bioavailability and toxicity of metal nutrients during anaerobic digestion

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Bioavailability and Toxicity of Metal Nutrientsduring Anaerobic Digestion

Sérgio F. Aquino1 and David C. Stuckey2

Abstract: This paper investigates the effect of chelating agents on the bioavailability of Fe and Cu during anaerobic digestion. Theresults on metal speciation and methane production in anaerobic serum bottles showed that biomass was able to grow in the presence ofcitrate 1 mM and nitrilotriacetic acid �NTA� 1 mM, suggesting that the binding sites at the cell surface competed efficiently for the metalswith the chelating agents added. The presence of free ethylenediaminetetraacetic acid 1 mM inhibited methanogenesis, and this seemed tobe related to a loss in metal uptake capacity. Although the addition of soluble microbial products �SMP� did not change metal distributionin anaerobic systems, it caused an increase in the rate of methane production, and it is believed that direct uptake of Cu-SMP complexeswas responsible for this increase. The best protection against Cu toxicity occurred when stoichiometric amounts of NTA, which shouldcomplex and solubilize most of the Cu, was added, and it is likely that NTA prevented lethal concentrations of Cu from being adsorbedonto the cell and hence internalized.

DOI: 10.1061/�ASCE�0733-9372�2007�133:1�28�

CE Database subject headings: Metals; Adsorption; Anaerobic treatment; Nutrients; Toxicity.

Introduction

In anaerobic wastewater treatment systems, the availability ofmetals as nutrients or toxicants is affected by many factors. Thetotal metal concentration, the environmental conditions such aspH and redox potential, and the kinetics of precipitation, com-plexation, and adsorption are believed to play a key role �Mosey1976; Callander and Barford 1983a,b; Oleszkiewicz and Sharma1990�. Many researchers have suggested that metal uptake inbiological systems occurs in two steps: a passive adsorption ofmetal onto the biomass surface, followed by an energy-dependenttransport into the cell �Lawson et al. 1984; Rudd et al. 1984;Oleszkiewicz and Sharma 1990; Liu et al. 2001�. This well-accepted theory agrees with the nonspecific, chemiosmotic-drivenmechanism identified by biochemists/microbiologists and re-viewed by Nies �1999�. According to this review, microorganismsnormally employ two types of metal uptake mechanisms. One isfast, unspecific, constitutively expressed, and driven by the che-miosmotic gradient across the cytoplasmatic membrane of micro-organisms; whereas the other has high substrate specificity, isslower and inducible, often uses adenosine triphosphate �ATP�hydrolysis as the energy source, and is only used by a cell intimes of need, starvation, or special metabolic situation. Accord-

1Associate Researcher, Dept. of Chemistry, UFOP, Campus Morro doCruzeiro Ouro Preto, MG–35.400.000, Brazil �corresponding author�.E-mail: [email protected]

2Professor, Dept. of Chemical Engineering, Imperial CollegeLondon, Prince Consort Rd., London SW7 2BY, U.K. E-mail:[email protected]

Note. Discussion open until June 1, 2007. Separate discussions mustbe submitted for individual papers. To extend the closing date by onemonth, a written request must be filed with the ASCE Managing Editor.The manuscript for this paper was submitted for review and possiblepublication on January 21, 2005; approved on May 15, 2006. This paperis part of the Journal of Environmental Engineering, Vol. 133, No. 1,
January 1, 2007. ©ASCE, ISSN 0733-9372/2007/1-28–35/$25.00.
28 / JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / JANUARY

J. Environ. Eng. 200

ing to Nies �1999�, there is evidence showing that most cationsaccumulate by the fast chemiosmotic-driven system, and the maindrawback of such a nonspecific system is that, when a cell faces ahigh concentration of heavy metal, deleterious cations are unwill-ingly transported into the cytoplasm.

Metal adsorption by biomass has been shown to occur withliving or dead organisms, to be reversible, and to be dependent onboth pH and concentration of dissolved organic matter �DOM� inthe bulk phase, but not dependent on metabolism �Huang andMorehart 1990; Butter et al. 1998; Wang et al. 1999, 2003; Fowleand Fein 2000; Leung et al. 2000; Guo et al. 2001�. Adsorption isbelieved to occur due to electrostatic interactions and complex-ation between free metals and negative charged groups located atthe cell surface, such as carboxyl and phosphate groups associ-ated with extracellular polymers �ECP or EPS�. In addition, it isthought that the intraparticular diffusion of metal is the rate-limiting step of the adsorption process �Butter et al. 1998�, whichmay confer on adsorption slow kinetics when compared to metalprecipitation and metal complexation. As implied by Hering andMorel �1990�, for metal occurring as organic complexes or asprecipitates, such as in anaerobic treatment systems, if the rates ofcomplex and precipitate dissociation are slow as compared to therate of adsorption, then the metal uptake may be limited by abi-otic chemical factors. On the other hand, if the rate of adsorptionis too slow to ensure that essential cations are satisfactorily takenup by the general chemiosmotic-driven mechanism, then it is pos-sible that microorganisms use more sophisticated mechanisms toenhance metal solubilization and uptake, thereby avoiding a“metal crisis.”

One uptake mechanism that is deployed in times of metal scar-city and does not seem to depend on metal adsorption wasdiscovered for aerobic bacteria and involves the excretion of mi-crobial products called siderophores to scavenge iron from theenvironment �Neilands 1967; Emery 1982; Andrews et al. 2003�.Siderophores are of low molecular weight ��1,000 Da� and arecharacterized by their high specificity and affinity �Kaff�1030�
towards ferric iron. They are generally synthesized and secreted
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by bacteria in response to iron restriction and usually formhexadentated ferric complexes that are first recognized by anouter-membrane receptor and then actively transported into theperiplasm. Once internalized, the ferri-siderophore complex mustbe dissociated to liberate the complexed iron for use in cellularmetabolism and recycle the siderophores, and this process isthought to involve reduction of the siderophore-associated ironbecause of the relatively low affinity of siderophores and Fe2+

�Andrews et al. 2003�. Diels et al. �1999� remark that some ofthese siderophores also have high affinities for other heavy metalsand that, in the case of Pseudomonas aeruginosa and Alcaligeneseutrophus siderophores, synthesis was induced by heavy metalseven in the presence of high iron concentrations.

In a similar fashion to the way that the predominance of in-soluble Fe�OH�3 in aerobic environments has forced aerobicbacteria to come up with a more sophisticated iron uptake me-chanism, it is possible that the formation of insoluble sulphidesin anaerobic systems has also encouraged anaerobic microorgan-isms to evolve special mechanisms to scavenge scarce and essen-tial metals. This may be especially important in cases where theunspecific chemiosmotic-driven mechanism is compromised,such as when the kinetics of adsorption is slower than the kineticsof precipitation. It is well known that hydrogen ions �low pHs�and ions present in high concentration �Ca, Mg� compete withessential metals �Ni, Cu, Co� for the adsorption sites at the cellsurface. Therefore, the development of metallophore-based up-take systems could alleviate the deficiency of certain metals inconditions where their adsorption was not favored. This might beparticularly important for acidogenic bacteria in two-stage reac-tors where the pH can decrease to relatively low levels because ofthe production of volatile fatty acids �VFAs� in the first step. Thedeliberate excretion of metallophores is obviously of considerableimportance for anaerobic wastewater treatment systems, becausethese soluble microbial products �SMP� will constitute part of theeffluent COD, and the survival of microorganisms and the stabil-ity of systems operated under nutrient deficiency may rely on theproduction of specific SMPs.

Indeed, different research groups have showed that SMPhave chelating properties, suggesting that microorganisms mayexcrete specific microbial products that play a role in the uptakeof metal nutrients and/or in metal toxicity mitigation �Callanderand Barford 1983a,b; Kuo and Parkin 1996; Barber and Stuckey2000; Mirimanoff and Wilkinson 2000; Gonzales-Gil et al. 2003�.Recently, Gonzales-Gil et al. �2003� showed that the bioavailabil-ity of Ni and Co was dramatically increased by the addition ofyeast extract in anaerobic treatment. According to the authors, thiswas due to the formation of soluble bioavailable complexes,which favored the dissolution of metals from their insoluble sul-phides. Callander and Barford �1983a;b� stressed the fact that themeasured levels of soluble metals in anaerobic systems are muchhigher than those predicted by chemical equilibrium, suggestingthat the presence of soluble organic compounds caused a pro-found impact on the treatment process by complexing with metalsand making them more soluble. However, metals complexed withsoluble ligands are not necessarily more bioavailable. Huang andMorehart �1990� showed that soluble proteins released from cellsdecreased Cu �II� uptake capacity because they competed for themetal ions with the proteins attached to the cell wall. Wang et al.�1999, 2003� also showed that dissolved organic matter decreasedthe efficiency of metal uptake because of competition betweenthe kinetics of complexation and the kinetics of adsorption.Although chelating agents make metals commonly found in
wastewaters more soluble, the results on bioavailability are con-
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tradictory; hence, the objectives of this paper were to investigatethe effect of some chelating agents commonly found in industrialwastewaters �ethylenediaminetetraacetic acid �EDTA�, nitrilotri-acetic acid �NTA�, citrate� on methanogenesis and to discuss therole of SMP in metal bioavailability and toxicity mitigation.

Materials and Methods

Two anaerobic laboratory-scale continuously stirred tank reactors�CSTRs� were used as the source of biomass and soluble micro-bial products �SMP�. The CSTRs were placed in a thermostattedbath that maintained the temperature at 35±0.5°C and fed bymeans of peristaltic pumps �Watson and Marlow 205S�. The feedwas comprised of glucose ��10 g COD/L�, NaHCO3, essentialminerals, and vitamins, as is shown in Table 1 and described inmore detail elsewhere �Aquino and Stuckey 2003�.

For the biochemical methane potential �BMP� assay �Owenet al. 1979�, 130 mL serum bottles were inoculated in triplicate byadding 30 mL of seed, 75 mL of biomedia, and 3 mL of glucose�72 g/L� so that a final concentration of �2,000 mg L−1 wasobtained in each bottle. “Normal biomedia” �NB� contained allmetals in the soluble form of chlorides �Table 1�; for the bottlesthat received no biomedia, a phosphate buffer was used instead.Biomass from the CSTRs running at steady state was used asseed; it was first centrifuged and then resuspended using NB,phosphate buffer, or SMP, as shown in Table 2. In the bottlesinoculated with 30 mL of SMP, the seed was resuspended withCSTR supernatant containing SMP in a concentration rangeof 200–300 mg L−1. Biogas production was measured by wettedglass syringes and biogas composition determined using a

Table 1. Input Values for Theoretical Determination of Metal SpeciationUsing Visual MINTEQ

Parameter CSTR BMP bottlea

pH 7.0 7.0

Temperature �°C� 35 35

HCO3− �mg L−1�b 3,556 2,983

HS− �mg L−1�c 3.4 0.013

PO43− �mg L−1� 112.3 479.8

Fe2+ �mg L−1� 1.41 0.39

Mn2+ �mg L−1� 0.07 0.19

Co2+ �mg L−1� 1.24 0.035

Zn2+ �mg L−1� 0.12 0.024

Al3+ �mg L−1� 0.03 0.0007

Ni2+ �mg L−1� 0.06 0.0042

Cu2+ �mg L−1� 0.093 0.002

Ca2+ �mg L−1� 6.8 20.8

Mg2+ �mg L−1� 1.77 10.1

MoO42− �mg L−1� 0.16 0.019

NH4+ �mg L−1� 299.4 70.2

Cl− �mg L−1� 610.6 1,225

K+ �mg L−1� 79.3 333

Na+ �mg L−1� 1,374 1,152aFor BMP bottles, chemical concentrations used in Visual MINTEQallowed for a dilution factor of 0.69 �75 mL biomedia/108 mL totalvolume�.bValue obtained after run where solids were not allowed to precipitate.cPotential sulphide concentration, i.e., considering that all sulphate isconverted into sulphide.

Shimadzu gas chromatography–thermal conductivity detector

F ENVIRONMENTAL ENGINEERING © ASCE / JANUARY 2007 / 29

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�GC-TCD� fitted with a Porapak N Column �1,500�6.35 mm�.The carrier gas was helium set at a flow rate of 50 mL/min,column temperature was 28°C, detector temperature was 38°C,and injector temperature was 128°C. The peak area was calcu-lated and printed out on a Shimadzu Chromatopac C-R6A.Samples of 1 mL were collected using a plastic gas tight syringe,and calibration gases were accurate to ±5%. During the experi-ments a very low coefficient of variance �CV� in methane produc-tion was observed among the triplicate bottles, and in most casesa CV of ±5% was obtained.

The sequential extraction procedure originally proposed byStover et al. �1976� and modified by Lake et al. �1985� was usedin this study for the determination of Cu and Fe, as outlined inFig. 1. The technique uses KNO3, KF, Na4P2O7, EDTA, andHNO3 to fractionate metals in sludges into soluble/exchangeable,adsorbed, organically bound, carbonate, and sulphide forms,respectively. Extracellular polymers �ECP� were also extracted

Table 2. Protocol for Incubation of BMP Bottles �Triplicates�

BottlesBiomedia�75 mL�

First incubation set

Normal biomedia �NB� NB

NB+EDTA �1 mM� NB

NB+EDTA �1 mM�+Fe2+ NB

NB+citric acid 1 mM NB

NB+NTA 1 mM NB

Second incubation set


NB+30 mL SMP NB

NB+75 mL SMP Supernatant

NB+EDTA �1 mM�+SMP NB

NB+SMP �no glucose� Supernatant

Third incubation set


NB+Cu2+ �1 mM� NB

NB+Cu2++NTA �1 mM� NB

NB+Cu2++citric acid �1 mM� NB

NB+Cu2++75 mL SMP SupernatantaIndicates media used to resuspend biomass.bGlu=glucose stock solution �72 g/L�.cAll concentrations refer to stock solution.

Fig. 1. Sequential chemical procedure used to fractionate Fe and Cuin anaerobic samples.



from the biomass using the steam procedure described in Zhanget al. �1999� so that the metal content associated with ECPcould be determined. Cu and Fe were measured using induc-tively coupled plasma �ICP� equipment �Optima 2000 DV, PerkinElmer Instruments�, and the limit of detection for both metalswas 0.01 mg L−1. A CV lower than ±2% was observed for mostsamples.

Results and Discussion

Effect of Chelating Agents on Methane Production

Physicochemical phenomena such as precipitation and complex-ation with inorganic and organic ligands occur simultaneously,making it difficult to predict the metal distribution in systems ascomplex as biological reactors. The software Visual MINTEQwas used in this study to solve simultaneous chemical equilibriumand to calculate the theoretical concentration of metallic forms inanaerobic environments. Calculations were done based on thefeed composition and input values presented in Table 1, and dueto a lack of data on biosorption it was assumed that there was nointeraction between the biomass and the metal nutrients. Althoughthis assumption was incorrect, it was felt to be useful to demon-strate how the distribution of the metal would look in an anaero-bic environment without biomass. Furthermore, by comparingthese predictions with the actual metal distribution measured ex-perimentally, it was possible to see the changes caused by thebiomass and microbial chelators, and this allowed us to drawinferences about the mechanisms by which biomass take up es-sential metals.

As the total amount of polyvalent metals equals 0.96 mM,the addition of 1 mM of EDTA should complex most metals,and indeed according to the Visual MINTEQ software and input

massa

mL�Feedb

�3 mL�Otherc

�1 mL each�

B Glu —

B Glu EDTA 0.11 M

B Glu EDTA and Fe2+ 0.11 M

B Glu Citric acid 0.11 M

B Glu NTA 0.11 M

B Glu —

rnatant Glu —

B Glu —

rnatant Glu EDTA 0.11 M

B Water —

B Glu —

B Glu Cu2+ 0.11 M

B Glu Cu2+ and NTA 0.11 M

B Glu Cu2+ and citric acid 0.11 M

B Glu Cu2+ 0.11 M

Bio�30

N

N

N

N

N

N

Supe

N

Supe

N

N

N

N

N

N

values from Table 1, the addition of EDTA so that its con-

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centration inside BMP bottles was 1 mM should solubilize allmetals apart from Cu, which becomes 2% soluble due to a totalcomplexation of Fe and consequent displacement of sulphide.Fig. 2�a� shows that BMP bottles inoculated with 1 mM of EDTAhad their methane production rate reduced by �70%, and Fig. 3shows that addition of 1 mM EDTA on day 2 increased theamount of soluble iron and significantly reduced the amountof iron more strongly bound to the cell �Na4P2O7 extract�. Theseresults suggest that EDTA is stronger than the chelating siteson the cell surface; hence, it is possible that addition of 1 mMEDTA made metals less available for microorganisms becausethe kinetics of complexation prevailed over the kinetics ofadsorption.

Although adsorption seems to be a very fast process, it may beslower than complexation and precipitation. In high ionic strengthsystems such as anaerobic reactors, the diffusivity of ions may beslowed, increasing the chance of a metal reacting with a precipi-tating or chelating agent before adsorbing to the biomass. For thetypical situation with bioreactor media, assuming a population of2�109 cells per cm3, the average separation between individualcells is computed to be 10,000 �m. This leads to an averageseparation of less than 10 �m, and using the data on diffusivity ofdivalent cations presented in Cussler �1997�, characteristic diffu-sion times for such distances are on the order of 10−1 s. Such slowionic diffusivities could encourage the precipitation of metal nu-trients in anaerobic systems unless microorganisms ensure thatthe required nutrients are not “available” for reaction in the bulksolution. It is hypothesized that one way the cell does this is by

Fig. 2. Effect of EDTA and other chelating agents on methaneproduction

excreting chelating microbial compounds that complex metal

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nutrients, so that the metal complexes are later recognized byspecific cellular surface sites or directly absorbed.

In order to verify the nature of inhibition observed in thepresence of 1 mM EDTA, a stoichiometric spike of Fe2+, whichshould complex nearly all the EDTA, was added to the bottle“NB+EDTA 1 mM” a week after incubation �on day 7 as in-dicated by the arrow in Fig. 2�a��. This did not result in anyimprovements in methane production, suggesting that the impair-ment was not reversible. It was expected that the stoichiometricaddition of Fe2+, which has a high complexation constant withEDTA, should be able to displace the complexed metal nutrients,hence making them more bioavailable. To further investigate thenature of EDTA inhibition, another experiment was devised. A setof BMP bottles was inoculated with 1 mM EDTA, with 1 mMFe2+ being added at the time of incubation. Fig. 2�b� shows thatthe inhibitory effect was not observed when Fe2+ was added withEDTA at the beginning of the experiment, suggesting that inhibi-tion could be avoided as long as a high concentration of noncom-plexed EDTA was not present in the solution. Fig. 2�b� also showsthat the addition of 1 mM citric acid or NTA did not cause areduction in the rate of methane production. At the end of theexperiment, the volume of methane produced in the bottles spikedwith citric acid was 8 mL higher, which corresponds to an extra17.7 mg COD degraded, and this correlates well with the amountof citric acid present in the BMP bottles at the time of incubation.This suggests that citric acid was degraded, and in those caseswhere metals complex with potential substrates, it is likely thatessential metals are indirectly taken up.

According to Visual MINTEQ, the addition of 1 mM NTAwould significantly increase the solubility of most metals. The

Fig. 3. Distribution of �a� Fe and �b� Cu in BMP bottles inoculatedwith normal biomedia and in presence of EDTA 1 mM

simulations indicate that nearly all the Al, Co, Fe, Mg, Ni, and Zn


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should be dissolved and at least 98% of the dissolved metalswould be complexed with NTA. If complexation does occur,then the results presented in Fig. 2�b� show that the biomasscan take up metal nutrients in the presence of mild �citrate�or medium-strong chelating agents �1 mM NTA�. This suggeststhat the biomass is able either to internalize the complexes�citrate-Me, NTA-Me� or to produce chelating sites that are stron-ger than NTA.

Effect of SMP Addition on Methane Production

As shown before, it seems that adsorption plays an important rolein metal uptake in biological systems. It is possible that the ad-sorption sites located on the cell surface, such as those associatedwith Na4P2O7 extracts, act as the main receptor sites of not onlyfree metals, but also Me-SMP complexes, as is believed for metaltransport mediated by metallophores �Nies 1999; Andrews et al.2003�. In other words, it is possible that some SMPs are deliber-ately excreted to scavenge certain insoluble metals, and thatSMP-Me complexes are recognized by surface sites that are stron-ger than the Me-SMP complex, so that the metal can be shuttledinto the periplasm and the SMP recycled. In this way, adsorptionand the excretion of SMP would complement each other. Theunspecific chemiosmotic-driven mechanism, which chiefly de-pends on adsorption, would still be the preferred route of metaluptake, and the release of specific SMPs would occur in certainsituations as a strategic response.

With these hypotheses in mind, it was decided to investigatethe effect of SMP addition on the bioavailability of metals in the

Fig. 4. Effect of SMP addition on methane production in differentBMP bottles.

presence of 1 mM EDTA. It was postulated that if a substantial



amount of metal was precipitated or complexed in the presence of1 mM EDTA, then the addition of extra SMP might increasemetal bioavailability. This is because the synthesis of SMP maytake some time; therefore, by adding SMP produced in the CSTRsby the same biomass used to inoculate the BMP bottles, higherrates of methane production might be obtained. Fig. 4�a� showsthat the addition of SMP, collected from anaerobic CSTRs, didnot increase the rate of methane production in the presence of1 mM EDTA. This result suggests that SMP are not sufficientlystrong chelating agents to counter the inhibition caused by EDTA;nevertheless, Fig. 4�b� shows that, in BMP bottles inoculated withnormal biomedia �NB� and SMP, the rate of methane productionincreased slightly when compared to the control bottles �NB�. Inthe control bottles, methane was produced at a rate of 4.8 mL/dduring the first 8 days, while the rate was 5.3 mL/d in the bottlesinoculated with 30 mL of SMP and 5.8 mL/d in the bottles in-oculated with 75 mL of SMP. Because the coefficient of variancefor the assays was relatively low, the differences observed werestatistically significant.

The addition of SMP in the BMP bottles did not change Feand Cu distribution significantly �Fig. 5�. Most of the Fe wasstill more strongly adsorbed onto biomass �Na4P2O7 extract� orprecipitated as a carbonate or sulphide salt �EDTA and HNO3

extract�, whereas most of Cu was precipitated as the sulphidesalt �HNO3 extract�. However, it was observed that there was10 times as much Cu in the residual ashes of the biomass col-lected in the bottles that received SMP as compared to the NBbottles, while there was no statistical difference in the residualamount of Fe when comparing the different bottles. These results

Fig. 5. Fe and Cu distribution in samples collected on days 3 and 7from BMP bottles inoculated with normal biomedia �NB� and withSMP �NB+75 mL SMP�

seem to indicate that the presence of SMP improved the rate of

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methane production because it boosted the internalization of Cu,and as the amount of Cu adsorbed onto the biomass did notchange in the presence of SMP, the results may also suggest thata direct uptake of the complex SMP-Cu might have occurred.These results tend to agree with the findings of Gonzales-Gil et al.�2003�, who showed that the addition of yeast extract dramati-cally increased the bioavailability of Co and Ni in anaerobic re-actors, and these authors suggested that the complexation of Coand Ni with yeast extract amino acids was responsible for anincrease in metal solubility and bioavailability. However, the re-sults presented by Gonzales-Gil et al. �2003� suggest that metals“hitchhiked” into the cells because they were complexed withsubstrates, whereas the results presented here suggest that somemicrobial compounds were deliberately excreted to play a role inmetal uptake.

Fig. 6. Effect of chelating agents in reducing Cu toxicity in BMPbottles

Fig. 7. Copper distribution in BMP bottles;

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It is also possible that the increased rate of methane productionobserved in this study was due to the degradation of SMP viahydrogenotrophic methanogenesis. However, as can be seen inFig. 4�a�, the presence of SMP as the only carbon source didnot result in any methane production. The experiment with SMPwas repeated and the results showed the same trend �data notshown�; i.e., bottles inoculated with SMP resulted in a rate ofmethane production �16% higher when compared to the bottlesinoculated with normal biomedia. In such an experiment if allSMP present in the bottles “NB+SMP �30 mL�” and in thebottles “NB+SMP �75 mL�” were degraded or cometabolized,then an extra 4.5 and 11.5 mL of methane would be produced,respectively. However, gas production results showed that on day5 the difference in methane production in the bottles inoculatedwith and without SMP was 5.5 mL and did not seem to be depen-dent on the amount of SMP added. In addition, analyses of H2

in the headspace of “NB+SMP �75 mL�” was not statisticallydifferent from that found in the NB bottles �data not shown�,suggesting that the hydrogen uptake in the presence of SMP wasnot higher than in the control bottles. Therefore, the degradationof SMP by the fast-growing hydrogenotrophic methanogenic mi-croorganisms did not seem to be the reason for the higher rate ofmethane production in the NB+SMP bottles. Furthermore, thepresence of SMP seemed to increase the amount of Cu internal-ized, and this may be an indirect evidence that the uptake ofSMP-Cu is also used by the biomass to complement the un-specific and chemiosmotic-driven mechanism that relies onadsorption as the first step. One advantage of the uptake mecha-nism involving SMP-Me is that specific metals that are scarceor cannot compete for adsorption sites with abundant metals �Ca,Fe, Mg� can be specifically targeted by the excretion of specificmetallophores.

Toxicity of Metal Nutrients

Because heavy-metal ions cannot be degraded or modified liketoxic organic compounds, there are only three possible mecha-nisms for a heavy-metal resistance system: metal efflux, metalcomplexation, and metal oxidation/reduction inside the cell �Nies1999, 2003�. According to Nies �1999, 2003�, the resistance to-wards heavy metals relies, directly or indirectly, on efflux sys-tems. Such systems involve the excretion of soluble proteins andmetallophores into the bulk solution, and this is an importantissue for wastewater treatment systems, because the tolerance to-wards heavy metals in biological treatment systems may occur atthe expense of the excretion of some SMP and a consequent re-duction in treatment efficiency.

ttles black bars; NB+Cu2+ 1 mM gray bars
NB bo

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The third series of BMP bottles was carried out to investigatethe effect of chelating agents in mitigating copper toxicity. Fig. 6shows that the presence of 1 mM of Cu2+ �63.5 mg L−1� severelyaffected methanogenic activity. It is important to highlight that thesymptoms of toxicity are similar to trace metal deficiency; i.e.,there is an accumulation of volatile fatty acids, mainly acetate,propionate, and butyrate, which may be followed by a drop in pHdepending on the buffer capacity of the system, resulting in adecrease in methane production. As shown in Fig. 6, the methaneproduction in the bottles spiked with Cu2+ was less than 5%of that observed in the control bottles �NB bottles�. Accordingto the Visual MINTEQ software, the addition of 1 mM Cu2+ inNB bottles would result in the precipitation of CuCO3 �malachite�because of the high concentration of HCO3

−, leaving a solubleCu2+ concentration of �3.310−6 �0.21 mg L−1�. The formationof a brown precipitate was observed when Cu2+ was added tothe BMP bottles, and metal distribution showed that the solubleconcentration of Cu2+ 1 day after incubation was �1 mg L−1 andthat most of the Cu was precipitated as a sulphide or carbonatesalt �HNO3 and EDTA extracts, respectively�. Results on metaldistribution �Fig. 7� revealed that, in the presence of 1 mM Cu2+

there was a large amount of Cu more strongly associated withbiomass �Na4P2O7 extract�, and this fact may be directly relatedto toxicity.

Babich and Stotzky �1983� showed that to decrease or eli-minate Ni toxicity to Nocardia thodochrous the sequence ofprotection offered was EDTA�NTA�aspartate�citrate. In thepresent study, Fig. 6 shows that the addition of a mild chelatingagent, 1 mM citric acid, did not result in any toxicity mitigation.Visual MINTEQ simulations suggested that 1 mM citrate wouldincrease the copper solubility by 30 times, from 0.3 to 9.8%,allowing for a soluble concentration of copper of �6.2 mg L−1,and 96% of the dissolved copper would be associated with citrate.Because 1 mM EDTA was shown to inhibit methanogenesis,another chelating agent, 1 mM NTA, was added instead andthe results were surprisingly different. According to the VisualMINTEQ software, addition of 1 mM NTA would ensure that99.9% of the copper was dissolved, of which 99% would be as-sociated with NTA. NTA addition would prevent CuCO3 forma-tion, but NTA is not strong enough to prevent CuS formation,which would be limited by sulphide concentration. Fig. 6�a�shows that the best protection against copper toxicity was actuallyoffered by 1 mM NTA, which should dissolve 99.9% of the cop-per and make it mobile.

When Kuo and Parkin �1996� used anaerobic reactors toinvestigate the effect of Ni on SMP production and to determinethe SMP chelating properties, they found that reactors receivingNi produced more SMP when compared to the control reactors.The authors suggested that, in an anaerobic system containing300 mg L−1 of SMP, approximately 0.75 mM �44 mg L−1 Ni�could be chelated. According to Kuo and Parkin �1996�, the in-crease in SMP production was because microorganisms producedmore chelators in the presence of Ni in response to metal toxicity,so that free Ni levels would be kept at subtoxic levels. In order toverify whether SMP could offer protection against copper toxic-ity, some BMP bottles were inoculated with 1 mM Cu and 75 mLof SMP ��230 mg L−1�, and Fig. 6�b� shows that methane pro-duction rates in the bottles “NB+Cu2+” were not statistically dif-ferent from those in the bottles “NB+Cu2++SMP ”. These resultsshow that SMP could not mitigate Cu toxicity and that toxicitywas more related to the amount of metal adsorbed onto the bio-mass �Na2P2O7 extract� than to the amount of soluble metal.

It has become widely accepted that free metallic ions in the



aqueous phase are directly responsible for toxicity �Mosey 1976;Oleszkiewicz and Sharma 1990�, and this does not contradict ourfindings, because the more free metal present in solution, themore metal should be adsorbed onto the biomass until a saturationpoint is reached. The fact that 1 mM NTA offered the best pro-tection is probably because it can stop lethal concentrations ofCu2+ from being adsorbed to the cell by competing efficientlywith the strong chelating sites located at the cell surface, hencereducing the amount of Cu that gained access to the unspecificchemiosmotic-driven metal uptake system. As this general uptakesystem cannot be stopped �Nies 2003�, one way of mitigatingtoxicity is to prevent toxic metals from being adsorbed onto bio-mass. If toxic concentrations of metals are adsorbed, they mightinevitably be taken up, and when this happens the cell relies onefflux systems to pump out the metals in excessive concentra-tions, which in turn leads to the production of SMPs.

Conclusions

The results presented in this paper showed that the presence offree 1 mM EDTA reduced the methane production rate, and thisinhibition seemed to be related to the unavailability of metalscaused by the complexation of metal nutrients with EDTA. Addi-tion of SMP did not change the metal distribution in anaerobicsystems despite increasing the rate of methane production, and itseems that the degradation of SMP via hydrogenotrophic metha-nogenesis was not responsible for this increase. The metal distri-bution in systems inoculated with SMP suggested that specificmicrobial compounds might have been excreted to play a role inmetal uptake, probably by delivering nutrient metals to specificbinding sites located on the cell surface and/or by increasing Cubioavailability through direct uptake of Cu-SMP complexes. Ad-dition of SMP did not reduce Cu toxicity, and the best protectionwas offered when stoichiometric amounts of NTA, which shouldcomplex and solubilize most of the Cu, was added.

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