study of microbial community and biodegradation efficiency for single- and two-phase anaerobic...
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Bioresource Technology 147 (2013) 193–201
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Bioresource Technology
journal homepage: www.elsevier .com/locate /bior tech
Study of microbial community and biodegradation efficiency for single-and two-phase anaerobic co-digestion of brown water and food waste
0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.08.038
⇑ Corresponding author at: Residues and Resource Reclamation Centre, NanyangEnvironment and Water Research Institute, Nanyang Technological University, 1Cleantech Loop, Singapore 637141, Singapore. Tel.: +65 67904100; fax: +6567927319.
E-mail addresses: [email protected] (J.W. Lim), [email protected] (C.-L.Chen), [email protected] (I.J.R. Ho), [email protected] (J.-Y. Wang).
1 Tel.: +65 65927760; fax: +65 67927319.
J.W. Lim a,b,1, C.-L. Chen a, I.J.R. Ho a, J.-Y. Wang a,b,⇑a Residues and Resource Reclamation Centre, Nanyang Environment and Water Research Institute, Nanyang Technological University, 1 Cleantech Loop, Singapore 637141, Singaporeb Division of Environmental and Water Resources, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore639798, Singapore
h i g h l i g h t s
� First study on microorganisms involved in brown water and food waste degradation.� Clear differences in bacterial communities between single- and two-phase CSTRs.� Methanosaeta was the main contributor for methane production in both CSTRs.� Firmicutes played an important role in solids reduction.
a r t i c l e i n f o
Article history:Received 5 June 2013Received in revised form 4 August 2013Accepted 6 August 2013Available online 14 August 2013
Keywords:Microbial community structureAnaerobic co-digestionBrown waterFood wasteSingle- and two-phase CSTR
a b s t r a c t
The objective of this work was to study the microbial community and reactor performance for the anaer-obic co-digestion of brown water and food waste in single- and two-phase continuously stirred tank reac-tors (CSTRs). Bacterial and archaeal communities were analyzed after 150 days of reactor operation. Ascompared to single-phase CSTR, methane production in two-phase CSTR was found to be 23% higher. Thiswas likely due to greater extent of solubilization and acidification observed in the latter. These findingscould be attributed to the predominance of Firmicutes and greater bacterial diversity in two-phase CSTR,and the lack of Firmicutes in single-phase CSTR. Methanosaeta was predominant in both CSTRs and thiscorrelated to low levels of acetate in their effluent. Insights gained from this study would enhance theunderstanding of microorganisms involved in co-digestion of brown water and food waste as well asthe complex biochemical interactions promoting digester stability and performance.
� 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Anaerobic digestion is a biochemical process that degradesbiomass biologically and produces biogas consisting mainly ofmethane, which is a valuable source of renewable energy. Foodwaste is a suitable substrate for anaerobic digestion due to its highorganic content. On the other hand, landfilling of food waste leadsto uncontrolled emission of methane, and incineration could beinefficient due to the low calorific value of wet food waste (Bernstadand Jansen, 2012). In comparison with landfilling or incineration,the anaerobic digestion of food waste was found to be a more suit-able and sustainable treatment method to address the growing
concern over large amounts of food waste generated worldwide.Therefore, the treatment of food waste by anaerobic digestion pro-cess has attracted increasing attention in recent years (Wang et al.,2002; Ike et al., 2010).
The addition of co-substrate (e.g. brown water) to food wastecould improve the anaerobic digestion process stability by provid-ing additional nutrients and maintaining buffer capacity. The ben-efits of co-digesting brown water and food waste was described byRajagopal et al. (2013). The authors observed higher biogas pro-duction and biodegradation efficiencies when brown water wasadded as a co-substrate to the anaerobic degradation of food waste.Production of methane via anaerobic digestion of organic pollu-tants not only provides a cheaper and greener alternative to foodwaste and brown water disposal, it also replaces fossil fuel-derivedenergy and reduces the impact of global warming (Abbasi et al.,2012).
Anaerobic digestion of organic matter is carried out syntrophi-cally by microbial communities consisting of both bacterial andarchaeal species. The degradation may be divided into three steps.
194 J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201
During the first step, hydrolysis bacteria degrade polymeric organ-ic matter into monomers, such as sugar and amino acid, which arefurther degraded in the second step by acetogenic bacteria intovolatile fatty acids (VFAs), such as acetate. In the last step, metha-nogens produce biogas mainly from formate, hydrogen andacetate.
In conventional applications, anaerobic digestion processesusually occur in a single reactor system. However, acid- and meth-ane-forming microorganisms have very different nutritional needs.When kept together in a single reactor system, some of such sys-tems gradually gave rise to reactor instability problems (Demireland Yenigun, 2002). The physical separation of acid- andmethane-forming microorganisms in different reactors was firstproposed by Poland and Ghosh (1971). Such systems providedoptimum environmental conditions for each group of organismsand thus led to enhanced stability and control of the overallprocess.
Studies on bacterial and methanogenic archaeal communitystructures in anaerobic digesters treating food waste have been re-ported recently (Ike et al., 2010; Wang et al., 2010). However, theunderstanding of microbial aspects for co-digestion of brownwater and food waste is still limited due to the lack of referenceson this topic. Comprehension of microbial community and its func-tion is necessary to improve the efficiency and process stability ofanaerobic digesters. 16S rRNA cloning and sequencing is the wellknown method used to characterize microbial community in ananaerobic reactor while fluorescent in situ hybridization (FISH) isa useful method to verify cloning findings and to visualize the dif-ferent cells in anaerobic sludge. Therefore, these two methodswere employed in the current study to determine the microbialpopulations. The objective of this work was to study the microbialcommunity and reactor performance for the anaerobic co-diges-tion of brown water and food waste in single- and two-phase con-tinuously stirred tank reactors (CSTRs). Insights gained from thisstudy would enhance the understanding of microorganisms in-volved in the anaerobic co-digestion of brown water and foodwaste as well as the complex biochemical interactions thatpromote digester stability and performance. These could aid theselection of seeding sludge for rapid startup in future applications.
2. Methods
2.1. Feedstock and reactor operation
Food waste was collected from canteens on campus whilebrown water was collected from a specially designed source-separation toilet, where urine with 0.3 L flush water (as yellowwater) and faeces with 2 L flush water (as brown water) werecollected in separate tanks. The feed for this study consisted of amixture of 300 g blended food waste and 2 L brown water, andhad an average pH of 6.23 ± 0.07. The characteristics of the feedare as shown in Table 1. Anaerobic co-digestion of brown waterand food waste was performed in laboratory scale (5 L) single-and two-phase CSTRs. The co-substrates were prepared daily andfed to the reactors, which included the acidogenic (RA) and metha-nogenic (RM) reactors of the two-phase CSTR system and thesingle-phase CSTR (RS), in batch mode. The working volumes ofRA, RM and RS were 1.2 L, 4.1 L and 5.3 L, respectively, and the con-tents were mixed continuously (mixing time: 5 min ON followedby 5 min OFF) at 80 rpm by an overhead mechanical stirrer asreported previously by Rajagopal et al. (2013). RA, RM and RS wereinitially inoculated with mesophilic anaerobic sludge collectedfrom a local wastewater treatment plant (Ulu Pandan Water Recla-mation Plant, Singapore). Reactor contents were gradually replacedby the brown water and food waste mixture. By the time this study
started, anaerobic sludge was completely replaced by the brownwater and food waste mixture. The single and two-phase CSTR sys-tems were operated in parallel for 150 days at 35 �C with hydraulicretention time (HRT) as shown in Table 1. HRT was reduced byadding increased volumes of the brown water and food waste mix-ture into the reactors of fixed working volumes. The organic load-ing rate (OLR) was maintained at around 0.5–0.8 g-VS L�1 d�1 inthis study. Both the single- and two-phase CSTRs were operatedin the same way and had the same overall reactor working volumeof 5.3 L. Both RA and RS were fed with brown water and food wastemixtures prepared daily while RM was fed with the acidified efflu-ent from RA during the study. The reactor performances for RA, RM,and RS were monitored weekly.
2.2. Chemical analysis
Biogas production was measured daily using a mass flow meter(McMillan Company, Model 50D-3E), while other parameters suchas pH, total (TS) and volatile (VS) solids, total and soluble chemicaloxygen demand (COD), VFAs and biogas composition were mea-sured weekly. The biogas composition (i.e., methane, carbon diox-ide, nitrogen and hydrogen contents) was analyzed by gaschromatograph (Agilent Technologies 7890A, USA) equipped witha thermal conductivity detector (TCD). pH value was measuredusing a compact titrator (Mettler Toledo) equipped with a pHprobe (Mettler Toledo DGi 115-SC). TS and VS were analyzedaccording to the Standard Methods (APHA, 1998). Total and solubleCOD were measured using COD digestion vials (Hach Chemical)and a spectrophotometer (DR/2800, Hach). Soluble COD was mea-sured using the supernatant of samples after centrifugation(KUBOTA 3700, Japan) at 12,000 rpm for 10 min. The determina-tion of VFAs was carried out using a gas chromatograph (AgilentTechnologies 7890A, USA), equipped with a flame ionization detec-tor (FID) and a DB-FFAP (Agilent Technologies, USA) column(30 m � 0.32 mm � 0.50 lm) and the samples were filteredthrough 0.45 lm cellulose acetate membrane filters (membranesolutions).
2.3. DNA extraction and construction of 16S rRNA gene clone libraries
Sludge samples were collected on day 150 and genomic DNAwas extracted from sludge using chemical lysis and phenol–chloro-form–isoamyl alcohol (25:24:1, v:v:v) purification protocol as de-scribed previously (Liu et al., 1997). Primer sets 530F (50-GTGCCAGC(A/C)GCCGCGG-30) and 1490R (50-GGTTACCTTGTTACG-ACTT-30) as well as Ar1F (50-TCYGKTTGATCCYGSCRGAG-30) and1490R were used to amplify 16S rRNA gene from the total-commu-nity DNA, targeting total prokaryotes and Archaea, respectively.The thermal program used for amplification of 16S rRNA genewas as follows: hotstart 94 �C for 3 min, 30 cycles of denaturation(30 s at 94 �C), annealing (30 s at 54 �C) and extension (45 s at72 �C) and a final extension at 72 �C for 5 min. TOPO TA cloningkit (Invitrogen, CA) was used for clone library construction accord-ing to the manufacturer’s instructions. Approximately 100 and 50clones were randomly selected from RA, RM and RS for the membersin the domain Bacteria (amplified by primer set 530F and 1490R),and Archaea (amplified by primer set Ar1F and 1490R), respec-tively. The amplified DNA insert was then PCR amplified with avector-specific primer set (i.e., M13F and M13R). Restriction frag-ment length polymorphism (RFLP) was used to screen the 16SrRNA gene fragments to further remove the possible redundantclones. The M13-PCR products were separately digested to comple-tion with tetramer restriction enzymes MspI and RsaI (NewEngland BioLabs, UK), and separated by electrophoresis in a 3%agarose gel. Gels were visualized using the FireReader gel docu-mentation (UVItec, Cambridge, UK) after staining with Gelred
Tab
le1
Ope
rati
onal
cond
itio
nsan
dre
acto
rpe
rfor
man
ce.
Peri
od1
(Day
s0–
55)
2(D
ays
56–1
09)
3(D
ays
110–
150)
Feed
aR A
R MR S
Feed
aR A
RM
RS
Feed
aR A
RM
RS
HR
Tb[d
]10
3040
1030
406
2935
Bio
gas
Prod
uct
ion
[Lg-
VS�
1d�
1]
n.d
.n
.d.
1.91
±0.
370.
96±
0.28
n.d
.n
.d.
1.15
±0.
380.
62±
0.27
n.d
.n
.d.
1.57
±0.
610.
65±
0.20
TS[g
L�1]
30.4
0±
9.99
23.1
7±
5.92
7.93
±0.
504.
75±
2.28
23.3
0±
6.25
12.2
1±
2.36
4.83
±1.
577.
76±
2.45
19.6
2±
10.3
711
.14
±1.
694.
04±
0.93
7.93
±1.
13TS
rem
oval
[%]
n.d
.n
.d.
72.3
±8.
7483
.61
±8.
26n
.d.
n.d
.76
.07
±15
.98
63.6
7±
18.2
5n
.d.
n.d
.76
.54
±8.
9953
.05
±17
.07
VS
[gL�
1]
26.2
1±
4.80
21.5
6±
5.60
5.39
±0.
433.
21±
2.02
22.0
7±
5.99
11.3
7±
2.95
3.27
±1.
405.
76±
2.11
18.2
9±
10.2
89.
63±
1.59
2.51
±0.
845.
87±
0.79
VS
rem
oval
[%]
n.d
.n
.d.
79.2
5±
4.40
85.4
0±
9.20
n.d
.n
.d.
76.0
7±
15.9
863
.67
±18
.25
n.d
.n
.d.
81.4
2±
6.09
56.5
0±
6.05
Solu
ble
CO
D[g
L�1]
15.4
7±
6.71
19.0
1±
12.6
30.
70±
0.32
0.65
±0.
2213
.01
±3.
2615
.39
±3.
751.
04±
1.02
0.53
±0.
098.
41±
1.36
18.6
0±
1.11
0.81
±0.
400.
49±
0.04
Solu
ble
CO
Dre
mov
al[%
]n
.d.
n.d
.94
.84
±2.
3495
.10
±2.
62n
.d.
n.d
.94
.02
±2.
3195
.59
±1.
28n
.d.
n.d
.91
.79
±3.
3094
.19
±0.
74TV
FA[m
g-C
OD
L�1]
815
±66
935
32±
2853
62±
1878
±37
2001
±65
814
173
±33
6880
1±
256
481
±19
512
34±
569
1936
7±
7799
1146
±40
121
5±
36
n.d
.:N
otde
term
ined
.a
300
gfo
odw
aste
+2
Lbr
own
wat
er.
bH
ydra
uli
cre
ten
tion
tim
e.
J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201 195
(Invitrogen, CA). Unique RFLP patterns were defined as a uniquesequence type of operational taxonomy unit (OTU).
2.4. Sequence analysis
The 16S rRNA gene of the representative clones with differentRFLP patterns were sequenced, by 1st BASE (Singapore), to deter-mine their phylogenetic affiliation. Nearly full-length 16S rRNAgene sequences of representative clones were compared to avail-able rRNA gene sequences in GenBank using the NCBI BLASTprogram. Chimeric artifacts were determined using DECIPHER(Wright et al., 2012) and phylogenetic trees were constructedwith MEGA5 program using the remaining clone sequences(approximately 80 for bacterial clones and 45 for archaeal clones)after removing the chimeric sequences. The Jukes–Cantor correc-tion was used for distance matrix analyses and the trees wereconstructed using the Neighbor-joining method. Archaeal andbacterial 16S rRNA partial sequences obtained in this study weredeposited in the nucleotide Genbank database, under the acces-sion numbers: KF169842–KF169904.
2.5. Fluorescence in situ hybridization (FISH)
Sludge samples from RA, RM and RS were collected towards theend of the operational period (on day 150) for FISH analyses. Thesludge samples were pretreated according to the protocol de-scribed previously by Amann et al. (1995), and fixed overnightwith 4% paraformaldehyde solution at 4 �C. Hybridization wascarried out at 46 �C for 3 h with hybridization buffer containing5 ng lL�1 of specific fluorescent probe. Two oligonucleotideprobes, EUBmix (i.e., EUB338, EUB338-II, EUB338-III) andARC915, were used to target the members of Bacteria and Ar-chaea, respectively (Daims et al., 1999; Amann et al., 1995). FISHhybridization was performed with 35% formamide concentrationfor both probes (EUBmix and ARC915) in the hybridization buffer.An Olympus BX53 epifluorescence microscope equipped with acooled CCD camera DP72 with a 100 W halogen bulb and fluores-cence filter sets (U-FGW and U-FF-Cy5) under �100 objectivelens (Olympus, Japan) was used to capture FISH-stained images.
3. Results and discussion
3.1. Reactor performance
The experimental results of this study were categorized intothree periods as shown in Table 1. The average biogas produc-tions for RM and RS throughout the study were 1.54 and0.74 L g-VS�1 d�1, and their average CH4 concentrations were60% and 50%, respectively. With no pH control in any of the reac-tors, the pH levels in the three reactors were stable throughoutthe study. RA, RM and RS had average pH levels of 3.72 ± 0.35,6.98 ± 0.14 and 6.98 ± 0.15, respectively. The pH of RM and RS
were not significantly different and were in the suggested opti-mal pH range suitable for methanogenesis to take place.
The overall average concentration of total volatile fatty acid(TVFA) for RA was 11,115 ± 7074 mg-COD L�1, representing anaverage of 9-fold increase in TVFA production as compared tothe feed. With average soluble COD values of 12.93 ± 5.67 and17.38 ± 7.66 g L�1, the degree of acidification (proportion of VFAin soluble COD) for the feed and RA effluent were 11% and 79%,respectively. This translated to a sevenfold increase in the degreeof acidification after treatment in RA of the two-phase system.Almost 80% of TVFA in RA comprised of acetate, propionate andbutyrate. Acetate was the dominant VFA throughout the studywhere it accounted for 40% of TVFA. The percentages of
196 J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201
propionate and butyrate in RA were on average 17% and 23%,respectively. The high degree of acidification as well as conversionof longer chain VFA to acetate suggested that the activities of acid-ogens and acetogens were high in RA.
In RM, VFAs fed from RA were mostly consumed and their totalcontent in RM was reduced to an average amount of 618 mg-COD L�1. Acetate was present in highest concentrations (averageof 101 mg-COD L�1) as compared to propionate (average of85 mg-COD L�1) and butyrate (average of 114 mg-COD L�1). Thelevels of TVFA in RM were approximately 60% and five times higherthan that in RS for periods 2 and 3 of the study, respectively. RM hadan average NHþ4 concentration of 689 mg L�1, which was almost 2times higher than that in RS (366 mg L�1). Despite the higher levelsof TVFA and NHþ4 in RM, there were no significant differences in thereductions of total and soluble COD between RM and RS. Bothachieved at least 74% total COD and 91% soluble COD removalrates. In addition, the higher levels of TVFA and NHþ4 did not leadto any observed inhibition effects by the two-phase system. Asshown in Table 1, the average TS and VS removal efficiencies forRM did not vary much throughout the study. On the contrary, TS re-moval efficiency for RS dropped from 84% to 53%, while its VS re-moval efficiency dropped from 85% to 57% as reactor operationproceeded from period 1 to 3. Overall, the performance of two-phase CSTR system (i.e., RA and RM) was better than that of sin-gle-phase CSTR (i.e., RS) in terms of higher biogas production,methane composition as well as solids reduction.
3.2. Microbial community characterization
Cloning and subsequent phylogenetic analysis were carried outto characterize the microbial community structures in both single-and two-phase CSTRs. Primer set 530F and 1490R were initiallyused to amplify the prokaryotic sequences from the extractedDNA. However, all the clones sequenced were affiliated withinthe domain Bacteria, indicating that bacterial cells were dominantin RA, RM and RS. Similar findings were also reported previously byTang et al. (2004). Therefore, an additional set of archaeal primersAr1F and 1490R was used in this study to construct the archaealrRNA clone libraries for RM and RS. Neighbor-joining trees showingthe phylogenetic identities of the 16S rRNA gene fragments wereconstructed and are shown in Figs. 1–4.
3.2.1. Bacterial community in acidogenic reactor of two-phase CSTR(RA)
As shown in Fig. 1, the bacterial community structure of RA wasexclusively composed of the phyla Firmicutes and Proteobacteria. In
BFABac 001_ (36%) BFABac 111_ (3%)
BFABac 009_ (8%)Lactobacillus amylovor Uncultured Lactobacillu BFABac 137_ (1%)Lactobacillus fermentumLactobacillus fermentum
BFABac 065_ (1%) Uncultured Clostridiu
BFABac 040_ (49%)Acetobacter peroxydan
BFABac 058_ (1%)Desulfobulbus propionicus
Sulfolobus acidocald
100
100
10091
9282
100
100 82
4532
100
0.05
Fig. 1. Phylogenetic tree of 16S rRNA gene sequences constructed for bacterial clones fromas the outgroup.
total, 7 bacterial operational taxonomic units (OTU) were identi-fied. The most detected OTU (BFABac_040), representing 49% ofthe total clones, was affiliated to Acetobacter peroxydans strainLMG 1633 (AJ419836) with 99% similarity. BFABac_001 was thesecond most detected OTU accounting for 36% of the clones. To-gether with BFABac_009 and BFABac_111, the second predominantgroup was affiliated with Lactobacillus amylovorus GRL 1112(NR_075048) with 99% similarity. BFABac_137 (1% of total clone)was affiliated with Lactobacillus fermentum strains IFO 3956(NR_075033) and JCM 8596 (AB690185). The two remaining OTUs(BFABac_065 and BFABac_058) each corresponded to 1% of thetotal clone and were closely related to uncultured Clostridium spe-cies (HQ183766) and Desulfobulbus propionicus DSM 2032(NR_074930), respectively.
3.2.2. Bacterial community in methanogenic reactor of two-phaseCSTR (RM)
The bacterial community in RM was found to be more diverseand a total of 28 bacterial OTU were identified and classifiedinto 13 different phyla. Fig. 2 shows that the dominant OTU in-cluded members affiliated within four different phyla: Bacteroi-detes, Chloroflexi, Proteobacteria and Firmicutes in proportions of40%, 13%, 10% and 8% of the bacterial clones, respectively. Withinthe 28 OTU, 5 were classified as Bacteroidetes, 5 as Chloroflexi, 5as Proteobacteria and 4 as Firmicutes. As shown in Fig. 2, theclosest matches for bacterial clones were mostly detected fromfood-processing, toluene-degrading and sulfate-rich wastewa-ters, human faeces, human oral cavities, animal waste treatmentand biogas plants, which were all related to anaerobicfermentation.
3.2.3. Bacterial community in single-phase CSTR (RS)The bacterial community in RS was slightly less diverse as
compared to RM where a total of 17 bacterial OTU were identi-fied, which could be classified into 8 different phyla. Fig. 3shows that the bacterial OTU were mainly affiliated with Fuso-bacteria, Bacteroidetes, Proteobacteria and Chloroflexi in propor-tions of 51%, 32%, 4% and 4% of the bacterial clones,respectively. Within the 17 OTU, 2 were classified as Fusobacte-ria, 6 as Bacteroidetes, 3 as Proteobacteria and 2 as Chloroflexi.BFSBac_073, the predominant OTU in RS, accounted for 35% oftotal bacterial count and was affiliated with uncultured Fusobac-terium species (FM242289). The closest matches for bacterialclones, as shown in Fig. 3, were detected from sources similarto that of RM.
GRL 1112 isolated from po cine faeces (NR 075048)us r _ sp. clone (HM218835)s
IFO 3956 (NR 075033)_ JCM 8596 (AB690185)
sp. clone (HQ183766)m
LMG 1633 (AJ419836)s
DSM 2032 (NR 074930)_ (NR 043400)arius
Firmicutes
Proteobacteria
RA. The 16S rRNA gene sequence of Sulfolobus acidocaldarius (NR_043400) was used
BFMBac 111_ Uncultured bacterium clone (GU389854)in AD of food processing waste BFMBac 136_ Uncultured bacterium clone (AF482439)fatty acid-oxidizing syntrophs in granular sludge Uncultured sp. clone (JQ079827)Syntrophobacter
strain Tb8106 (NR 043073)Syntrophobacter sulfatireducens , propionate-oxidizing syntrophs in UASB _ BFMBac 145_
bacterium enrichment culture (KC460267)Syntrophaceae BFMBac 079_
Uncultured organism clone (EU245608) BFMBac 018_ Uncultured bacterium clone (FJ769440)in sludge pretreatment Uncultured sp. clone (JQ723608)Geothrix
BFMBac 007_ strain CB-8 (JF496528)Aeromonas sharmana
BFMBac 038_sp. canine oral taxon 223 (JN713386)Propionivibrio
BFMBac 035_ Uncultured sp. clone (EU887787)Clostridium in Nisargruna biogas plant
bacterium canine oral taxon 067 (JN713232)Peptostreptococcaceae BFMBac 106_
sp. strain P2 (AY949856)Clostridium in UASB BFMBac 057_
sp. enrichment culture clone (HQ222293)Clostridium in phenolic biodegradation sp. (AB491207)Clostridium isolated from human faeces
BFMBac 138_ Uncultured bacterium clone (CU926291)Firmicutes in AD of sludge
BFMBac 028_ Uncultured bacterium clone (FJ437954)
strain A4T-83 (NR 041625)Luteolibacter pohnpeiensis _ BFMBac 105_ Uncultured bacterium clone (AB744095)acidogenesis Uncultured bacterium clone (AB780948)Verrucomicrobia in AD of corn straw
BFMBac 032_ Uncultured microorganism clone (JN387540)in sulfur spring
strain: JCM 16774 (AB558169)Leptotrichia goodfellowii sp. VNs100 (KC800693)Mesotoga
BFMBac 091_ Uncultured bacterium clone (FJ535538)in swine wastewater
BFMBac 051_ Uncultured bacterium clone (FN436169)
str. Buddy (AF357916)Sphaerochaeta globus Uncultured bacterium clone (AB175392) in anaerobic degradation of protein Uncultured 1 bacterium clone (CU921669)WWE in AD of sludge
BFMBac 082_ BFMBac 015_
Uncultured bacterium clone (JX575986)Bacteroidetes BFMBac 019_ Uncultured bacterium clone (CU918283)Bacteroidetes in AD of sludge BFMBac 143_
strain LIND7H (HQ020488)Macellibacteroides fermentans WB4 strain (NR 074577)Paludibacter propionicigenes _
BFMBac 042_ Uncultured bacterium clone (AB266963) in UASB treating food-processing wastewater Uncultured sp. clone (JQ815605)PaludibacterBFMBac 004_
Uncultured bacterium clone (CU926883)Bacteroidetes in AD sludge Uncultured Synergistetes bacterium clone OTU-X2-19 (JQ668564) BFMBac 139_ Uncultured bacterium clone (JQ624291)
Toluene-degrading methanogenic consortium bacterium Eub 4 (AF423184) BFMBac 008_ Uncultured clone (AB603823)Chloroflexi in acetate-utilizing synergistes bacterium BFMBac 092_
Uncultured bacterium clone (JQ996691) BFMBac 117_ Uncultured sp. clone (EU887788)Bellilinea in Nisargruna biogas plant
BFMBac 116_ Uncultured bacterium isolate CMW-14 (FR828730)hydrogen-producing Uncultured sp. clone (EU887779)Dehalococcoides in Nisargruna biogas plant BFMBac 023_
Toluene-degrading methanogenic consortium bacterium Eub 2 (AF423182) (NR 043400)Sulfolobus acidocaldarius _
100
10099
100
82100
100
100100
96100
100
100
100
99
100
99
47
53
100
8098
99
100
99
59100
100
100
100
100
100
100
97
56100
100
100
99100
100
100
100
99
100
100
66
685499
100
99
99
86
79
89
85
97
62
48
54
84
71
25
58
52
29
10
16
16
0.05
Deltaproteobacteria (8%)
Acidobacteria (1%)
Gammaproteobacteria (1%)
Betaproteobacteria (1%)
Firmicutes (8%)
Verrucomicrobia (7%)
Fusobacteria (8%)
Thermotogae (1%)
Spirochaetes (5%)
WWE1 (4%)
Bacteroidetes (40%)
Synergistetes (1%)
Chloroflexi (13%)
Fig. 2. Phylogenetic tree of 16S rRNA gene sequences constructed for bacterial clones from RM. The 16S rRNA gene sequence of Sulfolobus acidocaldarius (NR_043400) wasused as the outgroup.
J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201 197
3.2.4. Overview of bacterial communities in RA, RM and RS
The reactors in this study were fed daily with mixtures ofbrown water and food waste. Therefore, their bacterial communitystructures showed a close relationship to human sources such asgastrointestinal tract, oral cavity and faeces. Bacteroidetes andFirmicutes are known to be dominant phyla present in the human
gastrointestinal tract and adult faecal microbiota (Harmsen et al.,2002) while Fusobacteria was isolated from human oral cavities(Bennett and Eley, 1993). However, analysis of bacterial communi-ties in this study demonstrated clear differences in both dominantgroups and phylogenetic distribution between single- and two-phase CSTRs.
BFSBac 056_ sp. strain Z4 (AY949860)Bacteroides in paper mill wastewater
BFSBac 126_ strain LIND7H (HQ020488)Macellibacteroides fermentans in abattoir wastewater
sp. canine oral taxon 384 clone (JN713554)Paludibacter BFSBac 087_
Uncultured bacterium clone (AB266963) in food-processing wasewater BFSBac 024_
strain: JCM 15093 (AB547643)Bacteroides graminisolvens BFSBac 045_ Uncultured bacterium clone (AB780930)Bacteroidetes in corn straw
BFSBac 140_ Uncultured bacterium clone (HQ003606)Bacteroidetes
BFSBac 018_ Uncultured clone (JX575817)planctomycete in pit mud
Uncultured sp. clone (FM242289)Fusobacterium strain F0264 (FJ577259)Leptotrichia goodfellowii in human oral microbiome
Uncultured bacterium clone (GU472735)Fusobacteriales Uncultured microorganism clone (JN387540) BFSBac 026_
BFSBac 073_ BFSBac 112_ Uncultured bacterium clone (JX224706)in acetate amendment
Uncultured bacterium clone BXHB128 (GQ480156) Bacterium SUTW 81 (JN035218); D-lactic acid-producing bacterium
strain GPTSA-6 (NR 043470)Aeromonas sharmana _ BFSBac 129_
BFSBAc 061_ Uncultured organism clone (JN498907)
strain G13 (NR 043576)Geobacter pickeringii _ Uncultured bacterium clone (CU919618)Deltaproteobacteria in AD of sludge Uncultured sp. clone (EU921178)Syntrophus in compost BFSBac 059_
Uncultured bacterium clone (GU389881)in food-processing waste BFSBac 013_ Uncultured bacterium clone (AF482447)fatty acid oxidizing syntrophs in granular sludge
sp. (KC800693)Mesotoga BFSBac 033_
Uncultured candidate division TM7 bacterium clone (JN656764) Uncultured bacterium clone (EU266915) Chloroflexi ; anaerobic toluene degraders BFSBac 060_ Uncultured bacterium clone (JQ996691) Uncultured sp. clone (JX576078)Bellilinea in pit mud BFSBac 002_ Uncultured bacterium clone (HQ602791); acetoclastic sulfate reducers
(NR 043400)Sulfolobus acidocaldarius _
70100
100
100
8669
100
100
100
100
97
87
100
100100
100
100
93
79
100
100
9398
100
7690
99100
50
69100
100
100
100
82
97
74
34
85
30
66
15
18
0.05
Bacteroidetes (32%)
Planctomycetes (1%)
Fusobacteria (51%)
Unknown clone (1%)Gammaproteobacteria (1%)
Deltaproteobacteria (3%)
Thermotogae (1%)
TM 7 (1%)
Chloroflexi (4%)
Fig. 3. Phylogenetic tree of 16S rRNA gene sequences constructed for bacterial clones from RS. The 16S rRNA gene sequence of Sulfolobus acidocaldarius (NR_043400) was usedas the outgroup.
198 J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201
Lactobacillus species and A. peroxydans LMG 1633 representedthe exclusive dominant phylogenetic group (close to 98% by clon-ing analysis) in RA, suggesting a major impact of these bacteria onthe solubilization and acidification of brown water and food waste,at short retention time of 6–10 days and acidic pH conditions. Inthis study, Lactobacillus species was predominant in RA where thepH was around 4. However, it was not detected in RM and RS wherethe pH was around 7. This is in agreement with other studiesreporting that the presence and dominance of Lactobacillus wasdependent on pH values. According to Ye et al. (2007), Lactobacillusgrew intensively at pH 4–6, but slowly at pH 7–8.
The predominant Lactobacillus species in RA, L. amylovorus, is alactate-producing organism possessing amylolytic activity. There-fore it is able to metabolize starch directly to produce lactate andsmall amounts of acetate (Zhang and Cheryan, 1991). The otherpredominant species in RA – A. peroxydans was reported to containenzymes for the oxidation of lactate, pyruvate, ethanol and acetal-dehyde to acetate, through the transfer of electrons to oxygen. DeLey and Schel (1959) showed that lactate was oxidized to pyruvatefollowed by slow oxidation of pyruvate to acetate. Therefore, theco-existence of Lactobacillus species and A. peroxydans possibly re-sulted in the high levels of solubilization and acidification observedin RA.
All the reactors in this study were designed to operate undercompletely anaerobic conditions. Therefore, the predominance ofan obligately aerobic bacteria – A. peroxydans in RA was a surprisefinding. A. peroxydans was able to co-exist with Lactobacillus
species in RA since the latter is an aero-tolerant anaerobic bacteria.The exact experimental condition (i.e., anaerobic, microaerobic oraerobic) of RA was not determined by analytical methods such aslevels of dissolved oxygen or oxidation reduction potential. None-theless, the predominance and co-existence of obligately aerobic A.peroxydans and aero-tolerant anaerobic Lactobacillus species sug-gested RA was unintentionally operated at microaerobic conditions.The operation of RA under completely aerobic conditions washighly unlikely since the concentrations of TVFAs and solubleCOD in the effluent of RA were higher than that in the feed mixture(Table 1). However, further investigations are required to verifythat the predominance of A. peroxydans was due to the unintendedoperation at microaerobic conditions.
The predominant bacterial group present in RM was Bacteroide-tes (40% by cloning analysis), which is a major microbial compo-nent of anaerobic reactors. Another bacterial group present in RM
was Firmicutes (8% by cloning analysis), which are known to pro-duce cellulases, lipases, proteases and other extracellular enzymes(Levén et al., 2007). Therefore, the presence of Firmicutes reflectsthe ability of digesters to metabolize a variety of substrates includ-ing protein, lipids, lignin, cellulose, sugars and amino acids, whichare commonly found in food waste.
For RS, the predominant bacterial group present was Fusobacte-rium species (51% by cloning analysis), which are obligate anaerobicgram-negative bacilli found in large numbers in the mouth. It wasreported that Fusobacterium species weakly ferment simple sugarsto produce large amounts of n-butyric acid (Bennett and Eley, 1993).
BFMArc 004 (4 )_ 8% Uncultured archaeon clone (JN397877) Uncultured sp. clone (AB479394)Methanosaeta
BFMArc 039 (1 )_ 9% BFMArc 177 ( )_ 2%
Uncultured archaeon clone (CU916545)Methanosarcinales Uncultured archaeon clone (AB669270)Methanosaetaceae
BFMArc 103 (7 )_ % Uncultured sp. (GU179438)Methanoculleus
BFMArc 183 (2 )_ % Uncultured euryarchaeote clone (AB248621)
Uncultured sp. (JX560560)Methanoculleus BFMArc 134 ( )_ 21%
sp. strain dm2 (AJ550158)Methanoculleus(M59932)Methanopyrus kandleri
7999
96
100
50
100
100
7276
93
41
72
0.02
Methanosaetaceae
Methanomicrobiales
BFSArc 152 ( )_ 6% Uncultured euryarchaeote clone (AB248614) Uncultured sp. clone (AB077211)Methanosaeta Uncultured archaeon clone (AB669270)Methanosaetaceae BFSArc 034 ( )_ 3%
BFSArc 157 ( )_ 3% Uncultured archaeon clone (GU388805)
Uncultured sp. clone (AB479394)Methanosaeta Uncultured archaeon clone (JN397877) BFSArc 019 ( )_ 74% Uncultured s sp. clone (JX101963)Methanoculleu Uncultured archaeon clone (EU369613)
sp. strain dm2 (AJ550158)Methanoculleus BFSArc 061 (1 )_ 4%
(M59932)Methanopyrus kandleri3469
100
8299
64100
31
99
65
5675
Methanosaetaceae
Methanomicrobiales
(b)
(a)
Fig. 4. Phylogenetic tree of 16S rRNA gene sequences constructed for archaeal clones from (a) RM and (b) RS. The 16S rRNA gene sequence of Methanopyrus kandleri (M59932)was used as the outgroup in both (a) and (b).
J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201 199
In comparison to RM, the distribution of bacteria within the phy-lum Fusobacteria was higher, that of Bacteroidetes was lower, andFirmicutes was absent in RS. Since Firmicutes contain extracellularenzymes that carry out the solubilization of brown water and foodwaste, its absence could play a part in the poorer performance ofRS, in terms of lower solids reduction. In addition, the lack of Firmi-cutes could also suggest that there were large amounts of longchain fatty acids (LCFAs) in RS since LCFAs were reported to inhibitgram-positive bacteria such as Clostridia (Galbraith and Miller,1973).
There is a lack in studies on the microbial diversity of anaerobicdigesters treating brown water or mixtures of brown water andfood waste. On the other hand, several studies had reported thepredominance of Lactobacillus species in the fermentation of foodwaste (Wang et al., 2005; Ye et al., 2007), and that of L. amylovorus-related species in the first-stage reactor of a two-stage anaerobicdigestion system treating food waste (Shin et al., 2010). However,the diversity of Lactobacillus species in RA was lower as comparedto the above three references. In addition, none of the referencesreported the predominance of A. peroxydans, which represented49% of total clone count in RA. Therefore, the predominance of A.peroxydans was likely due to the unique operation (i.e. unintendedmicroaerobic conditions) of RA in this study. Shin et al. (2010) alsoshowed that the second-stage reactor consisted of membersaffiliated within four different phyla, Firmicutes, Proteobacteria,Spirochaetes, and Bacteroidetes. This is similar to the bacterialcommunity structure of RM in this study. Comparisons with exist-ing literature showed that the bacterial diversity of reactors treat-ing brown water and food waste shared some similarities with
those treating food waste only. They also suggested that the pre-dominance of Lactobacillus species in RA was largely due to the nat-ure of food waste.
3.2.5. Archaeal community in RM and RS
As shown in Fig. 4a and b, the diversity of archaeal clones in RM
and RS was limited to members of two orders: Methanosarcinalesand Methanomicrobiales with proportions of 69% and 30%, respec-tively for RM and 86% and 14%, respectively for RS. This indicatedthat methanogenesis took place preferentially via acetoclasticmetabolism for both single- and two-phase CSTRs. For RM, the mostdetected archaeal OTU (BFMArc_004), representing 48% of the totalclones, and the second dominant OTU (BFMArc_039), representing19% of total clones, were closely related to Methanosaeta species(AB479394) isolated from beer brewery effluent with 99% similar-ity. 30% of total archaeal count was composed of various speciesand clones within the genus Methanoculleus. They included BFM-Arc_134, BFMArc_103 and BFMArc_183 representing 21%, 7% and2% of total archaeal clones, respectively.
The predominant archaeal OTU for RS was BFSArc_019, whichrepresented 74% of total count, and had the same closest matchas BFMArc_004, the predominant archaeal OTU in RM. The seconddominant OTU (BFSArc_061), representing 14% of total clones,was affiliated with Methanoculleus species dm2 (AJ550158). Theremaining 12% of archaeal clones were closely related to Methan-osaeta species.
Methanogens can be categorized into two major groups accord-ing to the substrate they utilize. Acetoclastic methanogens con-sume acetate while hydrogenotrophic methanogens consume
200 J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201
hydrogen and formate for growth. Analysis of archaeal communi-ties in this study showed similar dominant groups and phyloge-netic distribution in single- and two-phase CSTRs. As shown inFig. 4a and b, Methanosarcinales and Methanomicrobiales, whichare acetoclastic and hydrogenotrophic methanogens, respectivelywere found in both RM and RS.
The order Methanosarcinales consist of genus Methanosarcinaand Methanosaeta. In this study, only Methanosaeta species weredetected and they formed the predominant archaeal group in bothRM (76% by cloning analysis) and RS (86% by cloning analysis). Theanalysis of archaeal communities in this study well agreed with areview of archaeal populations in anaerobic digesters where Meth-anosarcinales was reported to constitute more than 29% of the se-quences in all the studies, where sequences affiliated withMethanosaeta species were most frequently retrieved (Sekiguchiand Kamagata, 2004). The same study also found that the hydro-genotrophic pathway is commonly represented by Methanomicro-biales with proportions in a range of 1–29%.
As compared to Methanosarcina species, Methanosaeta specieshave higher substrate (i.e., acetate) affinity as well as lower maxi-mum specific growth rate (lmax) of 0.20 d�1 and half-saturationconstant (Ks) of 10–50 mg-COD L�1 (Vrieze et al., 2012). Accordingto Vrieze et al. (2012), Methanosaeta dominated at acetateconcentrations not exceeding 100–150 mg-COD L�1, whereasMethanosarcina became dominant at acetate concentrations above250–500 mg-COD L�1. In this study, the acetate concentrations inboth RM and RS were low, with average levels of 101 and 46 mg-COD L�1, respectively. This correlated with the dominance of Meth-anosaeta species, which are capable of scavenging acetate at lowacetate concentrations. The results in this study were in agreementwith earlier studies reporting the dominance of Methanosaeta inwell-operated mesophilic methanogenic systems with low effluentsoluble COD (Raskin et al., 1994; Ariesyady et al., 2007). Methan-osaeta was also shown to dominate reactors underfed with foodwaste (Williams et al., 2013). Hence, the predominance of Methan-osaeta and absence of Methanosarcina species suggested that RM
was at steady-state but working at less than optimum OLRs. TheOLR could be further increased to improve biogas production.
The analysis of archaeal communities in this study revealed thathigher levels of hydrogen-utilizing methanogens were present inRM (30%) than in RS (14%). This could be attributed to the feed ofRM, which contained higher levels of solubilized organic matteras compared to that of RS. Therefore, the greater extent of fatty acidfermentation in RM possibly led to increased hydrogen productionwhich encouraged the growth of more hydrogen-utilizing metha-nogens. Lerm et al. (2012) also detected a shift of hydrogenotrophicmethanogens due to increased VFA concentrations.
It was reported earlier that syntrophic degradation of propio-nate and butyrate is thermodynamically favorable, only when thehydrogen partial pressure is low enough (<10�4 atm) (Lowe et al.,1993; McCarty and Smith, 1986). Therefore, well establishedhydrogenotrophic methanogens possibly allowed the syntrophicVFA oxidizers to grow more quickly, and to degrade propionatemore rapidly, resulting in a more rapidly stabilizing digester(McMahon et al., 2001). The more diverse bacterial community inRM (28 OTU) as compared to RS (17 OTU) was also suggested to bedue to the higher prevalence of hydrogen-utilizing methanogens inRM. Similar findings were reported by St-Pierre and Wright (2013)where digesters with more hydrogenotrophic methanogens wereshown to support a greater level of phylogenetic diversity as com-pared to digesters predominated by acetotrophic methanogens.
3.3. FISH analyses
The FISH analyses of bacterial (green) and methanogenicarchaeal (red) populations in RA, RM and RS can be found in
Electronic annex 1. No methanogens and only bacterial cells offat and thin rods were detected in RA. This well agreed withthe predominance of Lactobacillus species and A. peroxydans inthe reactor, by cloning analysis, as shown earlier in Section 3.2.1.A mixed structure of bacterial and methanogenic archaeal popu-lations was observed in RM and RS as shown in Electronic annex1b and c, respectively. High bacterial diversity with differentmorphologies of small rods, fat rods, ovals, cocci, and thin fila-ments were detected in RM and RS. Bamboo-shaped Methanosae-ta-like and coccus-shaped Methanoculleus-like populations werealso observed in RM and RS. The morphologies of cells in RA, RM
and RS detected by FISH were consistent with the cloning andsequencing results.
3.4. Relationship between reactor performance and microbialcommunity structure
Although the effluent from RM of the two-phase CSTR con-tained higher levels of TVFA and soluble COD, it produced anaverage of 23% more methane as compared to the single-phaseCSTR. The main reason for higher methane production couldbe due to the greater extent of solubilization (represented bythe reduction of solids) and acidification in the two-phaseCSTR.
Through the determination of microbial diversity in the reac-tors, the greater degree of solubilization observed in the two-phaseCSTR was deduced to be attributed to the predominance and pres-ence of Firmicutes in RA and RM, respectively, and the lack of suchbacteria in RS. The unique operation (i.e., unintended microaerobicconditions) in RA possibly led to the co-existence of L. amylovorusand A. peroxydans, which could have enhanced the acidificationprocess in the two-phase CSTR. An earlier study showed thatmicroaeration resulted in greater degree of hydrolysis and acidifi-cation in anaerobic digesters (Lim and Wang, 2013). In addition, RM
had higher prevalence of hydrogen-utilizing methanogens as com-pared to RS. The syntrophic association between hydrogenotrophicmethanogens and fatty acid-oxidizing syntrophic bacterium couldhave also contributed to higher degree of acidification in the two-phase CSTR.
Methanosaeta species dominated the archaeal populations of RM
and RS and this correlated to low levels of acetate in their effluent.The greater distribution of hydrogen-utilizing Methanoculleus spe-cies in RM, and that of acetate-utilizing Methanosaeta species in RS
possibly led to the lower levels of acetate observed in RS as com-pared to RM. The higher prevalence of hydrogenotrophic methano-gens likely gave rise to increased bacterial diversity in RM. As aresult, RM became more resistant towards process disturbanceand shorten its recovery time.
4. Conclusions
The differences in biodegradation efficiencies for single- andtwo-phase CSTRs could be explained by their microbial commu-nity structures. Better reactor performance of two-phase CSTRcould be due to the presence of Firmicutes as well as greaterbacterial diversity and proportion of hydrogenotrophic methano-gens. The predominance of Lactobacillus, A. peroxydans andMethanosaeta, as well as possible syntrophic interactions betweenfermentative bacteria and hydrogenotrophic methanogens playedan important role in maximizing brown water and food wastedecomposition. The determination of microorganisms involvedin the co-digestion process would enhance the understanding ofthe complex biochemical interactions that promote digester sta-bility and performance.
J.W. Lim et al. / Bioresource Technology 147 (2013) 193–201 201
Acknowledgements
Authors are grateful to National Research Foundation (NRF),Singapore for financial support (NRF-CRP5-2009-02) as well asDr. Rajinikanth Rajagopal, for his advice and Ms. Mao Yu, Mr. AshiqAhamed and Mr. Bernard Jia Han Ng for their technical support inthis research.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.biortech.2013.08.038.
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