Thermophilic anaerobic digestion of thermal pretreated sludge: Role of microbial community structure and correlation with process performances

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    wat e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 9 8e5 0 9Available online at wScienceDirect

    journal homepage: www.elsevier .com/locate /watresThermophilic anaerobic digestion of thermalpretreated sludge: Role of microbial communitystructure and correlation with processperformancesM.C. Gagliano a, C.M. Braguglia a, A. Gianico a, G. Mininni a,K. Nakamura b, S. Rossetti a,*

    a Water Research Institute, IRSA-CNR, Via Salaria km 29,300, 00015 Monterotondo (RM), Italyb Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japana r t i c l e i n f o

    Article history:

    Received 17 May 2014

    Received in revised form

    7 October 2014

    Accepted 15 October 2014

    Available online 24 October 2014


    Thermophilic anaerobic digestion

    Thermal hydrolysis



    Fluorescence in situ hybridizationAbbreviations: AD, Anaerobic Digestion;Activated Sludge; TS, Total Solids; VS, VolaPolymeric Substances; FISH, Fluorescence In* Corresponding author. Tel.: 39 06 9067269E-mail address: (S. Ro 2014 Elsevier Ltd. All rights resea b s t r a c t

    Thermal hydrolysis pretreatment coupled with Thermophilic Anaerobic Digestion (TAD)

    for Waste Activated Sludge (WAS) treatment is a promising combination to improve

    biodegradation kinetics during stabilization. However, to date there is a limited knowledge

    of the anaerobic biomass composition and its impact on TAD process performances.

    In this study, the structure and dynamics of the microbial communities selected in two

    semi-continuous anaerobic digesters, fed with untreated and thermal pretreated sludge,

    were investigated. The systems were operated for 250 days at different organic loading


    16S rRNA gene clonal analysis and Fluorescence In Situ Hybridization (FISH) analyses

    allowed us to identify the majority of bacterial and archaeal populations. Proteolytic Cop-

    rothermobacter spp. and hydrogenotrophic Methanothermobacter spp. living in strict syntro-

    phic association were found to dominate in TAD process.

    The establishment of a syntrophic proteolytic pathway was favoured by the high

    temperature of the process and enhanced by the thermal pretreatment of the feeding

    sludge. Proteolytic activity, alone or with thermal pretreatment, occurred during TAD as

    proven by increasing concentration of soluble ammonia and soluble COD (sCOD) during the

    process. However, the availability of a readily biodegradable substrate due to pretreatment

    allowed to significant sCOD removals (more than 55%) corresponding to higher biogas

    production in the reactor fed with thermal pretreated sludge. Microbial population dy-

    namics analysed by FISH showed that Coprothermobacter and Methanothermobacter imme-

    diately established a stable syntrophic association in the reactor fed with pretreated sludge

    in line with the overall improved TAD performances observed under these conditions.

    2014 Elsevier Ltd. All rights reserved.TAD, Thermophilic Anaetile Solids; OLR, OrganicSitu Hybridization.7.ssetti).

    rved.robic Digestion; COD, Chemical Oxygen Demand; WAS, WasteLoading Rate; HRT, Hydraulic Retention Time; EPS, Extracellular


  • wat e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 9 8e5 0 9 4991. Introduction

    The Anaerobic Digestion (AD) of organic wastes still gathers a

    great interest due to the global needs for waste recycling and

    renewable energy production, in the form of biogas (Luo et al.,

    2013). AD has been evaluated as one of the most energy-

    efficient and environmentally beneficial technology for bio-

    energy production (Weiland, 2010). In a worldwide perspective,

    sewage sludge is far themost widespread substrate used in AD

    (Ahring et al., 2002). AD involves several microbial groups

    forming interdependent microbial consortia. Overall, four

    major steps can be distinguished. In the first hydrolysis step,

    both solubilization of insoluble particulate matter and biolog-

    ical decomposition of organic polymers take place. Acido-

    genesis and acetogenesis follow in the second and third step

    where awide variety of fermentation end-products are formed.

    Finally, in the last step, these products are transformed into

    methane by a methanogenic community. Hydrolysis is often

    limited to complex organic matter as Waste Activated Sludge

    (WAS); this requires the hydrolysis of particulate matter to

    soluble substrates (Pavlostathis and Giraldo-Gomez, 1991).

    Thermal, chemical, biological andmechanical processes, as

    well as combinations of these, have been studied as possible

    pretreatments to disintegrate sludge and accelerate hydrolysis

    (Ferrer et al., 2008). These pretreatments can disintegrate

    sludge flocs and cells allowing a significant solubilization of the

    organic matter, as extracellular polymeric substances (EPS).

    Thermal hydrolysis is a well-known and widely applied tech-

    nology used for WAS pretreatment at both laboratory and full-

    scale (Laurent et al., 2011). It allows the degradation of the gel

    structure and the release of linked water, improving the di-

    gestibility of the sludge (CarrereandDumas, 2010).Most studies

    report an optimal temperature range of 160e180 C and treat-ment times from 30 to 60 min, while the associated pressure

    may vary from 600 to 2500 kPa (Carrere and Dumas, 2010).

    Anaerobic processes operating under thermophilic condi-

    tions (50e55 C) are commonly applied throughout Europe fortreatment of the organic fraction ofmunicipal solidwastes and

    formanure treatment in large scale biogas plants (Ahring et al.,

    2002). Due to their apparent advantages, in recent years, Ther-

    mophilic Anaerobic Digestion (TAD) processes have attracted a

    great attention. These include enhanced organic matter

    removal, high methane production and foaming reduction (Ho

    et al., 2013). Moreover, TAD enhances the destruction of path-

    ogens, enabling effluent hygienization, which might be

    required in a short time for land application (EC, 2000).

    To deepen the investigation and control of the anaerobic

    digestion process, the identity and the metabolic potential of

    the microbial consortia driving the process need to be eluci-

    dated. There have been limited molecular-based studies of

    microbial communities in the AD systems, and most of these

    revealed mostly novel phylotypes (Pervin et al., 2013). Our

    knowledge about the microbial consortia involved in this

    process is indeed limited because of the lack of phylogenetic

    and metabolic data on these predominantly uncultivated


    Coprothermobacter proteolyticus is an anaerobic thermophilic

    microbeaffiliatedwith familyThermodesulfobiaceae,which is

    differently branched from families including most of amino

    acid degrading bacteria in the phylumFirmicutes (Sasaki et al.,2011). Nevertheless, Nishida et al. (2011) showed that Cop-

    rothermobacter represented a taxonomic group most closely

    related to Dictyoglomi and Thermotoga. Coprothermobacter spp.

    ferments proteins, and this proteolytic activity is largely re-

    ported (Ollivier et al., 1985; Etchebehere et al., 1998; Cai et al.,

    2011; Tandishabo et al., 2012; Lu et al., 2014b). Recently, Cop-

    rothermobacter spp. were identified in several studies focused

    on the analysis of microbial community structure selected in

    anaerobic thermophilic reactors treating sewage sludge

    (Kobayashi et al., 2008; Hatamoto et al., 2008; Lee et al., 2009;

    Luo et al., 2013; Pervin et al., 2013).

    Since only a few proteolytic anaerobic thermophiles have

    been characterized so far (Cai et al., 2011), this microorganism

    has attracted the attention of researchers for its potential

    applications in high temperature environments.

    Coprothermobacter activity is improved by the establish-

    ment of a syntrophywith hydrogenotrophicmethanogens like

    Methanothermobacter thermautotrophicus (Sasaki et al., 2011; Lu

    et al., 2014b), commonly found as component of methano-

    genic population in many thermophilic anaerobic systems

    (Yabu et al., 2011; Luo et al., 2013). Hydrogen is the primary

    energy source for this methanogen, even when in situ

    hydrogen concentrations are very low (Kato et al., 2008).

    The objective of this work was to investigate the structure

    and dynamics of microbial communities involved in TAD of,

    either untreated or thermally pretreated, waste activated

    sludge, and to correlate the biological data with process per-

    formances and operation parameters.2. Material and methods

    2.1. Reactors operation and performance

    2.1.1. SludgeSludge was sampled from the municipal Roma-Nord

    wastewater treatment plant, serving about 780.000 P.E. with

    an average flow rate of 4.1 m3/s. The average influent water

    quality was 250 mg COD/L, 20 mg NeNH4/L and 4 mg Ptot/L.

    The plant includes primary clarification and activated

    sludge secondary treatment without nutrients removal. Table

    S1 reports the average characteristics of WAS, collected

    directly from the oxidation tank operating at an average

    sludge age of 20 d. The anaerobic inoculumwas collected from

    the full-scale digester of the plant, fed with mixed sludge.

    2.1.2. Thermal pretreatmentThermal pretreatment was carried out on 400 mL of sludge

    sample using a bench scale autoclave Laboklav 25b, operating

    at T 134 C and p 312 kPa for 20 min, as described inGianico et al., 2013.

    2.1.3. Digester systemThe AD system operated for 250 days in semi-continuous

    mode at different Organic Loading Rate (OLR). Two jacketed

    anaerobic reactors (7 L) were completelymixed and kept at the

    constant temperature of 55 C. One reactor, as control unit,was fed with untreated WAS, and the second one, as experi-

    mental unit, was fed with the same sludge after thermal

    pretreatment (Fig. S1). Untreated or pretreated sludge samples

  • wat e r r e s e a r c h 6 8 ( 2 0 1 5 ) 4 9 8e5 0 9500were fed manually to the digesters once a day after with-

    drawing the same volume of digested sludge.

    The digestion period was divided in three phases on the

    basis of different operating parameters: phase #1 was carried

    out at HRT of 8 d and OLR of 1.8 g VS L1 d1; after 102 days theload was decreased to 1 g VS L1 d1 by increasing HRT to 15 d(phase #2, for 103 days). Finally, phase #3 was performed at the

    highest OLR, namely 3.7 g VS L1 d1 by reducing theHRT to 8 d.All phases were carried out using the same WAS; the first

    two phases were carried out with gravity thickened WAS

    (TS 20.8 g/L) while the last phase was carried out feeding adynamic pre-thickened sludge with total solids concentration

    up to 41 g/L. Pre-thickening of sludge was performed by

    centrifugation for 2 min at 1100 rpm.

    2.1.4. Biogas collection and analysisThe produced biogas was collected by acidified (pH 3)saturated NaCl water solution displacement in a biogas

    collection unit. The gas meter consisted of a volumetric cell

    for gaseliquid displacement, a sensor device for liquid level

    detection, and an electronic control circuit for data processing

    and display. Themethane content in the biogaswasmeasured

    using a PerkinElmer Auto System Gas Chromatographer

    equipped with a Thermal Conductivity Detector (TCD) as

    described in Gianico et al. (2013).

    2.1.5. Matter compositionTotal and Volatile Solids (TS and VS) were determined in

    triplicates according to standard methods (APHA, 1998). The

    pH was detected by a portable pH-meter (WTW, pH 330/SET-

    1). To analyse the soluble phase, the particulate sludge matter

    was removed by centrifugation (10 min at 5000 rpm), and the

    resulting supernatant was filtered through 0.45 mm pore size

    membrane filters.Table 1 e Clones number and affiliation for bacterial (a) and arc16S rRNA gene clonal analysis at the end of both the digestion

    Accession number Affiliation (accession no


    KF971872 Coprothermobacter proteolyticus (NR_074

    KJ626491 Anaerobaculum mobile (NR_102954.1)

    KJ626485 Clostridium sp. JC3 (AB093546.1)

    KJ626486 Uncultured Clostridium (FJ825462)

    KJ626490 Uncultured Clostridium (JF417907)

    KJ626484 Uncultured Thermoanaerobacteraceae (HQ

    KJ626496 Enterococcus faecium (CP006620)

    KJ626487, KJ626489 Uncultured Firmicutes (NR_029198.1)

    KJ626481 Uncultured Tumebacillus (JX110710)

    KJ626482 Dehalobacter sp. CF (NR_075066.1)

    KJ626483 Soehngenia saccarolythica (EU498369)

    KJ626488 Uncultured Planctomycetes (KC867694)

    KJ626492 Thermodesulfovibrio thiophilus (AUIU010

    KJ626493 Exiguobacterium aurantiacum (NR_11366

    KJ626494 Vagococcus fluvialis (NR_026489.1)

    KJ626495 Streptococcus equinus (KC699052)



    KF971874 Methanosarcina thermophila (AB973357)

    KF971873 Methanothermobacter thermoautotrophicu

    KF971875 Methanobrevibacter arboriphilus (NR_042

    TotalVolatile fatty acid (VFA)were quantified from0.2 mmfiltrate

    (soluble phase) by gas chromatography using PerkinElmer

    Auto System Gas Chromatograph with flame ionization de-

    tector (FID). The GC analyses were performed on a stainless

    steel column packed with 60/80 mesh Carboxen C, 0.3% Car-

    bowax (Supelco, USA), under the following conditions:

    injector 200 C, oven 175 C, detector 250 C. Nitrogenwas usedas a carrier gas at a flow rate of 30 mL/min.

    Soluble COD (sCOD) and soluble nitrogen were determined

    by Cell Test Spectroquant (Merck) as described in Gianico et al.

    (2013). Ammonia nitrogen was determined according to

    method 4500-NH3 C of APHA Standard Methods, 18th edition

    (1992). To analyse colloidal phase, sludge aliquots were

    filtered through glass filters with 1.2 mm pores (GF/C What-

    man); the supernatant was used for protein and carbohy-

    drates determination. Protein and carbohydrate contents

    were determined by colorimetric BCA and Dubois methods, as

    described in Braguglia et al. (2012).

    2.2. Microbial community analysis

    2.2.1. Sample collectionEffluent sludge samples were periodically collected from both

    reactors during start-up and at steady state operating condi-

    tions. Aliquots of 1.5 mL of mixed liquor were either imme-

    diately frozen at 20 C for further DNA extraction or fixedwith paraformaldehyde and ethanol for FISH analysis as

    described in Amann and Binder (1990).

    2.2.2. Genomic DNA extraction and PCR amplification of 16SrRNA geneDNA was extracted from z700 mg of thermophilic sludge

    collected at the end of operation of both systems following the

    protocol reported inRossetti et al. (2008). The concentration andhaeal (b) members of themicrobial population estimated byprocesses (250 d).

    .) Similarity (%) No. of clones

    653.1) 99 10

    99 3

    99 1

    99 1

    95 1

    183807) 99 2

    99 2

    87 2

    98 1

    92 1

    95 1

    97 1

    00004) 99 1

    6.1) 99 1

    99 1

    99 1


    99 34

    s (AE000666) 99 25

    783.1) 98 1


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