Bioresource Technology 102 (2011) 779–785

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Bioresource Technology

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Robustness of archaeal populations in anaerobic co-digestion of dairyand poultry wastes

Yan Zhang a, Esteban M. Zamudio Cañas a, Zhenwei Zhu a, Jessica L. Linville a, Si Chen a, Qiang He a,b,⇑a Department of Civil and Environmental Engineering, The University of Tennessee, Knoxville, TN 37996, USAb Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN 37996, USA

a r t i c l e i n f o

Article history:Received 21 June 2010Received in revised form 26 August 2010Accepted 27 August 2010Available online 21 September 2010

Keywords:CrenarchaeotaArchaeaAnimal wasteAnaerobic digestionMethane

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.08.104

⇑ Corresponding author at: Department of Civil andThe University of Tennessee, Knoxville, TN 37996, USA+1 865 974 2669.

E-mail address: qianghe@utk.edu (Q. He).

a b s t r a c t

The objective of this study is to investigate the responses of methanogen populations to poultry wasteaddition by comparing the archaeal microbial populations in continuous anaerobic digesters with orwithout the addition of poultry waste as a co-substrate. Poultry waste was characterized as anorganic/nitrogen-rich substrate for anaerobic digestion. Supplementing dilute dairy waste with poultrywaste for anaerobic co-digestion to increase organic loading rate by 50% resulted in improved biogas pro-duction. Elevated ammonia derived from poultry waste did not lead to process inhibition at the organicloadings tested, demonstrating the feasibility of the anaerobic co-digestion of dairy and poultry wastesfor improved treatment efficiency. The stability of the anaerobic co-digestion process was linked to therobust archaeal microbial community, which remained mostly unchanged in community structure fol-lowing increases in organic loading and ammonia levels. Surprisingly, Crenarchaeota archaeal popula-tions, instead of the Euryarchaeota methanogens, dominated the archaeal communities in theanaerobic digesters. The ecological functions of these abundant non-methanogen archaeal populationsin anaerobic digestion remain to be identified.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Animal waste generated from large-scale livestock operationsposes a major challenge to sustainable development, as the naturaldecomposition of animal waste releases large quantities of patho-gens, excess nutrients, organic matter, solids, methane, ammonia,and odorants to the environment (Jongbloed and Lenis, 1998;Sharpe and Harper, 1999; Topp et al., 2009). Anaerobic digestionprocesses, capable of both waste stabilization and the productionof biogas as a renewable fuel, have been established as an alterna-tive treatment technology for animal waste management (Bekker-ing et al., 2010; Chen and Cheng, 2005; Sakar et al., 2009).

However, the application of anaerobic digestion remains eco-nomically unattractive for the treatment of dilute dairy waste,which is characterized by low solids content (typically less than3%) and subsequently low biogas yield (Garrison and Richard,2005). In contrast, poultry waste has a much higher solids content(>50%) than other livestock wastes (Kelleher et al., 2002). Thus, theaddition of poultry waste as a co-substrate in the anaerobic diges-tion of dairy waste could potentially overcome challenges pre-

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sented by the low solids content and low biogas yield of dilutedairy manure. While the use of poultry waste as an organic-richco-substrate in anaerobic digestion has been tested for a numberof waste streams such as organic solid wastes and concentratedanimal manures (Güngör-Demirci and Demirer, 2004; Macias-Cor-ral et al., 2008; Mata-Alvarez et al., 2000; Misi and Forster, 2001),no studies in so far as we know have investigated the effectivenessof improving anaerobic digestion performance by supplementingdilute dairy waste with poultry waste.

The efficient utilization of the organic-rich poultry waste addedin anaerobic co-digestion, however, relies on the robustness of thecomplex anaerobic food web involved in anaerobic digestion, withthe main populations consisting of primary fermenters, secondaryfermenters, and methanogens (Ahring, 2003; Schink, 1997). Themethanogens, a group of microorganisms associated exclusivelywith Archaea, are responsible for methanogenesis, the terminalstep of methane formation from CO2/H2 or acetate in the anaerobicdecomposition of organic wastes (Madigan and Martinko, 2006).Methanogenesis is often considered as the limiting step in anaero-bic digestion processes under perturbation, due to both the slowgrowth rates and susceptibility to inhibitory substances character-istic of methanogens (Chen et al., 2008; Kotzé et al., 1969). Onesuch inhibitor is ammonia. Indeed, in anaerobic digestion,methanogens are among the microbial populations most sensitiveto ammonia, which could accumulate to levels toxic to methano-

780 Y. Zhang et al. / Bioresource Technology 102 (2011) 779–785

gens during the anaerobic conversion of nitrogen-rich substrates,leading to the blockage of methane formation and subsequentinstability of the entire anaerobic digestion process (Batstoneet al., 2002; Hansen et al., 1998; Kayhanian, 1994). Thus, the re-sponses of methanogen populations are useful indicators of theperformance of anaerobic digestion processes under theseconditions.

Since poultry waste is rich in organic nitrogen, the addition ofpoultry waste as a co-substrate may produce high levels of ammo-nia in anaerobic conversion, resulting in potential inhibition of theanaerobic digestion process (Kelleher et al., 2002; Krylova et al.,1997). Given the susceptibility of methanogens to such inhibitoryeffects, the responses of the methanogen population could be usedto evaluate the impact of poultry waste on the performance ofanaerobic co-digestion processes. Therefore, to assess the feasibil-ity of poultry waste as a supplemental substrate for the anaerobicdigestion of diary waste, the objective of this study is to investigatethe responses of methanogen populations to poultry waste addi-tion by comparing the archaeal microbial populations in continu-ous anaerobic digesters with or without the addition of poultrywaste as a co-substrate.

2. Methods

2.1. Substrates

Dilute dairy waste was collected from the waste storage basinof a 600-head dairy farm located in Loudon County, Tennessee,USA. The dairy waste was a mixture of raw manure and wastewa-ter discharges from cleaning, cooling and milking operations. Poul-try waste was collected from a broiler farm located in GreeneCounty, Tennessee, USA. The poultry waste was a mixture consist-ing of chicken feces, kiln dried wood shavings, spilled feed, andfeathers. All wastes were collected during the summer in a periodof 1 week to minimize large variations in substrate characteristics.Both waste substrates were stored at 4 �C in closed container be-fore use. To prevent clogging of tubings, the dairy waste slurryand the poultry waste were prepared by homogenization using ablender and subsequently passing through a 2-mm mesh sieve toremove large debris.

2.2. Anaerobic digester setup

Two sets of triplicate mesophilic continuous anaerobic digesterswere established prior to this study as described previously(Hashsham et al., 2000). All six digesters had a working volumeof 3.6 L and were operated in a constant-temperature room with

300

600

900

1200

1500

-40 -20 0 20 40 60Days

CH

4 Pro

du

ctio

n, m

l/day

Control Digesters

Co-Digesters

1st

2nd

Fig. 1. Methane production in the anaerobic co-digesters and control digesters.Arrows indicate the 1st and 2nd increases in organic loading rate in the co-digestersresulting from the addition of poultry waste. Data are means of triplicate digesters,with the error bars indicating the standard deviation.

the temperature controlled at 35 �C. The digesters were fed at4-h intervals and the hydraulic retention time was maintained at20 days throughout the study period. These digesters were initi-ated with inoculum from an operating laboratory dairy manureanaerobic digester and established using dilute diary waste asthe only feed. All digesters exhibited stable operation prior to theaddition of poultry waste with consistent pH, methane yield, andvolatile fatty acids (VFA) level (Fig. 1). The organic loading rate(OLR) was maintained at 1.0 g volatile solids (VS)/L/day in the trip-licate control digesters with no addition of poultry waste through-out this study. In contrast, the OLR was raised to 1.3 g VS/L/day inanother set of triplicate digesters (co-digesters) by the addition ofpoultry waste to the feed. The OLR in the co-digesters were raisedfurther to 1.5 g VS/L/day 35 days after the first OLR increase byadding more poultry waste to the feed. The feeding rate of dairywaste remained unchanged in all digesters studied.

2.3. Chemical analysis

Biogas production from the anaerobic digesters was determinedusing a water-displacement method described previously (Demirerand Speece, 1998). Methane content in biogas was analyzed usinga Hewlett Packard 5890 Series II gas chromatograph equipped witha thermal conductivity detector (TCD) and a Supelco packing col-umn (60/80 Carbonxen�-1000). Argon was used as the carrier gaswith a flow rate of 5 ml/min and the following temperaturescheme: oven 125 �C, injection port 150 �C, and detector 170 �C.Volatile fatty acids (VFA) in the aqueous phase were quantifiedusing a Hewlett Packard 5890 gas chromatograph equipped witha flame ionization detector (FID) and a Restek Stabilwax�-DA col-umn. Nitrogen was the carrier gas at 2.4 ml/min with a split ratioof 1:11. The following temperature scheme was used for VFA quan-tification: oven 110 �C, injection port 250 �C, and detector 250 �C.Chemical oxygen demand (COD), total alkalinity (TA), total solids(TS), volatile solids (VS), total Kjeldahl nitrogen (TKN), and totalammonia–nitrogen (TAN) were all determined according to stan-dard methods (APHA/AWWA/WEF, 2005): COD was measuredwith the ‘‘5220C” Close Reflux-Titrimetric Method; TA was quanti-fied using the ‘‘2320B” titration method with pH 4.5 as the endpoint; TS and VS were measured using the ‘‘2540 B&E” method,TKN was determined with the ‘‘4500-Norg C” Semi-Micro-Kjeldahlmethod; and TAN was quantified using the ‘‘4500-NH3 D” methodwith an Orion 9512 ammonia ion selective electrode (Orion Re-search Inc., Massachusetts, USA). Free ammonia was calculatedfrom TAN as a function of pH and temperature using a previouslydescribed formula (Hansen et al., 1998).

2.4. Clone library analysis of archaeal microbial populations

For molecular microbial community analysis, sludge sampleswere taken from the control digesters and co-digesters 56 days fol-lowing the addition of poultry waste to the feed. Samples werestored at �80 �C until analysis. Whole community DNA was ex-tracted and purified using a previously described method (Zhanget al., 2009). Archaeal 16S rRNA genes were subsequently amplifiedby polymerase chain reaction (PCR) using the Archaea-specificprimers Arch21F (50-TTCCGGTTGATCCYGCCGGA-30) and Arch958R(50-YCCGGCGTTGAMTCCAATT-30) (Delong, 1992). The PCR reactionmixture contained 0.4 lM of each primer, 200 lM dNTP, 2.5 U ExTaq DNA polymerase, the PCR buffer mix provided by the supplierof the Taq DNA polymerase (Takara, Madison, Wisconsin, USA), and10 ng DNA template. PCR was performed with the following ther-mal cycling program: 94 �C for 5 min; 25 cycles at 94 �C for 1 min,54 �C for 1 min, and 72 �C for 2 min; and a final extension at 72 �Cfor 6 min.

60

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100

N, m

g/L

Control Digesters

Co-Digesters

1st

2nd

Y. Zhang et al. / Bioresource Technology 102 (2011) 779–785 781

The amplified products were purified using the Qiagen PCRpurification kit (Qiagen, Valencia, California, USA) and cloned intothe pGEM-T Easy vector (Promega, Madison, Wisconsin, USA) fol-lowing the manufacturer’s instructions. Positive recombinantswere grouped by restriction fragment length polymorphism (RFLP)analysis to form operational taxonomic units (OTUs) using therestriction enzyme RsaI (Promega, Madison, WI, USA). After restric-tion digestion at 37 �C for 4 h, the banding patterns were comparedby electrophoresis and clones showing the same RFLP pattern weregrouped into a single OTU. Sequences of representative clones foreach OTU were subsequently obtained using M13 forward and re-verse primers.

The obtained sequences with chimeric artifacts were removedby the program Chimera Check at the Ribosomal Database ProjectII (Cole et al., 2003). The surviving 16S rRNA gene sequences weresearched for homology using the BLAST program at the NationalCenter for Biotechnology Information (NCBI), aligned with homol-ogous sequences using ClustalX (Thompson et al., 1997), and usedfor the construction of phylogenetic trees by the neighbor-joiningalgorithm (1000 bootstrap re-samplings) with Mega 4.0 (Tamuraet al., 2007). The chimera-checked 16S rRNA gene sequencesreported in this paper were deposited at GenBank under thefollowing Accession Nos: GU475150–GU475170, GU475172–GU475182, and GU475186–GU475192.

To compare the archaeal community structures of the anaerobicco-digesters and control digesters, the Shannon diversity index (H0)and evenness index (E) were calculated for each sample based onthe RFLP patterns of the clone libraries as previously described(Hill et al., 2003):

H0 ¼ �Xðpi ln piÞ;

pi ¼ni

N;

and

E ¼ H0

ln S;

where ni is the number of clones in the ith OTU, N is the total num-ber of clones in the library, and S is the number of OTUs in thelibrary.

3. Results and discussion

3.1. Substrate characterization

Poultry waste contained 55.7% of total solids, which was morethan 24 times the 2.3% solids content of dairy waste (Table 1).Since the volatile fraction of the solids content in poultry wastewas slightly higher than dairy waste, poultry waste evidently alsohad a much higher total VS content (33.8%) than that of dairy waste(1.3%), suggesting the significantly higher biogas potential of poul-try waste as compared to dairy waste.

Poultry waste also contained more nitrogen than that in dairywaste (Table 1). Since most of the nitrogen in anaerobic digestion

Table 1Characteristics of dairy and poultry wastes.

Parameter Dairy waste Poultry waste

Total solids (TS), % wet mass 2.3 55.7Volatile solids (VS), % TS 57.6 60.6Total COD (mg COD/g VS) 1460 389Total ammonia (mg N/g VS) 18.4 14.1Total Kjeldahl nitrogen (mg N/g VS) 56.1 70.7Total alkalinity (mg CaCO3/g VS) 270 83.0

is converted to ammonia nitrogen, which is toxic to microbialmetabolism (Chen et al., 2008), using poultry waste as a substratein anaerobic digestion could lead to a higher risk of process inhibi-tion. In contrast, dairy waste had high levels of total alkalinity,which would provide significant buffering capacity, a benefitimportant for maintaining process stability (Montusiewicz, 2008).

Therefore, the benefit of improved biogas yields with the addi-tion of poultry waste as an organic-rich co-substrate could be com-promised by the potential inhibitory effect of elevated ammonialevels resulting from the high nitrogen content of poultry waste.Thus, the impact of poultry waste addition on the anaerobic diges-tion of dairy waste was further evaluated in continuous digesters.

3.2. Performance of anaerobic co-digestion of dairy and poultry wastes

The impact of poultry waste on anaerobic digestion perfor-mance was evaluated in replicate continuous bench-scale anaero-bic co-digesters established with diary and poultry wastes aswell as control digesters with dairy waste as the sole substrate.

3.2.1. Methane productionAnaerobic co-digestion was initiated by the addition of poultry

waste after the establishment of stable methane production(833 ± 39 ml/day) in digesters fed with dairy manure as the onlysubstrate. Significant perturbation in methane production was ob-served immediately following each OLR increase: methane produc-tion first exhibited a sharp spike followed by a gradual decreasingtrend and stable methane production was again reached within20 days of operation Fig. 1.

When the OLR was first increased by 30% from 1.0 to 1.3 g VS/L/day in the anaerobic co-digesters, methane production stabilized at1079 ± 32 ml/day, representing a 29.5% increase. Subsequently,OLR was raised again from 1.3 to 1.5 g VS/L/day, representing a15.4% increase. Following this second OLR increase, stable methaneproduction was achieved again at 1243 ± 33 ml/day, correspondingto a 15.2% increase. Thus, the increase in methane production wasproportional to the increase in OLR. In contrast, methane produc-tion remained unchanged in the control digesters with no poultrywaste addition throughout the experimental period (Fig. 1). Over-all, a 50% increase in OLR by the addition of poultry waste resultedin a 49.2% increase in methane production. The proportional in-creases in methane production in response to the OLR increasessuggest that the microbial populations had successfully adaptedto the higher OLR exerted on the anaerobic co-digesters.

0

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NH

3-

Fig. 2. Free ammonia nitrogen (NH3–N) concentration in the control digesters andco-digesters. Arrows indicate the 1st and 2nd increases in organic loading rate inthe co-digesters resulting from the addition of poultry waste. Data are means oftriplicate digesters, with the error bars indicating the standard deviation.

782 Y. Zhang et al. / Bioresource Technology 102 (2011) 779–785

3.2.2. Ammonia nitrogen accumulationSince poultry waste was rich in not only organic matter but also

nitrogen, accumulation of free ammonia nitrogen (NH3–N), whichis toxic to anaerobic digestion at high concentrations (Kayhanian,1994), was monitored in the anaerobic digesters following poultrywaste addition as a co-substrate. The NH3–N level in the controldigesters averaged 34.5 ± 2.9 mg/L throughout the experimentalperiod. In contrast, the NH3–N concentration in anaerobic co-digesters rose significantly following the addition of poultry waste(Fig. 2). After the first increase in OLR, NH3–N levels in the anaer-obic co-digesters increased steadily, peaking at 54.6 mg/L. TheNH3–N level increased further following the second increase inOLR, reaching 74.2 mg/L, which was more than twice that of thecontrol digesters. Accordingly, the pH in the anaerobic digestersrose above 7.7 (data not shown), likely a result of the accumulation

C21

R8

C6

R65

99

99

9498

99

97

99

0.02

Fig. 3. Neighbor-joining phylogenetic tree showing the relationships of partial archaealThe numerical values at branch nodes indicate bootstrap values per 1000 re-samplings. Sethe control digesters. The scale bar represents the number of substitutions per sequenc

of ammonia nitrogen. In contrast, the pH in the control digesterswas relative stable ranging between 7.5 and 7.6.

The NH3–N levels reported to be inhibitory to anaerobic diges-tion vary considerably. Anaerobic digesters acclimatized to ammo-nia could tolerate free ammonia levels above 1 g/L (Abouelenienet al., 2010; Nielsen and Angelidaki, 2008). In contrast, anaerobicdigesters un-acclimatized to high ammonia, NH3–N levels as lowas 80–150 mg/L were reported to be inhibitory (De Baere et al.,1984; Heinrichs et al., 1990). While the highest NH3–N level inthe digesters of this study was still below the inhibitory range,the high pH values in the digesters could exacerbate the toxicity ef-fect of NH3–N level, as pH higher than 7 was shown to aggravateammonia inhibition of methanogens under mesophilic conditions(El Hadj et al., 2009). The microbial populations in the anaerobicco-digestion of dairy and poultry wastes, however, appear to be

C67

C80

R85

R5

C47

C24

C26

R73

R7

R22

R9

R49

R56

C2

C43

C35

C41

C10

C36

R1

C50

R92

R27

C48

R57

4

5

R60

R2

C87

R11

R37

R53

C4

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40

39

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99

Crenarchaeota

Euryarchaeota

16S rRNA gene sequences cloned from anaerobic co-digesters and control digesters.quence designation: R – clones from the anaerobic co-digesters; and C – clones from

e position.

Table 2Summary of archaeal OTUs in clone libraries from the anaerobic co-digesters and control digesters.

RFLP group Taxonomic identification Closest relative (GenBank Accession No.) Representative clonesa GenBank Accession No.

OTU1 Euryarchaeota Methanocorpusculum sp. (AY260434) R2 GU475176OTU2 Crenarchaeota Uncultured Crenarchaeote clone (CU916928) C50 GU475172OTU3 Euryarchaeota Methanosarcina barkeri (AF028692) C87 GU475181OTU4 Euryarchaeota Methanosaeta concilii (NR_028242) R10, R11, R37, R53, C4, C66, C98 GU475186–GU475192OTU5 Euryarchaeota Methanoculleus palmolei (NR_028253) R60, R84, C65 GU475173–GU475175OTU6 Crenarchaeota Uncultured Crenarchaeote clone (GU196174) R5, R7, R9, R22, R49, R56, R73, R85, C2,

C10, C24, C26, C35, C36, C41, C43, C47, C67GU475150–GU475167

OTU7 Crenarchaeota Uncultured Crenarchaeote clone (EF552166) R1 GU475170OTU8 Crenarchaeota Uncultured Crenarchaeote clone (AY464784) R27, R57, R92, C48 GU475177–GU475180OTU9 Euryarchaeota Methanomethylovorans sp. (EF174501) R65 GU475182OTU10 Crenarchaeota Uncultured Crenarchaeote clone (CU916760) C21 GU475169OTU11 Crenarchaeota Uncultured Crenarchaeote clone (GQ470595) C80 GU475168

a Clone designation: R – clones from the anaerobic co-digesters; and C – clones from the control digesters.

Control Digesters

OTU10 2%

OTU112%

OTU82%

OTU653%

OTU57%

OTU417%

OTU32%

OTU22%

OTU113%

Co-Digesters

OTU58%

OTU422%

OTU110%

OTU92%

OTU650%

OTU72%

OTU8%

A

B Control Digesters

OTU10 2%

OTU112%

OTU82%

OTU653%

OTU57%

OTU417%

OTU32%

OTU22%

OTU113%

Co-Digesters

OTU58%

OTU422%

OTU110%

OTU92%

OTU650%

OTU72%

OTU8%

A

B

Fig. 4. Relative abundance of identified archaeal groups (OTUs) as determined byRFLP analysis of partial archaeal 16S rRNA sequences from the anaerobic co-digesters (A) and control digesters (B). The characteristics of OTUs are described inTable 2.

Y. Zhang et al. / Bioresource Technology 102 (2011) 779–785 783

reasonably robust in response to the higher pH and NH3–N levelstested in this study, given the rapid stabilization of methane pro-duction in the anaerobic co-digesters (Fig. 1), demonstrating thefeasibility of anaerobic co-digestion as an alternative option forthe treatment of these two animal wastes.

3.3. Archaeal microbial community analysis in anaerobic digestion

Since methanogens as members of the Archaea are among themicrobial populations most sensitive to ammonia in anaerobicdigestion (Batstone et al., 2002; Hansen et al., 1998; Kayhanian,1994), the responses of archaeal microbial populations to the addi-tion of poultry waste as the co-substrate were further studied byclone library analysis to understand the potential linkage betweenthe methanogenic microbial community structure and anaerobicdigestion performance.

3.3.1. Similarities in Archaeal microbial communities in anaerobicco-digesters and control digesters

Digester samples were taken from both the triplicate co-digest-ers and control digesters 56 days following the increase in OLRwhen stable methane production was achieved (Fig. 1). Triplicatesamples were pooled and two archaeal 16S rDNA clone librarieswere constructed, one for the co-digesters and the other for thecontrol digesters. RFLP analysis was performed on 96 clones fromboth types of digesters to select representative clones (39) forsequencing. Phylogenetic analysis of the representative clone se-quences indicates considerable similarities in the archaeal commu-nity compositions of the co-digesters and control digesters withthe majority of phylogenetic groupings containing closely relatedsequences from both types of digesters (Fig. 3).

In order to quantify the relative abundance of closely relatedmicrobial populations, 16S rRNA sequences with similarity greaterthan 97% were further grouped into single operational taxonomicunits (OTUs). In total, 11 OTUs were identified in the two clone li-braries (Table 2). Three OTUs had relative abundance of 10% orabove: OTU1, OTU4, and OTU6, which accounted for over 80% ofthe archaeal abundance in both clone libraries (Fig. 4). For exam-ple, in the co-digester clone library, the relative abundance ofOTU1, OTU4, and OTU6 was 10%, 22%, and 50%, respectively. Sim-ilarly, the abundance of OTU1, OTU4, and OTU6 was 13%, 17%,and 53%, respectively, in the control digester library. Therefore, alarge majority of the archaeal community remained unchanged fol-lowing the addition of nitrogen-rich poultry waste as a co-sub-strate while maintaining a balanced anaerobic digestion process(Fig. 1).

Ecological diversity measures were also calculated for the twoarchaeal clone libraries. The Shannon diversity index (H0) was thesame for both clone libraries at 1.45. The Shannon evenness index

(E) was 0.37 and 0.38 for the anaerobic co-digesters and controldigesters, respectively. Both diversity measures show similaritiesbetween the archaeal communities in the two types of digesters.

784 Y. Zhang et al. / Bioresource Technology 102 (2011) 779–785

Since digestion performance remained healthy following an overall50% increase in OLR (Fig. 1), the stability of the archaeal commu-nity structure again indicates the robustness of the methanogenicpopulations in anaerobic co-digestion.

3.3.2. Abundance of Crenarchaeota ArchaeaOf the 11 OTUs identified in the anaerobic digesters, six repre-

sented the archaeal phylum Crenarchaeota and five representedthe Euryarchaeota (Table 2), which is surprising because all knownmethanogens belong to the archaeal phylum Euryarchaeota (Luoet al., 2009). Thus, contrary to the expected dominance of metha-nogens and Euryarchaeota in anaerobic digestion, a large portionof the archaeal community in the anaerobic digesters are not likelyto be methanogens.

As expected, all the Euryarchaeota OTUs had known methano-gens as the closest relatives and belonged to the class Methanomi-crobia, (Table 2), confirming their potential involvement inmethanogenesis in anaerobic digestion. The Crenarchaeota OTUs,however, were more phylogenetically diverse and only had closerelatives of uncultured clones lacking detailed physiological char-acterization (Table 2).

More importantly, in both digesters, Crenarchaeota comprisedthe dominant archaeal populations (Fig. 4). The most abundantCrenarchaeota group, OTU6, alone had a relative abundance of over50%. OTU6 is 99% similar to an uncultured crenarchaeote clone alsofrom an anaerobic digester (Table 2). Interestingly, recent findingshave suggested the broad distribution of mesophilic Crenarchaeotaand their roles in ammonia oxidation in the environment (Nicoland Schleper, 2006; Treusch et al., 2005). However, ammonia oxi-dation is unlikely the function of the Crenarchaeota dominant inthe anaerobic digesters of this study due to the lack of oxygenand the persistence of high ammonia levels in the digesters(Fig. 2). Given that Crenarchaeota were also found in other studiesas abundant populations in anaerobic digestion (Collins et al.,2005; Godon et al., 1997), more efforts are needed to understandtheir functions in anaerobic environments.

4. Conclusions

Poultry waste was characterized as an organic/nitrogen-richsubstrate. Supplementing dilute dairy waste with poultry wastefor anaerobic co-digestion to increase OLR by 50% resulted in im-proved biogas production. Elevated ammonia derived from poultrywaste did not lead to process inhibition at the OLRs tested, demon-strating the feasibility of the anaerobic co-digestion of dairy andpoultry wastes for improved efficiency. The stability of the anaer-obic co-digestion process was linked to the robust archaeal micro-bial community, which remained mostly unchanged following theincreases in OLR and ammonia levels. Surprisingly, Crenarchaeota,instead of the methanogens, dominated the archaeal communitiesin anaerobic digestion.

Acknowledgements

This work was partly supported by a US National Science Foun-dation Grant No. 0854332 as a graduate course project, and a USEnvironmental Protection Agency Grant SU-83431801.

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