fermoso-uasb

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Metal supplementation to UASB bioreactors: from cell-metal

interactions to full-scale application

Fernando G. Fermosoa, Jan Bartaceka,b, Stefan Jansenc,1, Piet N.L. Lensa,b,

aSub-department of Environmental Technology, Wageningen University, Biotechnion-Bomenweg 2, P.O. Box 8129, 6700 EV Wageningen,

The NetherlandsbPollution Prevention and Control core, UNESCO-IHE, P.O. Box 3015, 2601 DA Delft, The NetherlandscLaboratory of Physical Chemistry and Colloid Science, Wageningen University, Dreijenplein 6, 6703 HB Wageningen, The Netherlands

A R T I C L E D A T A A B S T R A C T

Article history:

Received 29 May 2008

Received in revised form

16 October 2008

Accepted 16 October 2008

Available online 16 December 2008

Upflow anaerobic sludge bed (UASB) bioreactors are commonly used for anaerobic

wastewater treatment. Trace metals need to be dosed to these bioreactors to maintain

microbial metabolism and growth. The dosing needs to balance the supply of a minimum

amount of micronutrients to support a desired microbial activity or growth rate with a

maximum level of micronutrient supply above which the trace metals become inhibitory or

toxic. In studies on granular sludge reactors, the required micronutrients are undefined and

different metal formulations with differences in composition, concentration and species are

used. Moreover, an appropriate quantification of the required nutrient dosing and suitable

ranges during the entire operational period has been given little attention. This review

summarizes the state-of-the-art knowledge of the interactions between trace metals and

cells growing in anaerobic granules, which is the main type of biomass retention in

anaerobic wastewater treatment reactors. The impact of trace metal limitation as well as

overdosing (toxicity) on the biomass is overviewed and the consequences for reactor

performance are detailed. Special attention is given to the influence of metal speciation in

the liquid and solid phase on bioavailability. The currently used methods for trace metal

dosing into wastewater treatment reactors are overviewed and ways of optimization are

suggested.

2008 Elsevier B.V. All rights reserved.

Keywords:

Metal supplementation

UASB

Granular sludge

Metal requirements

1. Introduction

Most industrial effluents contain trace quantities of a variety

of metals, of which many are classified as priority pollutants(EC, 2000). Microbial biofilms, natural or engineered, can be

used to remediate heavy metal pollution by biochemical

modification and/or the accumulation of toxic metal ions

(Muoz et al., 2006; Singh et al., 2006). An understanding of the

fate of metals in biofilms, as anaerobic granular sludge, is

crucial for the successful design of biological wastewater

treatment systems that are used for biodegrading organic

contaminants, which are frequently intermingled with metal

pollution (Singh et al., 2006).Most of these metals are also necessary for biological

growth or activity, and absence of sufficient quantities limits

the activity of the microbial population present in the

anaerobic bioreactors (Fermoso et al., 2008c). As technological

changes take place in themanufacturing or industrial process,

S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 7 ( 2 0 0 9 ) 3 6 5 2 3 6 6 7

Corresponding author. Sub-department of Environmental Technology, Wageningen University, Biotechnion-Bomenweg 2, P.O. Box8129, 6700 EV Wageningen, The Netherlands.

E-mail address: [email protected] (P.N.L. Lens).1 Present address: TNO/Deltares, Soil and Subsurface Systems, Princetonlaan 6, PO Box 85467, 3508 AL Utrecht, The Netherlands.

0048-9697/$ see front matter 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2008.10.043

a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m

w w w . e l s e v i e r . c o m / l o c a t e / s c i t o t e n v
mailto:[email protected]://dx.doi.org/10.1016/j.scitotenv.2008.10.043http://dx.doi.org/10.1016/j.scitotenv.2008.10.043mailto:[email protected]


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the compounds discharged and thus also the wastewater

characteristics change (Pons et al., 2004). The exact amounts of

trace nutrients needed varies for the different types of

wastewaters (Anon, 2000; Liang et al., 2007). Nowadays, trial

approaches are used to asses their benefit for anaerobic

processes (Opure BV, personal communication). The metal

dosingcan be optimized by a better fundamentalinsight in the

chemical, microbiological and engineering aspects determin-

ing the metal requirements of UASB reactors.

The Upflow Anaerobic Sludge Bed (UASB) process is the

most applied process for the anaerobic treatment of

industrial effluents (Van Lier, 2008). In this process, granular

sludge, a spherical biofilm developed by autoaggregation of

cells (Lettinga et al., 1979), is formed (Fig. 1A). The aim of

Fig. 1 (A) Multidisciplinary approach and (B) analytical infrastructure required for determining the interactions of metals with

the solid phase, liquid phase and the microorganisms present in anaerobic bioreactors, which leads to a more rational and

efficient metal supply to bioreactors. UASB reactor: Upflow anaerobic sludge bed reactor; AVS: Acid volatile sulfide; DMT:

Donnan membrane technique; SMA: Specific methanogenic activity.

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this review is to summarize the knowledge required to

understand the metal nutrient requirements and facilitate

design of metal supplementation in UASB reactors (Fig. 1).

The description of the interactions between metals and

microbes are given first. This allows to understand the role

that metals have in microbial systems, e.g. the role of metals

in enzymatic functioning and concepts of metal uptake. The

next step to gain a deeper knowledge of the trace metal

requirements in UASB reactors is the study of the possible

interactions between trace metals and UASB granules. The

role of metal speciation in the reactor liquid media is

evaluated as well. Metal precipitation, dissolution and

complexation with organic and inorganic ligands play an

important role in the fate of metals in UASB reactors. The

metal requirements of UASB reactors are then assessed by

describing the impact of trace metal limitation on the

microbial activity on the one hand and the toxic effects

caused by an excess of them on the other hand. Subse-

quently, different trace metal dosing strategies are re-

viewed and evaluated. Finally, recommendations for future

research in the field of trace metal supplementation are

given.

2. Metalsmicrobe interaction

2.1. Role of metals in the enzymatic system

Metals are important in all biochemical processes in any kind

of living organism. Of the 95 naturally occurring elements of

the periodic table, no less than 25 have an essential biologicalfunction (Frnzle and Markert, 2002). Some of these, e.g. zinc,

nickel, copper, selenium, cobalt, chromium, molybdenum,

tungsten, manganese or iodine are required in small amounts

andare termed essential trace elements.Most of them arepart

of the active site of enzymes. Table 1 lists the role of some

essential trace elements in various enzymes catalyzing

anaerobic reactions and transformations (Oleszkiewicz and

Sharma, 1990; Zandvoort et al., 2006b).

The presence of high metal concentrations in a bulk liquid

does not mean that microorganisms take the metal up and

incorporate it into the catalytic centers of their enzymes

(Ermler, 2005). For instance, microorganisms take up metals

also in the form of complexes, such as e.g. Vitamin B12(Ferguson and Deisenhofer, 2004), Co-citrate (Krom, 2002) or

bound to siderophores (Worms et al., 2006). More in general, in

between the presence of metals in the bulk liquid and the

actual biological effects, complex processes as biouptake and

biosynthesis play an important role. Some of these processes

are discussed in more detail later in this review.

Heavy metals act as inhibitors by blocking enzyme func-

tions. The toxic effect of heavy metals is primarily non-

specific and reversible (Nies, 1999). This type of inhibition is

characterized by the reversible binding of the inhibitor with

either the enzyme or the enzyme-substrate complex. Less

frequently, metals act as competitive inhibitors (they compete

with the substrate). This type of inhibition depends on the

affinity of the metal and enzyme, as well as on the relative

concentrations of the competing metals (Oleszkiewicz and

Sharma, 1990).

2.2. Metal uptake into the cell

It is generally accepted that the speciation of metals has a

huge impact on their uptake (and ensuing biological effects),

as has been demonstrated in detail for algae and some

microorganisms (Hudson, 1998; Slaveykova et al., 2004;

Sunda and Huntsman, 1998).

In general, the transport of metal ions is to a great extent

determined by the properties of the transport systems (Braun

et al., 1998). For instance, transport of cobalt and nickel is

reported to proceed either via specific cobalt and/or nickel

transporters, or via magnesium transporters, e.g. for cobalt

(Degen et al., 1999; Kobayashi and Shimizu, 1999; Komeda

et al., 1997; Pogorelova et al., 1996; Saito et al., 2002); and nickel

(Eitinger and Mandrand-Berthelot, 2000; Mulrooney and Hau-

singer, 2003; Watt andLudden, 1999). The uptakeof metal ions

by these specific transporters can be described by Michaelis

Menten kinetics, where the bioavailable metal ion is first

bound by a transporter site and subsequently taken up. The

binding properties determine the affinity of the transporter to

themetal, while theamount of thetransporter determines the

maximum uptake rate. Both of these parameters can change

Table 1 Role of some essential trace elements in variousenzymes involved in anaerobic reactions andtransformation.

Element Functions Element Functions

Cu Superoxide

dismutase

Ni CO-dehydrogenase

Hydrogenase

(Facultative

anaerobes)

Acetyl-CoA synthase

Nitrite reductase

Methyl-CoM

reductase (F430)

Acetyl-CoA

synthase

Urease

Stabilize DNA, RNA

Hydrogenase

Co B12-enzymes Se Hydrogenase

CO-

dehydrogenase

Formate

dehydrogenase

Methyltransferase Glycin reductase

Fe Hydrogenase W Formatedehydrogenase

CO-

dehydrogenase

Formylmethanofuran-

dehydrogenase

Methane

monooxygenase

NO-reductase

Aldehyde-

oxydoreductase

Superoxide

dismutase

Antagonist of Mo

Nitrite and

Nitrate reductase

Nitrogenase

Mn Stabilize

methyltransferase

in methane-

producing

bacteria.

Zn Hydrogenase

Redox reactions

Formate

dehydrogenase

Superoxide dismutase

Mo Formate

dehydrogenase

V Nitrogenase

Nitrate

reductase

Chloroperoxydase

Nitrogenase

Bromineperoxydase

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with changing chemistry or biology. Different metal ions can

compete for the same uptake site, thus affecting the condi-

tional affinity (Sunda and Huntsman, 1998). Furthermore, the

organism can actively decrease or increase the number of

transporters in response to its environment (Worms et al.,

2006), thus affecting the maximum uptake rates.

To quantify metal toxicity on biological systems,Pagenkopf

(1983) proposed the Gill Surface Interaction Model (GSIM),

while Morel (1983) formulated the Free-Ion Activity Model

(FIAM), schematically demonstrated in Fig. 2. Both the GSIM

and FIAM are the theoretical predecessors of the Biotic Ligand

Model (BLM) (Niyogi, 2004).The BLM has been proposed as a

tool to evaluate quantitatively the manner in which water

chemistry affects the speciation and biological availability of

metals in aquatic organisms, such as fish or Daphia magna

(Niyogi, 2004). This is an important consideration because it is

the bioavailability and bioreactivity of metals that control

their potential to cause adverse effects (Paquin et al., 2002).

The BLM is based on the hypothesis that toxicity is not simply

related to the total aqueous metalconcentration, butthat both

metalligand complexation and metal interactions with

competing cations at the site of toxic action need to be

considered (Meyer et al., 1999; Pagenkopf, 1983).

TheBLM model relieson theassumptionthat the processes

outside the organism are at equilibrium. This simple model

has been criticized, since its applicability has been shown to

be limited (Campbell et al., 2002; Hassler et al., 2004). For

instance, diffusion limitation, as expected to occur at low

concentrations, in combination withhigh affinity uptake sites,

will certainly cause deviations (Hudson, 1998). In these cases,

labile species will also be bioavailable (Van Leeuwen, 1999).

Secondly, not only free metal ions, but also lipophilic

complexes, and sometimes even hydrophilic complexes,

have been reported to be taken up as well (Phinney and

Bruland, 1994). Furthermore, biological responses can have a

significant impact on the metal speciation and bioavailability

in the exposure media due to excretion and changes of the

uptake properties (Slaveykova et al., 2004).

These toxicity models were established and verified for

natural waters and higher organisms such as freshwater algae

Chlorella kesslerii (Hassler et al., 2004), Urchin larvae (Lorenzo etal., 2006) or mussels (Mytilus galloprovincialis) (Beiras et al., 2003).

Thus far, this type of models has never been applied to the

anaerobic consortia present in biofilms or granules from waste-

water treatment reactors.

3. Metalsbiofilm interaction

3.1. Metal bioavailability in granular sludge

Metal sorption by anaerobic granules and similar aggregates

has been studied by many authors (Artola et al., 2000;Gonzalez-Gil et al., 2001; Gould and Genetelli, 1978; MacNicol

and Beckett, 1989; Osuna et al., 2003; Zandvoort et al., 2004).

Overall, these studies confirm strong sorption of metal ions in

granules due to precipitation, coprecipitation, adsorption and

binding by Extracellular Polymeric Substance (EPS) and

bacterial cells. EPS are major components of granular matrix,

and up to 90% of the dry biomass is EPS material ( Gao et al.,

2008), which bind metals (Guibaud et al., 2008). Furthermore,

the bacterial interface itself can also serve as an important

metal binding surface (Aksu et al., 1991).

X-ray analysis of UASB granules has confirmed the

existence of copper, iron, zinc and nickel sulfide precipitates

(Fang and Liu, 1995; Gonzalez-Gil et al., 2001; Kaksonen et al.,

2003; Liu and Fang, 1998). The bioavailability and mobility of

essential trace metals in the UASB reactors are mainly

controlled by the sulfide chemistry (Van der Veen et al.,

2007). On the basis of stability constants from literature

(Martell & Smith, 1989) and the composition of the anaerobic

wastewater environments, metal ions are expected to pre-

cipitate with sulfide, carbonate and phosphate in the pore

water present in the granular matrix. Metal sulfide precipita-

tion is expected to be the most important process. The

predominating role of sulfides in metal fixation in anaerobic

granules is supported by the high acid volatile sulfide (AVS)

content and the high metal content in the oxidizable (contain-

ing bothsulfidic andorganic bondingforms) fraction presentin

UASB systems (Van der Veen et al., 2007).

As metal sulfides have a very low solubility product

(Martell and Smith, 1989), it would be expected that these

metals are non-bioavailable to the methanogenic consortia.

Jansen et al. (2007) proposed nevertheless that in most cases,

the dissolution rates of cobalt and nickel sulfides do not limit

the methanogenic activity in anaerobic wastewater treatment.

It should, however, be noted that this was based on batch

experiments with methanogenic enrichments and freshly

prepared metal sulfide precipitates. Ageing of sulfidic pre-

cipitates, which takes place in the sludge during reactor

operation, will lower thedissolution rates and might therefore

lower the metal bioavailability (Gonzalez-Gil et al., 2003).

Fig. 2 Conceptual framework of the FIAM and BLM, including

(1) mass transport of the free metal (Mz+) and a hydrophilic

complex (ML) in solution; (2) dissociation/complexation with a

ligand in solution; (3)specific (M-Rcell) or non-specific(M-Acell)

adsorptionof the metal tothe surfaceof theorganism; (4) metal

transport intothe organismcharacterizedby an internalization

rate constant (kint) and a subsequent reaction an intracellular

ligand (MLbio); (5) expression of the biological effect. The box

identifies thereactions that are taken into account in the FIAM

and BLM (AfterHassler et al., 2004).



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The bioavailability of retained metals in granular sludge

can be assessed from the bonding form distribution, although

the fractions are operationally-defined and not phase-specific

(Van Hullebusch et al., 2005). The fractions possess a decreas-

ing solubility/reactivity from the first to the last step. This

decreasing solubility can be used as a measure of potential

bioavailability (Jong and Parry, 2004), beginning with the

exchangeable fraction, the most bioavailable, the carbonatefraction, the organic matter/sulfide fraction and the last

fraction, residual, the least bioavailable.

3.2. Metal toxicity in granular sludge

There is only a small difference between the optimal and toxic

free cobalt concentration: the optimal concentration is approxi-

mately 7 mol L1 with an apparent KM value of 0.9 mol L1

(based on totalcobalt addition) (Zandvoort et al., 2002a), whereas

a free cobalt concentration of only around 18 mol L1 is already

significantly (50% inhibition) toxic (Bartacek et al., 2008). This

means that in case only a small amount of complexing agents is

present in the medium, a relatively low total cobalt concentra-

tion will cause operational problems with anaerobic reactors.

Metal toxicity cannot be evaluated solely based on the

metal precipitated/sorbed in methanogenic granular sludge,

butrequiresalso thedetermination of the speciation of metals

present in the bulk solution (Paquin et al., 2000; Plette et al.,

1999; Shuttleworth and Unz, 1991). Although not only the free

cobalt species can be transported into a microbial cell,

Bartacek et al. (2008) suggested that toxicity of cobalt can be

assessed based on the free cobalt concentration. The latter

statement is only valid when the process conditions such as

pH and concentration of competing metals (namely calcium or

magnesium) are kept constant.

The donnan membrane technique (DMT) was successfully

used for measurement of the free cobalt (Co2+) concentration in

anaerobic granular sludge (Bartacek et al., 2008). Similarly,

Temminghoff et al. (2000) obtained a good agreement between

calculated and measured (with the DMT) free metal concentra-

tions for copper and cadmium with and without inorganic or

(synthetic as well as natural) organic complexation. A practical

problem of the DMT setupas used by Bartacek et al. (2008) is the

long time required to obtain experimental data (4 days for

equilibrium establishment, five days for DMT measurement).

This disables DMT for measurements of free metal concentra-

tions under dynamic conditions in bioreactors.

4. Metalsliquid phase interaction

4.1. Particulate metal binding

As for the pore water in the granular biofilm, metal sulfide

precipitation is expected to be the most important process in

metal precipitationin theanaerobic media presentin theUASB

reactor: the low solubility product of metal sulfides will result

in extremely low free metal ion concentrations (Martell and

Smith, 1989). However, onehas to be careful in tryingto predict

free metal ion concentrations from these solubility products.

In the first place, to know the solubility product, the crystal

structure of the metal sulfide precipitate should be known.

Secondly, the errors in the literature values for the solubility

products are very large, and different literature sources give

different values. Thirdly, precipitation equilibrium is actually

not reached in many cases due to kinetic limitations. And

finally, size and ligand effects can affect precipitation. Apart

from precipitation in the form of well defined precipitates,

coprecipitation and adsorption are alsoimportantphenomenain a mixture of metal ions. These processes have been studied

in sediments, typically containing excess of iron over other

metals(Cooper andMorse, 1999; Huerta-Diaz et al., 1998; Morse

andLuther, 1999). Considerableadsorption andcoprecipitation

of cobalt and nickel on FeS (mackinawite) occurs (Morse and

Arakaki, 1993). Kinetic factors can further influence coprecipi-

tation (Morse and Luther, 1999).

4.2. Liquid phase speciation

Sulfide is not only important because of the formation of

metal precipitates, but also because of the formation of

dissolved metal complexes. Dissolved metal sulfide species

have only been studied over the last 20 years, and theoretical

estimates date back only some 20 years (Dyrssen, 1988), while

measurements are available only since 15 years (Al-Farawati

and Van Den Berg, 1999; Luther et al., 1996; Zhang and Millero,

1994). Although the data vary, they demonstrate that dis-

solved metal sulfide complexes are very strong.

Other important inorganic ligands are CO32 and PO4

3.

Although the complexes are less strong than the metal sulfide

complexes, the importance of carbonate and phosphate

complexes was demonstrated for anaerobic media (Bartacek

et al., 2008; Callander and Barford, 1983). Carbonate is very

important because of its high concentration in wastewaters,

and its strong binding with metal ions, especially with Ni(II).

Unfortunately, somedisagreement aboutthe binding constant

exists in the literature (Hummel and Curti, 2003; Turner et al.,

1981). The ligands OH, SO42 and Cl are also present at large

concentrations, but are relatively unimportant because of

their weak binding.

Metal binding by Soluble Microbial Products (SMPs) was

demonstrated for nickel (Kuo and Parkin, 1996; Kuo etal., 1996)

and copper (Bender et al., 1970). Strong zinc complexing

ligands were detected by voltammetry in media after growth

of sulfate reducing bacteria (Bridge et al., 1999). The results

suggest that excretion of metal binding SMP can serve as a

mechanism to reduce metal toxicity. It is still an open

question whether the microorganisms in anaerobic waste-

water treatment systems actively excrete organic metal

ligands to bind metals in order to overcome limitation, as is

known for iron (Neilands, 1995) and cobalt (Saito et al., 2002).

Apart from organic ligands produced by microorganisms,

sometimes organic ligands are added. First, some organic

substrates can have metal binding properties, e.g. in the case of

acetate. However, most of these complexes are weak compared

to the strong complexes with sulfide (Martell and Smith, 1989).

Furthermore,sometimes synthetic ligandssuch as EDTA, NTAor

citrate are added. In some cases they are added deliberately, for

instanceto keep the metals dissolved (Bretler and Marison, 1996;

Hartung, 1992), in other cases they are present in the waste

stream, possibly as waste products (Nowack, 2002).



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Organic ligands might influence precipitation. In general,

in their presence, the concentration of dissolved metal is

increased. However, conflicting data are also found. For

instance, in a system containing Ag+ and amorphous FeS,

dissolved Ag+ was smaller than predicted in the presence of

high concentrations of the thiol compounds 3-mercapto-

propanoic acid and cysteine (Adams and Kramer, 1998). A

possible explanation for this could be the binding of the Ag-thiol complexes to FeS (Adams and Kramer, 1998). In

another study, the solubility of CuS was increased by thiols,

while that of PbS and CdS was not (Shea and MacCrehan,

1988a,b). Furthermore, organic ligands can affect the size

(Pommerenk and Schafran, 2005) and kinetics (see below) of

precipitation.

4.3. Kinetics of precipitation and complex formation

Within the dynamic bioreactor environment, relative rates of

the various processes can be of great importance, in particular

the kinetics of particulate formation and dissolution. Pre-

cipitation can be divided into 5 stages (Nielsen, 1964):

nucleation, growth of nuclei, aggregation, formation of

irreversible aggregates and formation of larger crystals at the

expense of smaller ones. In parallel with step 5, precipitates

age, generally transforming from amorphous precipitates to

more stable crystalline forms. Metal sulfide dissolution in near

neutral pH rangeshas only been studied for mackinawite (FeS)

(Pankow and Morgan, 1979, 1980) and for similar systems such

as sediments (Harper et al., 1998; Motelica-Heino et al., 2003).

Besides precipitation, the kinetics of adsorption onto and

desorption from precipitates can be very important, as exempli-fied by the coprecipitation/adsorption kinetics of copper, zinc,

lead and cadmium to FeS (Davis et al., 1994). The kinetics of

precipitation and dissolution are influenced by organic ligands:

they can decrease the precipitation rate (Helz and Horzempa,

1983; Shea and Helz, 1987) or increase the dissolution rate, e.g. in

case of siderophores (Cervini-Silva and Sposito, 2002; Kraemer

and Hering, 1997; Liang et al., 2000). Besides the reaction kinetics

involving theparticulate matrix,therates of processes withinthe

dissolved metal fraction can be important. In aqueous solutions,

therate of metal complexformationlargelydepends on thewater

loss rate constantsof the metal ions (Morel,1983), independent of

the nature of the ligand. From this so called Eigen mechanism,

the order of the reaction rates of some of the relevant metal ions

is given as: Fe2+NCo2+NNi2+. Especially for Ni2+, complexation

reactions are known to be relatively slow (Margerum et al., 1978).

Fig. 3 (A) Boundary conditions for metal addition to keep the UASB reactor efficiency optimal. (B) Effect of cobalt concentration

on methanogenic activityusing methanol as substrate (pH 7.0; 30 C) of anaerobic granular sludge present in the UASB reactors.

() 5 M cobalt (optimal), () 0 M cobalt (limitation) and () 100 M cobalt (toxicity).



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Furthermore, in solutions containing high concentrations of

competing ions such as Ca2+and Mg2+, exchange reactions canbe

inherently slow (Hering and Morel, 1989, 1990).

5. MetalsUASB interaction

Metal supplementation in UASB reactors is a compromisebetween achieving the maximal biological activity of the

biomass present in the reactor, while minimizing the costs of

the supplied metal andthe metal losses into theenvironment.

The boundary conditions to keep a stable reactor operation

vary between nutrient deficiency due to lack of essential

metals and toxicity due to their excess (Fig. 3). The metal

addition strategy affects the metal losses and costs of the

added metal in order to achieve the optimal metal concentra-

tion inside the UASB reactor. In literature, different metal

formulations have been used in studies on UASB reactors, but

they differ in composition, concentration and the forms in

which they are supplemented to the feed (Singh et al., 1999;

Tiwari et al., 2006; Zandvoort et al., 2004). Also, the appropriatequantification and suitable ranges in which nutrient dosing is

required during the entire operational period are poorly

understood (Zandvoort et al., 2006b). Thus, knowledge about

the effect of metals in the biomass and the metal distribution

through the UASB system is necessary in order to obtain a

proper strategy for metal supplementation.

5.1. Metal limitation in UASB reactors

5.1.1. Methanol fed bioreactors

Omission of a divalent cation metal (cobalt, nickel or zinc)

from the feed of methanol-fed anaerobic granular sludge

bioreactors leads to a reduced specific methanogenic activity

(SMA) on methanol and, consequently, to methanol accumu-

lation in the effluent, followed by the enhanced formation of

acetate as shown in Fig. 4 (Fermoso et al., 2008b,c,d). Thus,

during methylothrophic methanogenic degradation of metha-

nol, the decrease of SMA of the sludge with methanol as the

substrate is the variable to follow for predicting possible

methanol accumulation, further acetate accumulation and

ultimately complete reactor acidification.

The time period required to achieve nickel limitation

(140 days of operation, (Fermoso etal., 2008d)),as also observed

by Zandvoort et al. (2002b), was much longer compared to that

required for cobalt limitation, which is already achieved in

Fig. 4 (A)Proposedoutline of themechanism of induction of metal limitationin methanol-fedUASB reactors. Numbers indicate

predominant process. (1) methylotropihic methanogenesis. (2) acetogenesis. (3) acetotrophic methanogenesis. SMA: specific

methanogenic activity with methanol as the substrate (AfterFermoso et al., 2008a,b,c). (B) Decay of SMA with methanol as the

substrate over time due to absence of cobalt addition in the feed of a methanol-fed UASB reactor (pH 7.0; 30 C; Organic loading

rate: 10 g COD L1 d1). Day 0 indicates the pulse addition of cobalt (5 M, cobalt in vitamin B12) to the bioreactor; days 15 to 0

clearly illustrate a trace metal (cobalt) limited reactor operation.



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15 days (Fermoso et al., 2008c). Kida et al. (2001) observed that

the methanogenic activity with acetate as the substrate (pH 7,

37 C), as well as the F430 and corrinoid concentrations in

methanogenic mesophilic biomass from a sewage treatment

plant decreased with decreasing amounts of Ni2+ and Co2+

supplied. Theomission of nickelmight thus resultin a reduced

amount of coenzyme F430, thereby decreasing the SMA of the

sludge. The reduced SMA in the absence of nickel was notobserved until day 129 (Fermoso et al., 2008d). The initial

concentration of nickel in the seed sludge (43 g g TSS1) was

probably enough to support the enzymatic activity and

synthesis of coenzyme F430 for a considerably long time, even

if the size of the Methanosarcina population increased. More-

over, many organisms respond to a lower metal bioavailability

by an increased synthesis of metal transporters (Sunda and

Huntsman, 1998). This type of adaptation to low nickel

concentrations might also have contributed to the longer

time-period before a decrease in methylotrophic activity is

observed (Zandvoort et al., 2002b).

Only a few papers have addressed deprivation of other

essential metals during methanogenesis, as zinc or iron.

Osuna et al. (2002) studied metal deprivation in VFA fed

UASB reactors. The latter authors found that when adding

solely zinc to the medium, the SMA with acetate as the

substrate of metal deprived sludge increased by 36%. Sauer

and Thauer (1997) foundin enzymatic studies thatthe transfer

of the methyl group from CH3-MT1 to coenzyme M by MT2

involved in methanol degradation depends on the Zn2+

concentration. The KM value of this transfer reaction

amounted to 0.25 nM free Zn2+. This KM value is around 240

times lower than the dissolved zinc concentration in the

synthetic wastewater of a zinc limited methanol fed UASB

reactor (60 nM) at the moment zinc limitation has developed

(Fermoso et al., 2008b). The metalbiofilm interactions induce

differences between the Km value found by Sauer and Thauer

(1997) (with enzymes) and the dissolved zinc concentration at

the time of zinc limitation found by Fermoso et al. (2008b)

(with sludge granules).

Cobalt, nickeland zinc are componentsof different enzymes

involved in anaerobic methanol degradation (Fermoso et al.,

2008b,c,d). This suggests that also other metals present in the

enzyme system involved in methanol degradation may follow

the same pattern as well, e.g. iron which is present in

heterodisulfide reductase (Deppenmeier et al., 1999) or copper

which is present in Acetyl-CoA synthase (Seravalli et al., 2003).

5.1.2. Other substrates in anaerobic bioreactors

Zandvoort et al. (2006a) screened four full-scale bioreactors for

their response to trace metals, using three methanogenic

substrates, viz. methanol, acetate and H2/CO2. The four

reactors treat alcohol distillery wastewater, paper mill waste-

water, groundwater contaminated with perchloroethane and

brewery wastewater. In accordance with Section 5.1.1.,

Zandvoort et al. (2006b) observed a significant response

using methanol as the substrate. However, no significant

response to the supply of metals was found in activity tests

with acetate or H2/CO2 as the substrates. The trace metals

initially presented in each of the granular sludge inocula used

are sufficient for attaining the optimal activity on acetate and

H2/CO2 for all four sludges tested (Zandvoort et al., 2006b).

Propionate is a key intermediate in the conversion of

complex organic matter under methanogenic conditions (De

Bok et al., 2004). Propionate oxidation can only proceed if the

product H2 and formate are removed by methanogens or other

H2 or formate utilizing bacteria (Stams, 1994). Molybdenum,

tungsten and selenium are essential trace metals and are

often essential for enzymes catalyzing reactions, such as

formate dehydrogenase (FDH), which catalyze formate pro-duction by propionate oxidizers (Dong et al., 1994). In defined

cocultures of Syntrophobacter fumaroxidans and Methanospiril-

lum hungatei, grown on propionate, molybdenum and tungsten

limitation lowered the methane production rate and the FDH

activity in cell extracts of each organism (Jiang, 2006). There-

fore, in case of insufficient amounts of molybdenum and

tungsten are present in theinfluent, a limitation of propionate

oxidation is likely to develop in full scale reactors with

propionate as a key-intermediate.

5.2. Metal toxicity in bioreactors

Trace metals at a too high concentration can cause inhibition

of the methanogenic process (Bhattacharya et al., 1995; Ram

et al., 2000). Although heavy metal toxicity in UASB reactors

has been studied abundantly, reported values of toxic

concentrations vary considerably between different authors

(Fang, 1997; Fang and Hui, 1994; Karri et al., 2006; Lin and

Chen, 1997, 1999). In view of the importance of nickel and

cobalt in methanogenic processes, toxicity studies of these

metals are of high importance. The inhibition of methano-

genic activity from anaerobic sludge by nickel and cobalt has

been studied abundantly and high differences in toxic

concentrations were found (Table 2). This is due to the fact

that most authors report the total metal concentration in the

liquid phase, and do not consider metal speciation. Therefore,

the observed toxic concentrations are affected by differences

in medium composition, which causes changes in metal

speciation (Jansen et al., 2007; Worms et al., 2006).

5.3. Metal dosing

5.3.1. Metal dosing requirements

Bioreactor studiesindicate thatmetalsupplementation improves

in most cases the reactor efficiency, mainly measured as the

increase of methane production. However, metal supplementa-

tion toanaerobic wastewater treatment systems hasnot yetbeen

systematically researched (Table 3). Moreover, the approaches

used to study metal supplementation are hardly comparable.

Nutrient supply has also been studied at different tempera-

tures, ranging from 7 C (Li et al., 2007) up to 55 C (Paulo et al.,

2004). Different reactor configurations have been used in these

studies: such as UASB reactors (Espinosa et al., 1995; Shen et al.,

1993), fed-batch UASB sludges (Sharma and Singh, 2001),

anaerobic download fixed film reactors (Murray and Van den

Berg, 1981), anaerobic film expanded bed reactors (Kelly and

Switzenbaum,1984), upflow flocculent sludge reactors(Callander

and Barford, 1983) or continuous stirrer tank reactors (Percheron

et al., 1997).

Also different substrates have been fed to these metal

supplementation studies, including volatile fatty acids (Hoban

and Van Den Berg, 1979; Shen et al., 1993; Speece et al., 1983 ),



9/16

sulfate laden organics (Patidar and Tare, 2006), cane molasses

stillage (Espinosa et al., 1995), methanol (Florencio et al., 1993),

food industry wastewater (Oleszkiewicz and Romanek, 1989),

distillerywastewater (Sharma andSingh,2001), beanwastewater

(Murray and Vanden Berg, 1981),whey(Kelly and Switzenbaum,

1984), sugar cane fermentation waste (Callander and Barford,

1983) or molasses wastewater (Percheron et al., 1997).

5.3.2. Dosing protocol

In continuous methanol fed UASB reactors, cobalt supple-

mentation is required to maintain a high SMA, and therefore,

to prevent reactor acidification (Zandvoort et al., 2003).

Different cobalt dosing strategies have been studied in

methanol fed UASB reactors: continuous addition of a low

CoCl2 concentration to a methanol fed UASB reactor only

slightly enhanced the SMA of the sludge (Zandvoort et al.,

2002a). The same author studied the possibility of pre-loading

sludge by pre-incubating the inoculum in a 1 mM CoCl2solution for 24 hours. Pre-loading clearly overcame cobalt

limitation in the methanol fedUASBreactor(Zandvoort et al.,

2004). Pulse dosing of cobalt increased the SMA with

methanol as the substrate 4 times, clearly overcoming cobalt

limitation (Zandvoort et al., 2004). A drawback of these

dosing protocols is that high amounts of cobalt are lost with

the effluent.

5.3.3. Chemical species to be dosed

Repeated pulse addition of low amounts of cobalt is an efficient

dosing strategyto maintaina stable methanogenicmethanol fed

UASB reactor. Pulseadditionof cobalt(5 molescobalt perlitreof

reactor volume) in the form of CoCl2 creates a pool of cobalt in

the granular sludge matrix due to the high cobalt retention

(around 90%). In contrast, a much smaller cobalt pool is formed

when cobalt is dosed as Co-EDTA2: only 8% of the supplied Co-

EDTA2 is retained (Fermoso et al., 2008a). Additionally, cobalt

wash-out wasmuch less compared to the pulse addition of high

amountsof cobaltor thepre-loading cobalt strategiesreported in

previous studies with methanol fed UASB reactors. The cobalt

pool formed upon CoCl2 dosing was enough to keep stable

methanogenesis at an OLR of 8.5 g COD L1reactord1 for more than

15 days between the pulses. Additionally, pulse addition of CoCl2presents several advantages compared to Co-EDTA2. First of all,

as cobalt added as chloride is much more retained in the

granular sludge compared to cobalt bound to EDTA, its addition

frequency would be much lower, i.e. once per 15 days for CoCl2versusonceper 7 days forCo-EDTA2. Theside-effectsthatEDTA

Table 2 Toxic concentrations of cobalt, nickel and zinc in methanogenesis processes.

Metal Organism Reactor /technique

Toxic concentration Conditions Reference

Cobalt Mixed anaerobic

sludge

Anaerobic

toxicity

bioassays

Up to 35400 mg L1 (no

detectable inhibition)

The sludge was fed with nutrients and acetate or

glucose as the sole carbon source under oxygen-

free condition at 351 C.

Bhattacharya

et al. (1995)

600800 mg L1 (717%

inhibition)

950 mg L1 (100% inhibition)

15-day-old wet

slurry from

digested cattle

manure

Batch

bottles

120 g g dry matter1 The experiment was performed at 37 C. Jain et al.

(1992)

Nickel Anaerobic granular

sludge

UASB

reactor

81 mg L1 (50% inhibition of

VFA degradation)

The digesters were acclimated in a 13.5-liter UASB

reactor at 351 C. The HRT was 1 and 2 days.

Lin and Chen

(1999)


VFA degradation)

Anaerobic starch-

degrading granules

UASB

reactor

118 mg L1 (IC50 or 50%

inhibition of SMA)

The reactor was continuously fed with synthetic

wastewater composing starch as the sole organic

carbon source.

Fang and Hui

(1994)

Anaerobic granular

sludge

UASB

reactor

81 mg L1 (50% inhibition, bed

sludge)

The reactors were treating winery wastewater and

were acclimated in a 13.5-liters UASB reactor at 35

1 C. TheHRT was 1 day and the acclimation period

for the seed sludge was six months.

Lin and Chen

(1997)

78 mg L1 (50% inhibition,

blanket sludge)

Anaerobic granular

sludge from lab-

scale UASB reactor

UASB

reactor

118 mg L1 L (IC50) Granules were sample from four UASB reactors at

COD loading rate of 10 g COD L1 d1 for over six

months at 37 C.

Fang (1997)

Zinc Anaerobic granular

sludge from lab-

scale UASB reactor

Batch

bottles


methanogenic activity with

sludge operated at HRT 1 day)

The experiment was performed at 351 C

Winery wastewater as substrate.

Lin and Chen

(1999)


methanogenic activity with

sludge operated at HRT 2 day)

Anaerobic granular

sludge from lab-

scale UASB reactor

Batch

bottles


methanogenic activity)

The experiment was performed at 371 C

Starch synthetic wastewater as substrate.

Fang (1997)

UASB reactor: Upflow anaerobic sludge bed reactor.

CSTR: Completely stirred tank reactor.



10/16

causes damage to the granular sludge matrix and to the

microbial cells (Fermoso et al., 2008a) make EDTA an unreliable

ligand to use in full scale applications that require trace metal

supplementation. There are several ligands that could be even

more effective than chloride, such as vitamin B12, a complex

organic macromolecule with a cobalt ion as the catalytic centre,

which has been shown to enhance the methanogenesis of

carbon tetrachloride (Guerrero-Barajas and Field, 2005).

5.3.4. Quantity to be dosed

Calculation of therequiredamount of cobaltneeded perday to

support adequate methanogenesis is very important for

practice. With the data provided by Fermoso et al. (2008a),

the calculation of the mg of cobalt needed to be dosed per litre

of reactor and per day wasobtained empirically. Considering a

stable methanol removal period of 15 days after the CoCl2pulse additionand anOLR of8.5 g COD L1reactord

1, the required

cobalt amounted to 0.36 mol Lreactor1 d1 of CoCl2. In case of a

Co-EDTA2 pulse, considering a stable methanol removal

period of 7 days upon the pulse, the required cobalt amounted

to 0.77 mol Lreactor1 d1 of CoCl2. In a methanol fed UASB

reactor with an OLR of 2.6 g COD L1reactor d1, Zandvoort et al.

(2002a) estimated that 0.15 mol Lreactor1 d1 would be required

to maintain a stable methanol removal capacity when CoCl2was supplied continuously.

5.3.5. Timing of dosing

Fermoso et al. (2008a) showed that cobalt addition to cobalt-

limited methanol fed reactors has to be done before accumula-

tion of volatile fatty acids (VFA) in the effluent. If the addition is

doneafterVFAaccumulation,theVFA accumulationwill actually

be enhanced and reactor acidification will occur (Fig. 4A).

Fermoso et al. (2008a) have also shown that the addition of

methanol before VFA accumulation in a methanol-fed UASB

reactor enhanced methanogenesis. Therefore, a rational timing

of the cobalt addition as studied by (Fermoso et al., 2008a) hasto

be implemented in the strategies of metal addition for full-scale

anaerobic bioreactors.

6. Future perspectives

6.1. Mechanistic model of metal dynamics

A much more accurate quantification of the required amount

and dosing time of cobalt to be dosed can be obtained when a

mechanistic model for the CoCl2 dosing to methanol fed UASB

reactors is developed. The mechanistic modelling of the cobalt

uptake rate is, however, rather complicated because of the

complexity of the system under study, where the relation

between chemical speciation, biological uptake, biomass

Table 3 Stimulation of biological conversions in anaerobic bioreactors by metal supply.

Suppliedmetal

Reactor Temperature(C)

pH Substrate OLR(g CODL1d1)

Effect upon metal addition Reference

Fe CSTR 35 Acetate 0.25 Increase acetate removal Hoban and Van

Den Berg (1979)

Fe Upflow flocculent

sludge reactor

36 7.55 Cane fermentation

waste

0.750 Increase acetate removal Callander and

Barford (1983)

Ni, Co

and Fe

Fed-Batch pH-

stat

6.6 Acetate 4.3 Increase acetate removal Speece et al.

(1983)

Trace

metals

Anaerobic film

expanded bed

reactor

20, 25, 30

and 35

6.9 Whey Increase COD removal Kelly and

Switzenbaum

(1984)

Ni Poultry waste

digester

50 7.5 Chicken manure Increase biogas formation Williams et al.

(1986)

Ni, Co

and Fe

UASB reactor 35 6.9

7.3

Food industry

wastewater

15 Faster sludge growth and

better sludge retention.

Increase in COD removal

Oleszkiewicz

and Romanek

(1989)

Co UASB reactor 30 6.8 Methanol 8.5 Increase COD removal Florencio et al.

(1993)

Ni, Co

and Fe

UASB reactor 35 6.8 Volatile fatty acids 11 Increase COD removal Shen et al.

(1993)

Ni, Co

and Mo

UASB reactor 35 7.8 Cane molasses

stillage

21.5 Increase COD removal Espinosa et al.

(1995)

Co and Fe CSTR 35 7.8 Molasses

wastewater

2 Improve anaerobic digestion Percheron et al.

(1997)

Ni, Co

and Fe

Fed-batch

granular sludge

35 6.7

7.5

Distillery

wastewater

5.9 Improve methanogenic activity Sharma and

Singh (2001)

Co UASB reactor 30 7.0 Methanol 20 Increase SMA and methanol

removal

Zandvoort et

al. (2004)

Co UASB reactor 55 Methanol 4.38.9 High activity of methanogens Paulo et al.

(2004)

Trace

metals

UASB reactor 725 High concentrated

organic wastewater

Increase VFA removal Li et al. (2007)

UASB reactor: Upflow anaerobic sludge bed reactor.

CSTR: Completely stirrer tank reactor.



11/16

growth, hydrodynamics of the reactor and biofilm character-

isticshas to be takenintoaccount (Jansen, 2004). Some of these

aspectsare describedin the literature for biofilm systemsother

than granular sludge (Arican et al., 2002; Flemming, 1995;

Flynn, 2003; Wang et al., 2003; Weng et al., 2003; Worms et al.,

2006), but the aspects of greatest importance, i.e. chemical

interactions between cobalt and the granular sludge matrix, as

well as cobalt uptake and biomass growth kinetics are not yet

quantified (Van Hullebusch et al., 2003), which hampers the

development of these models.

6.2. Metal dynamics in living cells

An important prerequisiteforfurther understanding of themetal

dynamics in living cells is the ability to image the concentration

of trace metals in living cells in real time (Table 4). Fluorescence

microscopy is ideally suited for this purpose, and the develop-

ment of fluorescent probes that bind transition metal ions with

highaffinity andselectivityhasrecently becomean areaof active

research (Van Dongen et al., 2006), and can contribute to depict

the metal-microorganism interaction at single cell level in the

biofilm system.

Imaging of the element distribution in cells and biofilm

sections is becoming possible with sub-micrometer spatial

resolution and picogram-level sensitivity owing to advances in

laserablation MS(MassSpectrometry),ion beam andsynchrotron

radiation X-ray fluorescence microprobes (Gordon et al., 2008).

Progress in nanoflow chromatography and capillary electrophor-

esis coupled with element specific ICP MS (Saba et al., 2007;

Schaumlffel, 2007) and molecule-specific electrospray MS/MS

and Matrix-assisted laser desorption/ionization (MALDI) (Thar-

amani et al., 2008) enables speciation of elements in micro-

samples in complex biological environment as the anaerobic

granular sludge.

6.3. Metal dynamics and solid state speciation in anaerobic

granular sludge

The increasing sensitivity of EXAFS (Extended X-ray Absorp-

tion Fine Structure) and XANES (X-ray Absorption Near Edge

Table 4 Future research directions to improve the trace metal supplementation to UASB and anaerobic biofilm reactors.

Improvement in the characterization of Tool

Metalmicrobe interactions

To image the concentration of trace metals in living cells

in real time.

Fluorescence microscopy with advanced fluorescent probes that bind transition

metal ions with high affinity and selectivity.

Element distribution in cells and biofilm sections. Sub-micrometer spatial resolution and picogram-level sensitivity owing to

advances in laser ablation MS, ion beam and synchrotron radiation X-ray

fluorescence microprobes.

Speciation of elements in microsamples in complex

biological environment as the anaerobic granular sludge.

Nanoflow chromatography and capillary electrophoresis coupled with element

specific ICP MS and molecule-specific electrospray MS/MS and MALDI.

Metalsbiofilm interactions

To identify and to determine in situ the relative amounts

of functional sulfur species down to low concentrations

providing information about the oxidation state,

fingerprint speciation of metal sites and metal-site

structures.

EXAFS and XANES, owing to the use of more intense synchrotron beams and

efficient focusing optics, offers a unique non-destructive tool.

Non-invasively monitor metal transport processes and

biomass accumulation and distribution.

Nuclear magnetic resonance (NMR) methods.

Determination of cobalt taken up into the cell. Mapping radioactive metal isotopes previously supplied to the anaerobic

granule.

Metalsliquid phase interactions

To study the dissolved metal speciation. Techniques using semi permeable gels, such as DET, DGT or SOFIE.

Techniques using selective membranes, such as DMT (charge selective), and PLM

(ion selective).

Dynamic electrochemical techniques, such as voltammetries and (S)SCP for

metals that can amalgamate with mercury, or certain types of microelectrodes.

Combinations, such as GIME (porous gel in combination with voltammetry),

CGIME (a GIME with a layer of metal binding resin) or certain types of

microelectrodes.

MetalsUASB interactions

Trace metal source with a defined flux over the UASB. The DMT in a reversed set-up. The sensor and the dosing are then combined in

one unit.

To minimize the metal losses by manipulating the cobalt

dissolution rate.

Cobalt in slow release capsules.

Electrochemical release of cobalt.

Inclusion of trace metal measurement on the reactor

system as a controlled parameter.

Electrochemical sensors for trace metal analysis in water, using either

microlithographically fabricated- or screen-printed microelectrode arrays.

Implementation of metal pulse dosing as manipulated

parameter in process control algorithms.

Process control can alleviate fluctuations in metal loading rates and

subsequently increase the efficiency of anaerobic wastewater treatment plants.



12/16

Structure), owing to the use of more intense synchrotron

beams and efficient focusing optics, offers a unique non-

destructive tool to identify and to determine in situ the

relative amounts of functional sulfur species down to low

concentrations providing information about the oxidation

state, fingerprint speciation of metal sites and metal-site

structures (Jalilehvand, 2006; Lobinski et al., 2006). The

application of more elaborate sulfur extraction schemes foranaerobic granular sludge, yielding more specific fractions

including elemental sulfur and disulfides (Canheld et al., 1998;

Van der Veen, 2004), which have been successfully applied to

freshwater and marine sediments, can also contribute to a

better characterization of the sulfur pool in anaerobic granular

sludge. Nuclear magnetic resonance (NMR) methods provide

the ability to non-invasively monitor metal transport pro-

cesses and biomass accumulation and distribution (Seymour

et al., 2007). The further determination of cobalt taken up into

the cell, e.g. by mapping radioactive metal isotopes previously

supplied to the anaerobic granule, is another way to better

characterize this metal fraction (Lombi et al., 2001).

6.4. Analytical methods for speciation analysis

For studying the dissolved metal speciation, the low metal

concentrations and properties of the medium of anaerobic

bioreactors set very specific demands on the experimental

techniques chosen (Table 4). In general, a number of methods is

available, including: techniques using semi permeable gels,

such as diffusive equilibrium thin films (Bottrell et al., 2007),

diffusion gradient in thin films (Davison and Zhang, 1994),

sediment or fauna incubation experiment technique (Vink,

2002); Techniques using selective membranes, such as DMT

(charge selective) (Temminghoff et al., 2000), and permeation

liquid membrane (ion selective) (Parthasarathy et al., 2003);

dynamic electrochemical techniques, such as voltammetries

(Buffle and Tercier-Waeber, 2005) and stripping chronopoten-

tiometry at scanned deposition potential (Van Leeuwen and

Town, 2003) for metals that can amalgamate with mercury;

combinations, such as gel integrated microelectrodes (GIME)

(porousgel in combination with voltammetry) or CGIME (a GIME

with a layer of metal binding resin) (Buffle and Tercier-Waeber,

2005). Jansen et al. (2005) assessed the applicability of Compe-

titive LigandExchange Adsorptive Stripping Voltammetry (CLE-

AdSV) to analyze cobalt and nickel speciation in anaerobic

bioreactor media. This technique has the advantages of

selective detection of both cobalt and nickel at low concentra-

tions and the possibility of analyzing strong binding properties.

It combines bulk equilibrium ligand exchange with a two-step

electrochemical technique. Jansen et al. (2005) presented linear

calibration for both cobalt and nickel in anaerobic media down

to ca. 1 nM using CLE-AdSV and demonstrated the suitability of

the technique for distinguishing between strongly bound and

loosely bound/free fractions of metals.

6.5. Process monitoring and control

The DMT principle (Temminghoff et al., 2000) can be used in a

reversed set-up to control the addition of trace metals to

the reactor (Schrder, personal communication). Two general

set-ups are conceivable: by coupling the reactor via a DMT

membrane with a nutrient solution, buffered by the addition

of a strong ligand, the free ion activity of the reactor solution

can be controlled. The DMT cell can also be used as a trace

metal source with a defined flux over the membrane surface.

The sensor and the dosing are then combined in one unit,

which is a very elegant solution. Other kinds of cobalt dosing

strategies that minimize the metal losses by manipulating the

cobalt dissolution rate can be developed in further research,e.g. includingcobalt in slow release capsules (Im et al., 2005) or

electrochemical release of cobalt (Cosnier et al., 1994) (Table4).

Electrochemical sensors havebeen developedfor tracemetal

analysis in water, usingeither microlithographically fabricated-

or screen-printed microelectrode arrays (Nol et al., 2006; Xie

et al., 2005). The sensors are chiefly based on the principles of

stripping voltammetry (anodic, cathodic, and adsorptive vol-

tammetry) and stripping potentiometry (Ostapczuk, 1993). The

combination of microelectrodes and computer-controlled min-

iaturized instrumentation is very suitable for the development

of portable analytical instruments for in situ and on-site

measurement of heavy metals in reactors (Xie et al., 2005). The

inclusionof trace metal measurement in thereactor systemas a

controlled parameter will allow to implement metal dynamics

in process control design. Different applicable control strategies

include feedforward, supervisory, multivariable and adaptative

control features as well as sophisticated digital logic control

(Stephanopoulos, 1984). The implementation of metal pulse

dosing as manipulated parameter in such process control

algorithms can compensate process disturbances, such as

variation in incoming flows, wastewater characteristics or

temperature. Process control can thus alleviate fluctuations in

metal loading rates and subsequently increase the efficiency of

anaerobic wastewater treatment plants.

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