65

JOURNAL OF BIOSCIENCE AND BIOENGINEERING

Vol. 97, No. 1, 65–70. 2004

Effect of Ammonium Addition on Methanogenic Communityin a Fluidized Bed Anaerobic Digestion

SHIGEKI SAWAYAMA,1* CHIKA TADA,1 KENICHIRO TSUKAHARA,1

AND TATSUO YAGISHITA1

Biomass Research Group, Institute for Energy Utilization, National Institute of AdvancedIndustrial Science and Technology, 16-1 Onogawa, Tsukuba, Ibaraki 305-8569, Japan1

Received 1 September 2003/Accepted 28 October 2003

After immobilization of anaerobes on carbon felt in a fluidized-bed anaerobic digester at anammonium concentration of 500 mg N/l, the results of real-time PCR analysis indicated that thecell densities of the immobilized methanogens and bacteria increased compared with those of thefree-living methanogens and bacteria in the original anaerobically digested sewage sludge, respec-tively. The results of the clone analysis of the original sludge suggested that the major methano-gens were Methanosaeta sp. and the members of the order Methanomicrobiales, and that after im-mobilization, these were changed to Methanobacterium and Methanosarcina sp. The results ofreal-time PCR analysis also showed that the ratio of the Methanosaeta sp. in the methanogenicarchaea decreased from 58.2% to 0.3% after the immobilization. Methane production decreasedat ammonium concentrations of greater than 6000 mg N/l. The results of real-time PCR analysisindicated that the cell density of the immobilized archaea decreased at ammonium concentrationsof greater than 3000 mg N/l. On the other hand, the cell density of the immobilized bacteria didnot decrease at an ammonium concentration of 6000 mg N/l, but decreased at that of 9000 mg N/l.The major methanogenic clones immobilized on the carbon felt at an ammonium concentration of3000 or 6000 mg N/l were Methanobacterium sp. The present results indicated that methanogenswere relatively more sensitive to ammonium than bacteria.

[Key words: archaea, ammonium, anaerobic digestion, fluidized bed, immobilization, methane, methanogens,phylogeny, 16S rRNA, real-time PCR]

The anaerobic digestion of organic wastes has the advan-tages of relatively low sludge production, low energy con-sumption, and methane production, which makes this proc-ess widely studied and used (1). The anaerobic digestion ofrelatively high concentrations of organic waste is an attrac-tive technological direction to provide an efficient treatmentsystem.

For the anaerobic digestion process, the degradation rateof organic components is mainly dependent on the quantityof degrading microbes maintained in digesters. To maintaina high methanogen concentration in a digester, the immobi-lization of microbial cells onto various supports has beenstudied (2). The organic matter removal efficiency in fixed-bed reactors was reported to be directly related to the char-acteristics of the support material used for the immobiliza-tion of the anaerobes (3). The effect of the immobilizationon the methanogenic community has not yet been activelydiscussed.

Organic waste usually contains nitrogenous compoundssuch as proteins which are converted to ammonium by an-aerobic digestion. Although ammonium is a nutrient for thebacteria involved in the anaerobic digestion process, it in-hibits methanogenesis at concentrations exceeding approxi-

mately 100 mM (4). Such inhibition during the anaerobicdigestion of livestock is often caused by a high ammoniumconcentration (5). Thus, the dilution of raw materials withwater and the adjustment of the feedstock C/N ratio are themost common and effective methods for avoiding ammo-nium overload in anaerobic digestion (6).

It was reported that aceticlastic and hydrogenotrophicmethanogens contribute 70% and 30% of the methane pro-duction, respectively, in an industrial wastewater treatment(7). Ammonium at a concentration of 235 mM completelyprevented the growth of Methanobacterium formicicum in apure culture (8). It was reported that the total ammoniumand ammonia concentrations severely affect the growth ratesof hydrogen-utilizing methanogens, and acetate quickly ap-pears with increasing total ammonium and ammonia con-centrations (9, 10). At 350 mM ammonia, the maximumgrowth rate of hydrogenotrophic methanogens was also re-ported to decrease to almost half of the uninhibited value(11). It is difficult to determine which type of methanogensis relatively sensitive to ammonium.

The presence of counter ions in the growth mediumcaused an enhanced tolerance of methanogens to ammo-nium (4). Ammonia toxicity to the growth of all methano-gens can be correlated to alterations in their K+/NH

3 content

and/or to inhibitions of CH4 synthesis (12). Only limited in-

formation is available for the mechanism of ammonia toxic-* Corresponding author. e-mail: s.sawayama@aist.go.jpphone/fax: +81-(0)29-861-8184

SAWAYAMA ET AL. J. BIOSCI. BIOENG.,66

ity to the methanogens.Recently, advanced biotechnology has enabled us to ana-

lyze the microbial diversity and quantity during anaerobicdigestion (13). The diversity and distribution of microbes ingranules of upflow anaerobic sludge blanket (UASB) wererevealed (14). The determination of bacterial and archaealloads by real-time PCR using a broad-range Taq-man probeand primer set was reported (15, 16). Phylogenetic analysesof a microbial community immobilized on bed material dur-ing anaerobic digestion have not yet been actively carriedout.

In the present study, the effect of ammonium addition ona methanogenic community immobilized on carbon felt in afluidized-bed anaerobic digester was investigated by cloneand real-time PCR analyses.

MATERIALS AND METHODS

Reactor operation The anaerobic digester was a 1000-mlcylindrical glass vessel containing 200 pieces of carbon felt (10�10�5 mm), 40 ml of anaerobically digested sewage sludge from asewage treatment plant (Ibaraki) and 760 ml of a synthetic me-dium. The carbon felt (Japan Carbon Company, Tokyo) with a po-rosity of 92.2%, a specific surface area of 0.70 m2/g and a specificweight of 0.11 g/cm3 was used as a fluidized bed. The digester wasmaintained at 35�C. The acetate-and-glucose-based medium wassemicontinuously supplied at 400 ml/week without sterilization.The medium consisted of the following (g/l): 2 sodium acetate, 2glucose, 0.5–9 g N/l NH

4Cl, 0.016 KH

2PO

4, 0.025 CaCl

2�2H

2O,

0.025 MgCl2�6H

2O, 0.03 Fe–EDTA, 0.005 CoCl

2�6H

2O, 0.005

MnCl2�4H

2O, and 0.1 yeast extract. The ammonium concentration

of the medium was varied from 500 mg N/l to 3000, 6000, and9000 mg N/l.

Clone analysis of methanogens To study the clone distribu-tion of methanogens, DNA was extracted from the original anaero-bically digested sewage sludge (0.5 ml) and the microbes immobi-lized on carbon felt (0.5 cm3) at the ammonium concentrations of500, 3000, and 6000 mg N/l using a Fast DNA SPIN kit for Soil(Qbiogene, Carlsbad, CA, USA) and a Fast Prep FP120 instrument(Qbiogene) (17). After a hot start, the amplification followed athree-step PCR of 15 cycles with 60 s denaturation (95�C), 60 s an-nealing (50�C), and 120 s elongation (95�C). The PCR primer setfor the methanogens was S-P-MArch-0348-S-a-17 (5�-GYGCAGCAGGCGCGAAA-3�) and S-D-Arch-0786-A-a-20 (5�-GGACTACVSGGGTATCTAAT-3 �) (18, 19). S-P-MArch-0348-S-a-17 wasmodified using the Primer Express (Applied Biosystems, FosterCity, CA, USA) and BLAST search to limit the methanogens fromS-D-Arch-0348-S-a-17 (16, 20).

The PCR product was purified using a Microspin S-400 HR col-umn (Amersham Biosciences, Piscataway, NJ, USA) and clonedusing a TA Cloning kit (Invitrogen, Carlsbad, CA, USA) accordingto the manufacturer’s instructions. The cloned DNA was sequencedusing a dRhodamin Dye Terminator Cycle Sequencing FS ReadyReaction kit (Applied Biosystems) and an automated sequence ana-lyzer (model 377; Applied Biosystems). The sequence data werealigned using the CLUSTAL W package for phylogenetic analysis(21). A phylogenetic tree was constructed by the neighbor-joiningmethod using the MEGA V2.1 package (22).

Quantification of immobilized microbes using real-time PCR

DNA was extracted from the original sludge (0.5 ml) and microbesimmobilized on the previously described carbon felt (0.5 cm3). Thereal-time PCR was conducted using an ABI7000 (Applied Biosys-tems) and TaqMan Universal PCR Master Mix (Applied Biosys-tems). The quantitative measurement by the real-time PCR was

conducted in quadruplicate. The amplifying primer set of S-D-Bact-0348-S-a-17 (5�-AGGCAGCAGTDRGGAAT-3�) and S-D-Bact-0786-A-a-20 (5�-GGACTACYVGGGTATCTAAT-3�), and thedouble dye probe of S-D-Bact-0515-S-a-25 (5�-TGCCAGCAGCCGCGGTAATACRDAG-3�) were used for measuring the 16S rRNAcopy number of bacteria (16). The amplifying primer set of S-P-MArch-0348-S-a-17 and S-D-Arch-0786-A-a-20, and the doubledye probe of S-P-MArch-0515-S-a-25 (5�-TGCCAGCMGCCGCGGTAAYACCGGC-3�) modified from S-D-Arch-0515-S-a-25 wereused for the methanogens (16). The newly designed amplifyingprimer set of S-F-Msaet-0387-S-a-21 (5�-GATAAGGGRAYCTCGAGTGCY-3�) and S-F-Msaet-0573-A-a-17 (5�-GGCCGRCTACAGACCCT-3�), and the newly designed double dye probe of S-F-Msaet-0540-A-a-31 (5�-AGACCCAATAAHARCGGTTACCACTCGRGCC-3�) using the Primer Express (Applied Biosystems) andBLAST search were used for the Methanosaeta sp. (20). The real-time PCR amplification followed a three-step PCR (40 cycles)with 20 s denaturation (95�C), 20 s annealing (55�C), and 120 selongation (95�C). The annealing temperature only for the bacteriawas 50�C. The R2 range of the standard curves obtained by thereal-time PCR measurements was 0.993–0.995.

The standard DNA for the real-time PCR of bacteria was pre-pared by PCR with a 2 bp longer PCR primer set of S-D-Bact-0346-S-a-19 (5�-GGAGGCAGCAGTDRGGAAT-3�) and S-D-Bact-0786-A-a-22 (5�-GTGGACTACYVGGGTATCTAAT-3�). The stan-dard DNA for the methanogens and Methanosaeta sp. was pre-pared with Methanosaeta thermophila strain PT (DSM 6194) byPCR.

Clone analysis was conducted to check the range of the newlydesigned Methanosaeta-specific amplifying primer set. The aver-age 16S ribosomal RNA gene copy numbers of the bacteria (4copies/cell) and methanogens (2.5 copies/cell) were referred to theRibosomal RNA Operon Copy Number Database for the conver-sion of copy number to cell number (23).

RESULTS AND DISCUSSION

Ammonium inhibition on biogas production Theeffect of ammonium on the methanogens immobilized oncarbon felt in the anaerobic digester was studied. Methaneproduction from the fluidized-bed anaerobic digester de-creased at ammonium concentrations of greater than 6000mg N/l (Fig. 1). The methane concentration in the producedbiogas also decreased at ammonium concentrations of greaterthan 6000 mg N/l (Fig. 1). The methanogenic activity wasreported to decrease by 10% at ammonium concentrationsof 1670 to 3720 mg N/l, decrease by 50% at 4090 to 5550mg N/l, and dropped to zero at 5880 to 6000 mg N/l (24).The present results indicated that the methanogenic activityin the fluidized-bed mesophilic anaerobic digester drasticallydecreased at the ammonium concentration of 6000 mg N/l.

Comparison of methanogenic clone distributionsThe results of the phylogenetic analysis of the original an-aerobically digested sewage sludge for methanogens basedon the 16S rRNA gene sequence suggested that the majormethanogenic clones in the original sludge consisted ofMethanosaeta sp. (14/20 clones) and the members of theorder Methanomicrobiales (6/20 clones) (Fig. 2). This dom-inance of the Methanosaeta sp. in the anaerobically digestedsewage sludge coincides with the report of Raskin et al. (25)and McHugh et al. (26).

The major methanogenic clones immobilized on the car-bon felt at the ammonium concentration of 500 mg N/l were

AMMONIUM EFFECT ON IMMOBILIZED METHANOGENSVOL. 97, 2004 67

the Methanobacterium sp. (15/20 clones) and Methanosar-cina sp. (4/20 clones) (Fig. 3). The dominant methanogenicclones immobilized on the carbon felt were different fromthose of the original sludge. This could be caused by thechange in microbial environment from free living to immo-

bilization. The Methanosarcina sp. was reported to be themost abundant methanogen in the sample taken from labo-ratory acetate-fed chemostats (25). There is a possibilitythat this change was caused by the change in organic sub-strate from sewage sludge to acetate and glucose.

The major methanogenic clone immobilized on the car-bon felt at the ammonium concentration of 3000 mg N/l wasthe Methanobacterium sp. (13–16/20 clones) (Fig. 4). Themajor methanogenic clones immobilized on the carbon feltat the ammonium concentration of 6000 mg N/l was alsothe Methanobacterium sp. (13–17/20 clones) (Fig. 5). Themajor route of methane production was reported to bethrough a syntrophic relationship between acetate-oxidizingbacteria and hydrogen-utilizing methanogens (13). Furtherinvestigation is necessary to determine the major route ofmethane production at a relatively high ammonium concen-tration. There might be uncultured methanogens of the orderMethanobacteriales (UMAC211, UMAC323, UMAC320,and UMAC318) which are relatively tolerant to ammonium(Figs. 4 and 5).

With an increase in ammonium concentration, the num-ber of clones of the Methanosarcina sp. decreased. Thespecific growth rate of the aceticlastic methanogens washalved at the ammonium concentrations of 3.5 g/l, comparedto 7 g/l for the hydrogenotrophic methanogens (27). Theacetate-utilizing methanogenic consortium was reported tobe more affected by ammonium than propionate-utilizingconsortia (28). The present results of clone analyses sup-ported the result that the aceticlastic methanogens are moresensitive to ammonium than the hydrogenotrophic metha-

FIG. 1. Cumulative methane production from the carbon felt fluid-ized-bed anaerobic digester supplied with a medium of increasing am-monium concentration. The digester was maintained at 35�C and wassemicontinuously supplied with an acetate-and-glucose-based me-dium. Symbols: closed circles, methane production from the digester;open circles, methane content in the biogas.

FIG. 2. 16S rRNA-based phylogenetic relationship between theclones (UMAC001–UMAC015) amplified by PCR with the methano-gen-specific primer set from the original anaerobically digested sew-age sludge and recorded methanogens. Numbers in parentheses indi-cate clone numbers. Numbers at nodes represent bootstrap values (100replicates). The phylogenetic tree was constructed using the neighbor-joining method.

FIG. 3. 16S rRNA-based phylogenetic relationship between theclones (UMAC102–UMAC114) amplified by PCR with the methano-gen-specific primer set from the immobilized microbes at the ammo-nium concentration of 500 mg N/l and recorded methanogens. Num-bers in parentheses indicate clone numbers. Numbers at nodes repre-sent bootstrap values (100 replicates). The phylogenetic tree was con-structed using the neighbor-joining method.

SAWAYAMA ET AL. J. BIOSCI. BIOENG.,68

nogens.Quantitative change in methanogens The results of

real-time PCR analysis indicated that the methanogenic celldensity decreased at the ammonium concentrations of greaterthan 3000 mg N/l (Table 1). On the other hand, the bacterialcell density did not decrease at the ammonium concentra-tion of 6000 mg N/l, but decreased at 9000 mg N/l. The pre-sent results supported the finding that the methanogens wererelatively more sensitive to ammonium than bacteria. Thedecrease in biogas production was explained by the de-crease in the quantity of methanogens immobilized on thecarbon felt. The real-time PCR method enabled the quantifi-cation of the immobilized microbes on the bed materials inthe anaerobic digester to evaluate the effectiveness of thebed-type bioreactors.

The results of real-time PCR analysis indicated that themethanogenic cell density immobilized on the carbon feltwas higher than that of the free-living methanogens in theanaerobically digested sludge. The average 16S rRNA genecopy numbers of bacteria and methanogens were supposed

to be 4.0 and 2.5, respectively (23). The ratio of methano-gens to methanogens plus bacteria increased from 25% to48% by immobilization. This ratio change was caused bythe immobilization or the change in substrate supplied to thedigester.

The results of clone analysis indicated that the Methano-saeta-specific PCR primer set amplified only the Methano-saeta sp. (20/20 clones) (Fig. 6). The present primer set forthe quantification of the Methanosaeta sp. by the real-timePCR method was confirmed to amplify only the Methano-saeta sp.

The results of real-time PCR analysis showed that the de-crease in the immobilized cell density of the Methanosaetasp. had a similar trend to that of the methanogens with theincreased ammonium concentration. If the average 16SrRNA gene copy number of the Methanosaeta sp. was 2.5,the same as that of archaea, the Methanosaeta sp. was 58%dominant in the methanogenic community (23). The resultsof real-time PCR analysis also indicated that the dominant

FIG. 4. 16S rRNA-based phylogenetic relationship between theclones (UMAC204–UMAC221) amplified by PCR with the methano-gen-specific primer set from the immobilized microbes at the ammo-nium concentration of 3000 mg N/l and recorded methanogens. Num-bers in parentheses indicate clone numbers. Numbers at nodes repre-sent bootstrap values (100 replicates). The phylogenetic tree was con-structed using the neighbor-joining method.

FIG. 5. 16S rRNA-based phylogenetic relationship between theclones (UMAC302–UMAC323) amplified by PCR with the methano-gen-specific primer set from the immobilized microbes at the ammo-nium concentration of 6000 mg N/l and recorded methanogens. Num-bers in parentheses indicate clone numbers. Numbers at nodes repre-sent bootstrap values (100 replicates). The phylogenetic tree was con-structed using the neighbor-joining method.

TABLE 1. 16S rRNA gene copy numbers of immobilized microbes on carbon felt at ammonium concentrationsof 500, 3000, 6000, and 9000 mg N/l

DNA source Bacterial copy number

(copies/cm3)Methanogenic copy number

(copies/cm3)Methanosaeta copy number

(copies/cm3)

Anaerobically digested sewage sludge (3.4 �0.3a) �108 (7.2 �0.6) �107 (4.2 �0.2) �10 7

Immobilized microbes at 500 mg N/l (8.2�2.4)�108 (4.7�0.7)�108 (1.5�0.5)�106

Immobilized microbes at 3000 mg N/l (6.4�0.7)�108 (1.6�0.2)�108 (1.9�0.3)�106

Immobilized microbes at 6000 mg N/l (7.5�0.3)�108 (3.5�0.4)�107 (9.2�1.6)�105

Immobilized microbes at 9000 mg N/l (7.4�1.0)�107 Undetermined Undetermineda SD.The R2 range of the standard curves obtained by the real-time PCR measurements was 0.993–0.995.

AMMONIUM EFFECT ON IMMOBILIZED METHANOGENSVOL. 97, 2004 69

methanogen of the original anaerobically digested sewagesludge was the Methanosaeta sp., and that the immobilizedcell density of the Methanosaeta sp. decreased from the un-immobilized cell density of the original sludge. These re-sults coincide with those of clone analyses; therefore, cloneanalyses of the methanogens with 15 PCR cycles could pro-vide preliminary information about the methanogenic com-munity.

A DNA standard representing bacteria most likely to pre-dominate in a given habitat was reported to be important fora more accurate determination of the total bacterial load inthe real-time PCR due to variations in the 16S rRNA genecopy number (15). The present data of the 16S rRNA genecopy numbers of bacteria and methanogens were lower thanthose of the actual 16S rRNA gene copy numbers based onthe DNA extraction efficiency. Further data accumulationsof the 16S rRNA gene copy number and efficiency of theDNA extraction are necessary to determine the real metha-nogenic cell density.

Accession numbers The sequences determined in thepresent study have been deposited in the DDBJ/EMBL/GenBank databanks under the accession numbers fromAB112676 to AB112700. The organisms, together with theirGenBank and EMBL accession numbers, whose 16S rRNAsequences were used for the phylogenic analysis were asfollows: Methanobacterium bryantii, AF028688; M. congo-lense, AF233586; M. curvum, AF276958; M. formicicum,AF028689; M. palustre, AF093061; M. subterraneum,X99044; Methanobrevibacter arboriphilus, AB065294;Methanosphaera stadtmanae, M59139; Methanococcusvoltae, M59290; Methanomicrobium mobile, M59142;Methanofollis tationis, AF095272; Methanospirillum hun-gatei, M60880; Methanosarcina acetivorans, M59137;M. barkeri, AB065295; M. frisius, M59138; M. mazei,

AB065295; M. siciliae, U20153; M. thermophila, M59140;Methanococcoides burtonii, X65537; Methanolobus tindarius,

M59135; Methanomethylovorans hollandica, AF120163;Methanosaeta concilii, M59146; Methanosaeta thermoace-tophila, M59141; Sulfolobus acidocaldarius, D14053.

ACKNOWLEDGMENTS

We are grateful to Dr. Yoichi Kamagata and Dr. Yuji Sekiguchi,Institute for Biological Resources and Functions, National Instituteof Advanced Industrial Science and Technology, for helpful dis-cussions, and to Ms. Yuka Tsuchida and Ms. Mami Inoue for tech-nical assistance.

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