Bioresource Technology 106 (2012) 74–81

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Bioresource Technology

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Effect of continuous oleate addition on microbial communities involvedin anaerobic digestion process

Manel Garrido Baserba 1, Irini Angelidaki, Dimitar Karakashev ⇑DTU Environment, Technical University of Denmark, Building 113, DK-2800 Kgs. Lyngby, Denmark

a r t i c l e i n f o

Article history:Received 21 September 2011Received in revised form 5 December 2011Accepted 5 December 2011Available online 13 December 2011

Keywords:Anaerobic digestionOleateCommunity profileBacteriaArchaea

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.12.020

⇑ Corresponding author. Tel.: +45 45251600; fax: +E-mail addresses: mgarrido@icra.cat (M.G.

(D. Karakashev).1 Present address: ICRA – Catalan Institute for Water

Edifici H2O, Parc Científici Tecnològic de la UdG, E-170

a b s t r a c t

In the present study, the microbial diversity in anaerobic reactors, continuously exposed to oleate, addedto a manure reactor influent, was investigated. Relative changes in archaeal community were less remark-able in comparison to changes in bacterial community indicating that dominant archaeal compositionremained relatively stable. Majority of the analyzed bacterial amplicons were phylogenetically affiliatedwith uncultured bacteria belonging to Firmicutes, Bacteroidetes, Proteobacteria and Thermotogae phyla. Bac-terial community changes in response to oleate addition resulted in a less diverse bacterial consortiumrelated to functional specialization of the species towards oleate degradation. For the archaeal domain,the sequences were affiliated within Euryarchaeota phylum with three major groups (Methanosarcina,Methanosaeta and Methanobacterium genera). Results obtained in this study deliver a comprehensive pic-ture on oleate degrading microbial communities in high organic strength wastewater. The findings mightbe utilized for development of strategies for biogas production from lipid-riched wastes.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Long chain fatty acids (LCFA), the main component of oils andfats, are attractive for biogas production because of their high po-tential methane yield in comparison with other organic substances(Cavaleiro et al., 2007). However, utilization of LCFA for methaneproduction can be problematic due to their inhibitory effect tothe anaerobic digestion process (Pereira et al., 2005). As a conse-quence, wastes with high lipid-content can cause inhibition of thebiogas process due to potential accumulation of LCFA during hydro-lysis of the oil. Oleic acid is the most abundant LCFA present in avariety of industrial and domestic wastewaters (Pereira et al.,2002). It was also found to be the most toxic LCFA (Cirne et al.,2007). Process problems observed in biogas reactors were oftendue to alleged toxic/inhibitory effect of LCFA towards both aceto-genic bacteria, which are responsible for the b-oxidation of fattyacids (Lalman and Bagely, 2002; Kim et al., 2004), and Methanogenicarchaea, which convert the products (acetate, hydrogen) of b-oxida-tion into methane (Lalman and Bagely, 2000, 2001). Other reportedproblems in the anaerobic digestion (AD) process caused by lipidsare: disintegration of granules in upflow anaerobic sludge bed reac-tors, flotation and washout of biomass (Pereira et al., 2002; Amaralet al., 2004). These adverse effects could be explained by the

ll rights reserved.

45 45932850.Baserba), dbka@env.dtu.dk

Research, C/ EmiliGrahit, 101,03 Girona, Spain.

adsorption of LCFA onto microbial biomass particles and therebychanging their surface properties. LCFA absorption on the microbialsurface creates a physical barrier that hinders the transfer of metab-olites in/out of the cells. Several authors have reported adaptationof the AD process to LCFA (Alves et al., 2009; Cavaleiro et al.,2008; Pereira et al., 2004). The adaptation has been explained, bydevelopment of specialized microbial community able to fast de-grade the LCFA, and thus reducing the LCFA concentration belowinhibition levels.

The overall conversion process involves the concerted action ofLCFA-degrading anaerobic bacteria (proton-reducing acetogens)and M. archaea (acetoclastic and hydrogen-utilizing methanogens)that utilize hydrogen and acetate. Up until now, 14 acetogenic bac-teria able to utilize LCFA in syntrophic associations with hydrogenscavengers (mainly methanogens but also some sulfate-reducers)have been characterized (Sousa et al., 2009). They all belonged tofamilies Syntrophomonadaceae (class Clostridia) (Wu et al., 2006)and Syntrophaceae (class Deltaproteobacteria) (Jackson et al.,1999). Both, acetoclastic and hydrogenotrophic methanogens, werealso found to be affected by LCFA to some extent (Lalman and Bage-ly, 2001). Acetoclastic methanogens (family Methanosarcinaceaeawith genera Methanosarcina and Methanosaeta) was the most af-fected group (Lalman and Bagely, 2001; Kuang et al., 2006).

LCFA oxidation becomes thermodynamically favorable in anaer-obic environments when the consumption of reducing equivalents(hydrogen and/or formate) is coupled with hydrogenotrophicmethane formation (Sousa et al., 2009). Due to the syntrophicmetabolic interactions, development of suitable degrading

M.G. Baserba et al. / Bioresource Technology 106 (2012) 74–81 75

syntrophic microbial consortia is of main interest for optimizationof the AD process. In a previous study (Palatsi et al., 2010), the ef-fect of pulse oleate addition on process performance and microbialcommunities in an anaerobic reactor digesting manure was inves-tigated. Meanwhile, the oleate addition was not regular, whichmight have significantly influenced the adaptation capacity of theprocess, and the established microbial communities. Therefore, itis important to study continuous addition of oleate, which mightbe more suitable to sustain an LCFA degrading microbial consor-tium. In order to obtain a detailed overview of the effect of oleateon microbial communities, molecular methods for phylogeneticidentification need to be applied.

Anaerobic microbial communities involved in LCFA degradationcan be investigated by 16S rRNA gene-targeted PCR–DGGE fol-lowed by DNA sequencing. This technique is suitable cultivation-independent tool for the analysis of complex microbial communi-ties (Amann et al., 1995). Phylogenetic information in combinationwith reactor performance data can give a detailed picture of the ef-fect of LCFA on microbial communities involved in AD process.

The aim of the present study was to investigate changes in thebacterial and archaeal community structure of biogas reactors inresponse to continuous addition of oleate. This study brings newknowledge on the changes occurring in microbial ecology of LCFAloaded reactors under anaerobic conditions. Linking the microbialecology information with biogas process performance can provideuseful tools for designing AD systems for treatment of high lipidswastes.

2. Methods

2.1. Experimental set-up

Thermophilically (55 �C) digested material from a full-scaleplant, located at Vegger (Denmark), was used as inoculum in thereactor experiments. Vegger biogas plant is co-digesting mainlycattle manure (80%), together with different wastes from foodindustry (approx. 20%). Non-digested cattle manure was dilutedtwice with water and used as feedstock for the reactors at organicloading rate of 1.7 g VS L�1 day�1. Inoculum and feedstock charac-teristics are shown in Table 1.

Two continuously stirred-tank reactors (CSTR) were started up.Feedstock was fed into the reactors four times per day (200 mL perday) at 6-h intervals using peristaltic pumps with timer control.Reactor operational conditions are shown in Table 2. Reactor ROleate

was used to study the effect of continuous oleate addition at non-inhibitory levels (1.5 g oleate L�1 day�1 and 2 g oleate L�1 day�1).Reactor Rctr was used as control reactor (no addition of oleate).The operating temperature in the reactors was maintained at55 �C by circulating hot water through the space between the reac-tor glass walls. The effluents were collected in the effluent bottles.Biogas was measured daily.

2.2. Microbial community analysis

The composition of bacterial and archaeal communities wasstudied by DGGE fingerprints of the partial 16S rRNA gene ampli-cons from reactor samples. Samples were withdrawn from reactorsat different stages: sample substrate (non-digested manure), sam-

Table 1TS/VS and pH values of the inoculum and feedstock used.

TS (%) VS (%) pH

Inoculum 3.01 ± 0.55 2.06 ± 0.71 7.97 ± 0.12Feedstock 2.58 ± 0.127 1.82 ± 0.01 7.71 ± 0.34

ple I (inoculum, digested manure before oleate treatment); sampleC1 at day 43 (1 HRT after initiation of the continuous oleate addi-tion at day 28); sample C2 at day 58 (2 HRT after initiation of thecontinuous oleate addition at day 28).

2.2.1. PCR–DGGEDNA extraction, PCR and DGGE were performed as previously

described (Karakashev et al., 2009). DGGE profiles were comparedusing the Quantity One software package (version 4.6.0; Bio-RadLaboratories). Similarity indices (SI) of the compared profiles werecalculated from the densitometric curves of the scanned DGGE pro-files by using the Pearson product-moment correlation coefficient(Häne et al., 1993). DGGE profiles of communities were analyzedby subtracting the background fluorescence from each lane, andthen band intensities were normalized to the total intensity of allbands in a given lane, to give relative band intensities (%). Bandswith intensity <0.02 (2%) were excluded from the analysis. Relativeband intensity was used as parameter to estimate the relativeabundance of the corresponding microorganism in the mixed cul-ture. Clustering of patterns was calculated using the unweighted-pair group method using arithmetic mean (UPGMA).

2.2.2. Phylogenetic analysis

DGGE bands were excised from the polyacrylamide gel. DNAwas extracted in water, re-amplified by PCR and sent for sequenc-ing (MWG-Biotech, Ebersberg, Germany). The obtained partial 16Sribosomal DNA sequences were analyzed using the BLAST programof National Center of Biotechnology Information (NCBI). The se-quences were aligned to their nearest neighbors and phylogenetictrees were calculated using Ribosomal Database Project TREEBUILDER, using sequences aligned with RDPs. The final sequenceshave been submitted to the Genbank database under the accessionnumbers FJ227285–FJ227301 and FJ423172–FJ423180.

2.3. Analytical methods

Total solids (TS), volatile solids (VS) and pH were determinedaccording to standard methods (APHA, 1992). Volatile fatty acids(VFA), were measured by a gas chromatograph with flame ioniza-tion detector (FID) according to Liu et al. (2008).

The experimental biogas production was measured by an auto-mated displacement gas metering system with a 100 mL reversiblecycle and registration (Angelidaki et al., 1992). The water used ingas meter was acidified to pH 3.0 by HCl and NaCl was added toprevent CO2 dissolution. Methane content in the biogas producedin the reactors was measured with a GC with thermal conductivitydetector (TCD) (Liu et al., 2008). The samples injection volume was0.5 mL and a sample lock-gas-tight syringe was used.

3. Results and discussion

3.1. Process performance and microbial community changes

The reactors were started at day 0 and were operated for58 days. They were filled up with inoculum and were operated un-der identical conditions (feedstock, HRT, temperature, etc.) as thefull-scale biogas plant from where the inoculum was retrieved.Therefore, steady state performance of the reactors was achievedin relatively short time after initiation (in approx. two weeks), asindicated by stable methane production and VFA levels. Both reac-tors were operated at identical conditions for 27 days, before oleateaddition was initiated in reactor Roleate During the initial 27 days,both reactors (Roleate and R-ctr) were only fed with non-digestedmanure (Fig. 1a). The process parameters are summarized in Table

Table 2Operational conditions of the reactors.

Reactor Operating temperature(�C)

Total volume(L)

Working volume(L)

Oleateaddition

HRT(days)

Feed frequency (times/day)

Oleate loading rate(g L�1day�1)

Roleate 55 4.5 3 Added in feed 15 4 1.5; 2Rctr 55 4.5 3 No 15 4 0

76 M.G. Baserba et al. / Bioresource Technology 106 (2012) 74–81

2. During the initial period (day 0–27) the reactors were stable witha methane production rate of 250 ± 1.5 mL CH4 day�1 L�1, (348 mlCH4 g VS�1 added), a VFA concentration of approximately 10 mM± 4.5 and pH ranging from 7.6 to 8.2.

Introduction of oleate (loading rate 1.5 g oleate L�1 day�1) toRoleate at day 28 was followed by a sharp increase (7.6 times higherthan the value before oleate addition) of methane production and asimultaneous increase in VFA (8 times higher than the value beforeoleate addition) (Fig. 1b). At day 36 the influent oleate load in Roleate

was increased to 2 g oleate L�1 day�1 and approximately 10 daysafter that, the methane production stabilized at an average valueof 1900 ± 35 mL CH4 day�1 L�1(Fig. 1a). However, at the same timewhen oleate load was increased to 2 g oleate L�1 day�1, total VFAstarted to decline and became relatively stable at around 30 mM(Fig. 1b). Lack of process failure in Roleate is in contradiction with aprevious study (Nielsen and Ahring, 2006) reporting that a load of2 g oleate L�1 day�1 inhibited the anaerobic digestion process. This

Fig. 1. Process performance of Roleate and Rctr reactors. (a) Methane production (mL CH4 dreactors. Arrows indicate sampling for DGGE analysis. C1, sample taken after 1HRT fromcontinuous oleate addition.

disagreement could be explained by differences in the original inoc-ulum used in the two studies. The inoculum used in the currentinvestigation had probably some LCFA degrading capacity, as itwas also indicated by the presence of Syntrophomonas speciesfound in the feedstock (band 15, lane ‘‘Substrate’’, Fig. 2) and inthe reactor before initiation of the oleate addition (lane I, Fig. 2).Syntrophomonas-related bacteria are well-known LCFA-degradingsyntrophic microorganisms living under methanogenic conditions(Hatamoto et al., 2007; Sousa et al., 2007, 2010). Those bacteriaß-oxidize saturated fatty acids to acetate using protons as the elec-tron acceptor (Sousa et al., 2010), and their presence confirm thesyntrophic relation between acetogenic bacteria (hydrogen produc-ers) and hydrogenotrophic M. archae (hydrogen consumers) re-ported in LCFA degradation.

The analysis of DGGE band-patterns, from a sample (C1, Fig. 2)taken at day 43, after 15 days (1 HRT) of daily continuous oleateaddition, revealed a clear shift in the microbial community compo-

ay�1 L�1) from Roleate and Rctr reactors. (b) Total volatile fatty acids in Roleate and Rctr

the start of continuous oleate addition.C2, sample taken after 2HRT from the start of

Fig. 2. DGGE patterns of bacterial amplicons: substrate, non-digested manure; I, reactor sample before oleate addition; C1, sample taken after 1HRT from the start ofcontinuous oleate addition; and C2, sample taken after 2HRT from the start of continuous oleate addition. Corresponding similarity index dendrograms (UPGMA clustering)and similarity matrices are also presented. Reproducibility of PCR–DGGE data, for each sample, was verified in independent experiments (data not shown).

M.G. Baserba et al. / Bioresource Technology 106 (2012) 74–81 77

sition. Bacterial similarity indices (SI) between the reactor sampleexposed to continuous oleate addition (C1, day 43) and the reactorsample before oleate addition (I) was 67.7; i.e. 32.3% decrease insimilarity was registered. The SI became even lower (SI = 60.2, i.e.a decrease in similarity of approx. 40%) after 30 days (2 HRT) fromthe start of continuous oleate addition (C2, day 58, Fig. 2). Archaealcommunity profiles also exhibited a drop of similarity indices(from 100 to 77.2), showing a 22.8% (sample C1) change comparedto the original microbial community composition, which becameeven more pronounced with operational time of the reactor(change of 34.1% for sample C2) (Fig. 3). The finding that decreasein similarity indices (SI) was more pronounced for bacterial (Fig. 2)than for archaeal community (Fig. 3) is in accordance with anotherstudy (Pereira et al., 2002) focusing on effect of oleic acid on micro-bial communities in expanded granular sludge blanket (EGSB)reactors. In the present investigation abundance of some bacterialspecies, such as Bacteroidetes-related (band 10 and 12; Fig. 4a) de-creased with the time of oleate exposure (Fig. 2). This finding indi-cates that several bacteria, such as uncultured Bacteroidetes-relatedspecies (sequences corresponding to bands 3, 7, 10, 12; Table 3;Fig. 2) and Proteobacteria related species (sequence corresponding

to bands 2 and 8; Table 3; Fig. 2) were strongly inhibited by oleateand therefore, they either disappeared or their abundance wentdown in the reactors (Fig. 4a).

3.2. Bacterial community profile

The shift in the bacterial community composition in response tooleate addition in Roleate did not result in appearance of new species,but only in shift of the the relative abundance (Fig. 4a). The majorityof the analyzed bacterial amplicons in reactor Roleate after oleateaddition were phylogenetically affiliated with uncultured bacteriabelonging to phyla Firmicutes (8 out of 11 sequences) among whichmembers of the families Clostridiaceae (sequence corresponding toband 1, Table 3), Bacillaceae (sequences corresponding to bands 5and 6, Table 3) and Syntrophomonadaceae (sequences correspondingto bands 11, 14 and 15, Table 3) families represented 75% (6 out of 8sequences). A prevalence of organisms belonging to Firmicutes sug-gested that those species were the main bacterial players in thesyntrophic consortium mediating the methanogenic oleate degrada-tion. This is in agreement with previous investigation in microbiol-ogy of anaerobic LCFA degradation (Alves et al., 2009).

Fig. 3. DGGE patterns of archaealamplicons: Substrate, non-digested manure; I, reactor sample before oleate exposure; C1, sample taken after 1HRT from the start ofcontinuous oleate addition; and C2, sample taken after 2HRT from the start of continuous oleate addition. Corresponding similarity index dendrograms (UPGMA clustering)and similarity matrices are also presented. Reproducibility of PCR–DGGE data, for each sample, was verified in independent experiments (data not shown).

78 M.G. Baserba et al. / Bioresource Technology 106 (2012) 74–81

Bacteria belonging to phyla Bacteroidetes, Proteobacteria (withgenera Pseudomonas) and Thermotogae were also detected, thoughwith only one representative for each phylum (bands 12, 8 and 9,respectively). Bacteroidetes phylum was highly represented in theinoculum, which is coinciding with the abundance of Bacteroidetesin animal guts (Gross, 2007), and thereby also in biogas reactorsystem (Shigematsu et al., 2006a). Starting from four Bacteroide-tes-related species (sequences corresponding to bands 3, 7, 10and 12 presented in the consortium before oleate treatment, laneI, Fig. 2) only one was left at the end of oleate addition (C2 finger-print; Fig. 2) suggesting that Bacteroidetes-like species are the firstto disappear from the bacterial consortium. Disappearance of ole-ate sensitive species contributed to the lower microbial diversityin the oleate exposed reactor. Opposite to the results obtainedhere, increased abundance of Bacteroidetes sp. in response to LCFAaddition was observed by Shigematsu et al. (2006b). This differ-ence can probably be attributed first to particular LCFA (palmiticacid) used in their study, and secondly to the source of inoculum(digested sludge from sewage treatment plant). Some Bacteroidetessp. presented in the sludge (absent from manure used in thisstudy) might mediate the palmitic acid (not used as LCFA in cur-rent study) degradation resulting in increase of their relativeabundance.

Presence of bacterial species belonging to genera Thermotoga,not usually associated with LCFA degradation, was an interestingfinding. Many Thermotoga species have been isolated from oilsources (i.e. Thermotoga petrophila, Thermothoga naphtophila) (Sou-sa et al., 2009). This could be an indication that these microorgan-isms can survive under high LCFA concentrations, but up to datethere is no data showing how they could use these compounds.Their dominance in the oleate exposed reactor could have beendue to disappearance of other competing microorganisms.

Genera Pseudomonas, however, have already proved to showcertain abilities to degrade oleic acid (Pseudomonas putida) viathe original enoyl-CoA isomerize route (De Waard et al., 1993).Therefore, the persistence of Pseudomonas-related species on thebacterial fingerprints may be related to their ability to degradeoleic acid or some LCFA degradation by-product. Nevertheless, itis not clear if other microorganisms than members of Syntropho-monadaceae or Syntrophaceae families are able to degrade LCFA.

3.3. Archaeal community profile

All the retrieved 16S rDNA archael gene sequences belonged tothe phylum Euryarchaeota. They were mostly (4 out of 7) repre-sented by sequences closely related to those of acetoclastic metha-

(a)

(b)

0

5

10

15

20

25

Bac

teri

a r

elat

ive

band

inte

nsit

y (%

)

I C1 C2

Band 1

Band 2

Band 3

Band 4

Band 5

Band 6

Band 7

Band 8

Band 9

Band 10

Band 11

Band 12

Band 13

Band 14

Band 15

Band 16

0

5

10

15

20

25

Arc

haea

rel

ativ

e ba

nd in

tens

ity

(%)

I C1 C2

Band 17

Band 18

Band 19

Band 20

Band 21

Band 22

Band 23

Fig. 4. Relative band intensity for the microorganisms a: Relative band intensity forBacteria: I, reactor sample before oleate exposure; C1, sample taken after 1 HRTfrom the start of continuous oleate addition; and C2, sample taken after 2 HRT fromthe start of continuous oleate addition. Bands with intensity < 0.02 (2%) wereexcluded from the analysis. b: Relative band intensity for Archaea: I, reactor samplebefore oleate exposure; C1, sample taken after 1 HRT from the start of continuousoleate addition; and C2, sample taken after 2 HRT from the start of continuousoleate addition. Bands with intensity < 0.02 (2%) were excluded from the analysis.Data from triplicate analysis is presented. Standard deviations were below 5% (datanot shown).

Table 3Partial 16SrDNA gene sequences retrieved from Roleate.

Band SeqID Closest relatives (>1200 bp) % Ide

1 Oleate.band_1 Uncultured bacterium B55_K_B_F04 992 Oleate.band_2 Uncultured bacterium LE82 983 Oleate.band_3 Uncultured bacteroidetes bacterium clone G12 994 Oleate.band_4 Uncultured bacterium TG-57 995 Oleate.band_5 Uncultured bacterium clone biogas-DT-B16 996 Oleate.band_6 Uncultured bacterium clone A168 987 Oleate.band_7 Uncultured bacterium LE58 988 Oleate.band_8 Pseudomonas sp.91S1 999 Oleate.band_9 Uncultured Thermotogae clone SHBZ989 9710 Oleate.band_10 Uncultured bacterium SHBZ923 9911 Oleate.band_11 Uncultured bacterium clone hoa12_36D05 9812 Oleate.band_12 Uncultured bacterium clone A35_D28_L_B_A07 9913 Oleate.band_13 Uncultured bacterium B-2 9714 Oleate.band_14 Uncultured bacterium clone 3wk_4LB35 9915 Oleate.band_15 Syntrophomonas wolfei. Wolfei str. Goettingen 8916 Oleate.band_16 Uncultured bacterium 2d_1FB14 9817 Oleate.band_17 Uncultured methanothermobacter clone HALEY_A9 9418 Oleate.band_19 Methanoccocus maripaludis strain s2 9519 Oleate.band_18 Uncultured methanobacterium clone WA1 9920 Oleate.band_20 Methanosaeta thermophila PT 9521 Oleate.band_21 Methanosarcina barkeristr.Fusaro 9922 Oleate band_22 Methanosarcina mazei strain Goe1 9623 Oleate.band_23 Methanosarcina acetivorans str. C2A 99

M.G. Baserba et al. / Bioresource Technology 106 (2012) 74–81 79

nogens of the order Methanosarcinales. Furthermore, two sequenceswere clustered within the hydrogenotrophic Methanococcales andMethanobacteriales orders. Methanosarcina (sequences correspond-ing to bands 21, 22 and 23; Table 3) Methanobacterium (sequencecorresponding to band 19), Methanococcocus (sequence corre-sponding to band 18) and Methanosaeta (sequence correspondingto band 20) were the most predominant bands presented in theanalyzed samples. Methanogens phylogenetically related to Met-hanosarcina (band 22) and Methanococus (band 18) increased theirrelative abundance from 0 (lane I; Fig. 4b) to 16.8% (lane C2; Fig. 4b)and from 3.8% (lane I; Fig. 4b) to 23.7%, respectively (lane C2;Fig. 4b). Those archaea became the dominant methanogens in sam-ples exposed to continuous oleate addition (C1 and C2).

Prevalence of versatile hydrogen scavengers including both Met-hanococcus (strictly hydrogenotrophic) and Methanosarcina (assome members of Methanosarcina can utilize not only acetate butalso hydrogen) might be correlated to the functional specializationof the syntrophic consortium in respect to fatty acids degradation.Continuous addition of oleate seems to trigger the fast growing stricthydrogenotrophic methanogens such as Methanococcus-relatedspecies (relative abundance 23.7%, band 18, lane C2; Fig. 4b) to be-come dominant archaeal players in the syntrophic process. In addi-tion, low abundance of the strict aceticlastic methanogenMethanosaetacea (0% and 4.1%, bands 19 and 20, respectively, laneC2; Fig. 4b) suggests that hydrogenotrophic methanogenesis wasan important pathway for methane formation in oleate exposedreactors.

During oleate degradation, acetate was the main VFA detectedin both reactors in high levels (data not shown). According toZheng and Raskin (2000), low acetate concentrations are favorablefor the dominance of Methanosaeta. The release of large amounts ofacetate in the medium during LCFA degradation might createfavorable conditions for growth of Methanosarcina, which have ahigher growth rate than Methanosaeta at high acetate concentra-tions. Therefore, continuous oleate conditions enhanced the fastgrowing versatile methanogenic species, such as Methanosarcinacompared to Methanosaeta sp. Instead, Methanosaeta sp. becamevery faint in archael community profile C2 (sequences correspond-ing to bands 19 and 20, Fig. 3).

nt. Phylum GenBank accession number Predominant band presentin

I C1 C2

Firmicutes FJ227297 d d d

Proteobacteria FJ227286 d

Bacteroidetes FJ227300 d

Firmicutes FJ227292Firmicutes FJ227301 d d

Firmicutes FJ227290 d d

Bacteroidetes FJ227296 d

Proteobacteria FJ227287 d d d

Thermotogae FJ227288 d d d

Bacteroidetes FJ227295 d

Firmicutes FJ227293 d d d

Bacteroidetes FJ227294 d d d

Firmicutes FJ227291 d d d

Firmicutes FJ227289 d d d

Firmicutes FJ423178 d d d

Firmicutes FJ423179 d d d

Euryarchaeota FJ423172 d d d

Euryarchaeota FJ423176 d d

Euryarchaeota FJ423174 d d

Euryarchaeota FJ423175 d d d

Euryarchaeota FJ423173 d d d

Euryarchaeota FJ423177 d d d

Euryarchaeota FJ423180 d d d

80 M.G. Baserba et al. / Bioresource Technology 106 (2012) 74–81

In a previous study (Palatsi et al., 2010) with pulse addition ofLCFA mixture containing 40% oleate, microbial ecology (both diver-sity and community changes) of the process was also investigated.Comparison between pulse and continuous LCFA addition showedmajor differences in microbial ecology of the reactors. Only a singlepredominant methanogen (Methanosarcina thermophila) was foundin the archael community structure upon pulse LCFA addition whi-lea more diverse methanogenic community was established undercontinuous oleate addition in the present study. LCFA pulses mightresult in accumulations of fatty acids degradation products (hydro-gen, carbon dioxide and acetate) which will have more inhibitory ef-fect on the strict hydrogenotrophic methanogens, such asMethanococcus related species (detected in the present study),rather than on Methanosarcina related species (detected in bothstudies) which are more versatile and can utilize both acetate andH2/CO2. It is highly possible that Methanosarcina sp. have betteradaptation capacity (probably due to its wider substrate utilizationspectrum) both to accidentally (during pulses) and permanently(during daily addition) high levels of LCFA while Methanococcus sp.are more sensitive to pulse LCFA exposure resulting in substrate(H2/CO2) inhibition of those archae. However, as the similarity indi-ces were not presented in the investigation made by Palatsi et al.(2010) more detailed comparisons about microbial changes underdifferent modes of oleate exposure (continuous versus pulses) arenot possible.

Methanogenic LCFA degradation was investigated here with re-spect to process performance and microbial ecology. The knowl-edge obtained can provide useful tools for designing of processbiotechnology for treatment of lipids-containing residues.

4. Conclusions

Microbial community changes in response to continuous oleateaddition resulted in a new consortium composition as a result ofthe specialization upon LCFA exposure. Relative changes in archa-eal community were less remarkable in comparison to changes inbacterial community structure. Members of the bacterial phylumFirmicutes appeared to be important for oleate degradation,whereas less specialized LCFA-degradation phyla, such as Proteo-bacteria and Bacteroidetes, tended to disappear after extensive ole-ate exposure. Presence of Pseudomonas sp. as player in oleatedegradation was uncommon and could be linked to oleate degra-dation under specific conditions. Methanosarcina sp and Methano-coccus sp. were identified as the dominant methanogenic archaeupon exposure to continuous oleate addition.

The results obtained in this study clearly underline the impor-tance of continuous LCFA exposure for development of a stableand active reactor for anaerobic digestion of lipids-containingwastes. This finding might be utilized for development of strategiesfor high rate/yield of biogas production from lipid-riched wastes,or introduction of lipid containing wastes to biogas reactors.

Acknowledgements

This work was supported by the Danish Energy Council EFP-05Journalnr.: 33031-0029.

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