier transform infrared analysisFTIR spectroscopy was used to identify the chemical groups present in leaves. The samples were examined using JASCO FTIR 4000 spectrometer within range 400-4000 cm-1. KBr was used as background material in all the analysis. 0.0035 g leaves powder was mixed with 0.5 g KBr and pressed to form a pellet. FTIR spectra of leaves indicated the presence of hydroxyl, carboxyl, carbonyl, amino and nitro groups which are important sorption sites. There were also indications of presence of heavy metals and inorganic compounds below 870 cm-1. These metals were removed by treatment of leaves with nitric acid to improve the sorption capacity. FTIR spectra of acid treated ficus religiosa leaves and untreated ficus religiosa leaves are shown in Figure 1 and Figure 2 respectively.Results and DiscussionDetermination of equilibrium timeBatch experiments were carried out to find the equilibrium time for sorption of chromium and lead on ficus religiosa leaves. All experiments were performed three times and average values were used in all calculations. 1.0 g ficus religiosa leaves powder of 50 mesh sizes was mixed in 100 ml solutions of lead and chromium. The initial concentration of each solution was 100 mg l-1. It was shaken at 200 rpm and samples were collected at different time intervals. The pH for the experiment was taken as original pH of solutions which was 5.2 for chromium and 5.8 for lead. After completion of each batch of experiments the solution was filtered using vacuum filter assembly. Filtrate was analyzed using atomic absorption spectrophotometer to determine the amount of metal left after sorption. The amount of metal sorbed was calculated by material balance. Sorption capacity q was determined using the formula:Where C0 and C f are the initial and final concentrations of metal in solution, V is the volume of solution and m is the mass of adsorbent. As shown in Figure 3 about 80% removal was attained in first 15 min and concentration became almost constant after 45 min. The fast initial uptake was due to the accumulation of metal ions on surface of adsorbent which is a rapid step. More time was consumed on diffusion of ions to binding sites. It was concluded that one hr was sufficient for sorption to attain equilibrium. The equilibrium capacity obtained after one hr of sorption was 5.66 ± 0.43 mg g-1 and 16.95 ± 0.75 mg g-1 for chromium and lead respectively.Effect of adsorbent doseKeeping all other parameters constant adsorbent dose was varied from 1.0 to 50 gm l-1. It can be seen from Figure 4 that an adsorbent dose of 10 gm l-1 is sufficient for optimal removal of both metals. Increasing the dose further did not affect the percentage removal. The removal capacity was low at high dose rate and vice versa. This was due to metal concentration shortage in solution at high dose rates.Effect of pHKeeping the same operating conditions as mentioned previously, pH of solution was varied from 0.5 to 8 by the addition of 0.1 M nitric acid and 0.1 M ammonia solution. It was found that sorption of chromium was more at low pH where as lead has almost same sorption in the pH range of 3 to 5 (Figure 5). The optimal pH for lead and chromium was 4 and 1 respectively. At pH higher than 6 both metals were precipitated due to formation of hydroxides and removal due to sorption was very low. At low pH the concentration of proton was high and metal binding sites became positively charged repelling the Pb+2 cations and attracting the anions like HCrO-4 and CrO4-2. Thus at low pH removal of lead was low and removal of hexavalent chromium was high. Also more chromium removal at low pH was due to the reduction of Cr(VI) to Cr(III) (Park et al. 2006a), which was then precipitated at the biomass surface by forming Cr(OH)3. In this reduction H+ was consumed and increase in pH was observed at the end of experiments. The reduction of Cr(VI) to Cr(III) was confirmed by analyzing the solution forCr(VI) using 1,5 diphenylcarbazide in spectrophotometer. At pH close to 5 the binding sites became negatively charged due to presence of hydroxyl, carboxylic and amino groups. So at this pH Pb+2 was attracted by the adsorbent. A decrease in pH was observed at the end of experiments in case of lead sorption. This was due to the release of proton as result of ion exchange between Pb+2 and H+ ions. In case of hexavalent chromium there was an increase in pH which was due to exchange of HCrO4- with OH-.Effect of temperatureKeeping all other parameters constant temperature was varied from 20ºC to 50ºC. The sorption of chromium increased slightly with the increase in temperature up to 40ºC and then started decreasing, where as uptake of lead decreased considerably with increase in temperature from 30ºC to 50ºC (Figure 6). The temperature higher than 40ºC caused a change in the texture of the biomass and thus reduced its sorption capacity.Biomass contains more than one type of sites for metal binding. The effect of temperature on each site is different and contributes to overall metal uptake. The effect of temperature on biosorption also depends on the heat of sorption. Usually for physical sorption heat of sorption is negative; sorption reaction is exothermic and preferred at lower temperature. For chemisorption the overall heat of sorption is combination of heat of various reactions taking place at sorption sites. It depends on type of metal and adsorbent. That is the reason for having different behaviour of lead and chromium uptake with temperature.Effect of initial metal concentrationInitial concentrations of both metals were varied from 10 to 1000 mg l-1and quantity of adsorbent was kept constant at 10 gm l-1. It was observed that removal capacity decreased with decrease in metal concentration.Langmuir adsorption model was applied to data:This equation was rearranged to get:Where q e is the amount of metal sorbed per unit weight of biomass at equilibrium, C e is the residual equilibrium metal concentration left in solution after binding, q max is the maximum possible amount of metal ion adsorbed per unit weight of biomass and b is the equilibrium constant related to the affinity of the binding sites for the metals, lower is b more is the affinity of metal to biomass.Equilibrium concentration C e and equilibrium capacity q e were calculated for each initial metal concentration. C e was plotted against C e/q e and a straight line was fitted in the data. Correlation coefficient of 0.996 for chromium and 0.972 for lead indicate that sorption followed Langmuir model (Figure 7 and Figure 8). Values of Langmuir constants q max and b were calculated from slope and intercept of lines in Figure 7 and Figure 8 and are shown in Table 1.Low values of parameter b indicate that ficus religiosa leaves have high affinity for chromium and lead. The values of equilibrium relation parameter, R L were calculated for various initial concentrations for both metals. As shown in Table 2, R L values lie between 0 and 1 which indicate favourable sorption isotherm for both metals.Biosorption kineticsIn order to explain the kinetics of biosorption pseudo second order kinetics model was applied.This equation was integrated and arranged to get the following equation.Where k is the sorption coefficient, q e is the equilibrium capacity and q t is the sorption capacity at any time t.Time, t was plotted against t/q t and straight lines having correlation coefficient of 0.99 were fitted in the data for both metals. The sorption coefficient k and equilibrium capacity q e were calculated from the slope and intercept of lines in Figure 9 and are shown in Table 3.The values obtained for equilibrium capacity for both metals were very close to those obtained experimentally after one hr of sorption. It confirmed that one hr was sufficient for sorption to attain equilibrium and sorption followed pseudo 2nd order kineticsConcluding RemarksFicus religiosa leaves powder was found to be a very good adsorbent for hexavalent chromium and lead. It has good sorption capacity for both metals. The sorption capacity for hexavalent chromium was 5.66 ± 0.43 mg g-1 and for lead was 16.95 ± 0.75 mg g-1. The sorption was pH dependent and optimal pH was 4 and 1 for lead and chromium respectively. Biosorption of metals was temperature dependent. Optimal temperature was 40ºC for chromim and 25ºC for lead. Main removal mechanism was ion exchange between protons and metal cations in case of lead and between metal anions and hydroxyl ions in case of hexavalent chromium. This fact is indicated by the change of pH at the end of biosorption process. Physical sorption and precipitation also contributed to removal of metals. The biosorption process followed Langmuir model for both metals which indicated that ion exchange took place in a monolayer at the surface of adsorbent. The kinetics of biosorption process was represented well by pseudo 2nd order kinetics model.AcknowledgmentsFirst author is thankful to Dr. Shaiq A Haq for his valuable suggestions. Special thanks to Dr. Nadeem Feroz and Muhammad Umar for their cooperation and support.ReferencesBABEL, Sandhya and KURNIAWAN, Tonni Agustiono. Low-cost adsorbents for heavy metals uptake from contaminated water: a review. Journal of Hazardous Materials, February 2003, vol. 97, no. 1-3, p.219-243. [CrossRef]BROWN, Pauline; JEFCOAT, I. Atly; PARRISH, Dana; GILL, Sarah and GRAHAM, Elizabeth. Evaluation of the adsorptive capacity of peanut hull pellets for heavy metals in solution. Advances in Environmental Research, June 2000, vol. 4, no. 1, p. 19-29. [CrossRef]CIMINO, Giuseppe; PASSERINI, Amedeo and TOSCANO, Giovanni. Removal of toxic cations and Cr(VI) from aqueous solution by hazelnut shell. Water Research, August 2000, vol. 34, no. 11, p. 2955-2962. [CrossRef]CHONG, K.H. and VOLESKY, B. Metal biosorption equilibria in a ternary system. Biotechnology and Bioengineering, March 2000, vol. 49, no. 6, p. 629-638. [CrossRef]DAKIKY, M.; KHAMIS, M.; MANASSRA, A. and MER'EB, M. Selective adsorption of Cr(VI) in industrial wastewater using low-cost abundantly available adsorbents. Advances in Environmental Research, October 2002, vol. 6, no. 4, p. 533-540. [CrossRef]DEMIRBAS, Erhan; KOBYA, Mehmet; SENTURK, Elif and OZKAN, Tuncay. Adsorption kinetics for the removal of chromium (VI) from aqueous solutions on the activated carbons prepared from agricultural wastes. Water SA, October 2004, vol. 30, no. 4, p. 533-539.GUPTA, V.K.; MOHAN, D. and SHARMA, S. Removal of lead from wastewater using bagasse fly ash-a sugar industry waste material. Separation Science and Technology, June 1998, vol. 33, no. 9, p. 1331-1343.HOLAN, Z.R. and VOLESKY, B. Biosorption of lead and nickel by biomass of marine algae. Biotechnology and Bioengineering, May 1994, vol. 43, no. 11, p. 1001-1009. [CrossRef]JOHNSON, P.D.; WATSON, M.A.; BROWN, J. and JEFCOAT, I.A. Peanut hull pellets as a single use sorbent for the capture of Cu(II) from wastewater. Waste Management, August 2002, vol. 22, no. 5, p. 471-480. [CrossRef]KRATOCHVIL, David and VOLESKY, Bohumil. Advances in the biosorption of heavy metals. Trends in Biotechnology, July 1998, vol. 16, no. 7, p. 291-300. [CrossRef]KRATOCHVIL, David; PIMENTEL, Patricia and VOLESKY, Bohumil. Removal of trivalent and hexavalent chromium by seaweed biosorbent. Environmental Science and Technology, September 1998, vol. 32, no. 18, p. 2693-2698. [CrossRef]LEE, Hak Sung and VOLESKY, Bohumil. Interaction of light metals and protons with seaweed biosorbent. Water Research, December 1997, vol. 31, no. 12, p. 3082-3088. [CrossRef]MOHANTY, K.; JHA, M.; BISWAS, M.N. and MEIKAP, B.C. Removal of chromium (VI) from dilute aqueous solutions by activated carbon developed from Terminalia arjuna nuts activated with zinc chloride. Chemical Engineering Science, June 2005, vol. 60, no. 11, p. 3049-3059. [CrossRef]PAGNANELLI, Francesca; MAINELLI, Sara; VEGLIO, Francesco and TORO, Luigi. Heavy metal removal by olive pomace: biosorbent characterization and equilibrium modeling. Chemical Engineering Science, October 2003, vol. 58, no. 20, p. 4709-4717. [CrossRef]PARK, Donghee; YUN, Yeoung-Sang; JO, Ji Hye and PARK, Jong Moon. Biosorption process for treatment of electroplating wastewater containing Cr(VI): Laboratory-scale feasibility test. Industrial and Engineering Chemistry Research, July 2006a, vol. 45, no. 14, p. 5059-5065. [CrossRef]PARK, D.; YUN, Y.-S.; LIM, S.-R. and PARK, J.M. Kinetic analysis and mathematical modeling of Cr(VI) removal in a differential reactor packed with ecklonia biomass. Journal of Microbiology and Biotechnology, November 2006b, vol. 16, no. 11, p. 1720-1727.QUEK, S.Y.; WASE, D.A.J. and FORSTER, C.F. The use of sago waste for the sorption of lead and copper. Water SA, July 1998, vol. 24, no. 3, p. 251-256.REDDAD, Zacaria; GERENTE, Claire; ANDRES, Yves and LE CLOIREC, Pierre. Adsorption of several metal ions onto a low-cost biosorbent: Kinetic and equilibrium studies. Environmental Science and Technology, May 2002, vol. 36, no. 9, p. 2067-2073. [CrossRef]RAJI, C. and ANIRUDHAN, T.S. Chromium (VI) adsorption by sawdust carbon: kinetics and equilibrium. Indian Journal of Chemical Technology, September 1997, vol. 4, no. 5, p.228-236.SCHNEIDER, Ivo A.H.; RUBIO, Jorge and SMITH, Ross W. Biosorption of metals onto plant biomass: exchange adsorption or surface precipitation? International Journal of Mineral Processing, May 2001, vol. 62, no. 1-4, p. 111-120. [CrossRef]SEKHAR, K.C.; KAMALA, C.T.; CHARY, N.S. and ANJANEYULU, Y. Removal of heavy metals using a plant biomass with reference to environmental control. International Journal of Mineral Processing, January 2003, vol. 68, no. 1-4, p. 37-45. [CrossRef]SHARMA, Y.C. Cr(VI) removal from industrial effluents by adsorption on an indigenouslow-cost material. Colloids and Surfaces A: Physicochemical and Engineering Aspects, March 2003, vol. 215, no. 1-3, p. 155-162. [CrossRef]SINGH, D. and RAWAT, N.S. Adsorption of heavy metals on treated and untreated low grade bituminous coal. Indian Journal of Chemical Technology, January 1997, vol. 4, no. 1, p. 39-41.VOLESKY, B. and HOLAN, Z.R. Biosorption of heavy metals. Biotechnology Progress,May-June 1995, vol. 11, no. 3, p. 235-250. [CrossRef]VOLESKY, B. Sorption and Biosorption. Montreal-St. Lambert, Quebec, Canada, BV Sorbex Inc., 2003. 316 p. ISBN 0-9732983-0-8.。

碳纳米管对重金属吸附研究的英文文献

碳纳米管对重金属吸附研究的英文文献Research on Heavy Metal Adsorption Using Carbon NanotubesCarbon nanotubes (CNTs) have attracted significant attention for their potential applications in environmental remediation due to their unique properties, such as high surface area, large aspect ratio, and outstanding mechanical and electrical properties. Among various environmental pollutants, heavy metals play an important role due to their toxicity, persistence, and the potential risk to human health and the ecosystem. CNTs have been reported to be effective in removing heavy metals from aqueous solutions.In recent years, researchers have explored the use of CNTs as adsorbents for various heavy metals, including lead, cadmium, chromium, mercury, and arsenic. The adsorption of heavy metals by CNTs is mainly governed by physical and chemical interactions, such as electrostatic attraction, van der Waals forces, and chemisorption. The efficiency of heavy metal removal is influenced by several factors, such as the pH of the solution, the concentration of heavy metals, the surface functionalization of CNTs, and the presence of other ions.The adsorption capacity of CNTs for heavy metals is dependent on their surface properties, such as the presence of functional groups. For example, carboxylic acid groups on CNTs can enhance the adsorption capacity for heavy metals through ion exchange and electrostatic interactions. Inaddition, the modification of CNTs with other materials, such as polymers and nanoparticles, can improve the selectivity and efficiency of heavy metal adsorption.CNTs have been demonstrated to be effective in removing heavy metals from contaminated water and soil. However, further research is needed to optimize their performance and understand the mechanisms of heavy metal adsorption. The use of CNTs for heavy metal remediation holds great promise, and may provide a cost-effective and sustainable solution for environmental cleanup.。

生物冶金的工业化

生物冶金的工业化摘要：生物冶金作为一项最大极限地利用矿藏资源的绿色冶金技术，近年来其工业化应用得到了巨大的发展。

详细介绍了生物槽浸和生物堆浸两大类生物冶金工艺的工业化应用及最新进展。

关键词：生物浸出工业化生物槽浸生物堆浸Biological metallurgy industrializationAbstract：Bioleaching as a green metallurgical technology which could maximize the utilization of mineral resources,and its industrial app lication developed greatly in recent years. Industrial app lication and latest development of Tank Bioleaching and Heap Bioleaching these two kinds of bioleaching technolgy are presented in detail. Keywords:Bioleaching Industrializaton Tank Bioleaching Heap Bioleaching [引言]：随着我国工业化进程的加快，有色金属矿产资源消费急剧增加，供需矛盾日益突出，低品位复杂难处理矿和西部偏远地区矿已成为我国有色金属行业依赖的主体资源，而采用传统的选矿冶金技术难以经济利用。

生物冶金，也是生物浸出，通常也称生物氧化，即利用微生物、空气和谁等天然物质溶浸贫矿、废矿、尾矿和冶炼炉渣等，以回收某些贵重有色金属和稀有金属，达到防止矿产资源流失，最大限度地利用矿藏的一种冶金方法。

与传统冶炼方法相比，生物浸出通常基建投资少，操作成本低，对环境的污染小且可处理品位较低的矿物，因此被认为是绿色冶金技术]2][1[。

heavy metals

1Abstract Discharge of waste contaminated with heavy metals and related elements is known to have an adverse effect on the environment and solving this problem has for long been presented as a challenge. Nowadays, continuing demand for and increasing value of high-tech metals and rare earth elements makes efficient recy-cling technologies of utmost importance. In solving these tasks, the biosorption—sequestration of heavy metals, radionuclides and rare earth elements usually by non-living biomass—can be a part of the solution.Keywords Decontamination • Bacterial biosorbent • Fungal biosorbent • Algal biosorbent • Mechanism of biosorption • Modeling of biosorption1.1 B rief View on Conventional Waste Stream TreatmentsIndustrialization has long been accepted as a hallmark of civilization. The Boul-ton and Watt steam engine, the synonym of industrial revolution, propelled huge changes in mining, metallurgical technology, manufacturing, transport and agricul-ture. Since then, progressive metallurgy and the use of metals and chemicals in nu-merous industries have resulted in a generation of large quantities of liquid effluent loaded with high levels of heavy metals, often as bioavailable mobile and thus toxic ionic species (Calderón et al. 2003; Peakall and Burger 2003; Gadd 1992a ). Due to their elemental non-degradable nature, heavy metals always and regardless of their chemical form, pose serious ecological risk, when released into the environment. Not only is there the demand for cleanup of contaminated waste water to meet regulatory agency limits, but there is also increasing value of some metals which place a call for efficient and low-cost effluent treatment and metal recuperation technologies.P. Kotrba et al. (eds.), Microbial Biosorption of Metals, DOI 10.1007/978-94-007-0443-5_1, © Springer Science+Business Media B.V . 2011Chapter 1Microbial Biosorption of Metals—General Introduction Pavel KotrbaP. Kotrba ( )Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague, Czech Republic e-mail: pavel.kotrba@vscht.cz2P. Kotrba Conventional procedures for heavy metal removal from aqueous industrial ef-fluents involve precipitation, ion exchange, electrochemical methods and reverse osmosis. Another promising approach is solvent extraction. Conversion of metal ions to insoluble forms by chemical precipitation is the most common method, reducing the metal content of solution to the levels of mg l−1. The cheapest pre-cipitation technique relies on alkalization of the metal solution (usually with lime) to achieve formation of insoluble metal species, namely of hydroxides. Chemical precipitation could also be achieved by the addition of other coagulants, such as of potash alum, sodium bisuphite, sulphide or iron salts. Though it is cost effec-tive; such precipitation lacks the specificity, produces large volumes of high water content sludge and has low performance at low metal concentrations. Although adsorption using activated carbon is generally expensive (and not suitable for many metal species), it is an efficient method for the removal of metallic mercury following chemical reduction (e.g., with hydrazine) of mercuric ions in heavily contaminated process waters.Ion exchange employing manmade synthetic organic resins is the most common method. It becomes the method of choice especially for its capacity to reduce the metal contents to μg l−1 levels in relatively large volumes of effluent, some possi-bility to formulate metal-selective resin and well established procedures for metal recovery from and reuse of the ion exchanger. This method is, however, relatively expensive, which therefore makes the processing of concentrated metal solutions cost intensive. Precautions should also be taken to prevent the poisoning of ion exchanger by organics and solids in solution.Electro-winning, employing electro-deposition of metals on anodes is popu-larly used for the recuperation of metals in mining and metallurgical operations as well as in electrical industries and electronics. Electrodialysis involves the use of ion selective semi-permeable membranes fitted between the charged electrodes attracting respective ions (in the case of metal cations, the anode compartment is smaller to concentrate the metal in). The main disadvantage of electrodialysis operation is clogging of the membrane by metal hydroxides formed during the process. Like electrodialysis, reverse osmosis and ultrafiltration employ semi-permeable membranes which allow water to pass, while solutes, including heavy metals, remain contained in retentate. The advantages of membrane-based pro-cesses involve some selectivity of metal separation and tolerance to changes in pH. One disadvantage of membrane-based approaches is that they are cost intensive.Reactive two-phase extraction complexing extractants specifically (or preferen-tially) dissolved in organic solvents has been suggested as another technological alternative (Schwuger et al. 2001). This approach may provide a viable method for the selective recuperation of metals, e.g., of platinum group metals from spent cata-lysts (Marinho et al. 2010). Suitable extractants for platinum involve organophos-phorus compounds, aliphatic amines and ammonium quaternary salts. The main disadvantages of this process are the difficult recovery of metals from organic phase and the toxicity of extractants.3 1 Microbial Biosorption of Metals—General Introduction1.2 B io-based Methods for Waste Water Treatment and Environment RestorationThe natural capacity of microrganisms, fungi, algae and plants to take up heavy metal ions and radionuclides and, in some cases, to promote their conversion to less toxic forms has sparked the interest of (micro)biologists, biotechnologists and environmental engineers for several decades. Consequently, various concepts for “bio-removal” of metals from waste streams and bioremediations of contami-nated environment are being proposed, some of which were brought to pilot or industrial scale (Bargar et al. 2008; Macek et al. 2008; Muyzer and Stams 2008; Singh et al. 2008; Chaney et al. 2007; Sheoran and Sheoran 2006; Volesky 2004; Lloyd et al. 2003; Ruml and Kotrba 2003; Baker et al. 2000; Gadd 1992b; see also Chap. 2). The “bio” prefix refers to the involvement of biological entity, which is living organisms, dead cells and tissues, cellular components or products. The ultimate goal of these efforts is to provide an economical and eco-friendly tech-nology, efficiently working also at metal levels below 10 mg l−1. These are the features that living as well as dead biomass could be challenged for. There are generally three routes to follow considering “bio-removal” of metallic species from solutions. The first two rely on properties of living cells and involve active metal uptake—bioaccumulation (i.e., plasma membrane mediated transport of metal ion into cellular compartment) and eventual chemical conversion of mobile metal to insoluble forms. The later may occur in the cytoplasm, at the cell surface or in the solution by precipitation of metal ion with metabolites, via redox reac-tions or by their combination. The effectiveness of the process will depend on the (bio)chemistry of particular metal and on metabolic activity of eligible organism, which is in turn affected by the presence of metal ions. To this point, the use of metallotolerant species or physical separations of the production of metal-pre-cipitating metabolite from metal precipitation in contaminated solution produce viable methods. For their importance in the treatment of industrial liquid streams as well as of the environmental pollution are some of these approaches discussed in Chap. 9. Several of them are to various extents dependent on or involve the metabolism-independent metal uptake event at the cell wall by polysaccharides, associated molecules, and functional groups. This metal sequestration capacity is commonly known as biosorption, which itself represents the third potent way of “bio-removal” of metals from solution.Biosorption is a general property of living and dead biomass to rapidly bind and abiotically concentrate inorganic or organic compounds from even very di-luted aqueous solutions. As a specific term, biosorption is used to depict a method that utilizes materials of biological origin—biosorbents formulated from non-living biomass—for the removal of target substances from aqueous solutions. Biosorp-tion “traditionally” covers sequestration of heavy metals as well as rare earth ele-ments and radionuclides or metalloids, but the research and applications extended to the removal of organics, namely dyes (Kaushik and Malik 2009; see also some examples with magnetic biocomposite biosorbents in Chap. 13), and biosorption is4P. Kotrba being proposed for the recovery of high-value proteins, steroids, pharmaceuticals and drugs (V olesky 2007).Decades of biosorption research provided a solid understanding of the mecha-nism underlying microbial biosorption of heavy metals and related elements. It in-volves such physico-chemical processes as adsorption, ion-exchange, chelatation, complexation and microprecipitation. These depend on the type and ionic form of metal, the type of metal binding site available from microbial biomass, as well as on various external environmental factors (see Chap. 3). Accumulated knowledge resulted in the development of suitable modelling approaches comprehensively described in Chap. 4. When properly used, these models explain the equilibrium biosorption data, the kinetics in batch reactors and the dynamics in biosorption col-umns both for single and multimetal systems and provide a powerful tool for the de-sign and development of the actual biosorption process. It should be noted here that it was due to a poor understanding of mechanisms and kinetics of AlgaSORB TM and AMT-Bioclaim TM processes commercialized in the early 1990s that hindered the adequate assessment of process performance and limitations, and thus the expected widespread industrial application of biosorption.Biosorbents are derived from raw biomass selected for its superior metal-sequestering capacity. Investigated biomass types are of such diverse origins as bacterial, cyanobacterial, fungal (including filamentous fungi and unicellular yeasts), algal, plant or even animal (chitosan). This book covers development in major areas exploiting bacterial biomass (Chap. 5), fungal biomass (Chap. 6) and algal biomass (including macroalgae; Chap. 7) for the biosorption of heavy metal and radionuclides as well as for the sequestration of precious and rare earth elements (Chap. 8). It is noteworthy to add that the potential of plant-based biosorbents formulated from agricultural waste is attaining growing attention (Demirbas 2008; Sud et al. 2008; a few examples with magnetic biocomposite biosorbents are given in Chap. 13). The cheapest microbial biomass could be procured from selected fermentation industries as waste by-product or could be harvested from its natural habitat when it is produced in sufficient quantity there (e.g., marine macroalgae). Independent propagation of biomass under specific conditions optimizing its metallosorption properties is another option. Some ef-forts have been also devoted to modifications of yeast (Chap. 10) and bacterial (Chap. 11) cell walls through their genetic engineering, resulting in a surface display of particular amino acid sequences providing additional (even selective) metal-binding sites.When derived from dead raw biomass featuring high metal uptake, the biosor-bent for its practical application should exhibit some additional characteristics im-proving its stability and favoring hydrodynamic process conditions. To this end, biosorbent particle size, density and porosity, hardness, resistance to a broad range of physical and chemical conditions could be tailored by an appropriate immo-bilization method. Conventional strategies of biosorbent formulation from differ-ent types of microbial biomass are described in respective chapters as well as in Chap. 12. Chapter 13 further sets the biosorbent design forward to “smart materi-als”, the magnetically responsive biocomposites improving biosorbents applicabil-1 Microbial Biosorption of Metals—General Introduction5 ity by enabling their selective magnetic separation even from solutions containing suspended solids.1.3 F uture Thrusts in BiosorptionCompared with conventional or some biological methods for removing metal ions from industrial effluents, the biosorption process offers the advantages of low op-erating cost, minimization of the use of chemicals, no requirements for nutrients or disposal of biological or inorganic sludge, high efficiency at low metal concen-trations, and no metal toxicity issues. The operation of biosorption shares many common features with ion-exchange technology and, despite shorter life cycle and less selectivity options, biosorbents could be considered direct competitors of ionex resins. The high cost of the ion-exchange process limits its application. Not all industries producing metal bearing effluents have financial resources for such sophisticated treatment and most opt only for basic decontamination techniques to meet regulation limits. The accumulated knowledge already provides a solid basis for the commercial exploitation of biosorption processes. Huge markets already exist (V olesky 2007) and they may even grow with progressively stricter legisla-tion worldwide and increase demand on metal resources. Future efforts to improve selectivity and shelf life of biosorbents, further information on biosorption mecha-nisms and reliability and performance of biosorption models as well as more pilot scale demonstrations should bring convincing marketing arguments for large-scale applications. Biosorption also has the potential to find an industrial application in the future separation technologies with renewable biosorbents complementing con-ventional methods in hybrid or integrated installations.ReferencesBaker AJM, McGrath SP, Reeves RD, Smith JAC (2000) Metal hyperaccumulator plants: a review of the ecology and physiology of a biological source for phytoremediation of metal-polluted soil. In: Terry N, Bañuelos GS (eds) Phytoremediation of contaminated soil and water. CRC Press, Boca Raton, pp 85–107Bargar JR, Bernier-Latmani R, Giammar DE, Tebo BM (2008) Biogenic uraninite nanoparticles and their importance for uranium remediation. Elements 4:407–412Calderón J, Ortiz-Pérez D, Yáñez L, Díaz-Barriga F (2003) Human exposure to metals. Pathways of exposure, biomarkers of effect, and host factors. Ecotoxicol Environ Saf 56:193–103 Chaney RL, Angle JS, Broadhurst CL, Peters CA, Tappero RV, Sparks DL (2007) Improved under-standing of hyperaccumulation yields commercial phytoextraction and phytomining technolo-gies. J Environ Qual 36:1429–1443Demirbas A (2008) Heavy metal adsorption onto agro-based waste materials: a review. J Hazard Mater 157:220–229Gadd GM (1992a) Metals and microorganisms: a problem of definition. FEMS Microbiol Lett 79:197–203P. Kotrba 6Gadd GM (1992b) Microbial control of heavy metal pollution. In: Fry JC, Gadd GM, Herbert RA, Jones CW, Watson-Craik IA (eds) Microbial control of pollution. Cambridge University Press, Cambridge, pp 59–87Kaushik P, Malik A (2009) Fungal dye decolourization: recent advances and future potential. En-viron Int 35:127–141Lloyd JR, Lovley DR, Macaskie LE (2003) Biotechnological application of metal-reducing micro-organisms. Adv Appl Microbiol 53:85–128Macek T, Kotrba P, Svatoš A, Demnerová K, Nováková M, Macková M (2008) Novel roles for GM plants in environmental protection. Trends Biotechnol 26:146–152Marinho RS, Afonso JC, da Cunha JW (2010) Recovery of platinum from spent catalysts by liq-uid–liquid extraction in chloride medium. J Hazard Mater 179:488–494Muyzer G, Stams AJ (2008) The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol 6:441–454Peakall D, Burger J (2003) Methodologies for assessing exposure to metals: speciation, bioavail-ability of metals, and ecological host factors. Ecotoxicol Environ Saf 56:110–121Ruml T, Kotrba P (2003) Microbial control of metal pollution: an overview. In: Fingerman M, Nagabhushanam R (eds) Recent advances in marine biotechnology, vol 8. Science Publishers Inc., Enfield, pp 81–153Schwuger MJ, Subklew G, Woller N (2001) New alternatives for waste water remediation with complexing surfactants. Colloid Surface A 186:229–242Sheoran AS, Sheoran V (2006) Heavy metal removal mechanism of acid mine drainage in wet-lands: a critical review. Mineral Eng 19:105–116Singh S, Kang SH, Mulchandani A, Chen W (2008) Bioremediation: environmental clean-up through pathway engineering. Curr Opin Biotechnol 19:437–444Sud D, Mahajan G, Kaur MP (2008) Agricultural waste material as potential adsorbent for se-questering heavy metal ions from aqueous solutions—a review. Biores Technol 99:6017–6027 V olesky B (2004) Sorption and biosorption. BV-Sorbex Inc., St. LambertV olesky B (2007) Biosorption and me. Water Res 4017–4029。

POE-g-MAH_对PLA

第4期71李梦云，等：四环素和铜离子对生物除磷中微生物胞外聚合物的影响合物中蛋白质和多糖增加量三种配比下相对最高；四环素和铜离子配比为0.894时，四环素与铜离子浓度相当，混合物投加初期胞外聚合物中蛋白质和多糖增加量三种配比下相对最低。

三种配比混合物作用下，随混合物浓度增加，微生物胞外聚合物中蛋白质三维荧光强度逐渐减弱。

参考文献：[1] 李金璞，张雯雯，杨新萍.活性污泥污水处理系统中胞外多聚物的作用及提取方法[J].生态学杂志，2018， 37（9）：2825-2833.[2] Mohite B V，Koli S H，Patil S V. Heavy metal stress and its consequences on exopolysaccharide （EPS）-producing pantoea agglomerans[J].Applied Biochemistry and Biotechnology，2018，186（1）：199-216.[3] Grabert R，Boopathy R，Nathaniel R，et al. Effect of tetracycline on ammonia and carbon removal by the facultative bacteria in the anaerobic digester of a sewage treatment plant[J].Bioresource Technology，2018，267：265-270.[4] Li J Y，Du Q P，Peng H Q，et al. Spectroscopic investigation of the interaction between extracellular polymeric substances and tetracycline during sorption onto anaerobic ammonium-oxidising sludge[J].Environmental Technology，2021，42（11）：1787-1797.[5] Li W W，Yu H Q. Insight into the roles of microbial extracellular polymer substances in metal biosorption[J].Bioresource Technology，2014，160：15-23.[6] Li C X，Xie S Y，Wang Y，et al. Simultaneous heavy metal immobilization and antibiotics removal during synergetic treatment of sewage sludge and pig manure[J].Environmental Science and Pollution Research，2020，27（24）：30323-30332.[7] Zhang Y H，Liu S S，Liu H L，et al. Evaluation of the combined toxicity of 15 pesticides by uniform design[J].Pest Management Science，2010，66（8）：879-887.[8] Abbondanzi F，Cachada A，Campisi T，et al. Optimisation of a microbial bioassay for contaminated soil monitoring： bacterial inoculum standardisation and comparison with Microtox ® assay[J].Chemosphere，2003，53（8）：889-897.[9] 陶梦婷，张瑾，姜慧，等.3种农药对青海弧菌Q67的联合毒性作用特征[J].环境科学与技术，2019，42（6）：12-20.[10] Sutherland I W. Biofilm exopolysaccharides：a strong and sticky framework[J].Microbiology，2001，147（1）：3-9. L1-3* L1-5* L1-7* L1-9* L2-3* L2-5* L2-7* L2-9* L3-3* L3-5* L3-7* L3-9*图4 三种浓度配比混合物作用下生物除磷中微生物胞外聚合物的三维荧光光谱图（下转第91页）第30卷第4期2022年8月V ol.30 No.4Aug.2022安徽建筑大学学报Journal of Anhui Jianzhu UniversityDOI：10.11921/j.issn.2095-8382.20220412POE-g-MAH对PLA/PP共混材料界面状态及机械性能的影响陈鑫亮1，高尚1，樊炳宇1，刘瑾1，2，王平1，2（1.安徽建筑大学材料与化学工程学院，安徽合肥 230601；2.安徽省先进建筑材料国际联合研究中心，安徽合肥 230601）摘‌要：聚乳酸（PLA）与聚丙烯（PP）的相容性较差，界面相互作用较弱，导致PLA/PP共混材料的力学性能较差。

多孔材料吸附重金属英文

Porous Materials for Heavy Metal AdsorptionIntroductionPorous materials have gained significant attention in environmental science and technology due to their high surface area and unique structural properties. They possess interconnected networks of pores, which make them excellent candidates for various applications, including adsorption of heavy metals. Heavy metals, such as lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As), are highly toxic and can pose serious risks to human health and the environment. Therefore, the development of effective adsorbents for heavy metal removal is of utmost importance. In this article, we will discuss the characteristics of porous materials and their applications in heavy metal adsorption.Characteristics of Porous MaterialsPorous materials are solid substances that have internal pores or cavities. These pores can be categorized into three types based on their sizes: micropores (less than 2 nm), mesopores (2-50 nm), and macropores (larger than 50 nm). The presence of these pores provides a large surface area, which facilitates the adsorption of heavy metal ions. Additionally, the surface chemistry and functional groups of porous materials can be tailored to enhance their adsorption capacity.Types of Porous Materials1.Activated Carbon (AC): Activated carbon is a widely used porousmaterial due to its excellent adsorption properties. It is derived from various carbonaceous sources and possesses a highly porous structure with a largespecific surface area. The adsorption of heavy metal ions on activated carbon occurs through various mechanisms, such as ion exchange, electrostaticattraction, and complex formation.2.Metal-Organic Frameworks (MOFs): MOFs are a class of porousmaterials composed of metal ions or clusters coordinated with organic ligands.They exhibit high porosity, diverse structures, and tunable pore sizes, making them promising candidates for heavy metal adsorption. The metal centers in MOFs can interact with heavy metal ions through coordination bonds, enabling efficient removal.3.Mesoporous Silica: Mesoporous silica materials, such as SBA-15 andMCM-41, have ordered porous structures with uniform pore sizes. Theypossess high surface areas and large pore volumes, making them suitable for heavy metal adsorption. The surface of mesoporous silica can be functionalized with different organic groups to enhance the specific adsorption of heavy metal ions.Applications of Porous Materials in Heavy Metal Adsorption1.Water Treatment: Porous materials, such as activated carbon, arewidely used in water treatment processes to remove heavy metal ions. These materials can be employed in fixed-bed columns, batch experiments, ormembrane systems to effectively adsorb heavy metals from contaminatedwater sources.2.Industrial Effluent Treatment: Various industries generate effluentscontaining heavy metals, which need to be treated before discharge. Porousmaterials offer a cost-effective and efficient solution for the removal of heavy metal contaminants from industrial wastewaters.3.Soil Remediation: Porous materials can be used for soil remediationby immobilizing heavy metal ions, preventing their leaching into groundwater.The materials can be applied through in-situ injection or incorporation into soil amendments, allowing for the effective remediation of contaminated soils.ConclusionPorous materials possess unique structural characteristics and surface properties, which make them ideal for heavy metal adsorption. Activated carbon, metal-organic frameworks, and mesoporous silica are commonly used porous materials in heavy metal removal applications. Their high surface area, tunable pore sizes, and functionalizable surfaces enable efficient adsorption of heavy metal ions. Porous materials find applications in water treatment, industrial effluent treatment, and soil remediation. Continued research and development in porous materials for heavy metal adsorption will contribute to addressing the ongoing challenges associated with heavy metal pollution.。

PRECIPITATION OF HEAVY-METAL IONS

专利名称：PRECIPITATION OF HEAVY-METAL IONS 发明人：ENDRES, Helmut,WEHLE, Volker,HANSEN, Angela,WIECKEN, Barbara申请号：EP1991002147申请日：19911114公开号：WO92/009530P1公开日：19920611专利内容由知识产权出版社提供摘要：The invention concerns precipitating agents, methods of producing them and their use in the precipitation of heavy-metal ions from aqueous or water-containing solutions. The precipitating agents disclosed comprise metal hydroxosulphides and/or polysulphides of general formula (I): [M(II)1-xM(III)x(OH)y]Az. mH2O, in which M(II) is an at least bivalent metal ion, M(III) is an at least trivalent metal ion, A is an equivalent of a mono- and/or polybasic acid, but consists at least partly, or wholly, of sulphide ions (S2-) and/or polysulphide ions (S¿n?2-), x is a number from 0.01 to 0.5, 0 < y < 2.5, 0 < z < 2.5, the sum y + z = 2 + x, 0 « m « 1, and n is a whole number larger than or equal to 2.申请人：ENDRES, Helmut,WEHLE, Volker,HANSEN, Angela,WIECKEN, Barbara地址：DE,DE,DE,DE,DE国籍：DE,DE,DE,DE,DE代理机构：HENKEL KOMMANDITGESELLSCHAFT AUF AKTIEN更多信息请下载全文后查看。

土壤中重金属元素形态分析方法及形态分布的影响因素

当代化工研究Modern Chemical R esearch 132019•06综述与专论土壤中重金属元素形态分析方法及形态分布的影响因素*王高飞（海南省地质测试研究中心海南571400）摘耍：土壤中重金属的污染直接导致植物受到伤害，从而威胁到人类和动物的健康.因此，为了对这一环境污■染问题进行深入分析，制定切实可行的阻力和缓解措施，然后，有必要通过重金属元素形态来分析重金属形态分布对重金属污染的影响.建立风险预测机制以确定重金属的活动分类，存在状态和毒性.本研究从土壤中重金属元素形态分布、土壤中重金属元素形态分布测量方法以及澎响其分布的主要因素三个方面进行了简要的阐释.关键词：重金属元素；元素分析；元素形态分布中EB分类号：T文献标识码：ASpeciation Analysis Method of Heavy Metal Elements in Soil and Influencing Factors ofSpeciation DistributionWang Gaofei(Hainan Provincial Geological Testing Research Center,Hainan,571400)Abstract:The pollution of heavy metals in soil directly leads to plant injury,thus threatening the health of human beings and animals. Therefore,in order to deeply analyze this environmental p ollution problem andformulate f easible resistance and mitigation measures,it is necessary to analyze the influence of heavy metal speciation distribution on heavy metal pollution through heavy metal speciation analysis.Establish a risk prediction mechanism to determine the activity classification,presence status and toxicity of h eavy metal This study briefly explained the speciation distribution of h eavy metal elements in soil,the measurement method of t he speciation distribution of h eavy metal elements in soil and the main f actors affecting its distribution.Key words z heavy metal elements\element analysis\element speciation distribution1.前言虽然重金属的有效含量可以反映一定的生物利用度，但难以反映重金属的潜在危害以及不同形式的迁移转化特征；重金属形态的研究可以对重金属活性进行分类，揭示重金属在土壤中的存在状态，迁移转化，生物有效性，毒性和可能的环境影响。

Ecological risk assessment of heavy metals

Ecological risk assessment of heavy metals in sediment and human health risk assessment of heavy metals in ﬁshes in the middle and lower reaches of the Yangtze River basinYujun Yi a ,Zhifeng Yang a ,*,Shanghong Zhang ba State Key Laboratory of Water Environment Simulation and Pollution Control,School of Environment,Beijing Normal University,Beijing 100875,China bRenewable Energy School,North China Electric Power University,Beijing 102206,Chinaa r t i c l e i n f oArticle history:Received 1August 2010Received in revised form 18January 2011Accepted 6June 2011Keywords:Heavy metal Ecological risk Sediment Health risk FishYangtze Rivera b s t r a c tThe concentrations of heavy metals (Cr,Cd,Hg,Cu,Zn,Pb and As)in the water,sediment,and ﬁsh were investigated in the middle and lower reaches of the Yangtze River,China.Potential ecological risk analysis of sediment heavy metal concentrations indicated that six sites in the middle reach,half of the sites in the lower reach,and two sites in lakes,posed moderate or considerable ecological risk.Health risk analysis of individual heavy metals in ﬁsh tissue indicated safe levels for the general population and for ﬁsherman but,in combination,there was a possible risk in terms of total target hazard quotients.Correlation analysis and PCA found that heavy metals (Hg,Cd,Pb,Cr,Cu,and Zn)may be mainly derived from metal processing,electroplating industries,industrial wastewater,and domestic sewage.Hg may also originate from coal combustion.Signi ﬁcant positive correlations between TN and As were observed.Crown Copyright Ó2011Published by Elsevier Ltd.All rights reserved.1.IntroductionThe rapid development of industry and agriculture has resulted in increasing pollution by heavy metals,which are a signi ﬁcant environmental hazard for invertebrates,ﬁsh,and humans (Uluturhan and Kucuksezgin,2007).Signi ﬁcant quantities of heavy metals are discharged into rivers,which can be strongly accumu-lated and biomagni ﬁed along water,sediment,and aquatic food chain,resulting in sublethal effects or death in local ﬁsh pop-ulations (Megeer et al.,2000;Jones et al.,2001;Almeida et al.,2002;Xu et al.,2004).Suspended sediments adsorb pollutants from the water,thus lowering their concentration in the water column.Heavy metals are inert in the sediment environment and are often considered to be conservative pollutants (Wilcock,1999;Olivares-Rieumont et al.,2005)although they may be released into the water column in response to certain disturbances (Agarwal et al.,2005),causing potential threat to ecosystems (Chow et al.,2005;Hope,2006).Bottom sediments also provide habitats and a food source for benthic fauna.Thus,pollutants may be directly orindirectly toxic to the aquatic ﬂora and fauna.The effects of pollutants may also be detected on land as a result of their bio-accumulation and bio-concentration in the food web (Wu et al.,2005;Zhang and Ke,2004).Consequently,an analysis of the distribution of heavy metals in sediments adjacent to populated areas could be used to investigate anthropogenic impacts on ecosystems and would assist in the assessment of risks posed by human waste discharges (Hu et al.,2002;de Mora et al.,2004;Zheng et al.,2008).Under certain conditions,these metals may accumulate to a toxic concentration level which may lead to ecological damage (Jefferies and Freestone,1984).Methods used to evaluate the ecological risk posed by heavy metals in sediments include calcu-lation of the index of geo -accumulation (Porstner,1989),the potential ecological risk index (Håkanson,1980),and the excess after regression analysis (ERA)(Hilton et al.,1985),among which the ﬁrst two indices are the most popular.Several methods have been proposed for estimation of the potential risks to human health of heavy metals in ﬁshes.The risks may be divided into carcinogenic and non-carcinogenic effects.For carcinogenic contaminants,the observed or predicted exposure concentrations are compared with thresholds for adverse effects,as determined by dose-effect relationships (Solomon et al.,1996).The*Corresponding author.E-mail address:zfyang@ (Z.Yang).Contents lists available at ScienceDirectEnvironmental Pollutionjournal homepage:w ww.else /locate/envpol0269-7491/$e see front matter Crown Copyright Ó2011Published by Elsevier Ltd.All rights reserved.doi:10.1016/j.envpol.2011.06.011Environmental Pollution 159(2011)2575e 2585probability risk assessment technique has been adopted by a number of researchers(Solomon et al.,1996;Giesy et al.,1999; Cardwell et al.,1999;Hall et al.,2000;Wang et al.,2002)to fully utilize available exposure and toxicity data.However,these methods have only been used to quantify the health risks of carcinogenic pollutants.Current non-cancer risk assessment methods do not provide quantitative estimates of the probability of experiencing non-cancer effects from contaminant exposure.These methods typically are based on the Target Hazard Quotient(THQ).Although the THQ-based risk assessment method does not provide a quantitative estimate of the probability of an exposed population experiencing an adverse health effect,it does provide an indication of the risk level associated with pollutant exposure.This method of risk estimation has recently been used by many researchers(Chien et al.,2002;Wang et al.,2005)and has been shown to be valid and useful.This non-cancer risk assessment method was also applied in this study.In recent years,water pollution in the Yangtze River basin, associated with industrial and economic development,has attrac-ted increasing attention.More than25billion tons of wastewater are discharged annually into the Yangtze River,from industrial and mining enterprises,and from the sewage of nearby cities.The majority(80%)of wastewater and sewage discharged into the Yangtze River basin is untreated.Sixty percent of the length of the main river channel of the Yangtze River is affected by pollution and this is particularly widespread in the middle and lower reaches, which have high densities of industry and population(Liu and Wu, 2006).Heavy metals in sewage have been discharged into rivers and have accumulated in river-bed sediments,where they have affected the ecology of the river,with long-term impacts on planktonic and benthicﬁsh prey items.Heavy metals,which accumulate inﬁsh through the food chain,may subsequently pose a human health risk through consumption ofﬁsh.Although analyses of heavy metals in lake sediments have been used extensively for the purpose of pollution monitoring(Pekey et al.,2004;Liu et al.,2007),few researchers have focused on the routes of transmission of metals from sediment,toﬁsh,to humans.The pH,ionic strength,and water temperature of the Yangtze River provide ideal conditions for adsorption,ensuring a high capacity for self-puriﬁcation.Eighty-ﬁve percent of heavy metals entering the river are believed to be removed in10min from the water phase,mainly by adsorption and precipitation(Zhu and Zang,2001).Therefore,the sediment in this area highly concentrate pollutants in surface water,and it is important to further investigate the ecological effects of pollution and tech-niques for improving environmental conditions in this region.This paper studies the inﬂuence of heavy metal pollution onﬁsheries in the Yangtze River basin by evaluating the ecological risk of heavy metals in both the environment andﬁsh.By testing and analyzing the heavy metals in the water,sediments andﬁsh in the main river and in the lakes of the middle and lower reaches of the Yangtze, the health risks associated with heavy metals in sediments and ﬁsh were evaluated.2.Materials and methods2.1.Study sitesThe6300km long Yangtze River is the third longest in the world and the longest and largest river in China.Itﬂows from west to east and drains into the Eastern China Sea at Shanghai.The Yangtze River has a watershed of1.80million km2.In China,the river is called the Changjiang,and is divided into several reaches.The upper reach consists of the region between the source and Yichang(the Three-Gorges Dam),the middle reach runs between Yichang and Hukou(Poyang Lake mouth),and the lower reach runs between Hukou and Datong.All sections downriver from Datong are considered to be the estuary.In general,all sampling procedures were carried out according to internationally recognized guidelines(UNEP,1991).The samples were collected from the main river and lakes in the Yangtze River basin at17sites in the middle reach and from10sites in the lower reach,in2007(Fig.1).The middle and lower reaches of the Yangtze River,with many bends,are connected with many lakes.This region,referred to as the“Water Realm”is China’s major agricultural district.Several large and medium-sized cities are distributed along the river,forming a prosperous industrial belt.In contrast to the wide plains of the middle reach,the downstream plains alongside the river are long and narrow. Jianghan Plain lies in the north of the middle reach,while Dongting Plain and Poyang Plain are situated in the south.Between Hukou and Zhenjiang,a narrow alluvial plain extends on both sides of the main river.Along the middle and lower reaches of the Yangtze River,the river channelﬂows slowly and the plain alternates between wide and narrow.Below Datong station,tidal inﬂuences prevail,theﬂow becomes slower,and sediment deposition is enhanced.2.2.Sample collection and analysis2.2.1.Field samplingWater,sediment,ﬁsh,and crayﬁsh samples were collected from27sites in the middle and lower reaches of the Yangtze River watershed.Twelve sites were located in the middle reach of the main river,nine were in the lower reach,and six were in lakes in the middle and lower reaches(Fig.1).In total,27water and sediment samples and469ﬁsh samples were collected.Water samples were collected at the surface in40mL acid-washed polyethylene sample bottles,taking care not to incorporate sediment into the samples.The samples were acidiﬁed with10mL of1:1nitric acid:deionized water.Sediment and ﬁsh samples were collected into pre-cleaned polyethylene bags.Fish and crayﬁsh were collected by seine.To obtain a suitable mass of material for analysis,the soft tissues from1e18individual crayﬁsh were dissected,drained of excess liquid and stored in plastic bottles.Forﬁsh,100e300g of dorsal muscle from a single individual was dissected.All samples were frozen and stored atÀ18 C immediately upon returning from theﬁeld.2.2.2.Sample treatmentWater samples wereﬁltered using Whatman No.1ﬁlter paper and stored at 0e4 C.Sediment samples were freeze-dried and passed through a1mm clean plastic sieve to remove shell fragments.Sieved sediments were ground in an agate mortar.The powdered sediments were then transferred to a clean nylon membrane sieve(0.071mm)and shaken to obtain aﬁne homogeneous powder.A sample of 500e1000g of dried sediment material was weighed for digestion.Samples were microwave-digested in acid-cleaned Teﬂon vessels containing5mL of ultra pure nitric acid and2mL ultra pure,concentrated hydroﬂuoric acid(HF).Each digestion batch included at least one reagent blank and a representative reference standard and,typically,a sample replicate to check for homogeneity and process efﬁcacy. Samples were digested for30e40min at200 C.After cooling for at least1h,the vessels were opened and0.9g of boric acid was added to dissolve theﬂuoride precipitates.The vessels were resealed and placed back in the microwave digestion system for an additional20e30min.Following cooling for at least1h,the digested sample was transferred to a graduated plastic test tube with an additional0.5mL HF and brought up to volume(either15or50mL)with Milli-Q water.Samples ofﬁsh tissue(0.5Æ0.01g)were weighed directly into acid-washed Teﬂon digestion vessels.Ten milliliters of ultra pure nitric acid were added to each vessel which were then heated to100 C using an XT-9800pre-treatment heater, until almost all of the nitrogen dioxide was emitted.A4mL aliquot of concentrated HNO3:HF(1:1v/v)acid mixture was added before microwave digestion.Each digestion batch included at least one reagent blank,a representative reference standard and,typically,a sample replicate to check for homogeneity and process efﬁcacy.Microwave digestion consisted of three steps:0.5MPa for1min,1.0MPa for 2min,and1.5MPa for3min.After allowing at least1h for cooling,the digested sample was transferred to a graduated plastic test tube and the volume was made up to100mL with Milli-Q water.2.2.3.Sample testAll water and sediment samples were analyzed for Cd and Pb using an Induc-tively Coupled Plasma Mass Spectrometer(ICP-MS).Concentrations of Cr,Cu and Zn were determined by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES).As and Hg were measured using Atomic Fluorescence Spectrometry(AFS).The concentration of As and Hg in the tissue samples was measured by AFS. Tissue concentrations of Cu were measured using both ICP-MS and ICP-AES.Tissue Zn was measured exclusively by ICP-AES,whereas Cr,Cd and Pb were measured using ICP-MS.Sample digestates were typically diluted20Âwith2%nitric acid for analysis. Greater dilutions(up to10,000Â)were necessary for some analytes that were present at relatively high concentrations in sediment digestates(e.g.Cu).Results were quantiﬁed using an empirical calibration curve generated from the responses obtained from multiple dilutions of a multi-element calibration standard prepared from single-element standards(Alfa Aesar).Analytical quality control included analysis of a2%ultrapure nitric acid blank and a drinking water reference material, together with the procedural blank,reference material of a similar matrix,and a sample duplicate from the microwave digestion.Y.Yi et al./Environmental Pollution159(2011)2575e2585 25763.Results and discussion3.1.Distribution of heavy metals in sedimentsHeavy metal concentrations in sediments in the middle and lower reaches of the Yangtze River basin are summarized in Table 1.Total nitrogen (TN),total phosphorus (TP),and organic matter (OM)concentrations are also listed for later analysis.The lakes had the highest mean concentrations of Hg,Pb,Cr,Cu,Zn,and OM,while the lower reach of the main river had the highest mean concen-trations of Cd,As,TN,and TP.Mean concentrations of metals in the middle reach were relatively lower.This may be due to the downstream movement and deposition of suspended sediments containing heavy metals,combined with local pollution in the lower reaches.The heavy metal concentrations in the sediments were higher in the lakes than in the river.We suggest that this was due to the higher ﬂow disturbance in the river that led to re-suspension and downstream movement of pollutants.Table 1Summary statistics of heavy metal concentrations and nutrition elements in sediments in the middle and lower Yangtze River basin (dry weight).Hg (mg/kg)Cd (mg/kg)Pb (mg/kg)Cr (mg/kg)Cu (mg/kg)Zn (mg/kg)As (mg/kg)TN (%)TP (%)OM (%)IMinimum 0.0110.0642042224860.0150.037 2.55Maximum 0.5350.771109667.4350630.170.147.38Average 0.170.4039.3272.5444.75120.4216.730.060.07 4.39S.D.0.160.2923.9418.6515.0879.1819.670.050.03 1.56CV (%)95.8473.0760.8925.7033.7065.75117.6283.9750.2835.60IIMinimum 0.010.131356.721.7547.80.0130.041 3.42Maximum 0.550.7648.1196.475.1190.29820.380.197.55Average 0.190.4829.7774.8844.24107.6833.920.220.08 5.00S.D.0.210.2613.9013.7416.0347.4831.660.190.07 1.79CV (%)113.4154.3446.7018.3536.2344.1093.3585.2184.0935.83IIIMinimum 0.080.0816482245 6.90.0470.041 2.01Maximum 0.440.868812175750830.130.05711.83Average 0.220.3844.1784.1750.43218.3326.920.080.05 6.55S.D.0.150.3227.8427.9821.03270.0628.940.030.01 4.31CV(%)68.5283.8163.0333.2541.70123.69107.5338.5312.3465.77Notes:I Stream in middle reach;II Stream in lower reach;III Lakes in middle and lower reaches;TN is total nitrogen,TP is phosphorus,and OM is organicmatter.Fig.1.Map of study area,middle and lower reaches of the Yangtze River.(1)Yichang,(2)Yidu,(3)Jingzhou,(4)Gong ’an,(5)Haoxue,(6)Xinchang,(7)Baiji dolphin (Lipotes vexillifer)protection area,Jianli,(8)Dongting Lake;(9)Chenglingji;(10)Honghu Lake;(11)Yangtze River near Honghu Lake mouth,(12)Jiayu,(13)Paizhou Bay,(14)Wuhan,(15)Donghu Lake,(16)E ’zhou,(17)Liangzi Lake,(18)Jiujiang,(19)Poyang Lake,(20)Anqing,(21)Datong,(22)Wuhu,(23)Jiangning,(24)Nanjing,(25)Zhenjiang,(26)Jiangyin,(27)Haimen.Y.Yi et al./Environmental Pollution 159(2011)2575e 258525773.2.Heavy metals concentration in ﬁshesThe 469ﬁsh and cray ﬁsh samples together comprised 2classes,9orders,14families,33genera,and 41species.Their heavy metals concentration and habitats are listed in Table 2.The highest concentrations of Cu,Zn,and Hg were found in Eriocheir sinensis .Hemimyzon abbreviata had the highest concen-trations of Pb and Cd,Leptobotia elongata ,and Coreius guichenoti had the highest concentrations of Cr,and Rhinogobio typus had the highest concentrations of As.The benthic fauna (e.g.Eriocheir sinensis ),demersal ﬁsh (e.g.Hemimyzon abbreviata ,Rhinogobio typus and Leptobotia elongata ),and ﬁsh that inhabit the lower zone of the water column (e.g.Coreius guichenoti ),are likely to have higher heavy metal concentrations than ﬁshes that inhabit the upper water column.We hypothesized that their greater contact with polluted sediments and their greater uptake of heavy metal concentrations from zoobenthic predators would lead to higher heavy metal concentration in their tissues (Yi et al.,2008).The relationships among heavy metal levels in ﬁsh,benthic fauna,water,and sediments are analyzed in Section 3.3.Heavy metals concentrations in ﬁsh of this study and other literatures are listed in Table 3.Data from the open literatures showed that metal concentrations in muscles of ﬁsh varied widely depending on where and which the animals caught (Table 3).Chi et al.(2007)measured the concentrations of heavy metals in themuscles of different ﬁsh species from Taihu Lake,and reported 0.228e 1.89Cu,16e 130Zn,0.177e 0.287Pb,0.003-0.021Cd,ND e 0.387Cr mg/kg dry weight,metals concentration were lower than this study,except Zn.Bustamante et al.(2003)found that different ﬁsh species Kerguelen Islands contained 0.5e 2.5Cu,9.2e 32.2Zn,0.01e 0.1Cd,0.044e 1.19Hg mg/kg dry weight in the muscle,results generally were higher than present results,exceptHg.The results reported by Türkmen et al.(2005)from _IskenderunBay were generally lower than present results,except Cr.The concentrations of Cu,Zn and Pb in ﬁsh collected from the Yangtze River were higher than Pearl River on corresponding period,however,Cd,Hg,Cr and As concentrations were lower.Both Yangtze River and Pear River,heavy metal concentrations in ﬁsh were high in recent years than ten years ago.This demonstrate that heavy metals contaminant in Chinese river are becoming more and more serious.3.3.Relationships between heavy metal levels in ﬁsh and in the environmentHeavy metals entering the water body would be absorbed in sediments,and subsequently might migrate as a result of exchanges between water,sediment,and biota,through biological and chemical process.In the 1960s,serious mercury pollution occurred in Sweden (Jernelöv et al.,1975),and was later found inTable 2Average concentrations of heavy metals and habitat site for ﬁsh species (mg/kg,wet weight).Fish speciesCu Zn Pb Cd Hg Cr As Number of sample Habitat Mylopharyngodon piceus 1.197.60.290.0750.020.150.02219BottomCtenopharyngodon idellus0.87 4.20.250.0540.020.020.0130Middle-lower Squaliobarbus curriculus (Rich.) 1.39 6.680.770.0220.0030.0550.0192MiddleHypophthalmichthys molitrix 0.86 4.470.560.0730.020.1660.01849Middle-Upper Aristichthys nobilis (Rich.)0.789.860.7930.170.0250.190.01914Middle-Upper Cyprinus carpio Linnaeus 1.047.390.510.120.020.1780.02251Bottom Carassius auratus auratus 1.099.40.890.170.0140.330.01920Bottom Sinibrama macrops2.1814.870.050.00470.0210.0120.0193Middle Erythroculter ilishaeformis (Bleeker) 1.08 4.060.480.110.0020.1010.0153Up Erythroculter mongolicus (Basilewsky)0.3613.815 1.87e 0.0180.105e 1Up Hemiculter leucisculus (Basil.)1.548.950.160.0190.0220.0250.0196UpperPseudorasbora parve (Temminck et Schlegel)0.77 5.820.0110.0130.0340.0050.0223Upper-middle Sarcocheilichthys nigripinnis nigripinnis(Günther) 1.05 4.980.0250.0070.0310.0110.0235Middle-lower Coreius heterodon (Bleeker) 1.034 3.90.320.10.010.570.02956Low Coreius guichenoti1.67.330.560.10.0150.8050.02315Low Rhinogobio cylindricus Gunther1.2117.87 1.610.390.0060.0230.0252Bottom Rhinogobio ventralis Savage et Dabry2.22 4.73 1.160.067e e e 2Bottom Rhinogobio typus Bleeker3.414.040.0660.0970.0150.0330.0392Bottom Platysmacheilus longibarbatus Lo 1.280.793 1.650.2290.0060.0380.0232Bottom Leptobotia elongata (Bleeker)0.57 4.40.710.0880.0320.8050.028Bottom Misgurnus anguillicaudatus1.5310.790.0110.0090.0290.7320.021Bottom Hemimyzon abbreviata (güntheri)2.534.710.12e e e 1Bottom Pelteobagrus fulvidraco1.289.340.860.150.0380.540.01439Bottom Leiocassis longirostris Günther 0.78 4.930.650.0990.0110.430.02619Bottom Leiocassis crassirostris Regan 1.71 6.2 1.560.220.0160.640.0274Bottom Mystus macropterus (Bleeker) 1.7187.580.1840.110.0230.0170.0253Bottom Silurus asotus Linnaeus 1.39 5.82 1.140.2920.0360.2330.01940BottomCoilia nasus Schlegel2.2814.460.10.020.040.0310.0213Upper-middle Coilia brachygnathus Kreyenberg et Pappenheim 0.536 1.2520.10.0190.760.0163Upper-middle Coilia mystus (Linnaeus) 6.0115.770.2580.050.0230.1930.0154Upper-middle Lateolabrax japonicus2.75 6.830.0090.0010.0110.0050.0121Middle-lower Siniperca scherzeri steindachner 0.94 4.990.490.0350.0190.0240.0168Middle-lower Odontobutis obscurus 2.66 5.750.0190.0350.0150.0120.0194Bottom Channa argus (Cantor)0.94 2.980.0560.0010.0320.0130.0122BottomHemiramphys kurumeus Jordan et Starks 1.099.760.10.0020.0480.0330.0145Upper-middle collichthys lucidus (Richardson) 5.4411.0750.1750.0360.022e e 2BottomPampus argenteus 7.7613.860.440.070.03e e 1Upper-middle Cymoglossus robustus3.557.660.01660.020.023e e 5Bottom Palaemon (E.)carincauda Holthuis 5.548.90.1750.0650.0170.0330.0095Bottom Macrobrachium nipponense 10.7411.70.720.1840.0240.3730.02818Bottom Eriocheir sinensis18.7650.80.1420.2620.0540.060.0248BottomNotes:bold type means the highest concentration of each heavy metals,e means metals concentration below lower detection limit.Y.Yi et al./Environmental Pollution 159(2011)2575e 25852578Canada(Wheatley,1997).Following these events,researchers began to pay more attention to pollution by heavy metals.Accu-mulation of heavy metals inﬁsh results primarily from surface contact with the water,by breathing,and via the food chain.Uptake by these three routes depends on the environmental levels of heavy metals in the habitat of theﬁsh.Table4lists the heavy metal concentrations in these different media.Concentrations of heavy metals in the sediment were1000e100,000times higher than those in the water.A number of studies have reported a similar phenomenon(Enk and Mathis,1977;Anderson et al.,1978; Burrows and Whitton,1983;Barak and Mason,1989).The heavy metal concentrations were10e1000times higher inﬁsh and benthic invertebrates than those in the water,but lower than concentrations in the sediments(Table4).Heavy metals do not degrade in water but are generally not found in high concentra-tions,primarily due to deposition in sediments but also because of uptake by plants and animals(Yi et al.,2008).Heavy metals in the sediment enter the food chain via the feeding of benthic animals.The concentrations of heavy metals were highest in the sedi-ments,intermediate inﬁsh and lowest in the water.Demersalﬁsh and benthic fauna generally had higher levels of Cu,Cd,Pb,Zn,Hg, Cr and As compared with those inﬁsh at the upper-middle and middle-lower layers(Table4and Fig.2).Heavy metal concentra-tions were ranked as follows:sediments>demersalﬁsh and benthic fauna>pelagicﬁsh>water.Our results support the hypothesis that the sediment is the major sink for trace metal pollution and plays an important role in heavy metal uptake byﬁsh (Luoma and Bryan,1978).The heavy metals contained in the sedi-ment are then absorbed and stored in the tissues(Gao,2001).As a consequence,controlling the sources of contamination of water and sediments in the aquatic system is the key method for protection of theﬁsh resource.4.Ecological risk and health risk assessment4.1.Assessment of potential ecological riskHåkanson(1980)developed a methodology to assess ecological risks for aquatic pollution control.The methodology is based on the assumption that the sensitivity of the aquatic system depends on its productivity.The potential ecological risk index(R I)was introduced to assess the degree of heavy metal pollution in sediments, according to the toxicity of heavy metals and the response of the environment:R I¼XE i r(1) E i r¼T i r C i f(2)C if¼C i0=C i n(3) where R I is calculated as the sum of all risk factors for heavy metals in sediments,E r i is the monomial potential ecological risk factor,T r i is the toxic-response factor for a given substance,which accounts for the toxic requirement and the sensitivity requirement,as shown in Table5.C f i is the contamination factor,C0i is the concentration of metals in the sediment,and C n i is a reference value for metals (Table5).The risk factor R I was proposed by Hakanson based on eight parameters(PCB,Hg,Cd,As,Pb,Cu,Cr,and Zn).In this paper,PCBs were excluded.Based on the reference values for these elements, the adjusted evaluation criteria for the ecological risk index R I are listed in Table6.Using equations(1)e(3)and parameters listed in Table5,the potential ecological risk indices E r i and R I for each site were obtained and are listed in Table7.According to these data,Hg posed a considerable ecological risk atﬁve sites and a moderate risk at two.Additionally,Cd and As also posed relatively high ecological risks in these areas.The high ecological risks of these three heavy metals in freshwater ecosystems are consequences of their high toxic-response factors.In terms of their spatial distribution,sites with moderate or considerable potential ecological risk indices for Hg and Cd were located near to large cities(Wuhan,Nanjing,and Jiangyin),or ports(Chenglingji),or lakes with high human activity (Dongting Lake and Donghu Lake).The highest potential ecological risk indices for As were found in the lower reach,mainly down-stream of Poyang Lake.For other metals(Pb,Cu,Cr,and Zn),the potential ecological risk indexes were low.The potential ecological risk indices for single regulators(E r i)indicated that the severity of pollution of the seven heavy metals decreased in the following sequence:Hg>Cd>As>Cu>Pb>Cr>Zn.R I represents the sensitivity of various biological communities to toxic substances and illustrates the potential ecological risk caused by heavy metals.Two sites among twelve in the middle reach of the main river exhibited moderate or considerable ecological risk.Most of the lakes and sites in the lower reach of the main river posed moderate or considerable ecological risk.The R I values were clearly related to the degree of anthropogenic disturbance.For example, the Tian’ezhou wetland,which is a natural conservation area in China,had the lowest heavy metal concentrations and minimalTable3Comparison of heavy metal concentrations inﬁsh with values taken from the open literature.Sample area Cu Zn Pb Cd Hg Cr As ReferencesYangtze River b0.361e18.760.793e50.80.009e10.1ND e2ND e0.054ND e0.805ND e0.039This researchYangtze River b<2.0e14.920.24e1.750.03e0.18Chen et al.(2002)Pearl River a 1.17e6.72 2.62e20.20.05e1.94ND e33.20.98e7.86ND e5.360.17e1.46Xie et al.(2010)Pearl River a0.009e2.025ND e5.507ND e1.0490.005e0.079ND e0.772ND e1.23Wei et al.(2002)Taihu Lake b0.228e1.8916e1300.177e0.2870.003e0.021ND e0.387Chi et al.(2007)_Iskenderun Bay b 1.239e2.201 3.025e4.873 1.808e3.4740.831e1.341 1.309e2.719Türkmen et al.(2005) Kerguelen Islands b0.5e2.59.2e33.20.010e0.0860.044e1.19Bustamante et al.(2003)a Values represent the ranges or mean expressed as mg.kgÀ1dry wt.b Values represent the ranges or mean expressed as mg.kgÀ1wet wt.Table4Average heavy metal concentrations in water,sediment andﬁshes living in differentwater depths.Heavy metal Cu Zn Pb Cd Hg Cr AsWater0.00280.0310.0020.00040.000040.00130.00097Pelagicﬁsh 1.16 6.080.480.0720.0210.1230.016Demersalﬁshand benthicfauna2.248.050.570.1280.0220.3650.022Sediment45.7135.6370.4230.15576.424.7Notes:values are mg/kg wet weight forﬁsh tissue and water,and mg/kg dry weightfor sediments.Y.Yi et al./Environmental Pollution159(2011)2575e25852579。

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