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Research ArticleReceived: 19 April 2012 Revised: 30 May 2012 Accepted: 6 June 2012 Published online in Wiley Online Library: 20 July 2012

(wileyonlinelibrary.com) DOI 10.1002/jctb.3882

Application of anaerobic membranebioreactors for the treatment of protein-containing wastewaters under salineconditionsAlberto Hemmelmann, Alvaro Torres, Christian Vergara, Laura Azocarand David Jeison∗

Abstract

BACKGROUND: Anaerobic treatment of saline wastewaters may be hindered by problems related with biomass retention, sinceat high salt concentrations formation of biofilms and granules may not proceed well. This research studied the use of anaerobicmembrane bioreactors (AnMBR) as a way to promote complete biomass retention. A lab scale AnMBR fitted with a ceramictubular membrane was operated for 2 years.

RESULTS: Results showed that enhanced biomass retention produces conditions enabling anaerobic treatment of salinewastewaters. Despite the high resulting sludge retention time, no accumulation of a high proportion of dead cells was observed.Protein degradation and not methanogenesis was shown to be the rate limiting step for organic matter degradation, a fact thatis relevant for protein-containing wastewaters such as those from seafood processing industries. Only low levels of flux couldbe applied, in the region of 5 L m−2 h−1 due to reversible cake formation promoted by single cell growth.

CONCLUSION: Biomass retention provided by membrane filtration promotes conditions suitable for efficient treatment of salinewastewaters. However, operation may be restricted to low values of flux due to biomass development as single cells.c© 2012 Society of Chemical Industry

Keywords: anaerobic; salinity; membrane; biogas; bioreactor

INTRODUCTIONSaline effluents are generated during production processesrelated to fish and sea-food processing, chemical industries andtanneries.1 Physicochemical treatment is often applied to salinewastewaters. However, it involves continuous use of chemicalsand produces a sludge that on many occasions requires furthertreatment and special disposal. Biological processes represent afeasible treatment alternative for saline wastewaters, as long assufficient microbial activity can be ensured. Furthermore, for thosewastewaters containing a high amount of biodegradable organicmatter, anaerobic technology represents an interesting option,considering its low energy requirements, high loading capacitiesand potential bioenergy production in the form of biogas.

Low levels of sodium are beneficial for anaerobic microorgan-isms. Indeed, McCarty2 reported beneficial sodium concentrationsfor mesophilic anaerobic bacteria in the range 100–200 mg L−1

of sodium. However, when present at high concentration, sodiummay hinder anaerobic treatment due to inhibition of microorgan-isms involved in the conversion of organic matter. Different levelsof saline tolerance of anaerobic bacteria have been reported, de-pending on the conditions applied.3 Easily degradable substratesseem to increase salt tolerance, most likely as a result of high energyavailability, required to cope with the energetic requirements of

salt tolerance mechanisms.1 Several reports indicate that biomassacclimation may significantly increase the activity under salineconditions.4 – 7 However, reports are also available where no orlittle acclimation was observed.8 Then, selection rather than adap-tation is likely to be the mechanism providing high activity whenbig changes in salinity are imposed, requiring the presence ofsalinity-tolerant microorganisms in the inoculum.9 It is indeed acommon practice to use inoculums containing sources of salineresistant microorganisms, such as marine sediments.1

Considering available literature, it seems that even thoughsalinity inhibits anaerobic consortia, sufficient microbial activity isachievable over a wide range of saline conditions.10 Success ofanaerobic saline wastewater treatment would then be stronglydependent on the capacity of treatment systems to retain activebiomass. Biofilms and granules are the common way to providebiomass retention in high rate anaerobic systems. However,

∗ Correspondence to: David Jeison, Department of Chemical Engineering andScientific and Technological Bioresource Nucleus, Universidad de La Frontera,Casilla 54-D, Temuco, Chile. E-mail: djeison@ufro.cl

Department of Chemical Engineering and Scientific and TechnologicalBioresource Nucleus, Universidad de La Frontera, Casilla 54-D, Temuco, Chile

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problems related with biomass retention seem to be a commonfeature when saline wastewater treatment is considered. At highsalt concentrations, formation of biofilms does not proceed well.3

Jeison et al.10 observed that during the operation of a UASB reactorunder saline conditions, highly active biomass developed in smallflocs that could not be retained by settling, producing constantactive biomass washout. Indeed, anaerobic granules exposedto saline conditions normally show lower levels of mechanicalstrength than those cultivated in non-saline media.11 – 13 Thiswould be the result of, among other effects, Ca2+ leaching fromthe aggregates. Indeed, Ca2+ ion has been identified to play animportant role in biofilm formation.14

Anaerobic wastewater treatment of saline wastewater mayfail not necessarily as a result of biomass inhibition, but dueto biomass retention problems, when granular or biofilm basedsystems are considered. The application of a solid/liquid separationprocess capable of retaining active biomass, such as membranefiltration, may then contribute towards stable operation ofanaerobic digesters when treating saline wastewaters. Anaerobicmembrane bioreactors (AnMBR) may then be a viable solutionfor anaerobic saline wastewater treatment. AnMBR results fromthe combination of an anaerobic bioreactor with a membranefiltration unit, which ensures complete biomass retention. Sincebiomass washout is prevented, a high biomass concentration isfeasible, meaning higher loadings and therefore smaller reactors.An AnMBR system should provide conditions favorable to theretention and development of halotolerant and/or halophilicanaerobic microorganisms, and at the same time provide a treatedwastewater free of suspended solids.

When applying AnMBR technology to wastewater treatment,membrane fouling will determine filtration capacity, and thereforemembrane requirements. It seems reasonable to expectedmembrane fouling to be higher when saline conditions areapplied, for a variety of reasons: higher SMP concentrations, higherviscosity, higher scaling potentials, formation of more denselypacked cake layers.15 However, only a few reports are availablein the literature dealing with the application of AnMBR systemsfor the treatment of saline wastewaters. Jeison et al.10 operated amembrane assisted UASB reactor for the treatment of completelyacidified synthetic wastewater. Results showed a positive effectof membrane filtration. IC50 for acetotrophic methanogenesis was25 g Na+ L−1. Vyrides and Stuckey16 studied the application ofa submerged AnMBR for the treatment of saline sewage, usingparticulated activated carbon (PAC): 99% of dissolved organiccarbon removal was achieved at 35 g NaCl L−1 and 8 h hydraulicresidence time. The same authors also studied the formation andstructure of the cake layer formed over the membrane surface andits role on SMP retention, when operating an AnMBR with salinewastewaters.17

This research looks to advance the knowledge required tosuccessfully apply AnMBR systems to the treatment of salinewastewaters. It involves the long-term operation of an AnMBR(2 years) fed with a synthetic saline wastewater containingproteins. Analyses of biological activity of retained biomass, aswell as membrane performance are performed.

MATERIALS AND METHODSAnMBR setup and operationA 4.5 L laboratory scale AnMBR was operated for 700 days, with themembrane module placed outside the bioreactor. A single tubularceramic membrane was used (Attech Innovations, Germany),

with a pore size of 0.2 µm. New membrane resistance (RM) was8.96 × 1010 m−1. The membrane module was operated with gassparging inside the membrane tube. Biogas was recirculatedfor this purpose. Liquid circulation between the bioreactor andthe membrane module was the result of the gas-lift effect. Nosludge pumping was applied during reactor operation. Permeatewas collected by means of a peristaltic pump, that provided therequired trans-membrane pressure (TMP). AnMBR was operated at30 ◦C. AnMBR setup was equivalent to that previously reported.18

The AnMBR was fed with synthetic wastewater with a NaClconcentration of 25 g l−1. Chemical oxygen demand (COD) wasprovided by a mixture of peptone, yeast extract and ethanol,providing 45, 45 and 10% of the total COD, respectively. Thereactor was inoculated with sludge from a full scale UASBreactor treating wastewater from a styrene and propene-oxideproduction plant of Shell, Moerdijk, The Netherlands. DuringAnMBR operation, both physical and chemical cleaning procedureswere performed, as described by Torres et al.18 Before and aftercleaning procedures, resistance determinations were performedin clean water, recording TMP at increasing values of flux.

Critical flux determinationsA series of critical flux measurements were performed by the end ofreactor operation, at different gas and liquid superficial velocities(VS). A surface response methodology was used for this purpose,with a central composite design. Centre point was replicated fourtimes. During these experiments a peristaltic pump was used tocirculate liquid through the membrane, so gas and liquid velocitiescould be manipulated independently. The procedure was thesame as reported for an AnMBR treating non-saline wastewater.18

Critical flux was determined by a step increase in flux, as describedelsewhere.19

Determination of total and partial filtration resistancesTotal filtration resistance (RT ) was determined as a function of theapplied flux and resulting TMP:

RM = TMP

J × η(1)

where J is membrane flux and η is the permeate viscosity. Totalresistance (RT ) can be divided into partial resistances:

RT = RM + RF + RCR + RCI (2)

where RM represents membrane resistance, RF the resistance dueto internal fouling, RCR the resistance due loosely attached cakelayer (removable by short back-flushes) and RCI the resistance ofconsolidated cake layer formation, i.e. that needing physical orchemical cleaning for removal.19

Analyses and microscopic observationsVolatile suspended solids (VSS) and COD were determinedaccording to Standard Methods.20 Protein concentration wasdetermined by the Lowry method. Volatile fatty acids (VFA)were determined by gas chromatography with a FID detector(Clarus 400, Perkin Elmer). Sludge samples where observedusing an Olympus CX31 phase contrast microscope. Viability ofmicroorganisms in the sludge was determined qualitatively byconfocal laser scanning microscopy (CLSM) (Olympus Fluoview1000), using LIVE/DEAD BackLight viability bacterial kit (L13452,

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Molecular Probes), containing Syto9 and propidium iodide stains.Staining was applied following manufacturer indications on freshsamples (no preservation techniques were applied). An argon laser(488 nm) was used for stains excitation. Fluorescence was observedat 500 and 635 nm, for SYTO 9 and propidium iodide respectively.Resulting image histograms were obtained with commercial imageediting software. Histograms were used to compare the intensitiesof red and green channels, as a way to semi-quantitatively comparepotentially live and dead cells.

Specific cake resistance (α) was determined in unstirred dead-end filtration experiments, in a 30 mL batch filtration unit,using an outside/in microfiltration tubular membrane. Filtrationexperiments were performed at constant flux of 10 L m−2 h−1,recording resulting TMP by means of a pressure sensor locatedin the permeate line. During a dead-end filtration process, theflux is related to cake and membrane resistance through theresistance-in-series model:

J = 1

A

dV

dt= TMP

η

1

RM + RC(3)

where A represents the membrane area, V the permeate volume, tthe time, RC the cake resistance and RM the membrane resistance.During dead-end filtration, cake resistance is related to α throughthe amount of deposited particles:21

RC = V

Aα × C (4)

where C represents the solids concentration. If RC from Equa-tion (4) is substituted into Equation (3), and a constant flux isassumed, we obtain:

TMP = η × α × C × J

AV + η × RM × J (5)

The specific cake resistance is then determined through theevaluation of the slope of a plot of TMP against permeate volume.

Microbial activity determinationsThe specific methanogenic activity (SMA) was determined induplicate experiments performed in 120 mL serum bottles with50 mL of media, at 30 ◦C. Biomass concentration was 1 g VSSL−1. Acetate was used as substrate, at an initial concentrationof 1.5 g COD L−1. SMA was evaluated as the maximum specificmethane production rate, determined by pressure increase in theheadspace. Specific acidogenic activity (SAA) was determined atsimilar conditions to SMA, but with peptone as substrate. TheSAA was determined evaluating the maximum rate of proteinconsumption using peptone as substrate.

Protein degradation kinetics were determined applying theinitial rates method. Initial degradation rates were measured, atdifferent initial concentrations in the range 250–1500 mg L−1,using peptone as substrate. This method was applied to avoida potential inhibitory effect of acetate produced over proteindegradation. Such inhibition was reported at concentrations aslow as 250 mg acetate L−1 by Gonzalez et al.22 when studyinganaerobic protein degradation under saline conditions.

All activity determinations were performed at a NaCl concen-tration of 25 g l−1.

50%

60%

70%

80%

90%

100%

0

2

4

6

8

10

0 100 200 300 400 500 600 700

CO

D r

emov

al

OLR

(g

CO

D/L

d)

Time (d)

OLR COD removal

Figure 1. OLR and COD removal during AnMBR operation.

Table 1. SMA and SAA during reactor operation (measured at 30 ◦C)

Activity (g COD g–VSS d−1)

Substrate Day 311 Day 512 Day 675

Peptone (SAA) 1.7 1.7 1.7

Acetic acid (SMA) 0.59 0.58 0.51

RESULTS AND DISCUSSIONFigure 1 presents the applied organic loading rate (OLR) and CODremoval during reactor operation. Loading rate was increasedduring the first 150 days of operation, reaching close to 8 g CODL−1 d−1. Applied OLR was then gradually decreased to 4 g COD L−1

d−1, which was the result of an important decrease in operatingpermeate flux (Fig. 4), as a consequence of the increase of filtrationresistance. Attempts to keep OLR were made by increasing influentconcentration. However, inlet COD could not be increased beyond25 g l−1 to prevent inhibition by ammonia. OLR was close to6 g COD L−1 d−1 by the end of reactor operation. Biomassconcentration increased to 25 g VSS L−1 during first 450 daysof operation. No sludge was wasted from the reactor during thisperiod. From day 450 on, sludge was regularly wasted in orderto keep VSS concentration in the range 22–25 g l−1. Consideringfinal OLR and biomass concentration, applied specific loading ratewas 0.26 g COD g−1 VSS d−1.

Table 1 presents SMA and SAA analysis. Data show rather stablevalues throughout reactor operation. A wide range of SMA valueshave been reported in the literature, depending on factors such assalinity, inoculum, operation period, substrate, bioreactor type andtemperature. For example, Aspe et al.8 and Omil et al.23 reportedSMAs of 0.065 and 0.5–0.7 g COD g−1 VSS d−1, respectively, whentreating saline sea-food wastewaters, at 37 ◦C. Thus, SMA valuesof the biomass developed in the AnMBR may be considered ratherhigh, considering that values were obtained through batch testsperformed at only 30 ◦C. High SMA values may be regarded as theresult of active biomass retention provided by membrane filtration.Biomass yield (YX/S) was evaluated during reactor operation,considering COD fed and biomass produced. YX/S was in therange 0.2–0.3 g SSV g−1 COD, which seems low when comparedwith values expected for a non-acidified substrate under non-saline conditions. This is most likely the result of the applied salinecondition, since more energy is channelled to osmotic balanceand less to growth.15

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Decay rate (kd) is expected to be higher at increasing saltconcentrations. The combination of low YX/S, high kd , medium/lowspecific growth rates and total solids retention may result inthe accumulation of inactive biomass or cells debris inside thebioreactor. Such material may dilute active biomass, negativelyaffecting specific activity and filtration performance. Figure 2presents CLSM observation of a sludge floc at day 675, usingLIVE/DEAD BackLight viability bacterial kit. The intensities of greenand red fluorescence are related to the presence of viable and non-viable cells, respectively. Analysis of image histograms showedthat intensities of the green channel are 7 times higher thanthat of the red channel, indicating a high level of potentiallyactive cells. LIVE/DEAD BackLight kit is based on the integrityof cell membrane, which does not necessarily ensure actualmicrobial activity. Anyway, it represents a clear indication of a highproportion of potentially active cells. This result, combined withthe fairly stable microbial activities, suggests that accumulation ofdead cells did not happen on a large scale, despite the resultinglong solids retention time and the expected high decay rates.

By the end of reactor operation, COD concentration in thepermeate was in the range 400–600 mg L−1, proteins being themain contributor to permeate COD. VFA contribution to permeateCOD was lower than 35%. No differences were observed betweensoluble proteins concentration in the permeate and in the mixedliquor. The same was observed for soluble COD, indicating thatthe system membrane/cake layer did not retain soluble organiccompounds (SMP or other). This is the result of the use of amicrofiltration membrane, which is not expected to retain solubleorganic matter.

The presence of protein in the permeate was in principlesurprising, considering the high SAA shown in Table 1. However,SAA was measured at an initial protein concentration of 1.5 gCOD L−1, close to 4 times the protein concentration in the mixedliquor. Protein hydrolysis is usually represented by first-orderkinetics,24 so reaction rates are expected to be proportional tosubstrate concentration. Figure 3 presents the relation betweenprotein degradation rates and protein concentration, determinedby initial rates methodology. A kinetic constant of 1.4 h−1 canbe evaluated by linear regression, which is in the same rangeas that reported by Gonzalez et al.,22 when studying proteinhydrolysis under saline conditions. If a complete mix flow patternis assumed, then biomass was exposed to a protein concentrationof around 300–350 mg L−1, meaning a reaction rate in the range

0.0

0.5

1.0

1.5

2.0

0.0 0.5 1.0 1.5

dS/d

t (gC

OD

/L×

d)

Protein concentration (g/L)

Figure 3. Effect of protein (peptone) concentration over SAA of the AnMBRbiomass, by the end of reactor operation.

0.3–0.6 g COD L−1 d−1. Then, under anaerobic treatment of salinewastewaters containing high amount of proteins, like those fromfish and marine food processing facilities, acidogenesis and notmethanogenesis may be the rate limiting step if a complete mixpattern bioreactor is applied. Most of the research reported dealingwith anaerobic treatment of saline wastewaters is focused on theeffect of salinity on methanogenesis. Indeed, only few reports arededicated to the study of hydrolysis and/or acidogenesis. Moreresearch in that direction seems to be necessary.

Figure 4 presents the total filtration resistance (RT ) and theapplied flux during the operation of the AnMBR. During thefirst 150 days of operation, flux was reduced to 5 L m−2 h−1,as a result of strong increase in RT . Even though appliedflux was low, operation showed to be unstable with suddenincreases in RT , as a result of small or no apparent changes inoperational conditions. Both physical and chemical membranecleaning operations were performed during reactor operation(Table 2). The membrane was rinsed with tap water beforecleaning operations took place, a procedure that removed theloosely attached cake layer (represented by RCR). Therefore,resistance measurement before cleaning operations correspondsto the sum of RM, RF , and RCI. Observed values of RT duringreactor operation (Fig. 4) were several times higher than the sumRM + RF + RCI, determined before membrane cleaning operations(Table 2). Therefore, it is concluded that applicable flux wasrestricted mainly by reversible cake layer formation. This agreeswith our previous results regarding saline anaerobic wastewater

Green channel, viable cells Red Channel, non-viable cells

Figure 2. CLSM images, using LIVE/DEAD BacLight Bacterial Viability Kit.

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0

5

10

15

20

0.0E+00

5.0E+12

1.0E+13

1.5E+13

2.0E+13

2.5E+13

3.0E+13

0 100 200 300 400 500 600 700

Flu

x (L

/m2 ·h

)

Res

ista

nce

(m-1

)

Time (d)

Filtration resistance Flux

Figure 4. Filtration resistance (RT ) and permeate flux during the operationof the AnMBR.

Table 2. Filtration resistances measured in clean water before (Rbefore)and after (Rafter) membrane cleaning operations. Rbefore corresponds toRM + RF + RCI . RM : 8.96 × 1010 m−1

Day Type Rbefore (m−1) Rafter (m−1)

48 Physical 3.90 × 1012 1.30 × 1012

100 Physical 3.70 × 1012 9.39 × 1011

153 Physical 8.10 × 1012 3.98 × 1012

177 Physical 3.18 × 1012 2.62 × 1012

184 Chemical 2.62 × 1012 4.74 × 1011

254 Physical 1.18 × 1012 5.01 × 1011

289 Physical 2.76 × 1012 8.95 × 1011

319 Physical 1.22 × 1012 9.15 × 1011

336 Chemical 3.12 × 1011 2.17 × 1011

469 Physical 1.60 × 1012 3.55 × 1011

493 Chemical 9.16 × 1011 2.31 × 1011

treatment by AnMBRs.10 Other flux reducing phenomena werealso observed as is clear from data presented in Table 2. Physicalcleaning presented moderated levels of permeability restoration,indicating that RCI contribution to RT was significant. Table 2 alsoshows a reduction in physical cleaning effect, as is clear whencomparing Rafter values at days 254, 289 and 319. This may bethe result of the formation of a consolidated cake that could notbe completely removed by the erosion provided by the physicalcleaning procedure, or by internal fouling. Internal fouling wouldbe expected, considering protein concentration in the mixedliquor.

If RCR is mainly responsible for low flux operation, increases insurface shear should enable increases in operational flux. However,changes in gas VS in the range 0.2–0.5 m s−1 were not successfulin enabling operation at fluxes higher than 10 L m−2 h−1. DuringAnMBR operation, biomass suspension circulation through themembrane module was induced by gas lift effect, so changes ofgas VS induced also changes in liquid VS. In order to determine theindividual effects of gas and liquid VS over the critical flux, surfaceresponse methodology was used. Gas and liquid VS applied werein the range 0–1 m s−1. The latter range was selected since highervalues of gas VS resulted in a churn flow pattern inside the tubularmembrane, instead of slug flow. Liquid Vs higher than 1 m s−1

were not tested, in order to avoid the exposure of the biomass tohigh shear conditions. Results are presented in Fig. 5. Flux valueswere moderate/low, only reaching close to 14 L m−2 h−1 at the

Gas VS (m/s)Liquid VS (m/s)

15129630

0.2

0.40.6

0.8

1.0

1.00.8

0.60.4

0.2

Crit

ical

Flu

x (L

/m2 ·

h)

Figure 5. Surface response showing the effect of gas and liquid VS overthe critical flux.

highest tested values of liquid and gas VS. Gas VS showed littleeffect over critical flux within the range studied.

Low values of flux are most likely the result of insufficientlevels of surface shear. Previous research with a similar AnMBRconfiguration treating non-saline wastewater identified single celldevelopment as the phenomenon responsible for low operationalflux.18 Microscopic observation of the biomass revealed a highproportion of biomass developing as single cells (see Fig. 6).Suspension fractioning by centrifugation coupled with suspendedsolids analysis showed that over 30% of the biomass was in theform of single cells or flocs formed by only a few cells. Preventingsingle cell deposition requires high levels of surface shear, ifhigh levels of flux are of interest, due to the low particle size ofmicroorganisms. Low particle size also results in the formationof a cake layer with a high specific resistance. By the end of theAnMBR operation, biomass presented an α value of 2.7 × 1014 mkg−1. This means that the formation of a thin cake will producea high filtration resistance. Indeed, if an α value of 2.7 × 1014

m kg−1 is considered, a cake layer of only 100 µm would beenough to produce the highest filtration resistance observedduring reactor operation (around 2.5 × 1013 m−1). A high valuefor α suggests the formation of a dense and compact cakelayer. The Carman–Kozeny equation may be used to determinethe effect of floc size (dP) and porosity (ε) on α and then onfiltrability:

α = 180(1 − ε)

ρρd2ρε3

where ρP represents the density of the particles. If dP is asumed tobe 1 µm (roughly the size of a single bacterial cell), ε needs to belower than 0.1, in order to obtain an α value of 2.7 × 1014 m kg−1.Such a low value for ε is indicative of a compact cake, with littleporosity.

Even though only low levels of flux were achieved duringthis research, AnMBR operation showed that those flux levelsmay be sustained for long periods of time, in the absenceof membrane maintenance. During AnMBR operation severalcleaning procedures were performed during first 490 days ofoperation as a way to control filtration resistance. Cake layerformation and no irreversible fouling was the main factor limitingthe applicable flux. Therefore, if a low flux is applied, cake formationcan be minimized, enabling long-term operation. Proof of that isthe fact that the AnMBR was operated for 200 days (from day 500

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Figure 6. Phase-contrast microscopy picture of the biomass developed inthe AnMBR. Bar indicates 10 µm.

until day 700) in the absence of any physical or chemical cleaningprocedure.

CONCLUSIONEnhanced biomass retention promoted by membrane separationfacilitates the application of anaerobic digestion to the treatmentof saline wastewaters. Moderate/high levels of loading ratescould be applied under saline conditions as a result of completebiomass retention. Moreover, the high biomass retention timeresulting from low biomass yields would not result in theaccumulation of a high fraction of dead cells inside the bioreactor.Results indicate that increased care is necessary when treatingwastewaters containing high concentration of proteins, sinceprotein conversion and not acetogenesis or methanogenesis maybecome the rate limiting step for organic matter degradation.Despite the adequate performance of the AnMBR in terms oforganic removal, filtration performance may represent a drawbackof such systems, based on the low fluxes achieved. Single cellgrowth was identified as one of the key factors determiningmembrane filtration performance.

ACKNOWLEDGEMENTSThe authors want to acknowledge the financial support providedby CONICYT-Chile through FONDECYT projects 1080279 and3120171. The authors would also like to thank Bram Versprillefrom Biothane Systems International for providing the sludgeused as inoculum for the AnMBR.

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