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Microthrix parvicella and Gordona amarae inmesophilic and thermophilic anaerobic digestionsystemsMatina Marneri a , Daniel Mamais a & Efi Koutsiouki aa National Technical University of Athens, Faculty of Civil Engineering, Department of WaterResources and Environmental Engineering, 5, Iroon Polytechniou, Zografou, Athens 15780,GreeceVersion of record first published: 06 Apr 2009.

To cite this article: Matina Marneri , Daniel Mamais & Efi Koutsiouki (2009): Microthrix parvicella and Gordona amarae inmesophilic and thermophilic anaerobic digestion systems, Environmental Technology, 30:5, 437-444

To link to this article: http://dx.doi.org/10.1080/09593330902760631

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Environmental Technology

Vol. 30, No. 5, 14 April 2009, 437–444

ISSN 0959-3330 print/ISSN 1479-487X online© 2009 Taylor & FrancisDOI: 10.1080/09593330902760631http://www.informaworld.com

Microthrix parvicella

and

Gordona amarae

in mesophilic and thermophilic anaerobic digestion systems

Matina Marneri*, Daniel Mamais and Efi Koutsiouki

National Technical University of Athens, Faculty of Civil Engineering, Department of Water Resources and Environmental Engineering, 5, Iroon Polytechniou, Zografou, Athens 15780, Greece

Taylor and Francis

(

Received December 2007; Accepted January 2009

)

10.1080/09593330902760631

The scope of the study presented in this paper is to determine the fate of the filamentous bacteria

Gordona amarae

and

Microthrix parvicella

in anaerobic digestion operating under mesophilic and thermophilic conditions. In order todetect and quantify foaming bacteria in the anaerobic digesters, a fluorescent

in situ

hybridization (FISH) method wasdeveloped and applied. This paper presents the results of a laboratory-scale study that involved the operation of fourlab-scale anaerobic digestion systems operating in the mesophilic (35

°

C) and thermophilic (55

°

C) temperatureranges at 20 days’ detention time. According to the FISH counts of

G. amarae

and

M. parvicella

, it appears thatthermophilic conditions resulted in a higher destruction of both filamentous bacteria, averaging approximately 97%and 94% for the single thermophilic digester and the dual thermophilic/ mesophilic system, respectively. Within thecontext of this study, the overall performance of the four different anaerobic digestion systems was evaluated in termsof biogas production per mass of volatile solids destroyed, COD destruction, sludge dewaterability and foamingcharacteristics. The dual stage systems used in this study outperformed the single stage digesters.

Keywords:

M. parvicella

;

G. amarae

; foaming potential; anaerobic digestion; FISH

Introduction

The abundance of the filamentous bacteria

Gordonaamarae

(previously known as

Nocardia amarae)

and

Microthrix parvicella

is often associated with signifi-cant operating problems in activated sludge wastewatertreatment plants [1]. During these events, processcontrol of the sewage treatment line becomes extremelydifficult since a significant portion of the solids inven-tory is present in the foam. At the same time, the foam-ing problem is not restricted to the secondary systembut often initiates operating difficulties in anaerobicdigesters. Westlund

et al.

reported that three largewastewater treatment plants in the greater Stockholmarea have experienced serious digester foaming [2].Microscopic studies of the sludge from the foam phaseshowed a network of the filamentous organism

M.parvicella

. Another case of anaerobic digestion foam-ing caused by the abundance of the filamentous bacte-rium

G. amarae

is reported by Jones

et al.

[3]. Theauthors correlated the overgrowth of the filament in thesecondary system with foaming problems in the anaer-obic digesters. The produced foam reduced the activevolume of the digesters, resulting in interruption ofbiogas collection and reduced performance in both thedigestion and thickening processes.

Excessive levels of the filamentous bacteria

G.amarae

and

M. parvicella

in anaerobic digestion cancause foaming problems not only in the anaerobicdigestion process itself but in a preceding activatedsludge system as well. The presence of these filamen-tous foaming bacteria in the recycle stream from thedewatering facilities can seed the liquid treatmentprocesses and can cause them to persist in the activatedsludge [4,5] . Monitoring these filamentous bacteriaas well as estimating their viability could help toimprove control of the operating difficulties resultingfrom their overgrowth in activated sludge systems andmay even predict and avert foaming events. So far, themost common technique used to monitor the microbio-logical populations related to foaming is the Pitt–Jenkins technique [4] of Gram staining andintersections counting. This method is applicable toboth

G. amarae

and

M. parvicella

in activated sludgesince they are Gram-positive bacteria with uniquemorphology. Despite the fact that this technique isstraightforward and relatively fast it presents severalshortcomings, because it does not provide informationon the identity or on the viability of a bacterium.Furthermore, it has been observed that

G. amarae

willonly weakly retain a Gram-positive strain when

*Corresponding author. Email: mmarneri@mesogeos.gr

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M. Marneri

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subjected to competitive stress in anaerobic or anoxicselectors, and will completely lose their Gram stainingcharacteristics in anaerobic digesters because thestressful conditions of anaerobic digestion alter thecharacteristics of the cellular membrane [5]. Also, asreported by Westlund

et al.,

the morphology of thelong, coiled

M. parvicella

filament appears to beaffected by the anaerobic conditions where it becomesbroken up into shorter and thicker filaments [2]. There-fore, in order to study the fate of biological foaming inanaerobic digesters, other immunofluorescent or fluo-rescent

in situ

hybridization (FISH) methods have beendeveloped and applied.

Hernandez

et al.

[1] have developed an immunof-luorescent method to estimate the quantity and viabil-ity of

G. amarae

in anaerobic digestion based on theproduction of antibodies from rabbits injected with

G.amarae

emulsion. The bacterium was found tocomprise 13% of the volatile suspended solids in afoaming digester sludge, and viability was judged by2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazo-lium chloride (INT) reduction. The reduction of INTto INT-formazan crystals by bacteria has been devel-oped to examine their activity since INT acts as ahydrogen acceptor causing respiring (active) bacteriato accumulate water-insoluble red INT-formazan crys-tals.

Gordona amarae

was estimated to be 37–54%viable in mixed anaerobic digesters with hydraulicdetention time ranging from 14 to 28 days [7]. Astudy by de los Reyes

et al.

[6] compared the use ofantibody probes with oligonucleotide hybridizationprobes and due to the advantages the FISH methodpresents, especially with respect to the potential forobtaining information on metabolic activity, havedeveloped oligonucleotide probes for the identificationand quantification of

G. amarae

in anaerobic digesters[8]. In 1997, Erhart

et al.

developed fluorescent probesfor the identification of the microorganism

Microthrixparvicella

using FISH. In the same study they alsodeveloped a method for successful permeabilization ofthe bacterium employing mutanolysin [9]. In addition,Moter and Gobel [10] employed FISH to quantifyviable microorganisms. Therefore, within the contextof this study FISH was employed to provide an esti-mate of the viability of

G. amarae

and

M. parvicella

under anaerobic conditions.

The scope of the study presented in this paper wasto determine the fate of the filamentous bacteria

G. amarae

and

M. parvicella

in anaerobic digestion bymeans of FISH. The overall performances of fourdifferent anaerobic digestion systems were also evalu-ated in terms of biogas production per mass of volatilesolids destroyed, COD (chemical oxygen demand)destruction, concentration of volatile fatty acids, sludgedewaterability and foaming potential.

Materials and methods

Lab-scale set-up

The lab-scale systems consisted of six 5 L conical flaskswith 3 L operating volume. As shown in Table 1, onemesophilic reactor (35

°

C) and one thermophilic (55

°

C) reactor were operated at a detention time of 20 days,while two mesophilic reactors were operated in series,each at 10 days’ detention time. The fourth systemconsisted of a thermophilic reactor operating at ahydraulic residence time of eight days followed by amesophilic reactor with a residence time of 12 days. Alldigesters were initially filled with mesophilic digestedsludge from the Psyttalia Wastewater Plant in Athens(Psyttalia) and subsequently all anaerobic digesterswere fed daily with thickened primary and secondarysludge (1:1 volume-to-volume ratio) sampled fromPsyttalia. The feed of the digesters was supplementedwith foam from the secondary system of Psyttalia at apercentage of 10% w/w. The daily volume of sludgefeed was determined so that the residence time in alldigester systems was kept constant and equal to 20days. Microscopic observation of the foam showed thatat the beginning of the test period the dominant filamen-tous microorganism was

G. amarae

and

M. parvicella

was secondary

.

However, gradually the filamentousbacteria population changed and

M. parvicella

becamedominant.

Data collection commenced after 60 days of initialoperation while the units operated continuously for aperiod of 120 days. The sludge samples collected fromthe thermophilic and the mesophilic digesters wereanalysed for total and volatile solids (TS, VS), total andsoluble chemical oxygen demand (COD, sCOD:through membrane 0.45

µ

m) and volatile fatty acids(VFA). All the analyses were conducted according to

Table 1. Operating conditions of the four lab-scale anaerobic digestion systems (average values and 95% confidence intervals).

Single Mesophilic Digester

Single Thermophilic Digester

Dual Thermophilic / Mesophilic System Dual Mesophilic System

Detention time (d) 20 20 8 12 10 10Temperature (

°

C) 35.5

±

0.6 55.2

±

0.3 54.6

±

0.3 35.0

±

0.5 36.6

±

0.7 36.1

±

0.7pH 7.3

±

0.1 7.3

±

0.1 6.7

±

0.1 7.4

±

0.1 7.3

±

0.1 7.5

±

0.1

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439

Standard Methods [11]. Also, sludge dewaterability,foaming tests and FISH tests were performed weeklyaccording to the experimental protocols described in thenext paragraphs. Biogas was collected in invertedcylinders placed in a compartmentalized tank filled withacidified water, and sludge gas production rates weremeasured by the water displacement method.

Sludge dewaterabillity

One of the classical and most commonly used parame-ters for evaluation of sludge dewaterability is thespecific resistance to filtration (SRF) [12], whichmeasures the resistance of sludge to dewatering [13].Thirty millilitres of digested sludge were vacuum-filtered through Whatman filters (size 40) and thevolume of the filtrate over time was recorded until thevolume–time correlation shifted from linear to para-bolic. At that time the percentage of TS of the cakeformed on the filter and the slurry remaining in theBuchner funnel were measured.

The specific resistance was calculated using thefollowing formula:

where

r

is the specific resistance, m kg

−

1

;

P

is thevacuum pressure (76–80 kPa);

A

is the surface of thefilter, m

2

;

µ

is the viscosity of filtrate (taken as viscosityof water), N s m

−

2

;

b

is the slope of the linear correlationof

∆

t/

∆

V to V, s m

−

6

,

and

C

s

,

C

c

are slurry and cake suspended solids concen-trations, respectively, in per cent.

Foaming tests

The foaming potential and the foaming stability of thedigested sludge samples were measured with thecontrolled provocation of foaming in 1L volumetriccylinders by adding two Panadol Extra tablets in200 mL of digested sludge. The foaming potential wasequal to the maximum height of the foam layer gener-ated and was considered as an indication of the propen-sity of the sample to foam. The foaming stability wasequal to the time required for the generated foam tocollapse. This test is a variation of the Alka Seltzer testdeveloped by Ho and Jenkins [14] but due to the factthat Alka Seltzer tablets are commercially unavailablein Greece, Panadol Extra tablets, which contain the

same active substances (sodium bicarbonate and citricacid), were used instead. Both tests were conducted at37

°

C and at 1.5% total solids concentration.

Fluorescent

in situ

hybridization (FISH)

Cell fixation

Samples were fixed in a 1:1 volume-to-volume ratiowith pure grade ethanol (

≥

99.8%) fixative at 0

°

C for4 to 16 h, subsequently washed twice in phosphate-buffered saline (PBS) solution, and stored in 50% coldice ethanol in PBS at

−

20

°

C. The samples were blendedbefore their fixation for two minutes in order to open thefloc structure and obtain a better view of the filamentsduring microscopic observation.

In situ

hybridization

Teflon-coated 12-well slides (Precision Lab Products,LLC, Middleton, USA) were used in the study. Threemicrolitres of the fixed samples were spotted in eachwell, air dried and dehydrated in an ethanol series (50%,80%, and 99%) for three minutes each. Subsequently,mutanolysin (Sigma Aldrich, Cat. No. M9909) at aconcentration of 5000 U ml

−

1

in 0.1 M phosphate bufferwas used to make the cell wall of

G. amarae

and

M.parvicella

more permeable to the probes, and the slideswere incubated for 20 min at 4

°

C. After applying amixture of 1

µ

L of probe (50 ng

µ

L

−

1

) and 9

µ

L ofhybridization buffer (0.9 M NaCl, 0.1 M Tris-HCl,0.1% sodium dodecyl sulphate (SDS), 30% formamide,for

G. amarae

and 0.9 M NaCl, 20 mM Tris-HCl,0.01% SDS, 20% formamide for

M. parvicella

) on eachslide, the slides were incubated for four hours in ahumid chamber at 46

°

C. The probe used in this studywas the S-S-G.am-0205-a-A-19 (3

′

CGAAAACGC-CAGTCCCTAC 5

′

) developed by de los Reyes

et al.

[6] labelled with Cy3, which targets

G. amarae.

For theidentification of

M. parvicella

the probe MPA223 (3

′

GCCGCGAGACCCTCCTAG 5

′

) developed by Erhart

et al

. [9] labelled with Cy3 was implemented. Subse-quently, the slides were washed in prewarmed buffer for15 min at 48 °C, rinsed with ice-cold deionized waterand air dried. The washing buffer contained 20 mMTris-HCl (pH 7.2), 0.1% SDS, 0.215 M NaCl and 0.08M NaCl for M. parvicella and G. amarae, respectively,and 5 mM EDTA for G. amarae.

Microscopy

Slides were mounted with Citifluor solution (CitifluorLtd, Leicester, UK) and examined at 1000× magnifica-tion with a Nikon E50i microscope equipped with aspecific filter set for Cy3.

rP A b

w=

× × ××

21

2

µ( )

wC C

C Cc s

c s

=100( – )

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Results and discussion

Performance of the lab-scale digesters

Table 2 presents the performance characteristics of thedifferent anaerobic digestion systems. From the table itcan be seen that all digesters operated satisfactorily withaverage volatile solids reductions ranging from 25% to46%, total COD reductions within the range 21% to 47%and average biogas production rates from 0.42 to1.06 m3 biogas (kg VS destroyed)−1. The highestefficiencies were achieved by the dual mesophilicsystem, followed by the dual mesophilic–thermophilicand the single mesophilic digesters. This finding agreeswith both Cheunbarn and Pagilla [15] and Song et al.[16], who reported that the percentage destructions indual-stage digestion were considerably higher than insingle-stage digesters.

From Table 2 it can be seen that the soluble CODconcentration and the VFA levels were affected consid-erably by the digestion conditions. The soluble CODconcentration and the VFA level were higher in thesingle thermophilic digester than in the single meso-philic one, while they were reduced considerably in thedual stage digesters, a finding that agrees with resultsreported elsewhere [16]. The low biogas productionobtained in the thermophilic systems may be attributedto the high VFA concentrations that, according tothe literature [17–19], often cause inhibition ofmethanogens.

The dewaterabillity of the digested sludge can beestimated by calculating different parameters, such asthe polymer dose, the capillary suction and the specificresistance. In this study, the specific resistance wasemployed to assess the dewaterability of digestedsludge. The specific resistance is considered to beinversely proportional to the dewaterability of sludge.In order to have reference values for comparison, thespecific resistance values of the pre-thickened sludgefed to the lab-scale digesters, and the mesophilicdigested sludge from the full-scale digesters at Psyttalia,

were also calculated and were found to be 0.54 × 106

and 1.29 × 106 Tm kg−1.From the results it can be concluded that the sludge

of the single mesophilic digester had the best dewateringproperties, followed closely by the full-scale mesophilicdigester at Psyttalia and the dual mesophilic lab-scalesystems. On the other hand, the sludge from the singlethermophilic digester presented a higher specific resis-tance and therefore worse dewatering properties than allother digestion systems, a finding that agrees with otherstudies [18,20]. According to the study of Bivins andNovak, thermophilic anaerobic digestion creates orreleases colloidal materials that deteriorate the dewater-ing properties of digested sludge [21].

G. amarae and M. pervicella fate in anaerobic digesters

Average FISH counts and error bars at the 95% confi-dence level are shown in Figures 1 and 2 for all foursystems. It has to be noted that the G. amarae FISHcounts are lower than the M. parvicella ones becauseduring the time of the experiment the filamentous popu-lation shifted to the latter. As shown in the figures, thelowest concentrations of both G. amarae and M. parvi-cella were obtained in the two thermophilic systems(T20 and T8/M12).Figure 1. Average G. amarae FISH counts for each lab-scale digester.Figure 2. Average M. parvicella FISH counts for each lab-scale digester.Table 3 illustrates the average destruction of fila-mentous bacteria achieved by each system, calculatedaccording to the total quantities of G. amarae and M.parvicella being fed and withdrawn from each system,as determined by the FISH counts. According to theresults of FISH counts, the mesophilic digestersachieved destructions of 80.0–80.4% and 75.6–76.9%for G. amarae and M. parvicella, respectively. Gordonaamarae results appear to be in good agreement withprevious studies employing an immunofluorescentmethod, which reported an average viability decrease ofapproximately 63% at a 14 days solids retention time [1].

Table 2. Performance of the lab-scale digesters (average values and 95% confidence intervals).

Digester

20-day Mesophilic

20-day Thermophilic

8-day Thermophilic/12-day Mesophilic

10-day Mesophilic/10-day Mesophilic

Volatile solids reduction (%) 41.7 ± 2.7 25.3 ± 6.1 44.0 ± 5.1 45.6 ± 3.3Biogas production / mass VS destroyed (m3 kg−1)

0.95 ± 0.12 0.42 ± 0.22 1.06 ± 0.18 0.61 ± 0.12

COD reduction (%) 35.0 ± 7.2 20.7 ± 5.8 44.1 ± 4.3 46.7 ± 4.8Soluble COD (mg l−1)(SCODfeed= 3129 ± 235)

2,748 ± 367 3,980 ± 882 4,072 ± 546/591 ± 144

449 ± 185

VFA concentration (mg l−1) 860 ± 123 2,518 ± 403 2,854 ± 374/508 ± 80

473 ± 72

SRF (Tm kg−1) 1.1*106±2.7*105 3.2*106±8.4*105 1.4*106±1.8*105 1.6*106±3.2*105

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Significantly higher reduction rates were observed in thethermophilic digester and in the 8-day/12-day thermo-philic/mesophilic system, ranging from 93.2% to 97.8%and from 94.6% to 97.1% for G. amarae and M.parvicella, respectively. These results indicate that

thermophilic digestion or a combination of thermophilicand mesophilic digestion resulted in a higher decrease inthe viability of G. amarae and M. parvicella. Accordingto the results it appears that M. parvicella is slightlymore sensitive than G. amarae to anaerobic thermo-philic conditions.

Foaming results

The foaming potential and stability were measured bythe Panadol test, and the reduction percentages withrespect to the feeding sludge are presented in Figures 3and 4, respectively. From the results it can be concludedthat only the dual-stage systems and especially the dual

Figure 1. Average G. amarae FISH counts for each lab-scale digester.

Figure 2. Average M. parvicella FISH counts for each lab-scale digester.

Table 3. Destruction of G. amarae and M parvicella in thedifferent anaerobic digestion systems as determined by FISH.

M20 T20 T8/M12 M10/M10

Gordona destruction (%)

80.0 ± 3.3 97.8 ± 3.5 93.2 ± 3.4 80.4 ± 3.3

Microthrix destruction (%)

75.6 ± 2.4 97.1 ± 2.6 94.6 ± 2.6 76.9 ± 2.5

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thermophilic/mesophilic digesters lowered the foamingpotential and stability of the sludge significantly. Aclear conclusion, with respect to the effect of the ther-mophilic and the mesophilic conditions on the foamingcharacteristics of the anaerobically digested sludge,cannot be drawn. Apparently, the foaming potential andstability of the anaerobic digesters is a combined effectof both the concentration of the filamentous bacteriaand the concentration of foam-stabilizing materials inthe digested sludge. On the one hand, the concentrationof filamentous bacteria was higher in the mesophilicdigesters as determined by the FISH counts and, on theother hand, the concentration of foam-stabilizingmaterials is expected to be higher in the thermophilic

digesters due to the higher hydrolysis of organic matter,as reported in many studies [15,16,20]. The greaterdestruction of filamentous bacteria obtained in thethermophilic digesters may have further increased theconcentration of colloidal hydrophobic compounds dueto the release of compounds such as mycolic acid,therefore contributeing to the higher foaming potential.Figure 3. Reduction in foaming potential of the lab-scale digesters as measured by the Panadol foaming test.Figure 4. Reduction in foaming stability of the lab-scale digesters as measured by the Panadol foaming test.

Conclusions

In this study the performance of a 20-day mesophilicdigester was compared with that of a 20-day thermo-philic digester and with two dual-stage digestionsystems, the first consisting of an eight-day thermophilic

Figure 3. Reduction in foaming potential of the lab-scale digesters as measured by the Panadol foaming test.

Figure 4. Reduction in foaming stability of the lab-scale digesters as measured by the Panadol foaming test.

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digester followed by a 12-day mesophilic digester andthe second consisting of two 10-day mesophilic digest-ers in series. The performance was evaluated in terms ofbiogas production per mass of volatile solids destroyed,volatile solids and total COD reductions, soluble CODand VFA concentrations, sludge dewaterability andfoaming characteristics, as well as destruction of fila-mentous foaming bacteria. According to the results thehighest overall efficiencies were achieved by the dualmesophilic-thermophilic anaerobic digestion system.

Thermophilic conditions resulted in a high destruc-tion of filamentous foaming bacteria. According to G.amarae FISH counts, average destructions obtainedunder anaerobic mesophilic and thermophilic condi-tions were 80% and 95%, respectively. Similarly M.parvicella average destructions obtained under anaero-bic mesophilic and thermophilic conditions were 76%and 96%, respectively. It should be underlined,however, that although anaerobic conditions induced ahigher destruction of filamentous foaming bacteria,they did not improve the sludge foaming characteristicssignificantly. Apparently, the foaming potential andstability of anaerobic digesters is a combined effect ofboth the concentration of the filamentous bacteria andthe concentration of foam-stabilizing materials in thedigested sludge. Only the implementation of the dualthermophilic/mesophilic digesters significantly decrea-sed the foaming potential and stability of the sludge andresulted in a high destruction of filamentous foamingbacteria. Therefore, it can be concluded that dual ther-mophilic/mesophilic anaerobic digestion systemsappear to be able to minimize foaming problems as wellas to decrease the concentration of filamentous foamingbacteria significantly, subsequently resulting in adecrease in the filaments in the recycle stream from thedewatering facilities to the activated sludge process.

AcknowledgementsThe authors would like to thank Dr. Simona Rossetti, aresearcher in the Italian National Research Center in theWater Research Institute (CNR-IRSA) for her input withrespect to difficulties encountered with the application of theFISH method. The Project was co-funded by the GreekGeneral Secretariat for Research and Technology, PENED03D395.

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