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Energy 73 (2014) 866e874

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Energy

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Effects of operational shocks on key microbial populations for biogasproduction in UASB (Upflow Anaerobic Sludge Blanket) reactors

C.S. Couras a, 1, V.L. Louros a, 1, A.M. Grilo b, J.H. Leit~ao b, M.I. Capela a, c, L.M. Arroja a, c,M.H. Nadais a, c, *

a Environment and Planning Department, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugalb IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior T�ecnico, Universidade de Lisboa, Av.Rovisco Pais 1, 1049-001 Lisboa, Portugalc CESAM e Centre for Environmental and Marine Studies, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal

a r t i c l e i n f o

Article history:Received 22 December 2013Received in revised form10 May 2014Accepted 26 June 2014Available online 25 July 2014

Keywords:Biogas productionUASB reactorsIntermittent operationOperational shocksMicrobial populationsSyntrophomonadaceae

* Corresponding author. Environment and PlanninAveiro, Campus de Santiago, 3810-193 Aveiro, Portufax: þ351 234 370 309.

E-mail addresses: catiacouras@ua.pt (C.S. C(V.L. Louros), andre.grilo@tecnico.ulisboa.pt (A.M.ulisboa.pt (J.H. Leit~ao), icapela@ua.pt (M.I. Capela)nadais@ua.pt (M.H. Nadais).

1 Both authors contributed equally to the work.

http://dx.doi.org/10.1016/j.energy.2014.06.0980360-5442/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

This work compares the overall performance and biogas production of continuous and intermittent UASB(Upflow Anaerobic Sludge Blanket) reactors treating dairy wastewater and subjected to fat, hydraulic andtemperature shocks. The systems were monitored for methane production, effluent concentration, vol-atile fatty acids, and microbial populations of the Eubacteria, Archaea and Syntrophomonadaceae groups.This last microbial group has been reported in literature as being determinant for the degradation of fattysubstrates present in the wastewater and subsequent biogas production. Results show that bothcontinuous and intermittent systems supported the applied shocks. However, the intermittent systemsexhibited better performance than the continuous systems in biogas production and physical-chemicalparameters. Syntrophomonadaceae microbial group was present in the intermittent systems, but wasnot detected in the biomass from the continuous systems. Hydraulic and temperature shocks, but not thefat shock, caused severe losses in the relative abundance of the Syntrophomonadaceae group in inter-mittent systems, leading to undetectable levels during the temperature shock. The severity of the effectsof the applied shocks on the key microbial group Syntrophomonadaceae, were classified as:fats < hydraulic < temperature. Results from a full-scale anaerobic reactor confirm the effect of inter-mittent operation on the presence of Syntrophomonadaceae and the effect on reactor performance.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Interest in the development and utilization of non-petroleumbased renewable sources of energy has been fostered by concernson energy security and greenhouse gases emissions [1]. Amongrenewable energy resources, bioenergy is one of the fastestgrowing energy alternatives, with tremendous potential in manyregions of the world [2e5]. For example, the European strategy forrenewable energy sources identifies bioenergy as the most impor-tant renewable energy source for the future. The development of

g Department, University ofgal. Tel.: þ351 234 370 349;

ouras), vitorialouros@ua.ptGrilo), jorgeleitao@tecnico.

, arroja@ua.pt (L.M. Arroja),

bioenergy is an important measure to improve energy structure,safeguard energy security, protect the environment, and promote asustainable development [2]. In this context, the anaerobic treat-ment of dairy industry wastewater containing high concentrationsof complex organic matter is highly attractive because of highmethane yields and potential for energy production. Besides itscomplex chemical nature, dairy wastewater is characterized by awide variation in flow, organic matter composition and concen-tration, due to the cyclic nature of production and cleaning pro-cesses. In general, wastewater treatment plants include a flowequalization tank which reduces the flow and load fluctuations.Nevertheless, in real scale dairy plants, variations in flow, organicload and fat concentrations can reach an order of 100% compared tothe average values [6]. These fluctuations are important since themajor drawbacks of anaerobic technology are unstable operation,poor resistance to high load variations, and transient operationalconditions [7,8]. In dairy wastewater, the most problematic con-stituents for anaerobic treatment are fats and LCFA (Long Chain

C.S. Couras et al. / Energy 73 (2014) 866e874 867

Fatty Acids) that result from fat hydrolysis [9e11]. Several in-vestigations have been published in recent years concerning theanaerobic degradation of fats and LCFA and their effects on theoverall process performance [12,13]. Fats are degraded via meta-bolic pathways distinct from those of proteins and hydrocarbons. Ina first step, neutral fats are hydrolyzed (lipolysed) into free LCFAand glycerol, a process catalyzed by extracellular lipases. The freeLCFA are converted to acetate and H2 by syntrophic acetogenicbacteria through the b-oxidation process. Microorganisms of theSyntrophomonadaceae group have been found to play an impor-tant role in this step of LCFA anaerobic degradation [14e16], and aretherefore considered as a key microbial group for the anaerobicdegradation of fat-containing wastewaters (viz dairy wastewater)and subsequent biogas production. Since lipids and LCFA have highmethane potential, the Syntrophomonadaceae group is also a keymicrobial group for the maximization of biogas production. The laststep of the anaerobic process is the production of methane bymethanogens (Archaea microorganisms). The b-oxidation step hasbeen reported as the limiting step in the anaerobic degradation offats [11]. LCFA exert an inhibitory effect on the b-oxidation process,which was initially considered as permanent [9], but was laterdemonstrated to be reversible after a lag phase [17]. Recent studiesemphasize the importance of a well-balanced microbial commu-nity for the stability and good performance of anaerobic reactors[8,18e20], and for optimized biogas production. However, transientoperational conditions are known to alter this necessary equilib-rium [21]. It has been reported that anaerobic populations sufferedshifts caused by overloading [8,22] or by temperature changes [23].

Dairy wastewaters and other effluents, such as food processingeffluents, are subject to sharp fluctuations in composition whichmay affect anaerobic systems performance. Significant decreases inthe overall efficiency were observed by Schmidt and Ahring [24]when UASB (Upflow Anaerobic Sludge Blanket) reactors feedcomposition was changed. Variations in the carbon source presentin the wastewater were reported to cause gradual changes in thephysical structures, bacterial distribution and settling characteris-tics of the sludge in UASB reactors [25]. Research by Fukuzaki et al.[26] also demonstrated that variations of the carbon source presentin the wastewater caused changes on the physical structure,chemical contents (extracellular polymeric substrates) and bacte-rial distribution. Specifically in what concerns sudden overloadingof fatty substrates, serious drops of methanogenic activity andbiogas production have been reported as a result of inhibition,accompanied by deterioration of sludge quality [27]. Flotationfrequently results, due to the adsorption of LCFA at the sludgesurface [27,28]. Another effect is the disintegration of sludge ag-gregates, which can occur when lipids are present, due to thesurfactant effect of LCFA. The hydrophobic acetogens that candegrade LCFA are severely affected by this surfactant disaggregatingeffect [29].

The response of anaerobic reactors to hydraulic shocks hasbeen reported in the literature as resulting in the drop of removalefficiency [27], disaggregation and washout of filamentous or-ganisms [29,30], and VFA (volatile fatty acids) accumulation andinhibition of methanogenic bacteria with consequent drop inbiogas yield [31]. In this type of operational shock, an improve-ment effect has also been observed in the COD (chemical oxygendemand) concentration of the treated effluent, attributed tochanges in the structure of microbial populations inside thereactor [32], or to changes in the Monod half saturation constant[7]. Some authors suggest that the substrates diffusion rate in-crease with higher substrate concentration (Fick's law) anddecrease with a higher flow velocity [33], whilst other authors [34]found that the external mass transfer resistance can be decreasedby increasing the flow velocity. Brito and Melo [35] reported that

under conditions of turbulent liquid flow, and thus higher shearstress, the flow velocity had a pronounced effect on the biofilmthickness and compactness, leading to different mass transfercoefficients. If the bulk liquid suffers a shift in velocity, there is anincrease in internal mass transfer coefficient. In what concernsbiomass washout caused by hydraulic overload it was reported[36] that the microorganisms responsible for the degradation ofLCFA were the most susceptible to washout at low HRT (hydraulicretention time).

Operational temperature is a major factor on the performance ofanaerobic reactors [37,38]. Thermophilic operation has beenpointed out as presenting some advantages over mesophilic oper-ation, namely in terms of substrate degradation rates and biogasproduction [23,39]. However, mesophilic reactors present a higheroperational stability [38,40]. Significant methanogenic biomasswashout was reported by Khemkhao et al. [23] due tomesophilic tothermophilic transition of the operational temperature of a UASBreactor treating palm oil mill effluent. Other effects resulting fromthe rise of operational temperature include increased biogas pro-duction and lower methane content of the biogas. When the pro-cess is exposed to a sudden alteration of temperature, the processconditionsmay be unbalanced because of different responses of thevarious metabolic groups [41]. Van Lier et al. [42] found thatexposure of a UASB reactor to temperatures above 45 �C resulted inserious drop in the activity of mesophilic granular sludge due tohigh bacterial decay. Immediately after the temperature shock, araise in biogas production was observed, followed by a sharpdecrease. The microorganisms that oxidize propionate are the mostsusceptible to temperature raise, and it was also reported thatmethanogens are more susceptible to temperature shocks thanacidogens. It is also known that temperature variations can affectthe sludge retention capacity, since temperature affects viscosity,and consequently, the hydraulic shear forces exerted on the sludgeparticles [43].

UASB (Upflow Anaerobic Sludge Blanket) reactors are the mostwidely used anaerobic technology for the treatment of industrialwastewater [44,45]. The performance of these systems may belargely enhanced by a new form of operation named intermittentoperation, in which feed and feedless periods are combined to raisebiogas production [46]. The beneficial effect of intermittent oper-ation is ascribed to a forced adaptation of the anaerobic biomass tosubstrates resistant to degradation (fats and LCFA) which occursduring the feedless periods [47]. Studies on the microbial pop-ulations striving in continuous and intermittent UASB reactorstreating dairy wastewater have shown a significant raise in Syn-trophomonadaceae relative abundance in intermittent reactors ascompared to continuous reactors [47]. To date, no studies have beenpublished comparing the performance of continuous and inter-mittent UASB reactors submitted to operational shocks and theireffects on the Syntrophomonadaceae key microbial group.

2. Materials and methods

This work compares the performance of intermittent andcontinuous UASB reactors treating dairy wastewater at a load of12 g COD/L/day and subject to operational shocks. Two replicatelaboratory-scale UASB reactors (working volume of 6 L) wereoperated in a continuous mode and two were operated in anintermittent mode. The intermittent cycle consisted of 48 h feedfollowed by 48 h feedless [46]. The feed concentration for theintermittent reactors was always double the feed concentrationused for the continuous reactors, so that in a 96 h period the totalCOD mass admitted to each reactor was the same. The feed wascomposed of diluted semi-skimmed milk or whole milk, supple-mented with nutrients and alkalinity. The reactors were seeded

C.S. Couras et al. / Energy 73 (2014) 866e874868

with approximately 4 L of flocculent sludge adapted to dairywastewater from an industrial wastewater treatment plant. Theinoculation sludge had a SMA (specific methanogenic activity) of7.1 mL CH4/g VSS/day measured with sodium acetate at (35 ± 1) �C.Throughout the entire experiment, effluent from the reactors wascollected in 24 h composite samples. All physical-chemical de-terminations were made according to Standard Methods [48]. Theproduced biogas was measured by wet gas meters (Schlumberger).Methane content in biogas was monitored using a Shimadzu GC e

9a gas chromatograph equippedwith a SupelcoMolecular Sieve 5 Acolumn and a Thermal Conductivity Detector (T¼ 100 �C). Injectiontemperature was 45 �C and Helium was used as carrier gas(P ¼ 4.4 kg/cm2). Volatile fatty acids determination was carried outin a Chrompack CP 9001 gas chromatograph equipped with aChrompack CP e sil5 e CB column and a Flame Ionization Detector(T¼ 300 �C). The injection temperaturewas 270 �C and Heliumwasused as carrier gas with a volumetric flow of 8 mL/min. Biomasssamples for FISH (Fluorescence In Situ Hybridization) analyseswere collected immediately before each applied shock, at the end ofthe shock period, and 20 days after the end of the shock. Aftercollection, biomass samples were immediately frozen at�20 �C. For(FISH) experiments, samples were allowed to reach room temper-ature and were vortexed for 5 min to disrupt granules and otheraggregates, and subsequently fixed with formaldehyde (4% v/v).Fixed samples were washed 3 times with PBS (phosphate bufferedsaline) and stored at �20 �C in a mixture containing 50% PBS and50% ethanol. Samples were spotted onto the surface of glass slides(2 spots per slide) and allowed to dry for 30 min at 42 �C. Sampleswere further dehydrated by 3 incubations of 3 min each withincreasing concentrations of ethanol (50, 80 and 100% v/v) andfinally dried at room temperature. Each spot was covered with 9 mLof hybridization buffer (0.9 M NaCl, 20 mM Tris buffer, 10 mM EDTA(ethylenediaminetetraacetic acid), 0.01% SDS, pH 7.2) and 1 mL ofprobe working solution (50 mg/mL) of each probe. Probes usedwere EUB338 (50-GCGCCTCCCGTAGGAGT-30, [49]) specific forEubacteria, Arc915 (50- GTGCTCCCCCGCCAATTCCT-30, [50]) specificfor Archaea, and SYNM700 (50-ACTGGTN5TTCCTCCTGATATCTA-30,[51]) specific for Syntrophomonadaceae. These probes were chosento assess the microbial population composition, namely the totalbacterial population including acidogenic bacteria (EUB338), theArchaea populationwhich includes the methanogens (Arc915), andthe fatty acid degraders belonging to the Syntrophomonadaceae(SYNM700). Probes labeled with FITC (EUB338), CY3 (ARC915), orCY5 (SYNM700) were acquired from Eurofins MWG Operon (Ger-many). Hybridization was performed in a humidified chamber for3 h at 45 �C. After hybridization, slides were subsequently washedwith washing buffer (180 mM NaCl, 20 mM Tris, 5 mM EDTA, 0.01%SDS, pH 7.2) for 30 min at 48 �C. Slides were further incubated with10 mL of DAPI (2 mg/mL) in PBS at room temperature for 15 min,followed by 3washeswith distilledwater at room temperature. Theslides were analyzed with an AXIOPLANmicroscope equipped withan external UV light source and filters adequate for the fluorescentdyes under use. Images were captured by a CCD camera (RopersScientific) and processed with the Metamorph software. The ImageJ software was used to quantify the fluorescence intensity obtainedwith each probe. Results are the mean values obtained for at least 3slides, each spotted at least three times.

The reactors were operated in baseline conditions (HRT ¼ 12 h;OLR (organic loading rate) ¼ 12 g COD/L/d) for several monthsbefore the beginning of operational shock experiments. Three typesof operational shocks were applied, consisting in the raise of feedfat content, feed volumetric flow, or operational temperature.

The percentage of COD removal was calculated with thefollowing formula:

Removalð%Þ ¼ CODT�feed � CODS�effluentCODT�feed

� 100 (1)

The percentage methanisation was calculated with thefollowing formula:

Methanisationð%Þ ¼ COD� CH4

CODR� 100 (2)

Results for a full-scale anaerobic reactor from a milk processingand cheese production industrial plant are also presented in thiswork. The reactor was a mesophilic continuously stirred tankreactor with a working volume of 2500 m3. All the phys-icalechemical and microbiological determinations were performedin the same laboratory and with the same methods as for thebench-scale UASB reactors.

3. Results and discussion

3.1. Shock experiments with laboratory-scale UASB reactors

Three baseline periods were established, corresponding to thethree periods before each of the applied shocks. The performance ofthe intermittent systems operating under baseline conditions wasbetter than the observed for the continuous systems concerningmethanisation efficiency, in agreement with other reports[47,52,53]. Under baseline conditions, the average values of thisparameter ranged 86%e90% and 53%e58%, for the intermittent andthe continuous systems, respectively. The average COD removalefficiencies in baseline operation ranged 94%e96% for the inter-mittent systems, and 85%e90% for the continuous systems. Thesedifferences in operational results have been attributed to thefeedless phase of intermittent operation [54], in which the anaer-obic biomass is forced to adapt to complex substrates difficult todegrade (mainly fats and LCFA), by means of microbial populationshifts that raise the relative abundance of the Syntrophomonada-ceae group [47] involved in the degradation of fatty substrates. Infact, the relative abundance of the Syntrophomonadaceae group inthe microbial population developed in the intermittent systemsoperating at baseline conditions ranged from 17% to 20%, while thiskey microbial group was not present at detectable levels in thecontinuous systems throughout all the experiments performed inthis work.

Results for the fat, hydraulic, and temperature shocks, applied tocontinuous and intermittent UASB systems are discussed below.Baseline and post-shock periods were immediately before andimmediately after the shocks, respectively. Values for the baselineperiods and for the post-shock periods are averages of 20 days or 5intermittent cycles. Error bars represent standard deviations.

3.1.1. Fat shockThe applied fat shock consisted of a raise in average feed fat

content from 110 mg/L to 261 mg/L in the intermittent systems andfrom 63 mg/L to 130 mg/L in the continuous systems. The shocklasted for 20 days or 5 intermittent cycles. Average feed COD con-centration and volumetric flow were maintained in order to keep aconstant OLR during the experiment.

Fats are substrates with higher methane production potentialthan hydrocarbons and proteins. Theoretically, it would be ex-pected that a raise in feed fat content would cause a raise inmethane production if inhibition thresholds are not reached. Thefat concentrations during the fat shock were above the inhibitionthreshold value of 100 mg/L reported by Perle et al. [10]. Results inFig. 1a show that, opposite to the observed for the continuous

Fig. 1. Results from the fat shock on a) methane production; b) COD removal; c) TSS in treated effluent. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

Fig. 2. Methanisation with the fat shock experiment. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

Fig. 3. VFA in effluent from the fat shock experiment. I ¼ Intermittent; C ¼ Continuous.B ¼ Baseline; S ¼ Shock; PS¼Post-Shock.

C.S. Couras et al. / Energy 73 (2014) 866e874 869

systems, methane production in the intermittent systems raisedslightly when the feed fat content was raised. This difference in thesystems response to fat shock is probably due to the improvedsubstrate degradation occurring in the intermittent reactors as aconsequence of biomass adaptation to the complex substratespresent in the feed. The intermittent systems did not present sig-nificant variations on the effluent COD removal efficiency (Fig. 1b)or in the TSS (total suspended solids) washout (Fig. 1c) as aconsequent of the fat shock. However, the fat shock resulted in alowering of COD removal efficiency in the continuous systems. Thiswas probably due to the improved substrate degradation occurringin the intermittent systems, resulting in an anaerobic sludge withunaffected adsorption capacity, and leading to COD removal effi-ciency similar to that observed for the baseline. This effect was notobserved in the continuous systems, where the uninterruptedfeeding did not allow the complete degradation of the adsorbedcomplex substrates [55].

In the continuous systems, the effect of fat shock on suspendedsolids (Fig. 1c) was more pronounced than the effect on effluentsoluble COD. The raise in feed fat content caused an increase ineffluent suspended solids of the continuous systems from anaverage of 0.79 g/L to 1.30 g/L. A heavy accumulation of floatingbiomass was observed at the top section of the continuous reactors.This biomass flotationwas also observed, although to a much lesserextent, in the intermittent systems, since the feedless periodsdiminished biomass flotation and washout.

The methanisation of the removed COD differed for the twotypes of systems (Fig. 2). The continuous system suffered a loss inmethanisation efficiency, from an average of 59%e34% during theshock period, whereas the intermittent systems present a slightincrease in methanisation efficiency during the shock period, froman average of 90%e92%. After the shock period, the intermittentreactors presented a methanisation efficiency similar to theobserved before the shock. The continuous reactors attained anaverage value of 48%, lower than the observed before the shock.Important differences were found in the VFA content of both sys-tems subjected to the fat shock (Fig. 3). In baseline operation, aceticand propionic acids were the predominant acids in both systems,although total VFA concentration was higher in the continuoussystems. The fat shock caused a sharp rise in propionic acid con-centration from 28 to 105 mg/L (as acetic acid) in the continuoussystems. The surge of propionic acid is consistent with inhibitoryeffects of fats and fatty substrates upon anaerobic biomass. Otheracids present in the effluent from continuous systems were caproicand n-butyric acids. In the intermittent systems, the fat shockcaused the appearance of n-butyric and i-valeric acids, and to aminor extent, of caproic acid.

FISH results (Fig. 4a and b) show that the Syntrophomonadaceaegroup, a key microbial group for the degradation of fatty substratesand maximizing biogas production, was absent in the continuousreactors before, during, or after the shock. In these reactors, adecrease on the relative abundance of Archaea (methanogenic)microorganisms was also observed, during, and after the fat shock.

In the intermittent systems, the fat shock caused a slight raise in therelative abundance of the Syntrophomonadaceae group, during andafter the shock. A slight raise in the relative abundance was alsodetected for the Archaeamicrobial group. These results support theobserved performances for biogas production of the continuousand intermittent reactors.

3.1.2. Hydraulic shockThe hydraulic shock lasted for 12 h or half of the feeding period

of intermittent operation. This consisted in decreasing the averageHRT from 11.3 h to 6.1 h in the intermittent systems, and from 12.0to 6.1 h in the continuous systems. During the hydraulic shock theCOD of the feed was halved in order to maintain a constant OLRthroughout the experiment: 10.9 g COD/L/day and 11.3 g COD/L/dayfor the intermittent and continuous systems respectively. The hy-draulic shock was severe for both continuous and intermittentsystems. Methane production (Fig. 5) in the intermittent systemsdiminished from an average of 0.89 L/h to an average of 0.26 L/h,equivalent to a 70% decrease. In the continuous systems the effectwas even more severe, with methane production decreasing by76%, from an average of 0.50 L/h to an average of 0.12 L/h. Theslightly higher resilience of methane production in the intermittent

Fig. 4. Microbial populations in the fat shock experiment; a) continuous system; b) intermittent system. Eubacteria excluding Syntrophomonadaceae; Syntrophomonadaceae;Archaea. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.

Fig. 5. Results from the hydraulic shock a) methane production; b) COD removal; c) TSS in treated effluent. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

C.S. Couras et al. / Energy 73 (2014) 866e874870

system may be attributed to the presence of microorganisms fromthe Syntrophomonadaceae group (Fig. 8) and to a lower biomasswashout (Fig. 5c). The heavy TSS washout observed in the contin-uous system was attributed to the combined effect of the highupflow velocity and the presence of accumulated substrates on thebiomass surface, a typical result of continuous operation of UASBreactors [55]. As in the intermittent reactors the biomass flocs werenot so heavily surrounded by adsorbed fatty substrates, the effectsof high upflow velocity were not so severe. The clean biomassresulting from the intermittent operation also results in a highercapacity for adsorption of the wastewater substrates, thusexplaining the higher COD removal efficiencies (Fig. 5b). It is worthnoting that the effluent COD for the continuous system decreasedslightly after the hydraulic shock as compared to baseline. This isprobably due to the effect of the shear forces of the ascendingliquid, resulting in the liberation of adsorbed substrates andconsequent higher availability of adsorption sites for substrateremoval. The effect of higher upflow velocity upon mass transfer[35] is also to be considered.

In both systems, the methanisation efficiency was negativelyimpacted by the hydraulic shock (Fig. 6), decreasing from 89% to

Fig. 6. Methanisation with the hydraulic shock experiment. Baseline; Shock;Post-Shock. I ¼ Intermittent; C ¼ Continuous.

53% in the intermittent systems, and from 56% to 32% in thecontinuous systems. During the hydraulic shock, the VFA concen-tration in the effluents increased from 42 to 125 mg/L and from 69to 182 mg/L (as AcH), in the intermittent and continuous systems,respectively. In the intermittent systems, the major acids produceddue to the shock were n-butyric, i-butyric and i-valeric, whilst inthe continuous systems the major resultant acids were propionic,n-butyric and n-valeric (Fig. 7). After the shock, the values of totalVFA concentration were 54 and 132 mg/L (as AcH), respectively forthe intermittent and continuous systems.

The results of biomass monitoring by FISH in the intermittentsystems (Fig. 8b) show that the microorganisms from the Syntro-phomonadaceae group suffered washout, resulting in a decrease oftheir relative abundance from 17% to 5%. According to Hwu et al.[36] the bacteria that degrade LCFA are the most sensible towashout caused by low HRT. After the shock, the relative abun-dance of the Syntrophomonadaceae group raised to 10%. In thecontinuous systems the microbial group that suffered washout dueto the hydraulic shock was the Archaea (methanogenic) group(from 59% to 50%). After the shock, a slight recovery of relative

Fig. 7. VFA in effluent from the hydraulic shock experiment. I ¼ Intermittent;C ¼ Continuous. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.

Fig. 8. Microbial populations in the hydraulic shock experiment; a) continuous system; b) intermittent system. Eubacteria excluding Syntrophomonadaceae; Syntrophomo-nadaceae; Archaea. Shock; PS ¼ Post-Shock.

Fig. 9. Results from the temperature shock a) methane production; b) COD removal; c) TSS in treated effluent. Baseline; Shock; Post-Shock. I ¼ Intermittent; C ¼ Continuous.

C.S. Couras et al. / Energy 73 (2014) 866e874 871

abundance (to 55%) was observed for this microbial group. Theseresults correlate with the methane production observed in thereactors.

3.1.3. Temperature shockThe temperature shock lasted for 12 days or 3 intermittent cy-

cles and consisted in a one step raise of the operating temperaturefrom (35 ± 1) �C to (55 ± 1) �C. The shock led to an increase inbiogas production in both systems (results not shown). A similareffect was reported by Van Lier et al. [42]. However, the methanepercentage in the biogas was lowered in both systems. Thisreduction was more severe in the continuous systems (67%e52%),as compared to the intermittent systems (75%e67%). It would beexpected that a raise in the operational temperaturemight improvethe overall performance and biogas production of both systems,since thermophile reactors have been described asmore effective intreating complex wastewaters compared to mesophile systems[23,39]. However, the performance of both systems decreased,considering all parameters monitored (Figs. 9e11). The most severeeffect was observed for the TSS washout, which may have beencaused by disaggregation of biomass flocs due to high temperature

Fig. 10. Methanisation with the temperature shock experiment. Baseline; Shock;Post-Shock. I ¼ Intermittent; C ¼ Continuous.

[43] and due to the turbulence effects of the initial increase inbiogas production.

The temperature shock also resulted in a severe loss of micro-organisms from the Syntrophomonadaceae group in the intermit-tent reactors (Fig. 12b). The temperature shock generatedconditions that led to a reduction of the relative abundance of theSyntrophomonadaceae group from an initial value of 20% to valuesundetected by the FISH methodology used. Remarkably, after themesophile operation was resumed, the relative abundance of thismicrobial group reached detectable levels (9%). This reduction ofthe Syntrophomonadaceae group did not impact the biogas pro-duction of the intermittent reactor. These results suggest thatmethane production from substrates other than lipids/LCFA and/orviametabolic pathways other than b-oxidationwere affected by thetemperature shock at a more limited extent more limited than themicroorganisms from the Syntrophomonadaceae group. In thecontinuous systems the Archaea microbial group was the mostsusceptible to washout, with relative abundances declining from60% to 47%, and recovering to 52% after the shock.

Fig. 11. VFA in effluent from the temperature shock experiment. I ¼ Intermittent;C ¼ Continuous. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.

Fig. 12. Microbial populations in the temperature shock experiment; a) continuous system; b) intermittent system. Eubacteria excluding Syntrophomonadaceae; Syntro-phomonadaceae; Archaea. B ¼ Baseline; S ¼ Shock; PS ¼ Post-Shock.

C.S. Couras et al. / Energy 73 (2014) 866e874872

For each shock experiment performed in this work (fats, hy-draulic and temperature shocks), a ManneWhitney (non-para-metric) statistical test was performed to check the statisticalsignificance of the differences in relative abundance of the differentmicrobial groups between operating conditions for each pair ofreactors. For each pair of reactors all the differences in the relativeabundance of the various microbial groups from baseline, to shockor post-shock conditions were found to be significant for a signif-icance level of 5%.

Table 1 resumes the effects of the applied shocks upon therelative abundance of the Syntrophomonadaceae microbial groupin the intermittent systems. In terms of this key microbial group,the severity of the applied shocks may be classified as:fats < hydraulic < temperature.

Table 2Operational data for an industrial scale anaerobic reactor treating dairy wastewaterfrom a milk processing and cheese production industry.

Parameter Average SD Max Min n

Phase 1 (continuous)Volumetric flow (m3/d) 476.3 58.3 701 223 93O&G (g/m3) 130 26 204 92 113OLR (g COD/m3/d) 398 65.2 674 165 85COD removal (%) 71 14 89 52 87Methanisation of removed COD (%) 55 28 71 18 50

3.2. Full-scale anaerobic reactor

In other research works done by our group we used severalbiomass samples collected throughout a period of more than 3years from an anaerobic industrial reactor in a dairy wastewatertreatment plant located in the North of Portugal. Although thewastewater passed through an equalization tank some variationsoccurred in the operational conditions of the reactor. The func-tioning of the reactor can be divided in two phases (see Table 2). Inphase 1 the anaerobic reactor worked in a continuous mode andpresented several problems caused by the fatty materials in thewastewater, like encapsulation of biomass flocs and consequentbiomass washout. In the second phase, due to an alteration in theproduction schedule, the reactor was operated in an intermittentmode inwhich the feedwas interrupted during one day of theweek(Sundays). The data presented in Table 2 show that in phase 2 thereactor had an improved performance as compared to phase 1 inwhat concerns COD removal and methanisation efficiency. Theresults from FISH determinations on the biomass developed in theindustrial reactor during phase 1 showed that the Syntrophomo-nadaceae microbial group was not present in detectable levels inthe biomass and the relative abundance of Eubacteria and Archaeawere 63% (SD ¼ 12%, n ¼ 27) and 37% (SD ¼ 10%, n ¼ 27), respec-tively. The results from determinations on the biomass developedin the industrial reactor during phase 2 show that the

Table 1Effects of operational shocks upon detectable levels of Syntrophomonadaceae mi-crobial group in intermittent UASB reactors treating dairy wastewater (percentagesrefer to variations from baseline relative abundance).

Shock Shock effect (%) Recovery level (%)

Fats þ11.8 112Hydraulic �85.7 57Temperature �100 45

Syntrophomonadaceae was present with a relative abundance of2% (SD ¼ 1.7%, n ¼ 18), whilst Eubacteria excluding Syntropho-monadaceae and Archaea had relative abundances of 57%(SD ¼ 13%, n ¼ 18) and 41% (SD ¼ 17%, n ¼ 18), respectively. Theintermittent operation resulted in improved performance of thereactor (Table 2) and in the presence of the Syntrophomonadaceaemicrobial group, although in a lower relative abundance thandetected in the intermittent lab-scale UASB reactors. According topublished results [46,56] a longer feedless period in intermittentoperation improves biomass adaptation to fatty substrates andconsequently results in higher COD removal and methanisationefficiencies. The low frequency and the short duration of thefeedless periods in the industrial reactor (phase 2) explains thelower relative abundance of the Syntrophomonadaceae in com-parison to what was detected in the lab-scale UASB reactors.

These results show that the intermittent operation favors thedevelopment of the key microbial group Syntrophomonadaceae ina full-scale anaerobic reactor treating dairy wastewater subjectedto transient operational conditions.

4. Conclusions

In this work we have compared the performance of continuousand intermittent UASB reactors when subjected to fat, hydraulic, ortemperature shocks, with a special focus on its effects on the keymicrobial group Syntrophomonadaceae, detected only in theintermittent systems. This microbial group plays a key role in thedegradation of methane-rich substrates, like lipids and LCFA. Syn-trophomonadaceae are also considered a key microbial group for

VSS (g/L)a 11.9 1.3 14.1 8.8 33Phase 2 (intermittent)Volumetric flow (m3/d) 552.5 77.4 886 337 196O&G (g/m3) 145 49 211 72 97OLR (g COD/m3/d) 468 98.3 850 190 123COD removal (%) 92 11 96 84 110Methanisation of removed COD (%) 80 16 97 63 98VSS (g/L)a 16.5 3.2 23.3 10.3 55

SD ¼ standard deviation; Max ¼ maximum value; Min ¼ minimum value;O&G ¼ oils and greases; n ¼ number of observations.

a VSS measured at the bottom sampling port of the reactor.

C.S. Couras et al. / Energy 73 (2014) 866e874 873

the maximization of biogas production in anaerobic reactorstreating fatty substrates. The relative abundance of this microbialgroup increased slightly with the increase of fat content of the feedof the intermittent systems. This group of microorganisms provedto be extremely sensitive to hydraulic and temperature variations.Contrary to the fat shock, the hydraulic and the temperature shockscaused a loss in the relative abundance of the Syntrophomonada-ceae group on the populations developed in the intermittent sys-tems, this leading to undetectable levels during the temperatureshock. In terms of this key microbial group, the severity of theapplied shocks may be classified as: fats < hydraulic < temperature.

As a final conclusion, our results show that the intermittentoperation has a better performance than the continuous systemboth in baseline operation and in transient operational conditions.Full scale bioreactors treating complex wastewaters containing fats,such as dairy effluents, are often subject to transient conditions(hydraulic and load fluctuations) which turns them ideal candi-dates for intermittent operation in order to achieve a long termstable process with high efficiency. This resilience of the intermit-tent system is apparent in methanisation and COD removal effi-ciencies, which correlate with the presence of theSyntrophomonadaceae group in the microbial populations of thissystem.

Nomenclature

AcH acetic acid (g or mg/L)CCD charge-coupled deviceCOD chemical oxygen demand (g or mg/L),COD-CH4methane COD (g)DAPI 40,6-diamidino-2-phenylindoleDNA deoxyribonucleic acidEDTA ethylenediaminetetraacetic acidHRT hydraulic retention timeFISH fluorescence in situ hybridizationLCFA long chain fatty acidsMax maximum valueMin minimum valuen number of observationsO&G oils and greasesOLR organic loading rate (g COD/L/day or g COD/m3/day)PBS phosphate buffered salineSD standard deviationSDS sodium dodecyl sulfateSMA specific methanogenic activity (mL CH4/g VSS/day)TSS total suspended solids (g or mg/L)UASB upflow anaerobic sludge blanketVFA volatile fatty acids (mg as AcH/L)

SubscriptsT totalS solubleR removed

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