Journal of Bioscience and BioengineeringVOL. 111 No. 3, 312–319, 2011

www.elsevier.com/locate/jbiosc

Thermophilic two-stage dry anaerobic digestion of model garbage withammonia stripping

Hironori Yabu,1,2 Chikako Sakai,1 Tomoko Fujiwara,1 Naomichi Nishio,2 and Yutaka Nakashimada2,⁎

⁎ CorrespondE-mail add

1389-1723/$doi:10.1016/j

Food Technology Research Center, Hiroshima Prefectural Technology Research Institute, Hijiyama-Honmachi 12-70, Minami-ku, Hiroshima 732-0816,Japan1 and Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama 1-3-1,

Higashi-Hiroshima, 739-8530, Japan2

Received 8 June 2010; accepted 19 October 2010Available online 19 November 2010

To avoid the inhibition of methane production by ammonia that occurs during the degradation of garbage, anaerobicdigestion with prior ammonia production and subsequent stripping was investigated. In the ammonia production phase, themaximum ammonia concentration was approximately 2800 mg N/kg of total wet sludge in the range of 4 days of sludgeretention time, indicating that only 43% of total nitrogen in the model garbage was converted to ammonia. The model garbagefrom which ammonia was produced and stripped was subjected to semi-continuous thermophilic dry anaerobic digestionover 180 days. The gas yield was in the range of 0.68 to 0.75 Nm3/kg volatile solid, and it decreased with the decrease of thesludge retention time. The ammonia–nitrogen concentration in the sludge was kept below 3000 mg N/kg total wet sludge.Microbial community structure analysis revealed that the phylum Firmicutes dominated in the ammonia production, but thecommunity structure changed at different sludge retention times. In dry anaerobic digestion, the dominant bacteria shiftedfrom the phylum Thermotogae to Firmicutes. The dominant archaeon was the genus Methanothermobacter, but the ratio ofMethanosarcina increased during the process of dry anaerobic digestion.

© 2010, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Ammonia stripping; Garbage wastes; Biomass; Anaerobic digestion]

In Japan, 19.4 million tons of food wastes were produced byhousehold and industrial activities in 2004. To accelerate the recyclingof food waste, the “Act on Promotion of Recycling and RelatedActivities for Treatment of Cyclical Food Resources” (the Food WasteRecycling Law) came into force in 2001 in Japan. Although this law haschanged the process of food waste disposal in Japan, its implemen-tation remains insufficient at present. In 2006, although 48% ofrecycled foodwastes were used for composting and 45%were used forlivestock feed, most of food wastes is still disposed of throughincineration and landfills (1,2).

On the other hand, since food waste is a biomass resource that iscarbon neutral, an anaerobic digestion has been recently noted as ameans of energy recovery. In the anaerobic digestion, organic matteris degraded to small particles and subsequently converted to volatilefatty acids (VFAs). The VFAs are converted into acetate and H2, andfinally CH4 and CO2 are produced by methanogens (3). Anaerobicdigestion has been widely used for treatment of organic wastesincluding food wastes. The anaerobic digestion process is classified onthe basis of the content of total solids (TS) as either wet process (b20%TS) or dry process (N20% TS) (4). In Japan, wet process digestion hasbeen widely used for wastewater treatment in the food and livestockindustries. In contrast, the use of dry process digestion is very limitedalthough it has several advantages over wet process, including a

ing author. Tel./fax: +81 82 424 4443.ress: nyutaka@hiroshima-u.ac.jp (Y. Nakashimada).

- see front matter © 2010, The Society for Biotechnology, Japan. All.jbiosc.2010.10.011

smaller reactor size and a lower volume of effluent per amount oforganic matter loaded. Because of these advantages, the use of dryprocess has been increasing in Europe recently (5).

Carbohydrates in foodwastes are a favorable substrate for anaerobicdigestion, while the accumulation of ammonia released during thedegradation of proteins and amino acids is very toxic for methanogens.In dry process digestion, the inhibition of methane production byammonia is a significant problem because the concentration ofammonia readily exceeds the critical level atwhich ammonia inhibitionoccurs due to the low water content (6–8). Indeed, during a long-termthermophilic anaerobic digestion experiment using model garbage (TS8.44%, TN 3400 mg N/kg), the final concentration of ammonia–nitrogenaccumulated in the fermenter was 4000 mg N/l, and methane produc-tion was inhibited (9).

In our previous report, thermophilic dry anaerobic digestion ofdehydrated waste-activated sludge (DWAS) with 80% water contentshowed that the release and stripping of ammonia prior to anaerobicdigestion can prevent the inhibition of methane production by a two-stage process (10). This two-stage anaerobic digestion process candecrease the cost of further treatment of digested sludge and canrecover a high concentration of ammonia nitrogen effectively. Stabledry anaerobic digestion was also performed by recycling ammonia-stripped biogas using chicken manure containing a large amount ofnitrogen (11–13). If these processes can be used to treat garbage andfood industrywastes, dry anaerobic digestionwill bemore convenientand effective for treating biological organic wastes with low water

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TABLE 1. Composition of model garbage.

Classification Component Wet weight content (%)

Vegetables Cabbage 12Potato 12Carrot 12

Fruits Apple 10Grapefruit (rind) 5Orange (rind) 5Banana (rind) 10

Meat and fish Ground meat 5Fish (with born) 5Egg 4

Staple foods Rice 10Bread 5Noodle 2.5Chinese noodle 2.5

FIG. 1. Reactor system for ammonia stripping from model garbage. (1) Stirring motor,(2) stirring device, (3) inner rotating vessel, (4) heater, (5) thermocouple, (6) 4 Nsulfuric acid, (7) pump.

DRY ANAEROBIC DIGESTION OF MODEL GARBAGE 313VOL. 111, 2011

content. Thus, in this study, the performance of thermophilic dryanaerobic digestion of model garbage pretreated for the productionand removal of ammonia was investigated.

MATERIALS AND METHODS

Model garbage Model garbage was prepared using the typical components ofhousehold food waste in Japan (14) as shown in Table 1. The components were shreddedand mixed using a food processor without adding water. The characteristics of the modelgarbage are shown in Table 2.

Methanogenic sludges To initiate anaerobic digestion in the model garbage,thermophilic methanogenic sludge was collected from an anaerobic digester used fortreating waste-activated sludge in Hiroshima, Japan. The sludge was anaerobicallyincubated at 55°C for 60 days prior to use in order to remove residual organic matter.The microbial seed sludge used to induce ammonia production in the model garbagewas collected from another sewage treatment facility in Hiroshima. The sludgecharacteristics are shown in Table 2.

Ammonia production from model garbage For the experiments to knoweffect of SRT on ammonia production with repeated batch culture, the seed sludge andmodel garbage were mixed at a ratio of 1:1 (v/v) to initiate ammonia production in themodel garbage, and the initial pH was adjusted to within the range of 7 to 8 usingNaHCO3. After the mixed sludge was transferred to a polypropylene bottle with a screwcap, the headspace of the bottle was purged with N2 gas. After 10 days of culture, arepeated batch operation was started. The sludge retention times (SRT) for ammoniaproduction were set at 2, 4, and 6 days. For the repeated batch operation, about half ofthe sludge was drawn out of the bottle halfway through the SRT period. Then the samevolume of freshmodel garbagewas added, and the initial pHwas adjusted to within therange of 7 to 8 using NaHCO3. This operation was repeated 10 times at each SRT. Theexperiments were performed under the static condition at 55°C.

To prepare the substrate for experiments of ammonia stripping from ammoniaaccumulated garbage and dry methane production from ammonia-stripped garbage,the seed sludge for ammonia production and themodel garbageweremixed at the ratioof 2:1, and the initial pH was adjusted to within the range of 7 to 8 using Ca(OH)2. Theammonia production occurred under the static condition at 55°C anaerobically. Aboutone-third of the sludge was drawn out and replenished with new model garbage whenapproximately 50% of the total nitrogen (TN) was converted to ammonia in the modelgarbage. This procedure was repeated at SRT 12 days for efficient conversion of TN toammonia.

Ammonia stripping from model garbage after ammonia production Theammonia stripping apparatus used in this study is shown in Fig. 1 (10). This apparatushad a working volume of 5 l (11 l of total volume) equipped with a band heatersurrounding the outer vessel to keep the sludge temperature at the desired level. Tostrip the ammonia from the sludge after ammonia production, the pH of the sludge was

TABLE 2. Characteristics of seed sludge and model garbage.

Parameter Unit Methanogenicsludge

Acclimatedmethanogenic

sludge

AcclimatedDWAS

Modelgarbage

TS % 22 22 9 22VS %TS 52 47 87 94pH – 7 8.4 6.7 5.9TN mg N/kg t.w.s. 6900 7000 11000 6300NH4

+ mg N/kg t.w.s. 140 1500 7000 20Total VFA mmol HAc/kg t.w.s. 0 0 630 18

t.w.s. means total wet sludge.DWAS means dehydrated waste activated sludge.

raised to approximately 11 using Ca(OH)2 and the reactor temperature was raised to85°C. The sludge was then agitated by rotation of the inner vessel and stirring device,whichwere both rotated at 16 rpm in opposite directions. Simultaneously, fresh air wasput into the headspace of the reactor continuously using an air pump at the flow rate of10-l/min. Free ammonia released into the air was trapped with 4N H2SO4.

Semi-continuous dry anaerobic digestion The apparatus for semi-continuousdry anaerobic digestion used in this study is concisely illustrated in Fig. 2 (10). Theamount of sludge in the fermenter was 5 kg (19 l of total volume). A water jacket wasequipped to keep the temperature at 55°C. The sludge was agitated by inner rotatinghorizontal blades that were rotated at 3 rpm. The pH was not controlled.

To prepare substrates for dry methane fermentation, ammonia production (SRT12 days) and ammonia stripping were carried out mentioned the above. TN conversionof garbage to ammonia used in the experiment was 47%, and ammonia removal was34% for TN. After the ammonia stripping, the pH of the sludge was adjusted to withinthe range of 7 to 8 using HCl, and the TS was adjusted to 20% using deionized water. Toinitiate the semi-continuous culture, the sludge was fed into 5 kg of acclimatedmethanogenic sludge after the ammonia stripping. The same procedure was carried outonce a day according to the predetermined SRT. The treated sludge in the reactor waswithdrawn every 2 to 3 days to keep the total sludge weight at 5 kg. The SRT waschanged from 80 days to 30 days according to the substrate loading rate.

Analyses TS and VSwere determined by the JSWA standardmethod (15). The pHof the sludge was determined using a suspension that was ten-fold diluted by deionizedwater and subsequently homogenized with a high-speed blender. The suspension wascentrifuged at 17,800 ×g for 10 min at 4°C, and the clear supernatant was used todetermine the concentrations ofVFAsand ammonia–nitrogen.VFAsweredeterminedbyaLC-2000 plus system (JEOL, Tokyo, Japan) equipped with an Aminex HPX-87H column(Bio-Rad, Tokyo, Japan) and the refractive index detector RI-2031 (JEOL) using 5 mMH2SO4 as a carrier solvent at 65°C. The total ammonia–nitrogen was determined using theAmmonia Test Wako kit (Wako, Osaka, Japan). TN was determined by the Kjeldahlmethod. The biogas production was determined by a wet gas meter. The CH4 and CO2

FIG. 2. Schematic drawing of apparatus for semi-continuous methanogenic fermenter.(1) Fermentation chamber, (2) stirring device, (3) outlet port, (4) water bath, (5) feedtank, (6) feed pump, (7) gas sampling port, (8) desulfurizer, (9) gas flow meter.

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314 YABU ET AL. J. BIOSCI. BIOENG.,

compositions were determined using the GC8A gas chromatograph (Shimadzu, Kyoto,Japan) equipped with an activated carbon column and a thermal conductivity detector(TCD) using argon gas as the carrier gas at a pressure of 245 kPa. The temperature of theTCD was set at 100°C. The temperature of the column oven was increased stepwise from40°C to 200°C from the beginning of the measurement.

DNA extraction Samples were collected from the repeated batch ammoniaproduction experiment and semi-continuous thermophilic dry anaerobic digestion.Bacterial and archaeal DNAwere extracted from the sludges using the Fast DNA SPIN Kitfor Soil (Qbiogene, CA, USA). The quality of the extracted DNA was checked by agarosegel electrophoresis. The concentration of extracted DNA was measured using the UV-1200 spectrophotometer (Shimadzu).

Construction of 16S rRNA gene clone library The 16S rRNA gene fragmentswere amplified by PCR with the primer set 341 F (5′-CCTACGGGAGGCAGCAG-3′)–907R(5′-CCGTCAATTCCTTTRAGTTT-3′) (annealing temperature [T*]=57°C) for bacteria (16)and 344 F (5′-ACGGGGYGCAGCAGGCGCGA-3′) - 915R (5′-GTGCTCCCCCGCCAATTCCT-3′)(T*=61°C) for Archaea (17). PCR amplification was performed with ExTaq (TAKARA,Shiga, Japan) according to the manufacturer's instructions. The PCR protocol was asfollows: 94°C for 5 min; 30 cycles at 94°C for 30 s, T* for 30 s, and 72°C for 1 min, 72°C for7 min, and then 4°C until further processing. The PCR products were purified using aQIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and cloned into pGEM-T EasyVector (Promega, WI, USA). The ligation products were used to transform Escherichia coliJM109 cells (TAKARA, Shiga, Japan). Colonies were assayed overnight on LB agar platescontaining Ampicillin (30 mg/l), X-gal (40 mg/l), and IPTG (24 mg/l) at 37°C. The whitecolonies were randomly picked and grown overnight on LB agar plates containingAmpicillin, X-gal, and IPTG at 37°C. Thewhite picked cloneswere checked for the length ofthe insertion fragment by colony PCR using T7 and SP6 primers. The colony PCR protocolwas as follows: 94°C for 5 min; 30 cycles at 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min;72°C for 7 min; and then 4°C until further processing. The colonies harboring the correctinsertion fragment were used for the analysis of the microbial community. The 16S rDNAfragments amplified by colony PCR were grouped according to their restriction patternswith RsaI and HaeIII enzymes (TOYOBO, Osaka, Japan). The 16S rRNA gene fragments ofeach group were extracted by the QIAquick PCR Purification Kit (QIAGEN).

Sequencing and phylogenic analysis The DNA sequencing reactions of 16SrRNA gene fragments were carried out by PCR with a BigDye Terminator v1.1 CycleSequencing Kit (ABI, CA, USA). The sequences were automatically analyzed on an ABIPRISM 310 Genetic Analyzer (ABI) and were thereafter corrected manually. Thesequences were compared with NCBI databases and DDBJ databases using the BLASTprogram. Operational taxonomic units (OTUs) were identified by BLAST analysis usingsearch results of at least 97% similarity.

RESULTS AND DISCUSSION

Ammonia production in acidogenesis of model garbage Toelucidate the ammonia production in model garbage, repeated batchculture was carried out at various SRTs (Fig. 3). The main VFA productwas n-butyrate regardless of the SRT (data not shown). The ammoniaproductionwas relatively constant from 2nd to 6th cycles of repeatingbatch, inwhich the average ammonia concentrationwas approximately2140–2400 mg N/kg total wet sludge (t.w.s.) in the range of 2 to 6 daysof SRT, indicating that 34–38%of TN in themodel garbagewas converted

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FIG. 3. Ammonia and VFA production in repeated batch culture at SRT 2, 4, and 6 daysusing model garbage. Symbols: gray bar, SRT 2 days; white bar, SRT 4 days; black bar,SRT 6 days.

to ammonia. The maximum ammonia ratio of TN was about 43% at 5thcycle of SRT 4 day. However, the ammonia production was decreasedafter 7th cycle at all SRTs.

In this experiment, the ammonia yield for TN was much lower thanthat of DWAS, in which typically 60% or more of the organic nitrogen(=TN–total ammonia–nitrogen) in DWAS was converted to ammoniawithin 5 days (10). The ratio of VFA in treated model garbage for VS inraw model garbage that reflected the degradation efficiency of VS was3.12 mmol HAc/g-VS while that in DWAS was 4.52 mmol HAc/g-VS,suggesting that the VS degradation efficiency of model garbage lowerthan that of DWAS during ammonia production processmight decreaseammonia yield. In the culture with DWAS, the pHwas stable because ofthe balanced production of VFAs and ammonia. On the other hand, thepH rapidly dropped below 6 in the early stage of every repeated batchculture in the model garbage (data not shown). The model garbagecontained more carbohydrates than protein. The carbohydrates wererapidly degraded to produce more organic acids than ammonia fromprotein in the early stage of the culture, resulting in the rapid drop of pH.The decrease of pH that gave negative effect formicrobial activitymightdecrease VS degradation efficiency and ammonia yield for TN after 7thcycle of repeated batch.

In this experiment, the ammonia concentration in garbage wastewas lower than 3000 mg N/kg t.w.s., a level at which a little inhibition ofmethane production was observed. However, if direct anaerobicdigestion of the model garbage was carried out, the ammoniaconcentration should exceed the 3000 mg N/kg t.w.s. level because itwas reported that the VS degradation of the model garbage was 76% to78% in anaerobic digestion (14) while less VS degradation was observedvisually in this experiment. Thus, it was speculated that ammonia re-moval from ammonia-producing model garbage accelerated the meth-ane yield.

Ammonia stripping from model garbage that had accumulatedammonia The ammonia stripping was carried out under highalkaline and temperature conditions since the equilibriumof ammoniamoved from ammonium ions to free ammonia in water under theseconditions, resulting in increased efficiency of ammonia stripping(18). A time course of ammonia removal from the model garbage thataccumulated ammonia is shown in Fig. 4. The ammonia concentration

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FIG. 4. Typical time course of ammonia stripping from model garbage containingammonia. Symbols: open square, pH; closed square, ammonia concentration; opencircle, lactate; closed circle, acetate; and open triangle, butyrate.

DRY ANAEROBIC DIGESTION OF MODEL GARBAGE 315VOL. 111, 2011

and the pH showed logarithmic decreases during the ammonia-stripping process (Fig. 4A and B). The rapid drop of pH appeared to becaused by the decrease of ammonia from the model garbage. Duringthis process, approximately 80% of the ammonia in the ammonia-accumulated model garbage was removed. The concentration of VFAswas maintained during this experiment (Fig. 4C), indicating that theammonia stripping did not cause the substrate for anaerobic digestionto be lost.

Semi-continuous thermophilic dry anaerobic digestion Ther-mophilic dry anaerobic digestion using the ammonia-stripped modelgarbage was performed semi-continuously using the laboratory-scalereactor shown in Fig. 2. The methanogenic seed sludge was adapted tothe ammonia-strippedmodel garbage for 160 days at SRTs ranging from80 to 60 days before this experiment (data not shown). A performanceprofile over the 180 days of dry anaerobic digestion is shown in Fig. 5.During the operation, the pH was not controlled but was stable atapproximately 8 (Fig. 5D). The ammonia concentration decreased withshortening SRT and remained at less than 2000 mg N/kg t.w.s. (Fig. 5E).Although the gas production rate increased with the VS loading rate(Fig. 5B), a slight decrease was observed in the middle of a 40-day SRT.Since it was reported that the addition of mineral nutrients wasdesirable for the efficient anaerobic digestion of garbage (14,19),mineral nutrients (FeCl2, NiCl2, and CoCl2) were added at 10 mg/kg t.w.s. after 125 days (Fig. 5B arrow). After the addition of mineral nutrients,

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FIG. 5. Time course of semi-continuous dry anaerobic digestion using ammonia-stripped modsquare, gas yield; open diamond, pH; closed diamond, ammonia concentration; open triangle,

the gas production rate was recovered and the accumulation of VFAsdisappeared (Fig. 5F).

The accumulation of VFAs, mainly propionate, increased 50 mmolHAc/kg t.w.s. when the SRT was changed to 30 days, but the con-centration of VFAs decreased after subsequent operation. The averagebiogas yields at each SRT were in the range of 0.68 to 0.8 Nm3/kg VS(Fig. 5C). The percentages of CH4 and CO2 in the biogas were stable,and H2 was not determined during the operation. The average CH4

and CO2 contents in the biogas were 60.9% and 39.1%, respectively.The biogas yield and composition were in agreement with previousreports on thermophilic anaerobic digestion using model garbage(9,14). The average biogas yield produced by the two-stage anaerobicdigestion method using ammonia-stripped DWAS was in the range of0.5 to 0.6 Nm3/kg VS (10). Therefore, garbage was a more efficientsubstrate for biogas production than DWAS.

The behavior of VS and nitrogen in the process To determinethe behavior of VS and nitrogen in model garbage in each componentprocess of two-step thermophilic dry anaerobic digestion, calculationwas performed based on the results for 137 to 150 days at an SRT of40 days in this operation. During the ammonia production, 21% of VSin the model garbage decreased (Table 3). The majority of thedegraded VS was converted to VFAs and biogas composed of CO2 andH2. H2 made up approximately 50% of the biogas. During the ammoniastripping, there was no VS removed. During the anaerobic digestion,

90 120 150 180

ime (d)

40d 30d

el garbage. Symbols: solid line, VS loading rate; open square, gas production rate; closedtotal VFAs; closed triangle, propionate; arrow, starting point of mineral nutrient addition.

TABLE 3. The behavior of VS and nitrogen in garbage at each process.

Unit Initial garbage Garbage afterammonia production

Garbage afterammonia stripping

Garbage aftermethane production

TN mg N/kg w.w. 6300 (100) 6300 (100) 3920 (62.2) 3920 (62.2)NH4

+ mg N/kg w.w. 12.5 (0.2) 3090 (49.0) 712 (11.3) 1320 (21.0)VS g VS/kg w.w. 202 (100) 160 (79.2) 156 (77.2) 47.7 (23.6)

Parentheses in table represent percentage for initial TN or VS in model garbage.

316 YABU ET AL. J. BIOSCI. BIOENG.,

69% of VS was removed. The majority of the degraded VS wasconverted to CH4 and CO2 since there was little accumulation of VFAs.The average CH4 and CO2 contents were 60% and 40%, respectively.This means that 76% of the VS was removed during the whole process.

Regarding nitrogen balance, during the ammonia production, theamount of ammonia in the model garbage increased to 49% of TNbecause of treatment time extended to 12days of SRT (Table 3). Duringthe ammonia stripping, 77% of the ammonia in themodel garbagewasremoved as ammonia gas, which corresponded to 38% of TN in themodel garbage. During the methane fermentation, the amount ofammonia increased to 21% from 11% for initial TN the model garbage.This indicated that 59% of TN was converted to ammonia during thewhole process. Since the decreased pH in the ammonia productionprocess reduced ammonia conversion, it is expected that the improve-ment of this process increased the ammonia removal efficiency.

Because the amount of ammonia released from garbage reached3090 mg N/kg w.w. after ammonia production at 12 days of SRT in thisexperiment, the concentration of free ammonia was calculated to be810 mg N/kg t.w.s. using the Østergaard formula (8) under theexperimental conditions (55°C and pH 8). This value was higherthan 700 mg N/l that was the threshold level for the inhibition ofmethane production by free ammonia (7). Furthermore, assuming thatthe 59% of TN in model garbage (6300 mg N/kg t.w.s) was converted toammonia–nitrogen in the case without ammonia stripping as same aswith ammonia stripping, although culture conditions such as SRT, pHprofile, and possibly microbial community were different, it was cal-culated that the concentration of ammonia reached 3700 mg N/kg t.w.s.in themethane fermentation reactor, which resulted in 1030 mg N/kgt.w.s. of free ammonia. These calculations suggest that the ammoniaproduction and the following ammonia stripping were effective foravoiding the inhibition causedbyammonia indrymethane fermentationusing model garbage.

Bacterial community structure during ammonia produc-tion Because the ammonia production in the ammonia productionstep was the important process for effective removal of ammonia in theammonia striping step, the bacterial community was determined toknow efficient condition for stable ammonia production frommicrobialproperty. Clone libraries of the bacterial 16S rRNAgenewere constructedfrom sludge with high ammonia productivity (HAP), which was taken

TABLE 4. Distribution of 16S rRNA gene clones de

Putative division No. of clones (No. of OTUs) Closest

HAP LAP

Firmicutes, Clostridia 46 (1) 1 (1) Garciella s19 (2) 22 (2) Sporanaer10 (1) 3 (2) Clostridium4 (2) 6 (2) Clostridium

10 (2) Clostridium14 (2) Clostridium1 (1) Clostridium

Firmicutes, Bacilli 3 (1) 5 (2) Bacillus thUnknown 2 (2)Total 84 (9) 62 (14)

HAP means high ammonia productivity (Run 5 of SRT 4 days).LAP means low ammonia productivity (Run 10 of SRT 2 day).*Means belonging to the Clostridium Cluster XII.

from the culture in 5th cycle of repeated batch culture at SRT 4 daysshown in Fig. 3 and low ammonia productivity (LAP) from that in 10thcycle of repeatedbatchof SRT2 days. Ammoniaproduced for TNwas46%in HAP and 24% in LAP. The distribution of 16S rRNA gene clones inammonia production is shown in Table 4. In HAP, 84 clones wereobtained and belonged to 9 OTUs. In LAP, the 62 clones tested belongedto 14OTUs. InHAP, themajorOTUwas related toGarciella sp. EMZY-1. InLAP, the major OTU was related to Sporanaerobacter acetigenes. Thedominant phylum of ammonia production was Firmicutes.

In HAP, most of the clones affiliated with Firmicutes fell into theClostridium cluster XII (69 clones, 82% of total clones in the library) (20).It was reported that the bacteria belonging to the Clostridium cluster XIIcarried out protein degradation in BSA-fed chemostat cultivation (21).Among them, however, although a relative of Garciella sp., EMZY-1 wasdetected as the most abundant population, protein degradability of thisgenus was not reported so far. Since Sporanaerobacter acetigenes andClostridium sporogenes have the ability to degrade protein and aminoacid (22,23), relatives of these bacteria might also function in ammoniaproduction. In LAP, The ratio of Clostridium cluster XII in LAP librarydecreased to 63% (39 clones) from 82% in HAP library. The OTUs relatedto S. acetigenes,C. cochlearium, and C. ultunensewere frequently detectedinstead of the OTU related to Garciella sp. In the ammonia productionusingmodel garbage, the pH in the early stage of repeated batch culturerapidly decreased below 6. Since C. ultunense can grow at an initial pHvalue of 5.0 (24), the decrease of pH might induce a shift to bacteriacommunity, which have the ability to grow in low pH conditions such asC. ultunense. Thus, this difference in microbial communities mightinfluence stable ammonia production. Therefore, the pH control duringrepeated batch culture seems to be important for stable ammoniaproduction using model garbage.

Bacterial community structures during dry anaerobic diges-tion Anaerobic digestion of organic matter consists of hydrolysis,acidogenesis, acetogenesis, and methanogenesis phases (25). Becausethese phases were carried out different groups of microorganism,understanding the microorganism community structures involved inanaerobic digestion is essential to the control of bioreactors. In order toinvestigate microorganism community involved in thermophilic dryanaerobic digestion using the ammonia-stripped model garbage, thebacterial and archaeal communities were determined. Clone libraries of

tected in sludge during ammonia production.

microorganism Accession no. Sequence identity (%)

p. EMZY-1 * EU275367 92obacter acetigenes * GQ461827 99sporogenes NR_029231 100filamentosum * X77847 97ultunense * GQ487664 96cochlearium M59093 99sp. H2-13 FM865925 97

ermoamylovorans GU125643 100

TABLE 5. Distribution of bacterial 16S rRNA gene clones detected in methane fermentor.

Putative division No. of clones (No. of OTUs) Closest microorganism Accession no. Sequence identity (%)

23 days 150 days 186 days

Thermotogae, Thermotogales 29 (1) 2 (1) 1 (1) Uncultured Thermotogae bacterium clone QEEA1CH09 CU918433 100Synergistetes, Synergistia 5 (2) 6 (2) 10 (2) Anaerobaculum mobile NR_028903 100Firmicutes, Clostridia 3 (2) 7 (1) 2 (1) Thermacetogenium sp. Sp3 EU386162 98

1 (1) 2 (1) Coprothermobacter proteolyticus CP001145 1005 (2) 5 (3) Clostridium caenicola AB221372 984 (2) 1 (1) Tepidanaerobacter syntrophicus AB106354 992 (1) 1 (1) Syntrophomonas wolfei DQ666176 97

1 (1) Clostridium sufflavum AB267266 964 (1) Clostridium populeti X71853 96

1 (1) Clostridium thermopalmarium AF286862 100Firmicutes, Bacilli 1 (1) Leuconostoc mesenteroides GU138560 99

1 (1) Desemzia incerta GQ497930 99Bacteroidetes, Bacteroidia 2 (1) 2 (1) Bacteroidales bacterium 28bM GU129116 96Actinobacteria, Actinobacteridae 1 (1) Mycobacterium elephantis GU142939 99Unknown 7 (5) 14 (11)Total 40 (8) 40 (17) 40 (24)

DRY ANAEROBIC DIGESTION OF MODEL GARBAGE 317VOL. 111, 2011

bacterial 16S rRNA genes were constructed from the sludges in themethanogenic fermenter on days 23, 150, and 186. The number ofanalysis clones was 40 in each sample. The distribution of bacterial 16SrRNA gene clones in dry anaerobic digestion is shown in Table 5.

On day 23, the detected OTUs were affiliated with Thermotogae,Synergistetes, and Firmicutes. Five OTUs belonged to the phylumFirmicutes (6 clones, 15% of total clones in the library), and 4 of thesebelonged to the class Clostridia and other to the class Bacilli. Themostdominant phylum was Thermotogae (29 clones, 72.5% of total clonesin the library). It was known that Thermotogae produced H2 viafermenting a variety of organic compounds (26). Thermophilic,proteolytic bacteria such as relatives of Anaerobaculum mobile andCoprothermobacter proteolyticus (27,28) and cellulolytic bacteriasuch as relatives of Clostridium populeti and Clostridium sufflavum(29,30) were also detected.

The kinds of OTUs detected on day 150 were similar to thosedetected on day 186. On day 150, the detected OTUs, other than theunknowns, were affiliated with Thermotogae, Synergistetes, Firmicutes,and Bacteroidetes. The most dominant phylum was Firmicutes (23clones, 57.5% of total clones in the library), to which 8 OTUs belonged.On day 186, the detected OTUs, other than the unknowns, were affili-ated with Thermotogae, Synergistetes, Firmicutes, Bacteroidetes, andActinobacteria. Themost abundant OTUwas affiliatedwith Synergistetes,while themost dominant phylumwas Firmicutes (12 clones, 30% of totalclones in the library), which was observed in 8 OTUs which belonged tothe class Clostridia. The phylum Firmicutes was widely distributedthroughout various anaerobic digestion reactors (21,31–33). The domi-nance of this organism is likely due to the ability to degrade a broadspectrum of substrates.

The major OTU on day 150 was affiliated with the genusThermoacetogenium (7 clones, 17.5% of total clones in the library),which is known to include thermophilic, syntrophic, acetate-oxidizingbacteria (34). Thermoacetogenium oxidized acetate in co-culture withthermophilic hydrogenotrophic methanogen (35). Other syntrophicbacteria that were also detected included relatives of Syntrophomonaswolfei and Tepidanaerobacter syntrophicus (36,37). It is known that

TABLE 6. Distribution of archaeal 16S rRNA gen

Putative division No. of clones (No. of OTUs)

23 days 150 days 186 days

Euryarchaeota, Methanobacteria 38 (1) 38 (1) 31 (2) MethEuryarchaeota, Methanomicrobia 1 (1) Meth

1 (1) Uncu1 (1) 9 (2) Meth

Total 40 (3) 39 (2) 40 (4)

syntrophic bacteria degrade long-chain fatty acids, alcohol, andaromatic fatty acids. The conversion of these substrates through a β-oxidation reaction by syntrophic bacteria involved the cooperation ofmethanogens (25). This cooperationwas important for themethanogeniccommunities because an excess of these substrates inhibited methaneproduction (38).

It should be noted that the number of detected OTUs increasedfrom 17 on day 150 to 24 on day 186, although the number of kindsdetectedwas similar in both samples. This suggested that the bacterialcommunities became complicated in order to be suitable for high VSloading rate.

Archaeal community structures during dry anaerobic diges-tion Clone libraries of archaeal 16S rRNA genes were also constructedfrom the same sludgesduring dry anaerobic digestion. The distributionofclones is shown in Table 6. The numbers of analyzed clones were 40, 39,and 40 on days 23, 150, and 186, respectively. The detected OTUs wereaffiliated with the genera Methanothermobacter and Methanosarcina ineach sample.

The most dominant archaea was the genusMethanothermobacter,which is known as hydrogenotrophic methanogen (39). However,the ratio of Methanothermobacter decreased during the operation.This decrease seemed to influence the bacterial communitiesgreatly, by inducing, for example, the decrease of Thermotogae andThermoacetogenium, as these bacteria degraded substrate syntrophicallywith hydrogenotrophic methanogen (34,35,40). On the other hand, theratio of Methanosarcina was increased from 2 clones (5% of total clonesin the library) to 9 clones (22.5% of total clones in the library) duringthe operation. Methanosarcina is known as an acetoclastic methanogen(41). It was reported that the archaeal community was affected by theVFA concentration (42,43), it seems that the increase of acetoclasticmethanogen as Methanosarcina was caused by butyrate, which wascontained in ammonia-stripped model garbage, and propionate, whichwas accumulated during days 164 to 177.

In this study, the VFA that was mainly produced in the ammoniaproduction phase was butyrate. The degradation of butyrate consists ofthree different processes (25). The first process is butyrate conversion

e clones detected in methane fermentor.

Closest microorganism Accession no. Sequence identity (%)

anothermobacter thermautotrophicus EF100758 96anosarcina barkeri AF028692 99ltured Methanosarcina sp. KT19 AM418687 99anosarcina thermophila M59140 99

318 YABU ET AL. J. BIOSCI. BIOENG.,

to acetate and hydrogen catalyzed by syntrophic bacteria. The sub-sequent processes catalyzed by methanogen are CO2 reduction to CH4

and acetate cleavage to CH4 and CO2. Therefore, the cooperation ofhydrogenotrophic methanogen and acetoclastic methanogen plays asignificant in butyrate degradation (44). It was reported that theaccumulation of acetate inhibited butyrate degradation (45), suggestingthat acetoclastic methanogen plays an important role in the butyratedegradation process. The degradation scheme of propionatewas similarto butyrate (25). In experiment with propionate-fed chemostats, thecooperation of acetoclastic and hydrogenotrophic methanogen wasimportant for propionate degradation (46).

This clearly indicated that dry anaerobic digestion using modelgarbage proceeded stably, because the utilization of not only hydrogenbut also VFAs is important for the stable degradation of model garbage.This can be accomplished through the cooperation of the bacteriaand archaea communities. Moreover, it seems that the increase ofaceticlastic methanogen was induced by decrease of ammonia con-centration in this study, because in general, the inhibitory effect wasstronger for aceticlastic than for hydrogenotrophic methanogen(43,47,48). This increase of aceticlastic methanogen may be character-istics of archaeal community using ammonia-strippedmodel garbage. Itwas reported that the aceticlastic methanogen predominated inmesophilic BSA-fed reactor (21) and mesophilc UASB reactor treatingvarious substrate (49). On the other hand, the hydrogenotrophicmethanogenpredominated in thermophilic digester for kitchen garbage(50) and thermophilic upflow anaerobic filter reactor treating Awamoridistillery wastewater (31). As mentioned above, the archaeal commu-nities seem to correspond with the reactor conditions, such as organicloading rate, difference of substrate, temperature, and cooperation ofbacterial communities.

In conclusion, this study demonstrated that thermophilic dryanaerobic digestion including ammonia stripping was an effectivetreatment for model garbage as well as DWAS (10). DWAS, garbage,and swine manure account for more than 80% of the total emissions ofbiological organic waste in Japan (51). The process described in thepresent report would not only improve the process of dry anaerobicdigestion but would also make the recycling of ammonia more readilyavailable, leading to more efficient use of biological organic waste.

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