Two-Stage vs Single-Stage Thermophilic Anaerobic Digestion:Comparison of Energy Production and Biodegradation EfficienciesAndrea Schievano,*,† Alberto Tenca,‡ Barbara Scaglia,† Giuseppe Merlino,§ Aurora Rizzi,§

Daniele Daffonchio,§ Roberto Oberti,‡ and Fabrizio Adani*,†

†Ricicla Group, Dipartimento di Produzione Vegetale, Universita degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy‡Dipartimento di Ingegneria Agraria, Universita degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy§Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, DISTAM, via Celoria 2, 20133 Milano, Italy

*S Supporting Information

ABSTRACT: Two-stage anaerobic digestion (AD) for integratedbiohydrogen and biomethane production from organic materials hasbeen reported to promise higher process efficiency and energy recoveriesas compared to traditional one-stage AD. This work presents acomparison between two-stage (reactors R1 and R2) and one-stage(reactor R3) AD systems, fed with identical organic substrates andloading rates, focusing the attention on chemical and microbiologicalaspects. Contrary to previous experiences, no significant differences inoverall energy recovery were found for the two-stage and one-stage ADsystems. However, an accumulation in R2 of undegraded intermediatemetabolites (volatile fatty acids, ketones, amines, amino acids, andphenols) was observed by GC-MS. These compounds were thought tobe both cause and effect of this partial inefficiency of the two-stagesystem, as confirmed also by the less diverse, and thereby less efficient, population of fermentative bacteria observed (by PCR-DGGE) in R2. The extreme environment of R1 (low pH and high metabolites concentrations) probably acted as selector ofmetabolic pathways, favoring H2-producing bacteria able to degrade such a wide variability of intermediate metabolites whilelimiting other strains. Therefore, if two-stage AD may potentially lead to higher energy recoveries, further efforts should bedirected to ensure process efficiency and stability.

1. INTRODUCTION

The two-stage anaerobic digestion (AD) process has beenreported as a viable biotechnology to coproduce hydrogen andmethane (in two separated bioreactors in series) from a varietyof organic materials.1,2

Compared to the traditional single-stage AD process, thetwo-stage approach has been proposed by several authors as apossible solution to improve the overall process efficiency, interms of biodegradation rates/yields and overall energyproductivity. Some authors reported that splitting andseparately optimizing hydrolysis/acidogenesis and methano-genesis could enhance the overall reaction rate, maximizebiogas yields, and make the process easier to control, both inmeso- and thermophilic conditions.3,4 Some other authorsstated that enriching different microorganisms in eachanaerobic digester, the two-stage AD should extend thepossibility of processing different biomass species, enhancesubstrate conversion, improve the chemical oxygen demand(COD) reduction, and upgrade percent energy recovery.5,6

Moreover, the two-stage solution could increase the stability ofthe overall process: a controlled acidification process in the firstdigester should help in maintaining a constant composition ofthe methanogenic digester feed and, thus, in avoiding

overloading and/or inhibition of the methanogenic popula-tion.7

Furthermore, as typically only 15% of the energy containedin the organic substrate is obtained from the first stage in theform of H2, with relatively short retention times (RT = 1−4 d),while 80−90% of the initial chemical oxygen demand (COD)remains in the liquid phase, many efforts for enhancing theoverall process performances have been focused on optimizingand speeding up the second stage process, i.e. themethanogenic reactor.8 Many authors relied on the possibilityof enhancing methane production rates in the second stage,taking advantage of the fact that hydrolyzed and prefermentedorganic matter (OM) is more available to methanogens, ascompared to the untreated substrate.7,9 This would mean moreefficient biodegradation with the same RTs or similarbiodegradation yields with lower RTs.Unfortunately, methanogenic communities were widely

reported to be very sensitive to volatile fatty acids (VFA)

Received: April 11, 2012Revised: May 30, 2012Accepted: June 14, 2012Published: June 14, 2012

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concentrations in the liquid medium,9 so that the moresubstrates are soluble, easily fermentable, and rich in VFA, thelower can be their loading rate (LR) into digesters, as recentlysuggested by Schievano et al.10 This would limit possibleadvantages of two-stage, compared to single-stage AD.Furthermore, it remains unclear how the delicate equilibrium

among the wide variety of biochemical reactions involved inAD should work in separated stages. Conventional AD involvesan incalculable number of different biochemical pathwaysdriven by different microbial consortia, grouped into three maincategories: hydrolytic bacteria allow complex organic moleculesto be solubilized in the form of simple sugars, amino acids,organic acids, etc.; acidogenic bacteria break down monomersmainly into H2, CO2, and a variety of short-chain organic acids(VFA), such as acetic, propionic, butyric and lactic acids;alcohols and ketones are also commonly formed during thebreakdown of organic substrates by the acidogens, but in a well-operating process these products should be mostly converted toacetic acid and H2 by acetogenic bacteria. Contemporarily, anumber of methanogenic microorganisms convert mainly H2,acetic acid and/or simple alcohols directly to CH4 andCO2.

11,12 As volatile organic compounds (VOCs) representthe main intermediates (VFA, ketones, alcohols, etc.) of thevariety of biochemical reactions involved in AD andfermentations, their investigation can be useful to understandthe biochemical process in depth. For instance, highconcentrations of specific groups of VOCs may be the resultof process parameters such as feedstock composition,13

hydraulic retention time (HRT) and organic LR adopted,operational temperature and pH14,15 and could be indicative forspecific metabolic pathways. Many VOCs formed asintermediate metabolites by fermentative anaerobes (i.e.,phenols, alcohols, ketones, halogenated compounds, longchain fatty acids, alkenes) were indicated in the literature16 tobe possibly recalcitrant to further biodegradations and/orpossible inhibitors of methanogenic populations. Many of thosecompounds are formed differently when different pH, redoxpotential, other chemical environments, and predominantmicrobial populations are present in fermentation broths.16

For this reason, the separation of fermentative and methano-genic environments, imposed in two-stage anaerobic digestionsystems, may drive substantial changes in biochemical pathwaysand fermentation metabolites formation. Also, microbialspecies/subpopulations selected and the relative abundance ofthe same microorganisms in the consortium can differdepending on process operating conditions and strategies.16

It remains unclear whether all the combination of thesedifferent biochemical pathways involved in AD can be eitheroptimized or, on the other side, hampered by the separation ofacidogenic from methanogenic microbial consortia in a well-operated two-stage process. Even if these different microbialgroups differ in terms of physiology, nutritional needs, growthkinetics, and sensitivity to environmental conditions,17,18 inconventional AD they live altogether in the same environmentand the separation of acido/acetogenesis from methanogenesismay affect negatively syntrophic associations, above all bypreventing interspecies hydrogen transfer.19

To our knowledge, such chemical and microbiologicalaspects have not yet been clarified and there is the need ofdeeper efforts in comparing two- and single-stage ADprocesses. In this work, two AD lab-scale continuous systems(one-stage and two-stage) were identically fed and operated inparallel, registering their overall biogas production and energy

recovery and monitoring some interesting chemical/micro-biological parameters. A mixture of manure and fruit/vegetablewaste was chosen as feeding substrate, as it was before shown tohave interesting H2 production potentials (120−150 Sdm3 H2kg−1 TS and 300−400 Sdm3 CH4 kg

−1 TS).20

The aim was to more closely observe the biochemicalprocesses, the metabolite compounds produced, and the mainmicrobial communities involved in the two AD systems and togive a contribution to our knowledge about the possibleconveniences of adopting one approach instead of the otherone.

2. EXPERIMENTAL SECTION2.1. Apparatus and Process Operation. Three con-

tinuous flow stirred tank reactors (CSTR), in “wet” ADconditions (total solids <10% w/w), were used in this studyand the reactor designs are reported in Supporting Information(SI) Figure S1. The wet CSTR was chosen, as it is one of themost-used types of AD in full-scale applications. The two-stageprocess consisted of a 3-L hydrogen-producing reactor with 2-Lworking volume (R1) and an 18-L reactor with 14.7-L workingvolume for methane production (R2). The single-stage processconsisted of an 18-L reactor with 14.7-L working volume (R3).The same feeding mixture was added both to R1 and R3 afterthe removal of an equal amount (measured as wet weight) ofeffluent from the reactors. Hydraulic retention times (HRTs)were 3, 22, and 25 days for the reactors R1, R2, and R3,respectively (Figure S1). The feeding procedure was insemicontinuous regime, i.e. twice a day the digestate wasremoved and an equal volume of feeding mixture was insertedin each reactor. The operational HRT of R1 was chosenaccording to previous experience in optimized biohydrogenproduction from organic waste materials.20 HRTs of themethanogenic phases were chosen after applying a batchbiochemical methane potential test to the feeding mixture (seeSI and Figure S2). The overall HRT of two- and single-stageprocesses were equal (25 d) in order to make them comparable.The three digesters were simultaneously and continuouslymixed for 15 s every 45 s and kept at a temperature of 55 ± 2°C via water bath through water jackets surrounding thereactors.During the trial period the pH in the three reactors was not

actively controlled or adjusted and was dependent on theprocess natural conditions. Temperature and pH of thefermentative broth were measured continuously by threedifferent InPro 3253/225/pt1000 electrodes (Mettler-Toledointernational inc.). Gas flow-meters (adm 2000 model, Agilenttechnologies) were installed in each reactor to automaticallyrecord the gas production. Biogas volumes were registered ascumulated every minute, and daily (over 24 h) cumulatedamount was accounted. Biogas composition also wasdetermined daily, using a gas chromatograph: H2, CH4, andCO2 relative concentrations (v/v) were measured. Furtherdetails are reported in the SI. Methane and hydrogenproductions were calculated as daily cumulated productionvolume.

2.2. Inoculum, Feeding Materials, Process Startup,and Observation. The hydrogen-producing inoculum con-sisted of a digested material from a full-scale biogas plant,treating household source-separated biowaste and agro-industrial byproduct. Digestate before the use was heat-shockedat 100 °C for 2 h.4,9 The heat treatment was necessary, only atthe beginning of the experiment, to inactivate hydrogeno-

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trophic microorganisms and to harvest anaerobic spore-formingbacteria.4,12 The heat-shock was no longer applied during theexperiment, neither to R1, nor to the feeding material. Thesame digestate used for R1, without heat-shocking, was used asinoculum for the methanogenic stage of both two- and single-stage systems. After inoculation, each reactor was sparged for30 min at 0.1 L min−1 with N2 to remove dissolved O2 and toobtain anaerobic conditions.The feeding substrate was represented by a mixture of swine

manure (SM) and market biowaste (MBW). This kind oforganic material, which had been previously used in anoptimization study of the hydrogenic stage,20 is also similarto the feeding used in other studies concerning two-stageAD,4,9,11,13 so it was chosen here to perform a comparableexperiment. Fresh (age <1 week) SM was collected from 4different private farms near Milan (Italy) and then filteredthrough a stainless steel sieve (U.S. Mesh No. 10). MBWconsisted of fruits and vegetables residues, obtained from themunicipal fruits and vegetable market of Milan (Italy). Beforeuse, the MBW sample was shredded by a blender,homogenized, and then stored at −20 °C. The feeding, equallyused for the two- and the single-stage systems, was composedby mixing in the ratio 1:4 (on wet-weight basis) MBW and SM,respectively, so that the mix was liquid (bulk density of about1.1 kg/dm3).The two AD systems were fed and observed in parallel, until

stable biogas productions were reached in all reactors, i.e. whenthe gas evolution rate and the concentration of H2 or CH4 (v/v) in biogas were constant over at least 15 days. After this start-up period (which lasted approximately one month), productiondata were registered for at least 700 h (approximately oneadditional month). Therefore, the steady state lasted more than45 days. Considering the very homogeneous feeding andcontrolled conditions at lab scale (the feed was exactly weighted

every day), this steady-state time has been considered sufficientfor representing the process. At pilot or full-scale conditions,probably the same study should be carried out for a relativelylonger period.

2.3. Process Characterization. Liquid and gaseoussamples were characterized by various chemical analysismethodologies. Both digestion broths and biogas were sampledfour times (one sample a week) during the steady state process.Quantitative measurements of the total solids (TS), volatilesolids (VS), chemical oxygen demand (COD), total nitrogen(TN), and ammonia nitrogen (N-NH4

+) were performed onthe digestion broths, according to Standard Methods.21 VFAconcentrations in the fermentation broths were determined asreported by Schievano et al.10 Total alkalinity (TA) and totalVFA concentrations were determined in the bulk samples by a5-times-diluted solution of 2.5 g of wet sample, filtered to 0.45μm, according to the acid titration method.22

The biodegradability of the OM contained in fermentationbroths was determined by a respirometric test: the specificoxygen uptake rate (SOUR) test. The SOUR test wasperformed according to Schievano et al.10 and the cumulatedoxygen demand (OD) measured along 20 h test (OD20) wasused as parameter to measure short-term biodegradability(putrescibility) of OM. Details are remitted to the SI. Organicloading rates were calculated for R1, R2, and R3 and thecalculation included quantitative parameters (VS and COD)and a quanti-qualitative parameter (OD20), such as suggested ina recent work.10 The ultimate OM degradation efficiencies werealso determined for the overall two- and single-stage systems,striking a balance between the input and output flows of thefollowing parameters: TS, VS, COD, OD20.Intermediate metabolites were studied by volatile organic

compounds (VOCs) determination, both in gaseous and liquid

Figure 1. Observed trends over 700 h observation of production rates and volumetric contents in biogas of biohydrogen (for R1, panel a) andbiomethane (for R2 and R3, panels b and c, respectively). pH trends detected in R1, R2, and R3 (d).

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phases. GC-MS was used as previously reported by Orzi et al.23

All details are described in the SI.Bacterial and archaeal communities in both the two-stage and

single-stage reactors were analyzed at the steady state by PCR-DGGE and sequencing techniques, as previously reported.24,25

3. RESULTS AND DISCUSSION

3.1. Biogas and Energy Production Yields. The biogasproductions trends of the two- and one-stage AD systems arereported in Figure 1 and the average values in the observedperiod are shown in Table 1. The biogas production rateachieved by the hydrogenic stage (R1) was 3.51 Ndm3 L−1 d−1,with hydrogen content in the biogas of 44.9 ± 5.5% (v/v)(Table 1). No measurable CH4 was detected during theobservation period (Table 1), indicating that methanogenicmicrobial populations were substantially inhibited. This wasprobably the combined result of both the initial heat-shock andof the stable acidic (5 < pH < 6) conditions established in R1(Figure 1), as recently suggested by Tenca et al.20 This resultedin satisfactory hydrogen production rates (1.58 Ndm3 H2 L

−1

d−1 as average) and relatively high hydrogen yields (140 Ndm3

H2 kg−1VS‑added). Biogas productions rates from the methano-genic reactor (R2) resulted in 0.79 Ndm3 L−1 d−1 with anaverage CH4 concentration in biogas of 68.2 ± 1.7% (v/v).Methane production rates were, as average, 0.537 ± 0.071Ndm3 CH4 L−1 d−1 and the average methane yield was 351Ndm3 CH4 kg

−1VS‑added (referred to the VS added to R1, i.e., to

the two-stage system as-a-whole). The single-stage reactor (R3)produced 1 Ndm3 L−1 d−1 of biogas with average CH4

percentage of 54.5 ± 1.9%, i.e. an average methane productionrate of 0.545 Ndm3 CH4 L

−1 d−1, corresponding to an averagemethane yield of 404 Ndm3 CH4 kg−1VS‑added (Table 1). InTable 1 are also reported the variances (per hour) on averageproductions.The overall yields achieved by the two compared systems

were also calculated in terms of energy recovery by consideringH2 and CH4 superior calorific powers (Table 1). The two-stagesystem recovered 14.13 kJ kg−1VS‑added, considering that 1.79 kJkg−1VS‑added was recovered by the hydrogen-stage and 12.34 kJkg−1VS‑added was recovered by the methane-stage, while the

single-stage resulted in 14.21 kJ kg−1VS‑added energy recoveryyield.The hydrogen-stage (R1) gave a relatively partial contribu-

tion to the total energy yield achieved by the two-stage system(13% of the total energy produced). This was in agreementwith previous works, which demonstrated that hydrogeno-genesis from organic waste can contribute to total energeticproduction, typically up to 15%.8,26 The ultimate hydrogenyields and the H2 contents in biogas achieved were equal to orhigher than those obtained from similar materials, both inthermophilic and in mesophilic conditions, by severalauthors.4,27−29 Besides, R2 showed higher average CH4contents in biogas, as compared to the single-stage process(Table 1), again in agreement with previous experiences.4,29 Onthe other hand, approximately 13% lower CH4 yields wereachieved by R2, as compared to R3 (Table 1), contrarily towhat was found by Liu et al.,4 who recovered nearly 21% moremethane from the second-stage, treating mixed organic waste inmesophilic conditions. However, in a more recent contribution,Luo et al.29 achieved 11% more energy (overall H2 and CH4)from the two-stage process as a whole, compared to the one-stage applied to the same organic waste feeding in thermophilicconditions.In our case, no significant differences in energy production

could be found between the two- and single-stage AD systems(Table 1), even when ANOVA was applied on daily energyrecovery yields (n = 30, p < 0.05). As the AD systems were runin parallel, with exactly the same overall HRTs andexperimental conditions, these results suggested the hypothesisthat the two-stage AD systems may, in some case, bring noadvantage in terms of overall energetic-recovery yields. Thishypothesis is somehow in contrast with various previousliterature contributions,4,29 in which the evidence of a generalsupremacy of two-stage AD systems over one-stage wasassumed, both in mesophilic and thermophilic conditions. Inthis work, deeper analytical approaches helped in betterunderstanding the mechanisms that brought a different resultin the present case-study.

3.2. Organic Matter Degradation Efficiencies. The mainchemical characteristics of the feeding mixture and thedigestion broths (R1, R2, and R3) are reported in Table 2.

Table 1. Biogas, Bio-Hydrogen, and Bio-Methane Production Rates, Gas and Energy Production Yields, and BiodegradationYields Comparing the Two-Stage (R1 and R2) and the Single-Stage (R3) Processes

two-stage single-stage

R1 R2 R3

average (700 h) var. (per h) average (700 h) var. (per h) average (700 h) var. (per h)volumetric biogas production rate Ndm3 L−1dig d−1 3.51 0.58 0.79 0.10 1.00 0.08hydrogen content in biogas % v/v 44.9 5.5 <0.1 <0.1methane content in biogas % v/v <0.1 68.2 1.7 54.5 1.9carbon dioxide content in biogas % v/v 55.1 5.5 31.8 1.7 45.5 1.9volumetric hydrogen productionrate

Ndm3H2 L

−1dig d−1 1.58 0.27 udla udl

volumetric methane productionrate

Ndm3CH4 L

−1dig d−1 udl 0.537 0.071 0.545 0.042

hydrogen/methane productionyields

Ndm3H2/CH4

kg−1VS added

140 24 351 46 404 31

volumetric energy production rate kJ L−1dig d−1 19 4.9 17.4 2.2 18.5 1.3energetic yield MJ kg−1VS added 1.79 0.37 12.34 1.19 14.21 0.84

14.13 ab (R1 + R2) 1.24 14.21 ab 0.84audl = under detection limit. bNumbers in the same line followed by the same letter are not signinficantly different (ANOVA Tukey Test; n = 30, p< 0.05).

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Despite the insignificant differences in energy yields, thechemical characterizations suggested remarkable dissimilaritiesbetween the two AD systems, particularly evident from thecharacterization of the fermentation environments in R2 andR3. R1 achieved 13.9 ± 1.1%, 24.4 ± 0.9%, and 32.1 ± 1.5%removal efficiencies, in terms of TS, VS, and COD, respectively;R2 showed 35.6 ± 1.8%, 48.0 ± 1.2%, and 54.0 ± 1.3% furtherremoval efficiencies for the same parameters (Table 2). Theoverall two-stage process efficiencies were 44.6 ± 2.1%, 60.7 ±1.7%, and 68.8 ± 2.3%, while the single-stage process achieved55.7 ± 1.5%, 68.4 ± 2.1%, and 75.7 ± 1.9% removal efficienciesfor TS, VS, and COD, respectively (Table 2). In addition, theTS and COD contents were in fact significantly lower (n = 4, p< 0.05) in R3 as compared to R2. Considering these results, thetwo-stage process seemed to be slightly less efficient indegrading OM and this was linked to a partial inefficiency ofthe methanogenic reactor of the two-stage process (R2).Despite this, the energy produced was the same (Table 1), sothat the two-stage system, when run in optimal conditions,might have produced more energy.Other remarkable differences between R2 and R3 were

noticed in terms of biological stability of their output materials.The biodegradability of OM (measured as OD20) was nearlydouble in R2 as compared to R3 (Table 2). At the same time,the overall OD20 was consistently lower in R2 (79.3 ± 1.4%), ascompared to R3 (90.5 ± 2.6%). This means that a certainamount of relatively rapidly degradable OM was leftundegraded in R2, probably due to a partial inhibition of themethanogenic activity in this reactor.To better understand what may have occurred, we calculated

the OM loading rates (LRs) of R1, R2, and R3 in terms of VS,

COD, and OD20. The result was that R2, as compared to R3,presented lower VS-LR and COD-LR, but higher OD20-LR(Table 2). In fact, the acidogenic reactor (R1) gave only apartial abatement of the OM contents (see VS and CODreductions, Table 2) while, at the same time, an increase of theshort-term biodegradability, measured as OD20 (Table 2). Thismeans that the OM loaded into R2 was more rapidlybiodegradable than that loaded into R3 and probably richerin hydrolyzed and low-molecular-weight metabolites (VFAs,alcohols etc.) (Table 2). These molecules can typically beresponsible for partial inhibitions/inefficiencies of methano-genic communities when fed in relatively high concentrations.16

The detected chemical parameters supported this hypothesis,i.e., pH and VFA concentrations, which are considered two ofthe main environmental factors that regulate the metabolicpathways of anaerobic digestion.4,16

While the feeding pH was subalkaline (Table 2), due to astable VFA production during acidogenic phase, pH in R1 wasconstantly around 5.5 (Figure 1), with average of 5.52 and avariance (per hour) of 0.44, which fell in the range for optimalbiohydrogen production indicated by various authors.4,12,30 ThepH trend is reported in Figure 1. Contemporarily, R1 showed,for the whole period, relatively high tVFA contents (3840 ±745 mgCH3COOH kg−1), more than double than in the fedmaterial (Table 2). In particular, acetic acid was the main VFAspecie with more than 2500 mg kg−1, propionic acidapproximately doubled its concentration, while butyric acid,undetected in the fed mixture, showed the highest increase,reaching the average concentration of 960 mg kg−1 (Table 2).In the methanogenic reactor (R2), VFAs fed from R1 were

partially consumed and their total content in R2 was reduced to

Table 2. Characterization of the Input Mixture and the Three Digestate Materials

two-stage single-stage

feeding R1 R2 R3

TS g kg−1 39.5 ± 2.5 34.0 ± 14.7 21.9 ± 1.5 ab 17.5 ± 3 bb

VS g kg−1TS 854 ± 26 750 ± 81 605 ± 21 a 610 ± 11 aCOD g O2 kg

−1 85.9 ± 8.4 58.3 ± 7.4 26.8 ± 1.3 a 20.9 ± 3.2 bOD20 g O2 kg C−1

org h−1 298 ± 16 350 ± 40 111 ± 40 a 64 ± 22 b

TS reduction % of fed 13.9 ± 1.1% 35.6 ± 1.8%44.6 ± 2.1% (R1 + R2) 55.7 ± 1.5%

VS reduction % of fed 24.4 ± 0.9% 48.0 ± 1.2%60.7 ± 1.7% (R1 + R2) 68.4 ± 2.1%

COD reduction % of fed 32.1 ± 1.5% 54.0 ± 1.3%68.8 ± 2.3% (R1 + R2) 75.7 ± 1.9%

OD20 reduction −15.6 ± 0.6% 82.1 ± 2.2%79.3 ± 1.4% (R1 + R2) 90.5 ± 2.6%

VS-LR g VS L−1 d−1 11.24 1.16 1.35COD-LR g COD L−1 d−1 28.63 2.65 3.44OD20-LR g O2 L

−1 d−1 3.92 0.54 0.47pH 7.2 ± 0.1 5.52 var.(h) 0.44 7.61 var.(h) 0.06 7.94 var.(h) 0.25TVFA mgCH3COOH kg−1 1600 ± 115 3840 ± 745 756 ± 410 bb 75 ± 40 ab

Acetic acid mg kg−1 675 ± 520 2511 ± 345 455 ± 155 b 53 ± 35Propionic acid mg kg−1 172 ± 145 318 ± 140 299 ± 120 b udla

Butyric acid mg kg−1 udla 958 ± 270 udl a udl aa

TA mgCaCO3 kg−1 4708 ± 10 4050 ± 980 5050 ± 430 a 6480 ± 976 a

TVFA/TA ratio kgCH3COOH kg−1CaCO3 0.32 0.95 ± 0.06 0.15 ± 0.03 b 0.01 ± 0.01 aTN g kg−1 2.4 ± 1.1 2.85 ± 0.5 2.2 ± 0.3 a 2.1 ± 0.5 aN-NH4

+ g kg−1 1.5 ± 0.7 1.7 ± 0.3 1.6 ± 0.2 a 1.5 ± 0.4 aN-NH4

+ /TN % 64 ± 4 60 ± 4 72 ± 3 a 72 ± 6 aaudl = under detection limit. bANOVA (Tukey Test; n = 4, p < 0.05) was performed for comparing R2 vs R3. Numbers followed by the same letterin the same line are not statistically different.

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the amount of 756 ± 410 mgCH3COOH kg−1. Acetic acid keptbeing more concentrated (455 ± 155 mg kg−1 as average) thanpropionic (299 ± 120 mg kg−1 as average), while butyric wasreduced to undetectable concentrations (Table 2). In any case,VFA concentrations in R2, as well as the tVFA/TA ratio, werekept in acceptable ranges for successful methanogenesis, asaccording to various authors,13,28 so that microbial populationsdid not need any particular adaptation periods. On the otherhand, R3 showed tVFA concentrations approximately 10 timeslower than those in R2 (75 ± 40 mgCH3COOH kg−1), with aceticacid being the main component and both butyric and propionicacids concentrations were under detection limits (Table 2).Besides, the average pH of the two methanogenic environmentsresulted evidently more acidic in R2 (Table 2), as result ofhigher VFA concentrations. Furthermore, the tVFA/TA ratio(0.15 gCH3COOH g−1CaCO3) found in R2 is evidently higher ascompared to R3, and these characteristics are typically found in

partially efficient anaerobic digestion processes, as reported byseveral authors.16

The overall TA liberated in R2 was not significantly differentthan that in R3, as well as TN and ammonia contents (Table2). Ammonia was always under levels of possible inhibition ofthe methanogenic activity16 and ammonia/TN ratios wererelatively high in all digesters, due to the presence ofconsiderably high ammonia concentrations in the fed swinemanure. Further mineralization of organic nitrogen led tofurther increase of the N-NH4

+/TN ratios, both in R2 and inR3 (Table 2), as expected.31

3.3. GC-MS Determination of VOCs in Gas and LiquidPhases. The GC-MS characterizations of the gas and liquidphases of the three bioreactors are reported in Tables S1 and 3.The liquid phase of the feed-in material was also characterized(Table 3). Many different classes of VOCs were detected, aswell as a wide variety of compounds (Table 3). The mainclasses samples were, generally, aromatics, carboxylic acids,

Table 3. Concentrations (ppmv) of the Main VOCs Groups Measured in Reactors Liquid Phase

feed two-stage single-stage

R1 R2 R3

ppmv % of total ppmv % of total ppmv % of total ppmv % of total

main groups of VOCsalcohols 68 ± 1 Ab 6.9 79 ± 30 Ab 5.1 745 ± 99 bb 7.1 105 ± 56 ab 7.0aldehydes 7 ± 2 A 0.7 5.9 ± 2.1 A 0.4 42 ± 1 a 0.4 29 ± 30 a 2.0alkanes 11 ± 1 A 1.1 73 ± 29 A 4.7 451 ± 75 b 4.3 102 ± 89 a 6.8alkenes 19 ± 1 A 1.9 168 ± 86 A 10.9 557 ± 143 b 5.3 185 ± 141 a 12.3aromatic compounds 288 ± 1 A 29.3 435 ± 148 B 28.2 3738 ± 789 a 35.5 157 ± 116 a 10.5cycloalkanes 14 ± 1 A 1.5 68 ± 51 A 4.4 491 ± 79 b 4.7 106 ± 65 a 7.1cycloalkenes 7.9 ± 0.5 A 0.8 7.1 ± 3.5 A 0.5 udl aa 0.0 0.8 ± 0.7 a 0.1esters 33 ± 2 A 3.4 13 ± 1 A 0.9 60 ± 11 b 0.6 2.7 ± 3.1 a 0.2ethers udl Aa 0.0 15 ± 2 A 1.0 24 ± 2 b 0.2 udl aa 0.0halogenated compounds 11 ± 1 A 1.1 19 ± 9 A 1.2 udl aa 0.0 udl aa 0.0ketones 30 ± 1 A 3.0 122 ± 116 A 7.9 412 ± 66 b 3.9 132 ± 107 a 8.8nitrogenous compounds 31 ± 1 A 3.2 70 ± 30 B 4.6 1030 ± 706 b 9.8 56 ± 32 a 3.7siloxanes 159 ± 2 A 16.2 212 ± 58 A 13.7 584 ± 291 b 5.5 73 ± 36 a 4.8sulfur compounds 87 ± 3 B 8.8 1.9 ± 2.0 A 0.1 76 ± 10 b 0.7 12 ± 10 a 0.8terpenes udl Aa 0.0 udl Aa 0.0 13.4 ± 0.1 a 0.1 11 ± 14 a 0.7carboxylic acids 101 ± 1 A 10.3 1094 V 188 B 70.9 1071 ± 648 b 10.2 263 ± 291 a 17.5

carboxylic acidsvolatile fatty acids 58 ± 3 A 57.3 829 ± 199 B 75.8 463 ± 143 b 43.3 229 ± 251 a 86.9long chain fatty acids 51 ± 2 A 88.2 120 V 75 A 14.5 124 ± 142 a 26.8 77 ± 94 a 33.5VFA (% of total CA)

formic acid udl Aa 0.0 8.1 ± 8.1 A 0.7 84 ± 65 b 7.8 udl aa 0.0acetic acid 12.1 ± 0.3 A 12.0 199 ± 105 B 18.2 281 ± 40 b 26.3 61 ± 83 a 23.2propionic acid 13.9 ± 0.2 A 13.7 99 ± 38 B 9.0 72 ± 24 a 6.7 36 ± 25 a 13.8butyric acid 2.3 ± 0.9 A 2.3 62 ± 45 B 5.6 20 ± 5 a 1.9 69 ± 100 a 26.1pentanoic acid udl Aa 0.0 25 ± 21 A 2.3 5.8 ± 8.3 a 0.5 0.4 ± 0.8 a 0.2hexanoic acid 30 ± 1 A 29.3 437 ± 360 B 39.9 udl aa 0.0 udl aa 0.0

aromatic compoundsphenols 56 ± 1 A 19.6 330 ± 125 B 75.9 3285 ± 2507 b 87.9 33 ± 19 a 20.9indoles 140 ± 3 A 48.6 25 ± 12 B 5.7 176 ± 104 a 4.7 37 ± 29 a 23.5benzene compounds 3.6 ± 1.0 A 1.3 23 ± 9 B 5.2 9 ± 13 a 0.2 9 ± 11 a 5.8thiazole 3.4 ± 0.8 A 1.2 25 ± 15 B 5.7 45 ± 44 a 1.2 1 ± 2 a 0.7naphtalene udl Aa 0.0 4.4 ± 2.1 A 1.0 udl aa 0.0 udl aa 0.0toluene udl Aa 0.0 3. ± 5.4 A 0.9 67 ± 1 a 1.8 25 ± 15 a 16.2furan 0.4 ± 0.2 A 0.2 udl Aa 0.0 45 ± 2 a 1.2 5 ± 1 a 3.5total VOCs amount (ppbv) 984 ± 5 A 1544 ± 1397 A 10543 ± 2611 b 1500 ± 1206 atotal VOCs number 114 ± 9 134 ± 15 134 ± 13 157 ± 9audl = under detection limit. bANOVA (Tukey Test; n = 4, p < 0.05) was performed separately for Feed vs R1 (capital letters) and R2 vs R3 (low-case letters). Numbers followed by the same letter in the same line are not statistically different.

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amines, and ketones. These compounds are normally found inanaerobic degradation processes, such as recently indicated byOrzi et al.23 Zahn et al.32 reported the proponderance of VFAs,alcohols, ketones, and phenols in swine manure odor emissions.Even other kinds of biological environments (e.g., composting),when the organic substrate was similar, were reported33 withsimilar VOCs spectra, except for VFAs, which weresubstantially absent, probably due to the oxidative conditionsof composting process.R1 showed evident preponderance of carboxylic acids (over

70% of the total VOCs), both in gas and liquid phases (TablesS1 and 3, respectively). In particular, VFAs were around 93%and 75% of total carboxylic acids, in gas and liquid phases,respectively, while long chain fatty acids (LCFAs) and othercompounds represented the rest (Tables S1 and 3). Otherimportant groups of VOCs in R1 were aromatic compounds,alcohols, and ketones, both in gas and in liquid phases (TablesS1 and 3). Among VFAs, a severe prevalence of hexanoic acidwas revealed (around 58% and 40% of tVFAs, in gas and liquidphases, respectively), followed by butyric and acetic acids.Aromatic compounds were mostly benzene compounds(cymene was predominant) in the gas phase (Table S1) andphenols in the liquid phase (Table 3).Comparing the liquid phases of the feed material and of R1,

some groups of compounds were significantly (n = 4, p < 0.05)more concentrated in R1. In particular, carboxylic acids, VFAs(mainly acetic, propionic, butyric, and hexanoic acids),nitrogenous compounds, phenols, cymenes, and thiazoleswere found 2−15 times increased in R1, while sulfurcompounds and indoles significantly decreased (Table 3).R2 gas phase showed a wider variety of VOCs, with a

prevalence of ketones, carboxylic acids, and aromaticcompounds (27.1%, 15.5%, and 12.7% of total VOCs,respectively). The carboxylic acids were evidently reduced, incomparison to their high concentrations in R1 gas phase (TableS1). VFAs represented the majority of these carboxyliccompounds (78.2%), with a prevalence in hexanoic acid(55% of tVFAs) and the presence of acetic, propionic, andbutyric acids. For aromatic compounds, benzene compounds(mainly cymene) represented roughly 43% of them and seemedto remain undegraded after R1. Toluenes and phenols were alsofound in considerable concentrations (Table S1). The liquidphase of R2 was characterized by a considerable predominanceof aromatic compounds (35.5%) on other species, mainlyrepresented by phenols, which resulted in considerableconcentrations (Table 3) as compared to R1. Carboxylicacids (especially VFAs) were concentrated similarly than in R1(Table 3). R3, in the gas phase, showed a relativelyhomogeneous composition of VOCs, even if aromaticcompounds (mainly cymene) and ketones were found inconsiderable concentrations, representing approximately the20% of total VOCs (Table S1).Interestingly, remarkable differences were found between R3

and R2 gas phases: many VOCs groups (cycloalkenes, ketones,siloxanes, terpenes, carboxylic acids, VFAs, acetic acid, phenols,and thiazoles) were significantly (n = 4, p < 0.05) lessconcentrated in R3 than in R2 (Table S1). In the liquid phase,these differences were even more evident: total VOCsconcentration was found approximately 10 times higher andmany different VOCs groups were significantly (n = 4, p <0.05) more concentrated in R2 than in R3 (Table 3). Inparticular, phenols (which represented 87.8% of aromaticcompounds in R2) were 100 times more concentrated in R2

than in R3 liquid phases; at the same time, alcohols,nitrogenous compounds (aminoacids and amines), esters,ketones, sulfur compounds, and carboxylic acids weresignificantly (n = 4, p < 0.05) more concentrated (5−25times more) in R2 than in R3.These findings confirmed the hypothesis of partial

inefficiency of the methanogenic reactor of the two-stagesystem (R2). Many different intermediate metabolites weredegraded less efficiently in R2 than in R3, as above-described.The reasons for this can be found in the fact that R2 was fedwith the fermented material of R1, while R3 was fed with theraw material and the differences between these two materialswere very significant. VFAs were approximately 15 times moreconcentrated in R1 than in the feed, confirming what waspreviously measured by the OD20, i.e. that R2, as compared toR3, was fed by more rapidly degradable OM and richer inorganic acids, alcohols, and other intermediate metabolites. Assome authors stated,4,29 this might be an advantage for the two-stage system, as the whole process kinetics would be sped upand the methanogenesis would be favored by the presence ofmore readily available substrates (low-molecular mass acids andalcohols). On the other hand, it has been demonstrated that, atcertain concentrations of VFAs and alcohols, substrateinhibition effects may prevail on both fermentative andmethanogenic activities, causing partial inefficiencies ofsubstrate consumption and thereby negative feedback phenom-ena on the whole methanogenic process. Besides, othersubstances that have possible inhibiting effects (e.g., phenols,amines, ketones) were found in higher concentrations in R1liquid phase as compared to the feed (Table 3). Theseaccumulations in R2, more than in R3, probably contributed tothe observed partial inefficiency of fermentative/methanogenicprocesses in R2.Phenols and polyphenols are largely present in natural

environments and in vegetal biomass (fruits and vegetables),and they are also found in some essential amino acids (e.g.,tyrosine). For these reasons, they are frequently found inanimal manure.34 Simple phenols, as a result of biodegradationof more complex polymers and aromatic amino acids, can befurther anaerobically degraded by microorganisms to VFAs(mostly caproic acid and propionic acid), ketones (cyclo-hexanone), CH4, and CO2.

35 On the other hand, certainrecalcitrance to biodegradation, mainly given by the aromaticring, may drive accumulation of such compounds infermentation environments and their relatively high concen-trations may cause inhibiting effects of many microbial strains.16

Furthermore, acid pH and high concentrations of organic acidsin R1 (Tables 2 and 3) may have induced the liberation ofalkaline species, such as amines and other nitrogenousintermediates, by decarboxylation of aminoacids, whichsuccessively accumulated in R2 (Table 3) as reported inprevious studies on human and animal intestine metabolism.36

Macfarlane and Gibson36 demonstrated that acidic conditionscan induce aminoacids decarboxilations to amines, phenols, andindoles by various Clostridia and Bacteroides strains.These findings reveal that two-stage AD processes have

potential advantages in terms of energy recovery, with respectto one-stage, but, on the other side, they may drive thebiodegradation to inefficient metabolic pathways, reducing thisadvantage. This may depend strongly on the type of organicmaterial fed29 and this study should be considered as a first stepto get a wider knowledge on this topic. In addition, deeperstudies should be addressed at finding the optimal combination

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of process conditions (HRT, OLR, pH, temperature, etc.),separately for the hydrogenic and the methanogenic stages.3.4. Microbial Communities. Microbial communities

analysis gave bacterial and archaeal DGGE profiles for allreactors, shown in Figure 2a and 2b, respectively. Dominantbands were sequenced and their phylogenetic affiliations arereported in Table S2.

In both R1 and R2, all the identified microorganismsmatched with uncultured bacteria affiliated to the phylumFirmicutes, class Clostridia, whereas a greater diversity wasobserved in the R3, with bacteria belonging to three differentphyla: Bacteroidetes, Firmicutes, and Synergistes. This difference isnot surprising and suggests that (i) heat-treatment of the initialinoculum was efficient in selecting vegetative cells and activatedin R1 (and consequently in R2) the development of spore-forming microorganisms like Clostridia; (ii) the acid pH and thehigh concentrations of potentially toxic metabolites in R1(Tables 2, 3, and S1) probably contributed to a more strictmicrobial selection, which was reflected also in R2.In R3, higher bacterial diversity was observed, as compared

to both R1 and R2. Most of the sequenced dominant bandsresulted affiliated to Bacteroidetes, bacteria commonly found inanaerobic reactors.37,38 They can ferment carbohydrates orproteins; particularly, they first intervene in the degradation ofproteins and are able to ferment aminoacids to acetate.39

The relatively high bacterial diversity found in R3, ascompared to R2, could be the key point to explain its higherefficiency in degrading a number of intermediate metabolitesthat were found undegraded in R2 (Tables 2, 3, and S1).Interestingly, Bacterioidetes were reported to be more efficientthan Firmicutes in degrading phenols, amino acids, andamines.40 Furthermore, they are generally more tolerant tothe antimicrobial properties of phenols, as compared to

Firmicutes, because they can produce more glycan-degradingenzymes.40 The fact that bacterial population in R2 was lessdiverse than in R3 and mainly formed of Firmicutes, can be thecause of the partial accumulation of some potentially inhibitingsubstances (such as simple phenols, alcohols, ketones, amines),observed by GC-MS determinations in R2 (Table S1).Two-stage and single-stage processes showed well-estab-

lished archaeal communities, with the predominance ofacetoclastic methanogens in R2 and a more complex speciespattern composed of both acetoclastic and hydrogentrophicarchaea in R3 (Figure 2b), as expected. On the whole, thepartial inefficiency in degrading all intermediate metabolitesobserved in R2, seemed to be related more to fermentativebacteria communities, than to archaeal ones.The two-stage and one-stage AD systems considered in this

work, fed with the same organic mixture, with identical HRTsand OLRs, gave equal energy recovery yields. This is in contrastwith previous experiences which found better performances inthe two-stage approach. On the other hand, in this work, R2showed some residual biodegradable potential as compared toR3. An accumulation of undegraded intermediate metabolitesobserved in R2 seemed to be both the cause and theconsequence of the partial inefficiency observed for the two-stage system. This seemed to be related to a less diverse andthereby less efficient population of fermentative bacteria in thetwo-stage system, probably selected by stressing (acid pH andhigh VFA concentrations) environment of R1. Therefore, thetwo-stage system could have potentially led to higher energyrecoveries, but some inefficient fermentative pathways con-tributed to recalcitrant and potentially toxic metabolitesaccumulations.To clarify whether the two-stage system can be, in general, a

way to improve AD process, further efforts should be addressedat separately optimizing the two stages, in terms of processconditions (HRT, OLR, pH, temperature, etc.) to ensuremicrobial communities efficiency and stability.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information as mentioned in the text. This materialis available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: andrea.schievano@unimi.it (A.S.), fabrizio.adani@unimi.it (F.A.).

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe are grateful to Regione Lombardia, General Directorate ofAgriculture, for its financial support to project AGRIDEN.

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Figure 2. DGGE profiles of (a) bacterial and (b) archaeal microbialcommunities present in the reactors. R1, hydrogen reactor; R2,methanogenic two-stage reactor; R3, methanogenic single-stagereactor.

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