Waste Management 32 (2012) 1196–1201

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Waste Management

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High rate mesophilic, thermophilic, and temperature phased anaerobic digestionof waste activated sludge: A pilot scale study

David Bolzonella a,⇑, Cristina Cavinato b, Francesco Fatone a, Paolo Pavan b, Franco Cecchi a

a University of Verona, Department of Biotechnology, Strada Le Grazie, 15, 37134 Verona, Italyb University of Venice, Department of Environmental Sciences, Computer Science and Statistics, Dorsoduro 2137, 30123 Venice, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 May 2011Accepted 10 January 2012Available online 4 February 2012

Keywords:MesophilicThermophilicExtreme thermophilicTwo phase anaerobic digestionWaste activated sludge

0956-053X/$ - see front matter � 2012 Elsevier Ltd.doi:10.1016/j.wasman.2012.01.006

⇑ Corresponding author. Tel.: +39 045 8027965; faxE-mail addresses: david.bolzonella@univr.it (D. B

(C. Cavinato), francesco.fatone@univr.it (F. Fatone)franco.cecchi@univr.it (F. Cecchi).

The paper reports the findings of a two-year pilot scale experimental trial for the mesophilic (35 �C), ther-mophilic (55 �C) and temperature phased (65 + 55 �C) anaerobic digestion of waste activated sludge. Dur-ing the mesophilic and thermophilic runs, the reactor operated at an organic loading rate of 2.2 kgVS/m3dand a hydraulic retention time of 20 days. In the temperature phased run, the first reactor operated at anorganic loading rate of 15 kgVS/m3d and a hydraulic retention time of 2 days while the second reactoroperated at an organic loading rate of 2.2 kgVS/m3d and a hydraulic retention time of 18 days (20 daysfor the whole temperature phased system).

The performance of the reactor improved with increases in temperature. The COD removal increasedfrom 35% in mesophilic conditions, to 45% in thermophilic conditions, and 55% in the two stage temper-ature phased system. As a consequence, the specific biogas production increased from 0.33 to 0.45 and to0.49 m3/kgVSfed at 35, 55, and 65 + 55 �C, respectively. The extreme thermophilic reactor working at 65 �Cshowed a high hydrolytic capability and a specific yield of 0.33 gCOD (soluble) per gVSfed. The effluent ofthe extreme thermophilic reactor showed an average concentration of soluble COD and volatile fattyacids of 20 and 9 g/l, respectively. Acetic and propionic acids were the main compounds found in theacids mixture. Because of the improved digestion efficiency, organic nitrogen and phosphorus were sol-ubilised in the bulk. Their concentration, however, did not increase as expected because of the formationof salts of hydroxyapatite and struvite inside the reactor.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Sludge treatment and disposal has become a more pressingissue as sludge volumes are on the continual increase due to agrowing population and more stringent criteria for wastewatertreatment plants (WWTPs) effluents. The production of sludge inEurope stands at 10 million tonnes dry matter (Apples et al.,2008, whereas the figure for the US is reported to be around 6 mil-lion tonnes dry matter (Kargbo, 2010). The disposal of biosolids isone of the main costs for wastewater treatment works, togetherwith energy consumption and personnel, and may account for upto 50% of operating costs of WWTPs. Clearly, this is heavily depen-dent on specific local situations and sludge characteristics (i.e.,water, nutrients, and micropollutants contents).

This scenario highlights the urgent need to reduce the amountof sludge produced in the WWTPs. There are basically two waysthis can be done: directly, through the minimisation of sludge

All rights reserved.

: +39 045 8027929.olzonella), cavinato@unive.it, pavan@unive.it (P. Pavan),

production in the activated sludge process (Pérez-Elvira et al.,2006), or by improving the efficiency of the treatment technologiesin the sludge line of WWTPs. Since anaerobic digestion is the maintechnology for the treatment of waste sludge, major efforts havebeen dedicated in recent years to the study of sludge pre-treatmentby means of physical, chemical, and biological techniques (or com-binations of these). Research efforts have generally focused on theenhancement of the hydrolysis step (Carrère et al., 2010; Bragugliaet al., 2011). However, Boehler and Siegrist (2006) demonstratedthat the application of methods like mechanical disintegrationand ultrasounds or ozone application are convenient only whenthe costs for sludge disposal are higher than 450 euros per ton,while Schmelz et al. (2007) demonstrated by means of paralleltests that the application at full scale of various pre-treatmentprocesses (ball mill, ultrasounds, lysate centrifuge, and ozone)increased biogas production by only 20%. These values are veryoften insufficient to justify the investment and running costs forthe application of those pre-treatment techniques.

Recently, biological pre-treatments have received particularattention for industrial application due to their efficiency and rel-atively low cost compared to chemico-physical methods, which re-quire greater capital investment and have higher operating costs. A

Nomenclature

AD anaerobic digestionHAP hydroxyapatite phosphateHRT hydraulic retention timeMAP magnesium ammonium phosphate

OLR organic loading rateSRT solid retention timeTPAD temperature phased anaerobic digestionWAS waste activated sludge

D. Bolzonella et al. / Waste Management 32 (2012) 1196–1201 1197

particular kind of biological pre-treatment is the application of ahydrolysis step before methanisation in a two-step anaerobicdigestion process where the two stages generally operate at differ-ent temperatures (temperature phased anaerobic digestion, TPAD).In such a configuration different bacterial populations are providedwith the best conditions in which to operate (Lv et al., 2010). Two-phase processes have been widely applied in lab and pilot tests(Bhattcharya et al., 1996, Roberts et al., 1999; Song et al., 2004;Watts et al., 2005; Demirer and Othman, 2008) and full scale expe-rience has also been reported in literature (Oles et al., 1997;Cheunbarn and Pagilla, 2000). Extensive work on TPAD has beencarried out in the US with the aim of achieving class A biosolids(e.g., Vandenburgh and Ellis, 2002; Gray et al., 2006; Santhaet al., 2006).

A particular application of the temperature phased process isthe extreme thermophilic hydrolytic step, typically in the temper-ature range 60–80 �C, followed by the thermophilic (methanogen-ic) anaerobic digestion process (Wang et al., 1999; Gavala et al.,2003; Gray et al., 2006; Lu et al., 2008; Ferrer et al., 2009, 2010;Ge et al., 2011a,b).

In a previous paper (Bolzonella et al., 2007), we demonstrated atlab scale the benefits of this process when treating waste activatedsludge and its thermal sustainability. Here, we report the main find-ings from a three-year pilot scale experimentation which tested theperformance of the temperature phased anaerobic digestion ofwaste activated sludge at high solids concentration (6% dry matterin the feed). Results are compared with those obtained from meso-philic and thermophilic anaerobic digestion tests in terms of yields,nutrients fate, and specific resistance to filtration.

2. Materials and methods

The experimental architecture of the study consisted of a firsttrial (run 1) in mesophilic conditions (37 �C), a second (run 2) inthermophilic conditions (55 �C) and a third (run 3) in which ahydrolytic reactor working at 65 �C was added for the pre-treat-ment of waste activated sludge before going into the thermophilicanaerobic digester.

The waste activated sludge used in the experiment originatedfrom a 400,000 pe wastewater treatment plant that treats100,000 m3/d of municipal and (partially) industrial wastewatersin a denitrification–nitrification system. The applied solid reten-

Table 1Characteristics of the thickened waste activated sludge.

Avg Std Dev Min Max

pH (-) 6.4 0.1 6.3 6.7TS g/l 58.0 7.3 19.3 72.9TVS g/l 45.1 6.1 15.3 60.5TVS % 80 2 74 83COD/TVS (-) 1.6 0.48 0.7 3.2TKN % 7.5 1.20 4.7 9.8NH3–N gN/l 0.3 0.3 0.02 1.1TP % 1.9 0.5 0.2 3.1PO4–P gP/l 0.1 0.05 0.01 0.3

tion time in the activated sludge process is 15 days and the feedto microorganisms ratio (F/M) 0.2 kgCOD/kgMLVSS per day. Theaverage characteristics of waste activated sludge after centrifugethickening were those shown in Table 1.

The pilot scale anaerobic digester (methanogenic phase) oper-ated as a mesophilic reactor in phase 1 and a thermophilic reactorin runs 2 and 3 of the experimentation. It was a 1.3 m3 stirred reac-tor, initially inoculated with sludge from a full scale mesophilicreactor treating biological sludge characterised by a total solid con-centration of around 3%, 60% volatile. The specific methanogenicactivity on acetate of the inoculum was 0.08 gCOD-CH4/gVS perday (Angelidaki et al., 2009).

During run 3 the 1.3 m3 reactor was connected to an extremethermophilic (65 �C) anaerobic reactor for sludge pre-hydrolysis,with a total volume of 0.2 m3.

As for the operational conditions of the reactors, the methano-genic reactor operated with an HRT of 20 days in runs 1 and 2,18 days in run 3, and an OLR of 2.2 kgVS per m3 of reactor perday, while the hydrolytic reactor used in run 3 operated with anHRT of 2 days and an OLR of 15 kgVS per m3 of reactor per day(see Table 2).

All the analyses were carried out on grab samples according tothe Standard Methods (2005), while VFA were determined by GC ina Carlo Erba gas-chromatograph equipped with a flame ionisationdetector.

3. Results and discussion

The operational conditions applied to the reactors during themesophilic (run 1), thermophilic (run 2), and temperature phased(run 3) experimental trials are shown in Table 2: the HRT andOLR of the system were kept at 20 days and 2 kgVS per m3 of reac-tor per day in the three runs and only temperature varied.

The average characteristics of the waste activated sludge fed tothe reactors are shown in Table 1. The total solids after centrifuga-tion typically reached a 5.8% concentration (on average). Volatilesolids formed 80% of total solids and COD/VS ratio was 1.6. Thisfigure is higher than typical values reported in literature (e.g.,Bolzonella et al., 2005), and can be ascribed to the relatively highF/M ratio in the activated sludge process. The concentrations ofnitrogen and phosphorous were 7.5% and 1.9% respectively.

Table 3 shows the effluent characteristics, stability parametersand yields of the reactors during the experimentation. An increasein biogas production was observed when the process temperaturein the system was increased: biogas production rose from 0.88 m3/d in mesophilic conditions to 1.23 m3/d in thermophilic ones, andto 1.33 m3/d in the temperature phased system.

Table 2Operational conditions.

Run Temperature, �C HRT, d OLR kgVSfed/m3d

1 Meso 37 20 2.22 Thermo 55 20 2.33 2 phase – reactor 1 65 2 15.0

2 phase – reactor 2 55 18 2.3

Table 3Effluent characteristics, stability parameters, and yields for the three experimental runs.

Average values Phase 1 mesophilic Std dev Phase 2 thermophilic Std dev Phase 3 temperature phased Std dev

Effluent characteristicsTotal solids % 4.0 1.0 3.5 0.7 3.1 0.9Total volatile solids %TS 73 5.2 70 8.0 70 7.0pH (-) 7.8 0.1 7.8 0.15 7.9 0.1Total alkalinity (pH 4.3) mgCaCO3/l 8400 790 7970 750 8920 600Partial alkalinity (pH 5.7) mgCaCO3/l 6500 440 5980 512 6660 120VFA mg/l 570 400 710 540 830 170Total nitrogen (particulate) mgN/l 4210 311 3330 422 3440 375Ammonia mgN/l 2380 290 3130 250 3210 310Total phosphorus mgP/l 1180 530 1350 150 1130 90Phosphate mgP/l 200 62 230 15 230 22

YieldsVS removal (%) % 36 3.5 48 4.3 55 4.2COD removal (%) % 35 4.2 45 3.7 55 3.8Biogas production m3/d 0.88 0.25 1.23 0.18 1.33 0.22Biogas production rate m3/m3d 0.7 0.12 1.0 0.16 1.1 0.15Biogas productivity (on VSfed) m3/kgVSfed 0.33 0.08 0.45 0.05 0.49 0.06Biogas productivity(on VSrem) m3/kgVSrem 0.8 0.07 0.9 0.03 1.0 0.04CH4 content % 63 5 64 11 64 9

1198 D. Bolzonella et al. / Waste Management 32 (2012) 1196–1201

3.1. Mesophilic versus thermophilic trials

If we compare the results of the mesophilic and thermophilictrials, it is evident from the data reported in Table 3 that therewas a noticeable increase in terms of volatile solids and COD re-moval when the reactor temperature was raised, removal ratesincreasing from 36% to 48% and from 35% to 45% for VS and CODrespectively. As a consequence, biogas production went up from0.88 to 1.23 m3/d, or from 0.33 to 0.45 m3/kgVS in specific terms.The increase in biogas yield on destroyed volatile solids was lessappreciable: from 0.8 to 0.9 m3 per kgVS destroyed in the switchfrom mesophilic to thermophilic conditions. These results are con-sistent with those of previous studies reported in literature(Speece, 1988; Bolzonella et al., 2002, 2005). Methane content re-mained at a constant level: 63% and 64% in mesophilic and thermo-philic conditions, respectively. These values confirm previousfindings by de la Rubia et al. (2002, 2006), Gavala et al. (2003),and Nges and Liu (2010). Fig. 1 compares the different yields interms of VS and COD removal as well as specific biogas productionfor the two experimental runs.

As regards the stability parameters, pH was 7.8 in both cases,and total and partial alkalinity remained more or less stable. Thesame was true for VFA. Because of the increased hydrolysis of pro-tein material, the amount of ammonia in the bulk also increased,going from 2380 to 3130 mgN/l. There was, therefore, a corre-sponding high concentration of free ammonia as a consequenceof the combination of pH and temperature in the reactor. Despite

Fig. 1. Yields of the system along the different experimental runs.

this, the thermophilic process was stable with a higher degradationrates occurring at this temperature compared to the mesophilicconditions. Once again, the results concur with previous studies(e.g., Siegrist et al., 2002; de la Rubia et al., 2006). This experimen-tation therefore confirmed that it is possible to increase the reactortemperature from mesophilic to thermophilic conditions and ob-tain a considerable increase in biogas production (36%) withoutjeopardising the process stability (Dohanyos et al., 2004).

3.2. Temperature phased anaerobic digestion (TPAD) trials

During the last part of the experiment, sludge was first fed intoa 200 l stirred reactor working in extreme-thermophilic conditions(65 �C) prior to its digestion in the thermophilic reactor. The oper-ating conditions for the global system were the same as thoseadopted in previous experimental runs: a global HRT of 20 days(2 days in the first and 18 in the second reactor), and an OLR of2.2 kgVS/m3d were applied to the methanogenic phase of theprocess.

The hydrolytic reactor operated with a hydraulic retention timeof 2 days and an OLR of 15 kgVS/m3d. The characteristics of theeffluent sludge are those shown in Table 4. The biogas productionin this reactor was constantly lower than 0.06 m3 per kgVS fed, CO2

being by far the major component in the gas mixture. The presenceof methane was never observed at a level above 30%, while hydro-gen was detected at levels below 10%.

The concentrations of soluble COD and VFA were 19.6 and 7.9 g/l respectively, while corresponding specific yields were 0.33 and0.13 g per gVS fed. These results are compatible with a hydrolysisconstant of 0.1 day�1 already reported by the authors in a previouspaper (Bolzonella et al., 2007) and recently confirmed by Ge et al.(2011a,b). The main compounds found in the acid mixture wereacetic acid, propionic, and n-butyric acids, while iso-butyric andnormal- and iso-valerate were found at very low concentrations.The presence of n-butyrate, however, was surprising as thesemolecules have been reported to easily degrade to acetic acid viab-oxidation (Aguilar et al., 1995; Wang et al., 1999; Bolzonellaet al., 2007) while iso-forms of both butyrate and valerate havebeen reported to degrade slowly (Batstone et al., 2003; Pindet al., 2003).

These results concur with those we found in previous lab-scaleexperiments (Bolzonella et al., 2007). It is, then, interesting to notethat in our previous study at lab-scale, short chained VFA repre-sented 90% of the soluble COD, while in the current study they

Table 4Characteristics of sludge effluent the extreme thermophilic reactor (65 �C).

Parameter Average value Std dev Bolzonella et al. (2007)(HRT 1 d, 70 �C)

Std dev

pH 6.3 0.05 6.7 0.03TS g/l 56 2.1 30 1.9TVS g/l 46 2.0 22 1.6TVS %TS 79 8 74 6SCOD g/l 19.6 2.53 9.0 1.25SC-VFA g/l 7.9 0.72 7.0 0.31Acetic % on TVFA 37 7 34 4Propionic % on TVFA 25 6 20 2iso-Butyrate % on TVFA 6 2 9 1n-Butyrate % on TVFA 19 3 6 0.7iso-Valerate % on TVFA 6 1.7 16 1.8n-Valerate % on TVFA 1 0.2 1 0.1Alkalinity @ pH 4.3 gCaCO3/l 6.5 0.4 3.6 0.3Alkalinity @ pH 5.7 gCaCO3/l 1.5 0.1 1.2 0.1SCOD/VSfed 0.35 0.06 0.30 0.07

D. Bolzonella et al. / Waste Management 32 (2012) 1196–1201 1199

represent less than 50% of the soluble COD produced. This differ-ence could be a result of either the reactor operating conditionsor the sludge characteristics. In fact, the range of acidification prod-ucts observed and reported in literature is variable, depending onthe origin of the sludge and the operating conditions of the reactor(e.g., Gray et al., 2006; Ucisik and Henze, 2008; Zhang et al., 2009;Rubio-Loza and Noyola, 2010).

Because of the hydrolytic pre-treatment, the two-stage systemshowed a further increase in yields compared to the single-stagemesophilic and thermophilic trials. In fact, the percentage for bothVS and COD removal was 55%, slightly higher than those obtainedin thermophilic conditions (see Table 3). Biogas production rose to1.33 m3/d representing a 48% increase on that produced in the sin-gle-stage mesophilic reactor, and a 12% increase on that producedin the single-stage thermophilic one. However, methane contentremained at the same level as in previous trials (64% on average).Therefore, all the specific yields showed higher values than thoseobtained in mesophilic and thermophilic trials 1 and 2: SGP was0.49 m3/kgVSfed and SGP on destroyed VS reached 1.0 m3/kgVSrem,a value generally found in anaerobic digestion reactors treatingprimary or mixed sludge (Speece, 1988).

In this case, too, the process was very stable. It reached a steadystate condition immediately after the hydrolytic step was imple-mented, and all the stability parameters were at levels similar tothose observed in the single-phase thermophilic trials (see Table 3).

Fig. 1 compares the different yields in terms of VS and COD re-moval as well as specific biogas production for the three experi-mental runs.

It is then interesting to note that the yields from the tempera-ture phased configuration are sufficient to sustain the energeticbalance of the process even in the winter season as demonstratedby Bolzonella et al. (2007), Lu et al. (2008), and Nges and Liu(2010).

3.3. Nutrients behaviour and fate

The increased hydrolysis process of the TPAD configurationinevitably cause the extra release of ammonia nitrogen and phos-phate in the bulk and a consequent increase in terms of concentra-tions. However, while this was clearly observed for ammonia, itwas not for phosphorous. Ammonia concentration increased from2380 mgN/l in mesophilic conditions to 3130 mgN/l in thermo-philic conditions, and there was a further increase to 3210 mgN/lin the temperature phased system. The increase in concentrationtherefore was 31% and 35%, in the thermophilic and TPAD systemrespectively, compared to that produced in the mesophilic reactor.These figures are closely correlated with the increased destruction

of volatile solids, which rose from 36% in mesophilic conditions to48% in thermophilic conditions, reaching 55% in the temperaturephased system. The increase of ammonia concentration partiallycontributed to the rise in pH and alkalinity in the temperaturephased process (see data reported in Table 3).

Obviously, the increased presence of ammonia in the rejectwater from the dewatering station must be considered as an extracost when performing the economic assessment for the TPAD pro-cess implementation in a WWTP.

Not all of the solubilised organic nitrogen, however, was foundas ammonia in the bulk: part of it was involved in the precipitationof phosphorous salts like struvite (see below).

As regards phosphorous, it was observed that phosphate con-centrations in the soluble phase of the digestate remained at con-stant levels of 200–230 mgP/l despite the increased removal ofvolatile solids described above. Given the P concentration in sludge(1.9% on dry matter, Table 1), expected concentrations for phos-phate in the liquid phase of anaerobic effluents should be in therange 475–723 mgP/l on a mass balance basis.

It is therefore clear that phosphate is precipitated in the form ofinorganic salts in the reactor, hydroxyapatite (Ca5(OH)(PO4)3, HAP)and struvite (MgNH4PO4�6H2O, MAP) being likely forms. It hasbeen reported in fact that the high co-presence of Ca2+ and Mg2+

together with high concentrations of ammonia and phosphatescan lead to the formation of these salts (Battistoni et al., 2001;Pastor et al., 2008; Marti et al., 2008).

The presence of Ca2+ and Mg2+ were therefore determined in theliquid and solid phase of both the influent and effluent streams ofthe anaerobic digester. In the fed sludge, levels of Ca2+ and Mg2+ inthe liquid phase were 90 and 52 mg/l on average, respectively,whereas the presence of Ca2+ and Mg2+ in the sludge were 25and 6 g/kg dry matter respectively. In the case of Ca2+, however,only a minimal part is related to the biomass, typically 2 g/kg drymatter (Marti et al., 2008; Pastor et al., 2008), the remaining partbeing inorganic precipitates.

Given these levels of concentrations in sludge, expected concen-trations in the soluble phase of the digestate were 132–156 mg/lfor Ca2+ and 178–250 mg/l for Mg2+, respectively. Comparing thesevalues with those expected for phosphate, a molar ratio in theliquid phase being of 0.31 for Ca/P and 0.40 for Mg/P on averagecan be calculated. These values are compatible with the moderateprecipitation of phosphorus salts inside the reactor, in particular inthe form of hydroxyapatite (Battistoni et al., 2006).

Because of the salts precipitation, observed concentrations inthe liquid phase were therefore 55, 83, and 63 mg/l for Ca2+ and21, 68, and 29 mg/l for Mg2+ in the mesophilic, thermophilic andTPAD system, respectively.

1200 D. Bolzonella et al. / Waste Management 32 (2012) 1196–1201

The average percentages of precipitated P, Mg and Ca weretherefore 62%, 78%, and 51% respectively, clearly indicating theprobable formation of hydroxyapatite and struvite in the digester(Marti et al., 2008).

This phenomenon should be carefully considered and assessedin full scale applications and a specific process for struvite removalimplemented in specific cases (Battistoni et al., 2001; Marti et al.,2008).

4. Conclusions

The study reported in this paper considered the results of pilotscale trials on mesophilic, thermophilic, and temperature phasedanaerobic digestion of waste activated sludge at high rate (6%dry matter in the feed and organic loading rate of 2 kgVS/m3d).

The main findings of the study can be summarised as follows:

� The thermophilic and temperature phased processes showedclear increases in terms of organic matter removal and biogasproduction compared to the mesophilic process. In particular,the volatile solids removal increased from 36% in mesophilicconditions to 48% in thermophilic conditions, rising to 55% inthe temperature phased system. As a consequence, the specificbiogas production was 0.33, 0.45, and 0.49 m3/kgVS, respec-tively, with a 36% and 48% increase from the mesophilic tothe thermophilic and TPAD trials, respectively;

the increased hydrolysis of the organic matter determined animproved concentration of ammonia nitrogen and soluble COD inthe anaerobic supernatant (+33% and +100% respectively). This ex-tra-load needs to be treated in the wastewater treatment line andshould be taken into account when considering the application ofpre-treatment systems for sludge reduction. Moreover, the precip-itation of struvite and hydroxyapatite in the reactor was demon-strated on a mass balance basis – the higher the temperature thehigher the salts precipitation.

Acknowledgment

The Fondazione Cariverona (Scientific and Technological Re-search Program 2003) is gratefully acknowledged for the financialsupport of this research.

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