response of methanogen populations to organic load increase during anaerobic digestion of olive mill...

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:1556–1562 (2006)

Response of methanogen populationsto organic load increase duringanaerobic digestion of olive mill wastewaterAurora Rizzi, Mauro Zucchi, Sara Borin, Massimo Marzorati, Claudia Sorlini andDaniele Daffonchio∗Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM), Universita degli Studi di Milano, I-20133 Milano, Italy

Abstract: Process performances of an upflow anaerobic filter treating olive mill wastewater and the response ofmethanogenic Archaea to increasing volumetric organic load (VOL) were studied. At a VOL of 15 g chemicaloxygen demand (COD) L−1 day−1, 90% of the influent COD was removed. Following a VOL increase from 6 to 15 gCOD L−1 day−1, the polymerase chain reaction (PCR) titre of hydrogenotrophic Methanobacterium, determinedby magnetic capture of the target DNA and group-specific PCR based on the 16S rRNA gene, decreased from1011 to 108 cells g−1 sludge, while that of Methanomicrobiaceae and relatives increased from 104 to 106 cells g−1

sludge. Methanosaeta-like acetoclastic methanogens were less affected by VOL variation and dominated at highVOL with a 16S rRNA gene PCR titre of 109 cells g−1 sludge. Single-strand conformation polymorphism analysis ofthe PCR-amplified archaeal 16S rRNA gene showed a stable band pattern, indicating that VOL variation affectedthe methanogen PCR titre but not the archaeal community structure. 2006 Society of Chemical Industry

Keywords: olive mill wastewater; anaerobic digestion; methanogens; organic load; PCR monitoring; SSCP

INTRODUCTIONOlive mill wastewaters (OMWs) result from olive(Olea europaea) milling during oil extraction andare characterised by high chemical oxygen demand(COD, up to 200 g L−1) and low pH (<5).1,2 TheOMW disposal problem is particularly important inthe Mediterranean basin, where Italy, Spain, Greeceand Portugal together produce 75% of the world’solive oil.

Anaerobic digestion can be considered for OMWdisposal, since it allows energy recovery as methaneand results in low sludge production.3,4 However,process efficiency can be seriously reduced owing tothe presence of high concentrations (8–16 g L−1) ofphenolic compounds.5

During the degradation of complex organic mat-ter by anaerobic digestion, methanogenic Archaea arethe final catalysers of the food chain. By remov-ing the metabolic products of syntrophic acetogens,methanogens play a regulative role in maintainingthe overall efficiency of the process. An efficientmethanogenic consortium for OMW treatment shouldmaintain high methane production even in the pres-ence of high concentrations of phenolic compounds.However, methanogens are highly sensitive to thetoxicity of tannins and polyphenols.6,7 Hence thestudy of the response of the methanogenic com-munity to different OMW organic loads would helpthe identification of methanogenic consortia that are

adapted to high concentrations of toxic phenolic com-pounds.

The analysis of phylogenetic markers such asthe 16S rRNA gene may efficiently complementtraditional microbiological analysis by distinguishingthe different phylogenetic groups of prokaryotes.8,9

To our knowledge, only one study has described thebehaviour of methanogenic Archaea in the biofilmof an anaerobic reactor treating OMWs.5 Thatstudy was performed in a granular activated carbon(GAC) packed bed biofilm reactor and showedthat the main methanogenic Archaea in the biofilmwere related to hydrogenotrophic Methanobacteriumformicicum.

Here we describe the process performances of alaboratory-scale wood chip-packed anaerobic upflowfilter during OMW treatment at different volumetricorganic loads (VOLs). We used a 16S rRNAgene-based magnetic capture hybridisation (MCH)polymerase chain reaction (PCR) method to estimatethe abundance of different phylogenetic groupsof methanogens in the biofilm in response toincreasing VOL. The methanogen species diversity atdifferent organic loads was evaluated by single-strandconformation polymorphism (SSCP) of the amplified16S rRNA gene.10 The overall data indicated thatmarked changes occurred in the proportions ofdifferent groups of methanogens in the reactor biomassin response to different organic loads.

∗ Correspondence to: Daniele Daffonchio, Dipartimento de Scienze e Tecnologie Alimentari e Microbiologiche, Universita degli Studi di Milano, Via Celoria,I-20133 Milano, ItalyE-mail: [email protected](Received 1 October 2005; revised version received 5 December 2005; accepted 5 December 2005)Published online 12 July 2006; DOI: 10.1002/jctb.1558

2006 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2006/$30.00

Methanogen response during anaerobic digestion of OMW

MATERIALS AND METHODSReactor configuration, olive mill wastewater andoperating conditionsA laboratory-scale upflow anaerobic digester (Fig. 1)consisting of a cylindrical reactor (internal diameter15 cm, height 126 cm) composed of an upflow fixedbed digester in the lower region and a contact reactorin the upper region was used in the study. The filterpart (height 84 cm) was filled with wood chips as asupport matrix. The internal volume after wood chipaddition was 11 L (7.5 L lower filtration region, 3.5 Lupper contact reactor region). In the contact region thebiogas flow contributed to determine a sludge blanket.The plant was installed in a controlled environment at37 ◦C and the digester was connected to an AB1 gascounter (SIM Brunt, Milan, Italy). The reactor hadoperated in previous years with other agroindustrialwastewaters such as pyrolysis effluents,11 OMWs,12

wool-scouring wastes and winery effluents.13

The OMW was obtained from an olive mill locatednear San Dorligo, Trieste, Italy. The reactor influentwas prepared daily by diluting the OMW with tapwater to obtain an influent with the desired COD. Tocorrect the carbon/nitrogen ratio, NH4Cl was addedto the influent and NaHCO3 was supplemented as analkaline source to give a final influent pH >6.

The reactor was run at a VOL of 1 g CODL−1 day−1 for 2 months. After this acclimatisationperiod the VOL was increased progressively to 15 gCOD L−1 day−1 (Table 1).

Sampling, sample preparation and analyticalmethodsInfluent and effluent samples were filtered througha 0.45 µm cellulose nitrate filter and then analysed

for COD and volatile fatty acids. Biofilm sampleswere recovered from wood chips withdrawn from thetop of the filtration bed. Wood chips were placedin 50 mL of sterile phosphate-buffered saline (PBS)and vortexed for 10 min in the presence of glassbeads (3 mm diameter). The bacterial suspensionobtained was centrifuged to recover the detachedbiomass, which was then resuspended in 20 mL ofPBS and homogenised in a rotating homogenisersix times for 1 min each at 8000–24 000 rpm. Thefinal suspension was divided into two aliquotsthat were used for DNA extraction as describedbelow.

COD and methane in the biogas were determinedas described previously.13 Volatile fatty acids in theeffluent were determined by gas chromatography aftersteam distillation of the acidified sample.13

Methanogenic Archaea listed in Table 2 wereobtained from the German Collection of Micro-organisms (DSMZ, Braunschweig, Germany). Thestrains were cultivated anaerobically in the spe-cific media under the conditions for each strainreported in the DSMZ catalogue available atthe DSMZ web site (http://www.dsmz.de). Non-methanogenic facultative bacteria Bacillus cereus strainDSMZ 31T and Bacillus licheniformis strain ATCC14 580T were cultivated aerobically in nutrient broth(Difco, Milan, Italy) at 30 ◦C overnight. Lacto-bacillus helveticus V65SP was cultivated anaerobi-cally in MRS broth (Difco) at 37 ◦C. Desulfovib-rio desulfuricans strain DSMZ 1926 and Desulfovib-rio salexigens strain DSMZ 2638T were cultivatedin medium 272 as described in the DSMZ cata-logue.

Figure 1. Schematic diagram of the anaerobic digester used in the study.

J Chem Technol Biotechnol 81:1556–1562 (2006) 1557DOI: 10.1002/jctb

A Rizzi et al.

Group-specific amplification of methanogensand PCR sensitivityThe sequence of forward primers for group-specificamplification of methanogens was obtained by aligningthe 16S rRNA sequences of methanogens. As reverseprimers the specific sequences identified by Raskinet al.14 in different regions of the 16S rDNA genewere used. The primer sets were as follows: Mb, tar-geting Methanobacteriaceae, S-G-Mbac-0009-a-S-20(5′-CCGTTTGATCCTGGCGGAGG-3′) and S-F-Mbac-0310-a-A-22 (5′-CTTGTCTCAGGTTCCA-TCTCCG-3′); Mc, targeting Methanococcaceae, S-F-Mcoc-0009-a-S-20 (5′-CCGGTTGATCCCGCCG-GAGG-3′) and S-F-Mcoc-1109-a-A-20 (5′-GCAAC-ATAGGGCACGGGTCT-3′); Mm, targeting Metha-nomicrobiaceae, Methanocorpusculaceae and Methano-planaceae, S-O-Mmic-0009-a-S-20 (5′-CTGGTTG-ATCCTGCCAGAGG-3′) and S-O-Mmic-1200-a-A-21 (5′-CGGATAATTCGGGGCATGCTG-3′); Ms,targeting Methanosarcinaceae, Methanosaeta and Me-thanotrix, S-O-Mmic-0009-a-S-20 (5′-CTGGTTGA-TCCTGCCAGAGG-3′) and S-F-Msae-0825-a-A-23 (5′-TCGCACCGTGGCCGACACCTAGC-3′).Primer specificity was tested on pure cultures ofmethanogens. PCR tests were performed in a PTC100thermal cycler (MJ Research Inc., Watertown, MA,USA). Template DNA solution (2 µl) was mixed witha master mix solution (48 µl) containing 10 mmol L−1

Tris-HCl (pH 8.8), 50 mmol L−1 KCl, 0.1% Tri-ton X-100 (1× buffer DyNAzyme, Finnzyme OY,Espoo, Finland), 2 mmol L−1 MgCl2, 0.2 mmol L−1

each deoxynucleoside triphosphate (dATP, dTTP,dCTP and dGTP), 0.5 µmol L−1 each primer and1 U of DNA polymerase (DyNAzyme from Thermusbrokianus). The PCR mixture was overlaid with 55 µLof mineral oil (Sigma, Milan, Italy). A hot start proce-dure was used. Thirty-five cycles of amplification wereperformed in the denaturation step at 94 ◦C for 1 min.The annealing step was performed at different temper-atures for 1 min depending on the primer set: 70 ◦C,Mb set; 72 ◦C, Mc set; 69 ◦C, Mm set; 64 or 68 ◦C,Ms set. The elongation step was at 72 ◦C for 1 min.For each reaction a final elongation step at 72 ◦C for5 min was added. The PCR products were separatedby agarose gel electrophoresis.15

The sensitivity of the PCR methods, i.e. the lowestcell number giving a PCR signal, was determinedaccording to Wang et al.16 A 1 mL aliquot of theculture was pelleted by centrifugation, washed threetimes with PBS and suspended in 100 µL of lysissolution (0.1% w/v sodium dodecyl sulfate (SDS), 1%w/v Nonidet P-40 and 1% v/v Tween 20). Hencethe bacterial suspension was decimally diluted inthe lysis solution and lysed at 95 ◦C for 15 min. A2 µL aliquot from each dilution was used directlyfor PCR. PCR products were visualised by agarosegel electrophoresis. The cell number C (cells mL−1)in the original culture broth was determined bydirect microscopic counts with a Thoma countingchamber and a phase contrast microscope. At least 16

microscopic fields were monitored for each count.16

The PCR sensitivity S (cells) was calculated accordingto S = C/10D, where D is the last decimal dilutiongiving a positive PCR signal.

DNA extraction from the sludge anddetermination of the PCR titre of differentmethanogenic groupsAn MCH-PCR assay was used for analysingmethanogenic Archaea in the cell suspensions obtainedfrom the sludge as explained above. Two aliquots ofthe same cell suspension were used for MCH-PCR.The cells in PBS were centrifuged and resuspendedin 1 mL of lysis solution (0.1% w/v SDS, 1% w/vNonidet P-40 and 1% v/v Tween 20). Hence the cellswere decimally diluted and each dilution was treatedfor 15 min at 95 ◦C for cell lysis and centrifuged. Thesupernatant from each dilution was supplemented with1 volume of hybridisation buffer (1 mol L−1 NaCl, pH7.5), denatured at 95 ◦C for 15 min, cooled on ice andthen transferred to a tube containing 20 µL of biotiny-lated capture probes bound to streptavidin magneticbeads (Boehringer Mannheim, Milan, Italy), preparedas explained below. In order to allow hybridisationbetween the magnetic probes and the target DNA,the mixture was incubated for 1 h at room temper-ature with gentle shaking. Following incubation, thetube was placed in a magnetic particle separator (CPGInc., Lincoln Park, NJ, USA) to allow magnetic cap-ture of the beads, and the supernatant was removed.The beads were washed twice in 2 mol L−1 NaCland resuspended in 100 µL of distilled water. Afterdenaturing at 95 ◦C for 15 min, the supernatant con-taining the released single-strand DNA was separatedfrom the magnetic beads by magnetic capture andtransferred to a new tube. The process was repeatedonce more with 50 µL of distilled water. At the endof the procedure, each tube contained 150 µL. Two6 µL aliquots from each tube were used directly forduplicate PCR under the conditions given in the pre-vious subsection. The PCR products were separatedby agarose gel electrophoresis. To calculate the PCRtitre (cells g−1 sludge wet weight) of methanogensin the granular sludge, the following equation wasused: PCR titre = S × 10D−1 × (Vtot/Vdil) × Wsample,where S is the PCR sensitivity (cells) determined onpure cultures for each methanogenic group, D is thelast PCR-positive dilution, Vtot is the total volume(1000 µL) of the original sample after addition of thelysis solution, Vdil is the volume (10 µL) of the sampleplus the lysis solution used to prepare the first dilutionanalysed by PCR, and Wsample is the weight (0.5 g) ofthe sludge sample.

PCR-SSCP to analyse the diversity ofmethanogens in the sludgeA portion of the archaeal methanogenic 16S rRNA(positions 798–915) was amplified from pure cultureand sludge DNA by using the forward primer ArchF(5′-CCGGATTAGATACCCGGGTAG-3′) specific

1558 J Chem Technol Biotechnol 81:1556–1562 (2006)DOI: 10.1002/jctb


for most methanogenic Archaea and the reverse primerArchR (5′-GTGCTCCCCCGCCAATTCCT-3′)that is universal for Archaea. The primer ArchR wasphosphorylated at the 5′ end. PCR conditions werethe same as those described above for amplificationof the different groups of methanogens. The anneal-ing temperature was set at 60 ◦C. The phosphorylatedstrands were eliminated by λ-exonuclease digestion10

and the remaining strands were separated on a nativepolyacrylamide gel.17,18

RESULTS AND DISCUSSIONProcess performance of the anaerobic filterProcess performances of the anaerobic reactor arereported in Table 1 and Fig. 2. The efficiency of CODremoval was ∼90% under all operative conditionstested. The best performance was obtained whenthe reactor operated at a VOL of about 10 gCOD L−1 day−1, with COD removal reaching anaverage value of 92% (Table 1). In general, CODremoval was very stable despite the sudden changesin VOL (Fig. 2). At a higher VOL of 15 g COD

L−1 day−1, COD removal decreased to 89% and, ingeneral, a decrease in the main process parametersoccurred. Specific methane production decreased to0.16 L g−1 CODrem, while at other VOLs above 5 gCOD L−1 day−1 it was between 0.23 and 0.27 Lg−1 CODrem. At the highest VOL a higher amountof suspended solids was observed in the effluent,indicating that the reactor suffered a process imbalanceand that biomass accumulated in the reactor. With theVOL increase to 20 g COD L−1 day−1 the biomassexcess started to wash out, decreasing the reactorperformance.

Up to a VOL of 10 g COD L−1 day−1 the totalvolatile fatty acid (VFA) level was almost always<800 mg L−1, confirming the very good efficiency ofthe methanogenic biomass (Fig. 3). The main VFAdetected in the effluent was always acetate, whichrepresented 70–80% of total VFAs. Considering that1 g of acetate gives about 1 g of COD, it can be deducedthat, when the reactor operated at a VOL of 10 g CODL−1 day−1, most (>50%) of the COD in the effluent(1.6 g L−1; Table 1) was due to VFAs and hence theconcentration of residual phenolic compounds was

Table 1. Process parameters of OMW anaerobic digestion at different VOLs. For each process phase, average values and standard deviations of

the parameters were calculated for the number of samples n given in parentheses

VOL (g COD L−1 day−1)

Parameter 2.7 ± 0.47 (28) 5.7 ± 0.5 (13) 6.6 ± 0.9 (10) 10.1 ± 2.4 (11) 15.2 ± 3.7 (8)

Treatment time (days) 70 28 22 18 34Hydraulic retention time (days) 2.2 ± 0.3 (61) 2 ± 0.1 (26) 2 ± 0.1 (20) 2.2 ± 0.3 (18) 2 ± 0.1 (23)Influent pH 6.17 ± 0.12 (50) 6.38 ± 0.09 (23) 6.4 ± 0.07 (19) 6.42 ± 0.14 (16) 6.4 ± 0.1 (23)Effluent pH 7.77 ± 0.21 (43) 7.82 ± 0.3 (17) 7.8 ± 0.28 (18) 7.98 ± 0.35 (14) 7.76 ± 0.26 (24)Infl. sol. COD (g L−1) 5.9 ± 0.8 (30) 11.9 ± 0.8 (13) 13.2 ± 1.3 (10) 21.8 ± 2.7 (11) 31.3 ± 7.8 (8)Effl. sol. COD(g L−1) 0.96 ± 0.3 (23) 1.4 ± 0.4 (11) 1.4 ± 0.4 (7) 1.61 ± 0.48 (8) 2.92 ± 48 (6)Sol. COD removal (%) 84.1 ± 5.1 (23) 88.5 ± 3.6 (11) 88.7 ± 3.9 (7) 92.4 ± 1.6 (8) 89.2 ± 1.9 (6)Spec. CH4 prod. (L g−1 CODrem) 0.33 ± 0.08 (22) 0.26 ± 0.06 (11) 0.27 ± 0.04 (7) 0.23 ± 0.06 (8) 0.16 ± 0.04 (6)CH4 in biogas (%) 74.2 ± 2.5 (29) 71.9 ± 1.6 (15) 71.6 ± 1.4 (11) 69.6 ± 1.8 (13) 69.5 ± 3.4 (17)

Figure 2. Time course of soluble COD removal (�) and specific methane production (�) at different volumetric organic loads (♦) during anaerobicdigestion of olive mill wastewater.


A Rizzi et al.

Figure 3. Time course of total volatile fatty acids (�) and acetic acid (�) in the effluent of the anaerobic reactor at different organic loads (♦) duringanaerobic digestion of olive mill wastewater.

Table 2. Primer set specificity assayed on pure methanogen strain DNA and PCR sensitivity assayed on pure methanogen strain DNA in absence

(‘without’) and presence (‘with’) of exogenous bacterial DNA. Methanogen strains were from the German Collection of Micro-organisms (DSMZ) and

the number of each strain refers to the DSMZ strain code. Primer set Ms was used at (Ms1) 64 and (Ms2) 68 ◦C annealing temperatures. The lowest

PCR sensitivity recorded is reported for each group of species

Primer sets PCR sensitivity (cells)

Species (strains)a Mb Mc Mm Ms1 Ms2 Without With

Mb. formicicum 1535, 3637, 3636, 2639, 3722, 6299; Mb. ulginosum2956; Mb. ivanovii 2611; Mb. bryantii 863; Mb. palustre 3108; Mb.thermoalcaliphilum 3267; Mb. thermoautotrophicum 1053, 3720; Mb.thermoaggregans 3266; Mb. wolfei 2970; Mb. thermophilum 6529;Mbrev. arboriphilicus 1125; Msph. stadtmanae 3091

+ − − − − 104 104

Mc. maripaludis 2067; Mc. voltae 1537; Mc. vannieli 1224 − + − − − 103 103

Mg. cariaci 1497; Mcull. marisnigri 1498; Mspi. hungatei 864; Mcorp.parvum 3823; Mcorp. sinense 4274; Mp. limicola 2279

− − + − − 102 102

Msar. barkeri 800; Msar. mazei 2053; Msar. vacuolata 1232; Msar.acetivorans 2834

− − − + − 102 102

Mtrix thermophila 6194; Msaeta concilii 3671 − − − + + 104 104

a Abbreviations of methanogen genera: Mb., Methanobacterium; Mbrev., Methanobrevibacter; Msph., Methanosphaera; Mc., Methanococcus; Mg.,Methanogenium; Mcull., Methanoculleus; Mspi., Methanospirillum; Mcorp., Methanocorpusculum; Mp., Methanoplanus; Msar., Methanosarcina;Mtrix, Methanothrix; Msaeta, Methanosaeta.

rather low. This indicates that the microflora selectedin the reactor were well adapted to and efficientlyremoved high concentrations of phenolic compounds.

Evolution of the methanogenic population in thereactorBased on the probes specific for different groupsof methanogens,14 we have designed sets of primerpairs for the different groups of methanogens andoptimised the PCR conditions on a collection ofpure cultures of methanogens (Table 2). To testthe specificity of the primer sets and the selectedamplification conditions in the presence of non-targetDNA, the DNA of methanogens was amplified in thepresence of a mixture of bacterial DNA from B. cereus31T, B. licheniformis 14 580T, L. helveticus V65SP,D. desulfuricans 1926 and D. salexigens 2638T. Theprimer sets retained the specificity also when the DNAof the target strains was mixed with non-target DNA,which did not give any PCR product when used astemplate without the methanogenic DNA.

We determined the PCR sensitivity of each primerset for its target group, i.e. the minimum numberof cells that gave a positive PCR signal (Table 2).PCR sensitivities were retained also when the reactionwas prepared in the presence of a mixture of non-target DNA from B. cereus 31T, B. licheniformis14 580T, L. helveticus V65SP, D. desulfuricans 1926and D. salexigens 2638T. The PCR sensitivities werehence used to determine the 16S rRNA gene PCR titrein the biofilm of the reactor according to Wang et al.,16

as described above. In particular, magnetic captureof archaeal DNA using methanogenic DNA captureprobes after total sludge DNA extraction resulted aneffective method for methanogenic DNA enrichmentwhen compared with simple total DNA extractionfrom the sludge (data not shown).

Two sludge samples were analysed, the first whenthe reactor operated at a VOL of 5.7 g COD L−1 day−1

and the second at a VOL of 10 g COD L−1 day−1.The same PCR titres were obtained by analysing tworeplicate aliquots of cell suspensions derived from



Figure 4. PCR-SSCP of archaeal 16S rRNA gene (positions 798–915) following λ-exonuclease digestion of 16S rRNA gene amplified from totalDNA extracted from anaerobic biofilm recovered when the reactor operated at (lane 9) 5.7 and (lane 10) 10 g COD L−1 day−1. Lanes: 1 and 2,Methanococcus strains 1537 and 2067; 3, Methanobrevibacter strain 1125; 4–8, Methanobacterium strains 3108, 2956, 863, 2611 and 1535; 9,Methanosaeta strain 3671; 10, Methanothix strain 6194; 11–14, Methanosarcina strains 2053, 1232, 2834 and 800; 15, Methanospirillum strain 864;16, Methanogenium strain 1497. Species designation of each strain is given in Table 2.

the sludge samples. The 16S rRNA gene PCR titre ofhydrogenotrophic Methanobacteriaceae, predominatingin the biofilm at a VOL of 5.7 g COD L−1 day−1,decreased from 1011 to 108 cells g−1 sludge wetweight when the VOL was increased to 10 g CODL−1 day−1. The decrease in Methanobacteriaceae waspartially compensated by a moderate increase inMethanomicrobiaceae and relatives, whose 16S rRNAgene PCR titre increased from 104 to 106 cells g−1

sludge. Methanosaeta-like acetoclastic methanogenswere less affected by VOL variation and, under thefinal operative condition at 10 g COD L−1 day−1,dominated in the biofilm with a 16S rRNA gene PCRtitre of 109 cells g−1 sludge. A possible explanation ofthe high stability of the Methanosaeta-like communityis the localisation of this acetoclastic methanogen inthe biofilm. In the food chain of the methanogenicprocess, methanogens are the final substrate usersof the chain. It has been shown that, in sugar-fedreactors, methanogens are localised in the deepestparts of the biofilm structure.19–22 Methanosaeta ischaracterised by a high affinity for acetate and hencecan colonise the deepest niches in the biofilm or can besegregated in zones where the relative concentrationsof acetate are lower. It has been shown that, insome cases, Methanosaeta tends to be segregatedin specific cell clusters dispersed in the biofilm.23

Although the segregation in isolated cell clusters orin the deepest layers of a biofilm may negativelyaffect the rate of nutrient mass transfer, the highaffinity for the substrate allow the cells to survive andactively metabolise even at the relatively low substrateconcentrations. However, the deep position in thebiofilm allows the cells to be more protected fromthe effects of non-metabolised substrates or of toxiccompounds such as the polyphenols in OMW. Thisprotection from toxic polyphenols could explain theprevalence of Methanosaeta when the reactor operatedat the highest VOL.

The diversity of methanogens at VOLs of 5.7 and10 g COD L−1 day−1 was evaluated by SSCP analysisof a short fragment of the 16S rRNA gene specificallyamplified from methanogenic Archaea. The fragmentprofiles obtained were compared with those obtainedfrom pure strains (Fig. 4). SSCP of the amplified 16SrRNA gene at the two different VOLs showed thesame pattern type, suggesting that VOL variation did

not significantly affect the methanogenic communitystructure in the biofilm.

CONCLUSIONSThe upflow anaerobic filter described in this paperenabled effective anaerobic digestion of rather con-centrated OMW. The process performances werecomparable to those described by other authors.5 Incontrast to a recent study of a GAC biofilm reactortreating OMW,5 where the only methanogen foundwas Methanobacterium formicicum, here Methanobacte-riaceae and Methanosaeta/Methanothrix were the mainmethanogens in the reactor. These groups changedin relative abundance in response to increasing VOL.Apparently, VOL changes did not affect the archaealcommunity structure but rather the relative amount ofeach group in the sludge.

ACKNOWLEDGEMENTThis work is dedicated to the memory of Mauro Zucchi(1965–2001).

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