Bioresource Technology 101 (2010) 3925–3930

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Bioresource Technology

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High-rate anaerobic degradation of 5 and 6 carbon sugars under thermophilicand mesophilic conditions

C. Forbes *, D. Hughes, J. Fox, P. Ryan, E. ColleranEnvironmental Microbiology Research Unit, Department of Microbiology, National University of Ireland, Galway, Ireland

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

Article history:Received 8 September 2009Received in revised form 5 January 2010Accepted 9 January 2010Available online 4 February 2010

Keywords:Thermophilic ADPre-hydrolysisPectinCelluloseOFMSW

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.01.019

* Corresponding author. Present address: EnviroUniversity College Cork, Lee Road, Ireland. Tel.: +214901932.

E-mail address: c.forbes@ucc.ie (C. Forbes).

In this research paper, a comparison between thermophilic and mesophilic anaerobic degradation of avariety of the simple sugar components of carbohydrate rich biomass is presented. In order to investigatethe degradability of these basic sugars, three synthetic sugar based influents were supplied to two highrate upflow anaerobic hybrid reactors (UAHR) operated at 37 �C (R1) and 55 �C (R2). These influentstreams were: D-glucose/sucrose; L-arabinose/D-xylose and L-rhamnose/D-galacturonic acid. The reactorswere challenged in terms of influent composition rather than loading rate and were therefore operated ata maximum volumetric loading rate (VLR) of 4.5 gCOD l�1 d�1 during stable reactor performance. It wasfound that a switch from a D-glucose/sucrose synthetic influent to an influent composed of L-arabinose/D-xylose resulted in failure of the mesophilic reactor while the thermophilic UAHR was able to tolerate thechange of sugar influent at an unchanged VLR of 4.5 gCOD l�1 d�1. A subsequent phasing-in approach wasused to introduce new sugar influent streams and proved highly successful. The physiology of the bio-mass was assessed and it was noted that thermophilic anaerobic digestion (AD) involved the formationof acetate and H2, implying the involvement of homoacetogenic bacteria, while mesophilic AD proceededvia the formation of other intermediates.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

In unprocessed plant materials, carbohydrate may account forup to 60–95% of total dry weight. Plant-derived raw materialsare utilized widely in the food, beverage, paper and packagingindustries and thus constitute a significant portion of the wastefrom these industries. The waste generated from the food industryranges from relatively unprocessed food materials (surplus andspoiled or contaminated stock) to the by-products of food process-ing activities in an industrial context. In recent years, a number ofnovel approaches for the treatment of these wastes with concom-itant energy recycling have been investigated, including the pro-duction of bioethanol and biodiesel as well as biodegradableplastics (Callaghan et al., 1999). From both an environmental andeconomic perspective, however, anaerobic treatment is arguablythe best option for remediation of these wastes, simultaneouslyreducing their pollution potential while producing a value addedend product in the form of a renewable biofuel, methane gas.

There are limitations, however, to direct anaerobic treatment ofcarbohydrate rich materials which extend to the organic fraction of

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municipal solid wastes (OFMSW), food wastes and plant materialsused as energy crops. The effective and high-rate anaerobic treat-ment of solid wastes is impeded by the comparatively lengthy timetaken to hydrolyse complex polymers such as cellulose, pectin andhemi-celluloses, which comprise the major components of non-animal food wastes, to their constituent monomeric sugars (Noikeet al., 1985). Additionally, the biodegradability of solid wastes ishighly dependent on the composition of cellulose, hemicelluloseand lignin which are characteristic to the different componentsof OFMSW (Hartmann and Ahring, 2006). Successful treatment ofthese wastes in single stage reactors involves the operation of Con-tinuously Stirred Tank Reactors (CSTRs) with retention times in theregion of 10–30 days. A commonly employed method to circum-vent these problems involves pre-hydrolysis of these materials toyield more readily degradable short chain polymers and monomersprior to anaerobic treatment. By using pre-treatment strategies,improved hydrolysis of complex polysaccharide containing wastescan be achieved. As a result of this approach, the retention times ofwaste in anaerobic digesters can be considerably shortened, andhigher methane yields can be produced. A number of pre-hydroly-sis strategies have been investigated in order to eliminate the ini-tial rate limiting hydrolysis step. Pre-treatment methods includemechanical, chemical, and biological based approaches as well asvarious combinations of these. Those currently employed havebeen reviewed extensively elsewhere (Mata-Alvarez et al., 2000;

3926 C. Forbes et al. / Bioresource Technology 101 (2010) 3925–3930

Hartmann and Ahring, 2006). Another major limitation in the useof single stage anaerobic digestion of OFMSW and highly biode-gradable food wastes is the rapid acidification of these wastesresulting in pH decrease in the reactor, inhibiting the activity ofmethanogenic Archaea. These instability factors preclude the useof high-rate anaerobic digesters with retention times measuredin hours rather than days (Mata-Alvarez et al., 2000).

In the current trial, three distinct synthetic sugar influents weredevised in order to assess the anaerobic biodegradability of prehy-drolysed carbohydrate wastes. Each influent stream consisted oftwo simple sugars, which were applied to the UAHRs in tandemand designed to represent the carbohydrate macromolecule com-ponents of OFMSW. The first waste stream supplied to the reactorsconsisted of D-glucose and sucrose, both of which have been dem-onstrated to be readily degraded anaerobically (Forbes et al., 2009).Most carbohydrates contain D-glucose, often as their sole buildingblock, as in the examples of starch, glycogen, and most impor-tantly, cellulose. Sucrose is a disaccharide molecule, the structureof which inhibits further binding to other saccharide moieties. Itis normally extracted from either sugar cane or beet and is com-monly used as a sweetener and extensively employed in the bakingindustry and in food preservation. The second waste stream sup-plied to the reactors consisted of L-arabinose and D-xylose. Bothare pentose monosaccharides and found as part of more complexpolymers, such as xylans, hemicelluloses and pectins. The thirdinfluent stream was comprised L-rhamnose and D-galacturonicacid, the major monosaccharide components of pectin (Grohmannand Bothast, 1994; Renard et al., 1995). It was anticipated that thereactor sludges would adapt readily to these three synthetic influ-ents as these reactors had previously been employed in a 650 daystrial during which both had treated a variety of more complexhydrolysate influents (Forbes et al., 2009).

2. Methods

2.1. Reactor design and operation

Two laboratory scale UAHRs, both with an active volume of3.9 l, were used in the current trial. These reactors had previouslybeen inoculated and operated at 37 �C (R1) and 55 �C (R2) for670 days as described by Forbes et al. (2009), followed by a periodof 200 days of application of D-glucose and sucrose. On day 1 of thecurrent trial, both reactors were supplied with the first trial influ-ent, which consisted of D-glucose and sucrose on an equal COD ra-tio to a total VLR of 4.5 gCOD l�1 d�1. This was achieved byoperating the reactors at a 2 days hydraulic retention time (HRT),with an influent concentration of 9 gCOD l�1. The reactors weremaintained on this influent until day 91 when the influent compo-

Table 1Influent regime showing the contribution of each sugar to each influent stream, on a COD

Phase I II III IV

Start of phase (day of trial) 1 91 103 110

D-Glucose 4.5 – – –

Sucrose 4.5 – – –

L-Arabinose – 4.5 – 2.25

D-Xylose – 4.5 – 2.25

L-Rhamnose – – – –

D-Galacturonic acid – – – –

Total influent COD (g l�1) 9 9 0 4.5HRT (days) 2 2 390* 2VLR (gCOD l�1 d�1) 4.5 4.5 0 2.25

* Based on 10 mls of buffer per day being supplied to the reactors in order to obtain an

sition was changed to an equal ratio, on a COD basis, of L-arabinoseand D-xylose to a total of 9 gCOD l�1. Feeding with L-arabinose andD-xylose was continued at the same rate until day 103. Due toinstability, the feeding was then stopped for 6 days (Table 1; PeriodIII). During this period, the reactors were unfed and only 10 ml ofbuffer and associated nutrients were added on occasion to allowremoval of effluent samples for analysis. L-arabinose and D-xylosewere reintroduced on day 110, at a lower combined influent CODconcentration of 4.5 g l�1, at a VLR of 2.25 gCOD l�1d�1.

On day 126, the L-arabinose and D-xylose influent was replacedby D-glucose and sucrose at a VLR of 4.5 gCOD l�1 d�1 (Period V).This VLR was maintained until day 201.

L-arabinose and D-xylose were then phased back into theinfluent at a combined COD of 3 gCOD l�1 on day 202. TheVLR of 4.5 gCOD l�1d�1 was maintained by inclusion of D-glucoseand sucrose at an influent COD of 6 gCOD l�1 during the period(Period VI). On day 216, the influent L-arabinose and D-xylosewas increased to a combined gCOD l�1 of 6, while correspond-ingly reducing the D-glucose/sucrose in the influent to 3 gCODl�1 (Table 1; Period VII). On day 264, D-glucose and sucrose wereremoved totally from the influent and the combined COD con-centration of L-arabinose and D-xylose was increased to9gCOD l�1 d�1 (Table 1; Period VIII).

L-rhamnose and D-galacturonic acid were phased into the reac-tor influent, beginning on day 342. This was achieved by reducingthe combined COD of L-arabinose and D-xylose to 6 gCOD l�1 andintroducing L-rhamnose and D-galacturonic acid at 3 gCOD l�1 (Ta-ble 1; Period IX). The influent concentration of L-rhamnose and D-galacturonic was increased to 6 gCOD l�1 on day 363 (Table 1; Per-iod X). L-arabinose and D-xylose were withdrawn from the reactorinfluents on day 400 with a concomitant increase in the combinedinfluent of L-rhamnose and D-galacturonic acid to 9 gCOD l�1. Solefeeding of the latter two sugars was continued at a HRT of 2 daysand a VLR of 4.5 gCOD l�1 d�1 until day 430, at which point the trialconcluded.

Throughout the duration of the trial, the COD:N:P ratio of allinfluents was maintained at 1000:5:0.5 by supplementation withNH4Cl and KH2PO4 to the required concentrations. Buffering wascarried out by addition of NaHCO3 (12 g l�1) and influent wastestreams were supplemented with micronutrients (1 ml l�1), as rec-ommended by Shelton and Tiedje (1984).

2.2. Analytical techniques

Samples of reactor influent/effluent were routinely analysed forCOD (g l�1), VFAs (mg l�1) and pH as previously described (Forbeset al., 2009). Biogas was sampled for CH4 determination accordingto Standard Methods (APHA, 2001).

basis (gCOD l�1).

V VI VII VIII IX X XI

126 202 216 264 342 363 4004.5 3 1.5 – – – –

4.5 3 1.5 – – – –– 1.5 3 4.5 3 1.5 –

– 1.5 3 4.5 3 1.5 –

– – – – 1.5 3 4.5

– – – – 1.5 3 4.5

9 9 9 9 9 9 92 2 2 2 2 2 24.5 4.5 4.5 4.5 4.5 4.5 4.5

effluent sample.

Fig. 1. Percentage soluble COD removal by R1 and R2 during the trial. Differentoperational periods are denoted by arrows (Table 1). ( R1 R2).

C. Forbes et al. / Bioresource Technology 101 (2010) 3925–3930 3927

2.3. Specific substrate utilisation (SSU) tests

SSU tests were performed on days 194, 305 and 430 of the trial,corresponding with times at which the reactors were fully adaptedto each synthetic influent. SSU tests assessed the activity of fer-mentative species in the reactor sludges to degrade the varioussugars applied during the trial. Tests were carried out at 37 �Cand 55 �C in 60 ml serum vials containing sludge (2–5 gVSS l�1),anaerobic buffer and one of each of the trial sugars (2.5 gCODl�1) to a total of 30 ml. Samples were removed intermittently fromtest vials, were centrifuged at 10,000g for 10 min and the superna-tant analysed spectrophotometrically using the Dubois method forthe quantification of carbohydrates (Dubois et al., 1956). Briefly,one volume of sample was mixed with one volume of 5% phenolsolution, and the mixture cooled at 4 �C for 10 min. Five volumesof concentrated H2SO4 were rapidly added to the pre-cooled mix-ture, with continuous mixing, followed by boiling for 5 min. Sam-ples were then cooled and incubated at room temperature for30 min prior to determining the absorbance at 490 nm. In vial car-bohydrate concentrations were plotted against time to give a deg-radation profile for each sugar. Specific substrate utilisation rateswere calculated by using the period of most rapid carbohydratedegradation from each profile, and expressing this as the rate ofcarbohydrate depletion per gram of volatile suspended solids(mg carbohydrate g VSS�1 hr�1).

2.4. Specific methanogenic activity (SMA) tests

Specific methanogenic activity tests were also performed ondays 194, 305 and 450 of the trial. SMA measurements were car-ried out using the pressure transducer method developed by Coll-eran and Pistilli (1994). Briefly, the procedure involved themeasurement of the biogas pressure increase developing in sealedvials fed with the non-gaseous substrates ethanol (30 mM), propi-onate (30 mM), butyrate (15 mM) and acetate (30 mM), or of thedecrease in vials pressurised to 1 atm pressure with the gaseoussubstrate H2/CO2 (80:20). Tests were carried out in triplicate at37 �C and 55 �C in 20 ml serum vials containing sludge (2–5 gVSS l�1), anaerobic buffer (Hungate, 1966) and one of each of thetest substrates. Appropriate temperature conversion factors wereused to record the final results as ml CH4 gVSS�1 day�1 at standardtemperature and pressure (STP).

Fig. 2. Effluent VFA levels (mg l�1) from the mesophilic reactor, R1 (A), and thethermophilic reactor, R2 (B) throughout the trial period. Different operationalperiods are denoted by arrows (Table 1). Acetate propionate butyrate.

3. Results and discussion

3.1. Reactor operation

Prior to the start of the current study, both reactors had beenoperated for 900 days at 37 �C (R1) and 55 �C (R2). For the 200 daysimmediately preceding the trial, both reactors operated in a stablefashion on an influent composed of D-glucose/sucrose.

The current trial initially utilized the same substrates as previ-ously at a VLR of 4.5 gCOD l�1 d�1 and a HRT of 2 days for a 90 dayperiod (Table 1). Despite fluctuations it was clear that both reactorscould achieve >95% COD removal under the conditions imposed(Fig. 1). On day 91, the influent was changed to L-arabinose andD-xylose (on an equal COD basis), while maintaining the VLR andHRT (Table 1). This resulted in an immediate and dramatic de-crease in COD removal efficiencies (Fig. 1). The methane contentof the produced biogas also decreased to 14% from the mesophilicreactor and 12% from the thermophilic reactor. Within five HRTsthe performance of the thermophilic reactor had deteriorated to35% COD removal, with methane comprising only 16% of the biogasproduced. The response of the mesophilic reactor was even moresevere, with total failure in terms of COD removal efficiency and

methane production by day 100 (Fig. 1). There was a parallel in-crease in effluent VFA concentrations (Fig. 2A). By day 102, themesophilic effluent VFA concentrations were 1440, 3310 and3490 mg l�1 for acetate, propionate and butyrate, respectively(Fig. 2A). By contrast, the thermophilic effluent VFA concentrationswere considerably lower, with levels of 676 and 1709 mg l�1 foracetate and propionate, respectively, and with negligible levels ofbutyrate (Fig. 2B). These high concentrations of short chain fattyacids resulted in pH levels of lower than 5.5 in both reactors byday 100. It is noteworthy that Kim et al. (2002) defined reactor fail-ure in terms of an effluent discharge pH lower than 5.5.

Table 2Substrate utilisation rates for R1 (37 �C) and R2 (55 �C) sludges against sugarsubstrates used throughout the trial. Rates expressed in mg gVSS�1 hr�1.

Day of test 194 305 430

Temperature 37 �C 55 �C 37 �C 55 �C 37 �C 55 �C

D-Glucose 19.9 15.4 9.6 5.8 38.0 18.5

Sucrose 13.9 22.1 6.1 2.8 36.6 12.2

L-Arabinose 11.4 3.2 5.0 6.8 7.4 8.1

D-Xylose 6.6 5.2 5.7 5.2 10.0 7.4

L-Rhamnose 8.8 6.1 4.9 2.2 14.3 32.5

D-Galacturonic acid 13.9 13.9 5.2 2.2 29.7 16.0

3928 C. Forbes et al. / Bioresource Technology 101 (2010) 3925–3930

The influent supply to both reactors was discontinued on day103. An immediate reduction in effluent VFA concentrations wasnoted (Fig. 2), together with a decrease in effluent COD (Fig. 1). Thissuggested that the rapid acidification following introduction of L-arabinose and D-xylose had not caused complete inhibition ofsludge syntrophic and archaeal populations.

L-arabinose and D-xylose were reintroduced on day 110 (PeriodIV, Table 1) at a lower COD concentration of 2.25 gCOD l�1 for bothsugars. This resulted in a VLR of 2.25 gCOD l�1 d�1 at a 2 days HRT.Although the performance of the mesophilic reactor deteriorated,the thermophilic reactor performance showed greater stability,with 50% COD removal efficiency on day 126 (Fig. 1) and a biogasmethane content of 30%. The effluent VFA profiles (Fig. 2) confirmthe better performance of the thermophilic reactor during thisperiod.

To avoid total failure of the mesophilic reactor, on day 126 theinfluent to both reactors was changed to the D-glucose/sucroseinfluent at the previous VLR of 4.5 gCOD l�1 d�1 while maintainingthe HRT at 2 days (Table 1). As a result, an immediate improvementwas observed in the performance of both reactors. By day 202, bothreactors had fully recovered and regularly exhibited >90% COD re-moval efficiencies.

L-arabinose and D-xylose were reintroduced to the reactors onday 202 (influent concentration of 1.5 gCOD l�1 for each sugar)while maintaining a reduced combined D-glucose/sucrose influentof 6 gCOD l�1 (Phase VI, Table 1). This initial phasing in of L-arab-inose and D-xylose had no significant effect on the COD removalefficiency of either reactor, with COD removal efficiencies remain-ing between 92% and 98% in both (Fig. 1).

On day 216, the L-arabinose/D-xylose influent concentrationwas increased, with a corresponding decrease in the D-glucose/su-crose content of the influent (Phase VII, Table 1). This had minimaleffect on the performance of both reactors, although a minor per-turbation was noted for the thermophilic reactor on day 218(Fig. 1). This was accompanied by a short-term elevated dischargeof acetate and propionate in the R2 effluent (Fig. 2B). Omission ofD-gluose/sucrose and an increase in the L-arabinose/D-xylose influ-ent concentrations (4.5 gCOD l�1 d�1 and 2 days HRT) on day 264were accommodated readily by both reactors and were accompa-nied by increased biogas methane percentages to 85% from bothreactors and no significant effect on effluent VFA (Fig. 2).

L-rhamnose and D-galacturonic acid were introduced to bothreactors while gradually decreasing the L-arabinose/D-xylose influ-ent concentrations (Periods IX–XI, Table 1). Both reactors adaptedto the new influent sugars and, by the end of the trial, the COD re-moval efficiency was >95% (Fig. 1) and effluent VFA levels fromboth reactors were very low (Fig. 2).

3.2. Mesophilic versus thermophilic reactor performance

During initial introduction of L-arabinose and D-xylose from day91 onwards (Table 1: Period II), a severe decrease was noted in theperformance of both reactors within 1 day. The performance ofboth reactors, and the pH of the effluents reached levels catego-rised as ‘reactor failure’ by other authors (Kim et al., 2002). Itwas notable during this period of instability that the mesophilicreactor was more severely impacted by the sudden change to thenew influent stream. In most reports to date, thermophilic anaero-bic digestion has been reported as a more sensitive technology andassociated with high effluent VFA, particularly propionate, accu-mulation due to the poor substrate affinities of some organisms(van Lier et al., 1996, 1997). This was not the case in the currentstudy, however, and throughout the trial, effluent propionate dis-charge was a feature of the mesophilic reactor rather than its ther-mophilic counterpart. The deteriorated performance of themesophilic reactor during this time may have been due to the pro-

liferation of acetogens at the expense of methanogens. Acetogenicoxidation of arabinose by the mesophilic Clostridium scatologenesstrain SL1 has been shown to produce acetate and butyrate andmay account for the elevated levels of these VFAs and decreasedmethane production during reactor failure (Kusel et al., 2000).

During the phasing in of the sugar influents, both the thermo-philic and mesophilic reactors performed comparably, in terms ofCOD removal efficiency and % biogas methane. The phasing in ofnew influent constituents was a highly successful approach, afford-ing the biomass time to adapt to the new sugars, most likely by thedevelopment of new bacterial populations. All six trial sugars weredegraded under both thermophilic and mesophilic conditions. Theoverall average COD removal efficiencies during the final trial per-iod for both reactors were very similar �93.8% for R1 and onlyslightly better, 96.4%, for R2, with the % CH4 biogas of the two reac-tors highly comparable at 60.6% for R1 and 61.3% for R2. Thesefindings are in contrast to what has been observed in other studies,which have generally shown thermophilic anaerobic digestion tobe a more fastidious and sensitive process than mesophilicdigestion.

3.3. Biomass physiology

3.3.1. Specific substrate utilisation (SSU) testsSpecific substrate utilisation tests assessed the activity of the

various fermentative species found within the reactor sludge. Therate of degradation of each sugar by the sludges during the trialis presented in Table 2. It is evident that, by day 194, the reactorsludges had a high capacity to degrade D-glucose and sucrose,and the degradation rates for these sugars exceeded the rates forthe others tested. This is not surprising given that the reactor influ-ent prior to this test was composed solely of these sugar moities.

The biomass was again tested on day 305, by which time bothreactors had successfully adapted to L-arabinose and D-xylose assubstrates. From Table 2 it is clear that the SSU rates upon thesesugars were comparable at both 37 �C and 55 �C. SSU values forL-arabinose and D-xylose were similar for those of the other testssugars for the mesophilic sludge, with the exception of glucosewhich was almost twofold higher than mesophilic degradation ofL-arabinose. By contrast, degradation for L-arabinose and D-xyloseexceeded those of all other test sugars for the thermophilic sludge(Table 2).

The most significant trend evident from the data obtained onday 430 was that SSU rates for all substrates were markedly higherthan in the previous two tests, with rates as high as 38.04 mggVSS�1 h�1 being recorded for the mesophilic degradation of D-glu-cose (Table 2). Notably high SSU values were also observed forthermophilic degradation of L-rhamnose and mesophilic degrada-tion of D-glucose, sucrose, and D-galacturonic acid. It is not surpris-ing that a faster rate would be evident in the degradation of D-galacturonic acid, as this sugar had been applied in the reactorinfluent in the period preceding the test.

Table 3Specific methanogenic activity (SMA) profiles of R1 (37 �C) and R2 (55 �C) sludges cultivated throughout the trial. Biomass activity expressed in ml CH4 (STP) g�1VSS d�1. Valuesare the mean of triplicates ± standard deviation.

Day of test 194 305 430

Substrate 37 �C 55 �C 37 �C 55 �C 37 �C 55 �C

H2CO2 756.9 ± 46.0 284.7 ± 30.5 508.8 ± 21.8 231.7 ± 32.1 137.4 ± 11.2 225.9 ± 15.6Acetate 55.3 ± 3.8 117.1 ± 9.3 109.9 ± 11.7 36.8 ± 2.1 73.1 ± 11.3 53.1 ± 0.9Propionate 38.4 ± 3.4 22.1 ± 2.6 62.3 ± 3.2 4.6 ± 1.6 77.1 ± 7.8 7.8 ± 1.2Butyrate 21.2 ± 2.8 165.5 ± 15.6 40.2 ± 2.0 31.7 ± 1.1 9.6 ± 2.5 12.1 ± 3.1Ethanol 65.2 ± 3.7 89.3 ± 8.6 88.2 ± 1.5 22.2 ± 1.5 90.1 ± 3.5 14.1 ± 0.8

C. Forbes et al. / Bioresource Technology 101 (2010) 3925–3930 3929

It was of interest, however, that SSU values for D-glucose andsucrose increased (Table 2; day 430). Since D-glucose and sucrosehad not been included in the reactor influent since day 264, theSSU results suggest that fermentative species developed duringinfluent feed Periods of IX–XI (Table 1) were D-glucose and sucroseutilizers with a substrate range for the 6-carbon sugars, L-rham-nose and D-galacturonic acid. Reactor performance and SSU results(Table 2, day 305) suggest that this had not been the case for thepentose sugars, L-arabinose and D-xylose. It is likely that new fer-mentative species, which were present in the reactor at low levels,had to be developed in order to degrade L-arabinose and D-xyloseat the concentrations presented during the trial. From SSU resultson day 305, it does not appear that the 6-carbon sugar fermenterswere involved in the degradation of 5-carbon sugars. At the threetest times, SSU results were higher at 37 �C than at 55 �C for allof the sugars tested, with the exceptions of L-arabinose at 55 �Con day 305 and 430, and sucrose on day 194 (Table 2).

3.3.2. Specific methanogenic activity (SMA) testsThe SMA values calculated throughout this trial are presented in

Table 3. It is evident that on day 194, following reactor recovery,the mesophilic biomass exhibited markedly high activity on thegaseous substrate, H2/CO2. This is probably a direct result of thepreceding poor reactor performance which would likely have in-creased H2 pressures, and led to a proliferation in H2 utilisers asthe reactor recovered. It is evident that as the trial progressed,the activity of the biomass cultivated in the mesophilic reactor in-creased against the syntrophic substrates, propionate and ethanol.This indicated the improved ability of this reactor sludge to de-grade the intermediate organic acids that had accumulated and re-sulted in failure of this reactor in response to the introduction of L-arabinose and D-xylose on day 91. A lower activity on butyrate at37 �C was observed which may indicate that sugar degradationproceeded through the formation of other fatty acid intermediates.With respect to the activity of the methanogens within the R1sludge, a marked decrease in hydrogenotrophic activity was ob-served as the trial progressed. This, together with an increase inthe activity of the acetotrophic methanogens suggests a shift fromhydrogen based methanogenesis to acetotrophic methanogenesisas the successive influents were phased into R1.

By contrast, SMA values for the thermophilic biomass against alltest substrates was found to decrease as the trial progressed (Ta-ble 3). The only exception to this finding was a marginal increasein the SMA values against acetate and propionate on day 430.The comparatively low SMA values against propionate for the R2biomass were typical of thermophilic sludges (Ahring, 1994; Ahr-ing et al., 1993).

A marked decrease in the activity of the thermophilic biomass onbutyrate and ethanol was noted as the trial progressed on the differ-ent sugars (Table 3). It is possible that intermediates produced by thedifferent substrate fermenters, (e.g. formic and lactic acids etc.) maynot have been detected during the thermophilic trial. Alternatively,sugar substrates may have been degraded directly to acetate byhomoacetogenic bacteria growing heterotrophically. The role of

acetogens during thermophilic digestion, however, requires furtherinvestigation. Although mesophilic acetogenic oxidation of sugarshas been previously observed, heterotrophic involvement of aceto-gens at 55 �C remains unclear (Drake et al., 2006; Winter and Wolfe,1980). In both reactors, the decreased SMA values against H2/CO2

and acetate (Table 3) may reflect the relatively crude SMA measure-ment technique. Since the sugar fermentative species are likely togrow rapidly, the contribution of the slower-growing archaeal andsyntrophic organisms to the overall VSS is likely to decrease. Thisapparent reduction in SMA values would be more pronounced forthe slower-growing acetotrophs rather than the hydrogenotrophicArchaea.

4. Conclusions

The current study demonstrated that thermophilic AD wascomparable to mesophilic AD during the treatment of simple sug-ars. At the beginning of this trial, both processes were comparableon a D-glucose/sucrose influent. The introduction of 5 carbon sug-ars, however, resulted in near failure of both reactors. It is of inter-est that the thermophilic reactor recovered more rapidly when D-glucose and sucrose were reintroduced. The subsequent introduc-tion of novel influents at initial low and gradually increasing VLRswas accommodated by both reactors, with marginally better per-formance by R1 (mesophilic) for the duration of the trial. Given fer-mentative Bacteria may vary considerably with regard to theirsugar fermentative profiles, this study highlighted the importanceof gradual introduction of new sugar substrates to both mesophilicand thermophilic reactors.

Acknowledgements

The receipt of financial support from the Irish EnvironmentalProtection Agency is gratefully acknowledged.

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