Bioresource Technology 137 (2013) 245–253

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Biore source Technology

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Influence of bicarbonate buffer on the methanogenetic pathway during thermophilic anaerobic digestion

0960-8524/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.03.093

Abbreviations: AM, acetoclastic methanogenesis; ARISA, automated ribosomal intergenic spacer analysis; HM, hydrogenotrophic methanogenesis; SAO, syn- trophic acetate oxidation; VFAs, volatile fatty acids.⇑ Corresponding authors. Tel./fax: +86 21 6598 1383 (F. Lü), tel./fax: +86 21 6598

6104 (P. He).E-mail addresses: lvfan.rhodea@tongji.edu.cn (F. Lü), solidwaste@tongji.edu.cn

(P. He).

Yucheng Lin a, Fan Lü a,⇑, Liming Shao c, Pinjing He b,⇑a State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, PR China b Institute of Waste Treatment and Reclamation, Tongji University, Shanghai 200092, PR China c Centre for the Technology Research and Training on Household Waste in Small Towns & Rural Area, Ministry of Housing and Urban-Rural Development of PR China (MOHURD),PR China

h i g h l i g h t s

� High concentrated bicarbonate promoted hydrogenotrophic methanogenesis.� High concentrate d bicarbonate promoted acid production, lowered acid methanization.� Higher concentration of organics led to a larger carbon isotope fractionation effect.� The boundary concentration of bicarbonate serving as buffer was 0.15 mol/L.

a r t i c l e i n f o

Article history:Received 14 January 2013 Received in revised form 10 March 2013 Accepted 13 March 2013 Available online 21 March 2013

Keywords:BicarbonateMethanogenetic pathway Stable isotope technique Automated ribosomal intergenic spacer analysis

a b s t r a c t

To investigate the influence of bicarbonate on the metabolic pathway of methanogene sis, different con- centrations of bicarbonate (0–0.2 mol/L) were applied during thermophilic anaerobic digestion of 2.5 and 5 g/L glucose. The stable carbon isotopic results demonstrated that, as the bicarbonate concentr ation increased, the proportion of total CH4 generated from hydrogenotrophic methanogenesis general lyincreased. Furthermore, methane production rates and acetate degradation rates were seriously reduced under high levels of bicarbonate (0.15 and 0.2 mol/L). Meanwhile, carbon isotope fractionation was more prominent in treatment s with 5 g/L glucose than that of 2.5 g/L glucose. Increased concentrations ofbicarbonate altered the dominant methanogens and bacteria and increased the microbial diversity. The inhibitory effects of high concentrations of bicarbonate suggested that bicarbonate should be used cau- tiously as a buffer salt in anaerobic processes, especially when methanogenetic pathways were studied.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Due to energy crisis and greenhouse effect, anaerobic digestion of biomass into methane is receiving greater attention. Biomass isconverted through a complex process consisting of hydrolysis, aci- dogenesis, acetogenesi s, and methanogenesi s, with CH4 productionby methanogenic archaea being the terminal step. There are two pathways for methane production from acids: acetoclastic metha- nogenesis (AM) and a tandem reaction of syntrophic acetate oxida- tion (SAO) and hydrogenotr ophic methanogenesi s (HM). The role

of SAO-HM pathway has been highlighted in recent years, owing to the widespread prevalence of the organisms that mediate this pathway and their possible tolerance to hostile environm ents such as anaerobic digesters with high concentratio ns of acid or ammo- nium (Goberna et al., 2009; Hao et al., 2011a,b ).

pH is one of the key paramete rs regulating the anaerobic pro- cess. Usually, a neutral pH range (6.6–7.8) (Lay et al., 1997; Ward et al., 2008 ) is preferable. pH as low as 5.5 would extend the lag- phase of methane production from acetate and change the domi- nant pathway of methanogenes is (Hao et al., 2012 ). pH higher than 8 is also adverse (Gao et al., 2010 ). Therefore, a buffer such as bicar- bonate or phosphate is often added to maintain a moderate pH. Asreviewed in Appels et al. (2008), a molar ratio of at least 1.4:1 ofbicarbonate /VFAs should be maintained for a stable and well-buf- fered digestion process. Vavilin et al. (2008) applied 0.15 mol/L NaHCO3 and 0.15 mol/L K2CO3 in the degradation of 16 g/L solid waste. Paulo et al. (2003) used 72 mmol/L phosphate as a pH buf- fer. For easily biodegradab le biomass, the imbalance between fast

246 Y. Lin et al. / Bioresource Technology 137 (2013) 245–253

acidification and slow methano genesis may result in substantial accumulation of volatile fatty acids (VFAs) (up to tens in g/L) and a sharp drop in pH (low to 4.0–5.5) (Hori et al., 2006; Staley et al., 2011 ). Consequently, the amount of buffering salts needed would be even larger.

High concentratio ns of buffer should then be taken into ac- counts with concerns of their potential effects on anaerobic diges- tion. For example, phosphat e buffer at 21 mM was optimal for protease production by Bacillus firmus, but higher concentrations inhibited cell growth and repressed protease production (Moonand Paruleka r, 1991 ). Methanogenes is around rice roots via the AM pathway was inhibited by 20 mM phosphate (Conrad et al.,2000). Similarly, 70 mM phosphat e completely suppressed the acetoclastic methano gens in thermophili c anaerobic sludge (Pauloet al., 2005 ). Comparativ ely, the potential effects of carbonate buf- fer are less known. Liu et al. (2012) discovered that carbonate has apositive effect on both H2 production and acetic acid generation,and attributed it to its pH buffer ability. However, little is known about whether carbonat e would inhibit methanogenes is and espe- cially affect the methanogenet ic pathway. In consideration of car- bonate’s direct contribution to HCO 3�, a substrate for HM and aproduct for SAO and AM pathway, it might act on the pathways shifting among AM, SAO and HM.

The present work aimed at investigatin g the influence of bicar- bonate on thermophilic methanogenet ic pathway. Stable isotope technique and automated ribosomal intergenic spacer analysis (ARISA) were employed to explore methano genetic pathways and the succession of microbial communi ty, respectively .

2. Methods

2.1. Preparation of thermophilic inoculum

Anaerobic granular sludge used in this study was collected from a laborator y-scale (3.5 L) anaerobic sequenced batch reactor (ASBR). The ASBR was operated at 55 �C in the dark, using glucose and acetate (80%: 20%, calculated as COD) as the substrate at an or- ganic loading rate of 2 g-COD/L/ d. The inoculum was taken after being acclimated for more than 3 months. When being sampled,the ASBR was gently shaken to get a representative and homoge- nous inoculum. No bicarbonate was added during the process ofacclimation .

2.2. Experimenta l setup

The anaerobic batch experiments were conducte d in the serum bottles (1.1 L) with 500 mL basal medium containing (per liter):0.2 g MgCl 2�6H2O, 1 g NH4Cl, 0.1 g CaCl 2, 0.2 g Na2S�9H2O, 2.77 gK2HPO4, 2.8 g KH2PO4, 0.1 g yeast extract, 5 mL trace element solu- tion, and 2 mL vitamin solution. The stock trace element solution contained (per liter) 1000 mg Na2-EDTA�2H2O, 300 mg CoCl 4,200 mg MnCl 2�4H2O, 200 mg FeSO 4�7H2O, 200 mg ZnCl 2, 80 mgAlCl3�6H2O, 60 mg NaWo 4�2H2O, 40 mg CuCl 2�2H2O, 40 mg NiSO 4-

�6H2O, 20 mg H2SeO4, 200 mg HBO 3 and 200 mg NaMoO 4�2H20.The vitamin solution consisted of (per liter) 10 mg biotin, 50 mgPyridoxinHC l, 25 mg ThiamineHCl, 25 mg D-Calsium pantothenate,10 mg Folic acid, 25 mg Riboflavin, 25 mg Nicotinic acid, 25 mgP-aminobenzi c acid and 0.5 mg vitamin B1. The added phosphate was to provide basic buffer capacity. The sludge concentr ation was 3 g of volatile suspended solids per liter.

Glucose was used as the carbon source at 2.5 and 5 g/L. For each glucose level, sodium bicarbonate was added at a final concentra- tion of 0, 0.05, 0.1, 0.15, and 0.2 mol/L. Endogenous methanogene- sis from the inoculated sludge was studied in a blank without glucose and sodium bicarbonate addition. Therefore, there were

11 sets of experiments named L0, L0.05, L0.1, L0.15, and L0.2 for 2.5 g-glucose/L treatments at different bicarbonate levels; H0,H0.05, H0.1, H0.15, and H0.2 for 5 g-glucose/L treatments; and BLfor blank. Different amounts of sodium chloride were then added to get an identical Na+ concentr ation (about 0.2 mol/L) in every bottle. The initial pH was adjusted to 7.5 by either NaOH or HCl.

After closing each serum bottle with a butyl rubber and alumin- ium cap, the gas in the headspace of each bottle was exchanged bythree cycles of vacuuming and refilling with oxygen-free N2

(99.999%). All the reactors were then incubated at 55 �C in the dark. All eleven treatments were conducted in duplicate. Data are reported as the arithmetic mean ± standard deviation. Liquid, gas,and granular samples were periodica lly collected under anaerobic condition s until all the glucose was consumed.

2.3. Analysis of gas and liquid samples

Gas composition (CH4, CO2, and H2) was analyzed using a gas chromatogr aph (GC112A, Shanghai Precision and Scientific Instru- ment CO., LTD, China). Gas pressure in the serum bottles was deter- mined by a Testo 512 manometer to calculate biogas production.Gas samples were also collected periodica lly by vacuum tubes tomeasure the stable isotope signatures of CH4 (dCH4) and CO2

(dCO2). The isotopic analyses were performed using a GV isoprime mass spectrometer linked to an Agilent 6980N gas chromatogr aph with a CP-poraplot Q column (25 m � 0.32 mm � 20 lm) following the protocol used in Hao et al. (2011a).

Liquid samples were analyzed for pH, VFAs, and total organic car- bon (TOC). pH was measured immediately after sampling using a pHmeter (pHS-2F, Shanghai Precision and Scientific Instrument Co.,LTD, China). Liquid samples were filtered through 0.22- lm polyes- ter filters prior to VFAs and TOC measurement. VFAs (including for- mate, acetate, propionate, n-butyrate, iso-butyrate, n-valerate and iso-valer ate) were determined by a high performance liquid chro- matograp hy (LC-20AD, Shimadzu, Japan) equipped with a COD- 10Avp electrical conductivi ty detector, a CTO-10ASvp column oven,a SCL-10Avp system controller, a Shim-pack SPR-H analytical col- umn, a Shim-pack SPR-H(G) guard column and a LCsolutio n Ver 1.1 single workstation . The mobile phase used in the study was 4 mmol L�1 p-Toluensulfoni c acid. The buffer phase containe d16 mmol L�1 Bis–Tris, 4 mmol L�1 p-Toluens ulfonic acid and 100 lmol L�1 EDTA. The flow rate of the mobile phase and the buffer phase was 0.8 mL/min. The column temperature was 45 �C, while the temperature of the conductivity was 48 �C. TOC was measured with a total organic carbon survey meter (TOC-V CPH, Shimadzu,Japan) using the 680 �C combustion catalytic oxidation/ND IRmethod.

2.4. Data fitting to the modified Gompertz model

In this study, the modified Gompert z three-pa rameter model (Behera et al., 2010 ) was fitted to the cumulative CH4 productioncurves to determine the maximum CH4 production rate (Rmax)and the lag phase (k). The equation used had the following form Eq. (1):

MðtÞ ¼ P � exp � expRmax � e

Pðk� tÞ þ 1

� �� �ð1Þ

where, M(t) is the cumulative CH4 product ion (mmol CH4/g-glu cose)at time t; P is the maximu m CH4 potential (mmol CH4/g-glucose) atthe end of the incubation; t the time (d); Rmax is the maximum CH4

product ion rate (mmol CH4/(g-glucose�d)); k is the lag phase (d),and e is 2.71828.

Y. Lin et al. / Bioresource Technology 137 (2013) 245–253 247

2.5. Data processing of stable isotope signatures

The apparent carbon fractionati on factor (ac) was calculated using Eq. (2):

ac ¼dCO2 þ 103

dCH4 þ 103 ð2Þ

The fraction of methane produced from the HM pathway (fmc),defined as the fraction of CH4 derived from H2/CO2 relative to total CH4, was determined by Eq. (3):

fmc ¼dCH4 � dmadmc� dma

ð3Þ

where, dCH4 and dCO2 are the 13C isotope signatures of total CH4

and CO2; dma and dmc are the 13C isotope signatures of CH4 derivedfrom acetate and CO2, respective ly. Detailed information for fmc cal-culation was listed in the Section S1 of Supplementa ry materials .

2.6. DNA extraction and ARISA analysis of bacterial and archaeal community structure

Granular sludge samples from the serum bottles were stored at�80 �C before being further processed. Total DNA was extracted using the PowerSoil™ DNA isolation kit (Mo-Bio Laboratories Inc., CA). In the ARISA experiments, the extracted DNA was ampli- fied using primers 1389F (50-ACGGGCGGTGTG TGCAAG-3 0) and 71R (50-TCGGYGCCGA GCCGAGCCA TCC-3 0) for archaea (Qu et al., 2009 )and primers ITSF (50-GTCGTAA CAAGGTAGCCG TA-3 0) and ITSReub (50-GCCAAGGCATCC ACC-3 0) for bacteria (Cardinale et al., 2004 ).PCRs were performed in a thermal cycler (Bio-Rad C1000 TM,USA). The reaction mixture containe d: 1 � PCR buffer, 1 U of Taq DNA polymerase, 0.2 mmol/L (each) deoxynucle oside triphosph ate and 0.5 nmol/L (each) primer in a final volume of 25 lL. For ar- chaea, the mixture was held at 95 �C for 5 min, followed by 35 cy- cles of 94 �C for 1 min, 54.8 �C for 1 min, 72 �C for 2 min, and a finalextension at 72 �C for 10 min. For bacteria, the procedure was asfollows: 94 �C for 5 min, followed by 35 cycles of 94 �C for 1 min,54.5 �C for 1 min, 72 �C for 2 min, and a final extension at 72 �Cfor 10 min. ARISA of the PCR product was carried out using the Agi- lent DNA 7500 kit (Agilent Technologies Inc., CA) and an Agilent 2100 Bioanalyzer (Agilent Technologie s Inc.).

Gel-like images of ARISA were converte d to decimal data using the software Quantity One version 4.6.2 (Bio-Rad, USA) to obtain the band intensity in every lane. Both density and position of the bands were then subjected to Shannon–Wiener diversity index (H

0) calculation and principal component analysis (PCA) using the

software PAlaeontologi cal Statistics (PAST) version 2.17b accordin gto Hammer et al. (2001).

3. Results

3.1. Methanogen ic conversion of glucose

The data on methane production was shown in Fig. 1a and b and fit with a Gompert z model (Table 1). For 2.5 g-glucose /L treat- ments, methano genesis started immediatel y when glucose was added (Fig. 1a). During days 0–5, there was no apparent differenc ein CH4 generation under different concentrations of bicarbonate.After that, the generation rate of CH4 decreased as bicarbonate con- centration increased. The maximum rates in treatments L0, L0.05,L0.1, L0.15, and L0.2 were 2.68, 2.95, 2.02, 1.14, and 1.27 mmol CH4/(g-glucose�d), respectively, demonstrating slowed methane production at 0.15 and 0.2 mol/L bicarbonate. The ultimate meth- ane yields were 17.41–17.74 mmol CH4/g-glucose for treatments L0, L0.05, L0.1, and L0.15, and only 15.68 mmol CH4/g-glucose for

L0.2. Among treatments with 5 g-glucose/L, a similar trend ofmethane generation occurred (Fig. 1b), except that there was alag time for treatment H0, which lacked bicarbonate . The lag re- sulted from insufficient buffer capacity, which led to a lower pHto 4.7. The methane yield from the BL control was only 0.14 mmol,accountin g for no more than 1% of the yields in the glucose- amended treatments . Hence, the endogenous methanogenesi sfrom the inoculum was negligible.

These data demonst rated that, although high-concentra ted bicarbonate could provide more alkalinity and thus shorten the lag time of methane production. Methane production slowed down under such conditions. Smaller methane production rate also oc- curred at 5 g-glucose /L treatments compared with 2.5 g-glucose/L treatments. Comparativ ely, the ultimate methane yields were less affected by either bicarbonate or glucose.

VFAs are generated from glucose by acidogenesi s and further degraded into methane by acetogenesi s and methanogenesi s. Ace- tate was the dominant VFA in every treatment in the present study.Butyrate at maximum concentr ations of 8.9 and 4.9 mmol/L oc- curred in treatments H0 and H0.05, respectively . Other VFAs barely existed and thus could be neglected. VFAs were not detected in the BL control during the entire process. As shown in Fig. 1c and d, ace- tate rapidly accumulate d during the initial phrase of incubation.For 2.5 g-glucose/L treatments , the acetate concentr ation reached a maximum on day 2 at every level of bicarbonate. The value ofthe peak concentr ation of acetate rose with increasing bicarbonate concentr ations. Peak values of 8.7, 12.1, 14.5, 14.1, and 15.0 mmol/ L occurred in treatments L0, L0.05, L0.1, L0.15, and L0.2, respec- tively. The accumulate d acetate decreased to no more than 1 mmol/L in 7 days in treatments L0 and L0.05, whereas 11, 35,and 49 days were required in treatments L0.1, L0.15, and L0.2,respectively . For 5 g-glucose /L treatments, the values of the peak acetate concentratio n were 15.4, 13.9, 22.0, 25.1, and 26.8 mmol/ L in treatments H0, H0.05, H0.1, H0.15, and H0.2, respectively .The accumulated acetate decreased to no more than 1 mmol/L in11 and 9 days for treatments H0 and H0.15, respectivel y, whereas 13, 23, and 35 days were required in treatments H0.1, H0.15, and H0.2, respectively . For treatment H0, there was a slight increase in the acetate concentratio n on day 13, because the generate dbutyrate (up to 8.9 mmol/L) was degraded to acetate. For both con- centrations of glucose (2.5 and 5 g/L), the peak value of acetate concentr ation rose with the increase of bicarbonate , suggesting that bicarbonate promoted the fermentation of glucose. Compara- tively, the degradation of the accumulate d acetate slowed down with the increasing concentration of bicarbonate.

Since acetate was the dominant VFAs in the present study, VFAs concentr ation (Fig. 1e and f) had a similar trend to that of acetate.Peak values of 10.0, 12.1 14.5, 14.1 and 15 mmol/L (acetic acid equivalent) occurred in treatments L0, L0.05, L0.1, L0.15, and L0.2, respectively . For 5 g-glucose/L treatments , the values were 21.0, 23.6, 22.0, 25.1 and 26.8 mmol/L (acetic acid equivalent) intreatment H0, H0.05, H0.1, H0.15 and H0.2, respectively.

3.2. Methanog enic pathway analysis by stable carbon isotope signatures

The method of natural stable carbon isotopic signature is effec- tive to distingui sh the AM and HM methanogenic pathways (Haoet al., 2011a,b; Qu et al., 2009 ). Owing to the fact that isotope frac- tionation is much stronger for HM than AM (Conrad, 2005; Heet al., 2009 ), d13CH4 from HM is usually low and declines further,whereas the corresponding d13CO2 might increase significantly ow- ing to the high fractionation factor (about 1.08) for CO2 consump -tion in HM and the comparative ly low fractionation factor (about1.00–1.01) for CO2 production via SAO. In contrast, d13CH4 fromAM will usually increase with heavier acetate remaining, whereas

(a)

(c) (d)

Time (d)0 10 20 30 40 50

VFAs

(mm

ol/L

)

0

5

10

15

20

25

30

Time (d)0 10 20 30 40 50

Acet

ate

(mm

ol/L

)

0

5

10

15

20

25

30

Cum

ulat

ive

CH

4 Pro

duct

ion

(mm

ol/g

-glu

cose

)

0

5

10

15

20

(e) (f)

(d)(c)

(a) (b)

Fig. 1. Temporal change of the cumulative CH4, acetate and VFAs. (a, c, e) 2.5 g-glucose/L, (b, d, f) 5 g-glucose/L. Bicarbonate concentration (mol/L): d 0, s 0.05, . 0.1,4 0.15,j 0.2. The results are means of two replicates. Error bars represent the data range.

Table 1Calculated results using the modified Gompertz equation for methanogenesis under different conditi ons.

Treatment Lag time k (d) CH4 production rate Rmax [mmol CH4/(g-glucose d)] Ultimate CH4 yield P (mmol CH4/g-glucose) Coefficient R2

L0 1.41 ± 0.15 2.68 ± 0.08 17.70 ± 0.13 0.992 L0.05 0.93 ± 0.16 2.95 ± 0.09 17.41 ± 0.15 0.987 L0.1 0.25 ± 0.25 2.02 ± 0.09 17.73 ± 0.20 0.981 L0.15 0 1.14 ± 0.11 17.74 ± 0.37 0.952 L0.2 0 1.27 ± 0.11 15.68 ± 0.31 0.949 H0 3.12 ± 0.25 1.44 ± 0.06 16.76 ± 0.20 0.991 H0.05 1.50 ± 0.20 2.50 ± 0.10 17.64 ± 0.17 0.987 H0.1 0.64 ± 0.10 2.28 ± 0.04 17.20 ± 0.09 0.996 H0.15 0 1.31 ± 0.06 17.02 ± 0.20 0.986 H0.2 0 0.68 ± 0.09 18.39 ± 0.91 0.948

Note: The parameter standard error was estimated according to the weighted regression.

248 Y. Lin et al. / Bioresource Technology 137 (2013) 245–253

the correspond ing d13CO2 might stay relatively constant owing tothe low fractionation factor in AM (Methanos aeta 1.007–1.010, Met-hanosarcina 1.022–1.027) (Conrad and Klose, 2011 ).

For 2.5 g-glucose/L treatments amended with bicarbonate , dur- ing the first 5 days of incubation when 38–54% of CH4 was gener- ated, the dCH4 of the four was initially �65.6‰–�70.7‰ andincreased to �17.9‰–�32.9‰, whereas the dCH4 changed from �35.7‰ to �29.9‰ in treatment L0 lacking bicarbonate (Fig. 2a).These results suggested the AM pathway was more dominan t inthe initial period of methanogenes is in the four bicarbonate- amended treatments . From day 7 to day 18 (61–95% CH4 was

generate d), the dCH4 slightly decreased in the L0, L0.05, L0.1,L0.15 and L0.2 treatments, from �11.9‰, �20.6‰, �18.9‰, �31.0‰,�28.0‰, respectively, to �22.1‰, �39.1‰, �23.3‰, �45.7‰,�36.7‰. From day 21 to day 35 (84–100% CH4 was generated), the cor-responding dCH4 decreased faster, from �40.1‰, �40.8‰, �37.0‰,�69.1‰, �53.6‰ to �41.1‰, �54.9‰, �65.1‰, �78.3‰, �72.7‰,respectively. In general, the dCH4 values from the L0.15 and L0.2 treat-ments were lower than the values from treatments with lower bicar-bonate concentrations. The decrease in dCH4 suggested an increasingprevalen ce of HM relative to AM. In particular, the minimum value dCH4 was �78.3‰ and �72.7‰ for treatments L0.15 and L0.2,

δδ

α

(a) (b)

(c) (d)

(e) (f)

(g) (h)

Fig. 2. Temporal change of stable isotopic indicators dCH4, dCO2, ac and fmc. (a, c, e, g) 2.5 g-glucose/L, (b, d, f, h) 5 g-glucose/L. Bicarbonate concentration (mol/L): d 0, s 0.05,. 0.1, 4 0.15, j 0.2.

Y. Lin et al. / Bioresource Technology 137 (2013) 245–253 249

suggesting dominance of HM during the later period ofmethanogenes is.

For 5 g-glucose/L treatments, the trend was quite similar, but agreater isotope fractionation effect was observed, with a larger dif- ference between low and high bicarbonate levels (Fig. 2b). During the initiation period (day 1 to day 9), the dCH4 of the four bicarbon- ate-amended treatments quickly increased from �62.5‰, �66.8‰,�67.5‰, �67.3‰ to �14.4‰, �22.9‰, �42.3‰, �53.3‰, while the dCH4 of the H0 treatment lacking bicarbonate rose from �37.3‰ to�13.6‰. Subsequentl y, the dCH4 gradually decreased from the peak values to �38.9‰, �48.9‰, �64.6‰, �70.0‰ and �77.5‰,

respectively , in treatments H0, H0.05, H0.1, H0.15 and H0.2, with- out any delay. The dCH4 values significantly decreased with increasing bicarbonate concentratio n.

The dCO2 value gradually increased during the incubation peri- od, and the average dCO2 value decrease d with increasing bicar- bonate concentr ation (Fig. 2c, d). For treatments L0, L0.05, L0.1,L0.15 and L0.2 with 2.5 g/L glucose, dCO2 increased from �2.7‰,�23.4‰, �26.5‰, �27.4‰, �28.3‰ to 14.7 ‰, �1.5‰, �13.0‰,�12.9‰, �17.7‰, respectivel y. For the correspondi ng treatments with 5 g/L glucose, dCO2 increased from �0.9‰, �23.6‰,�26.5‰, �26.8‰, �28.5‰ to 11.1 ‰, �3.3‰, �2.2‰, �0.3‰,

Time (d)0 10 20 30 40

pH

5

6

7

8

9

pH

6.0

6.5

7.0

7.5

8.0

8.5

9.0

(a)

(b)

Fig. 3. Dynamics of pH. (a) 2.5 g-glucose/L, (b) 5 g-glucose/L. Bicarbonate concen- tration (mol/L): d 0, s 0.05, . 0.1, 4 0.15, j 0.2. The results are means of two replicates. Error bars represent the data range.

250 Y. Lin et al. / Bioresource Technology 137 (2013) 245–253

�2.0‰, respectively . It should be noted that the measure d dCO2

may have been seriously affected by the background isotopic value of the added bicarbonate.

The apparent carbon isotopic fractionation factor (ac) was also used to characteri ze the methanogeni c environments, with a high- er value representing a larger contribution of the HM pathway tototal methane production. Usually, ac > 1.065, ac < 1.025, and ac

around 1.045 were characteristic for HM, AM, and the combination of HM and AM, respectivel y (Conrad, 2005; Conrad and Klose,2011). As shown in Fig. 2e and f, for the treatments L0, L0.05,L0.1, L0.15, and L0.2, ac was around 1.035–1.045 during the initia- tion period (days 1–3), and then decreased to the minimal values of 1.019, 1.014, 1.001, 1.011, and 1.005, respectively , on day 5 or7, suggesting the dominan ce of AM pathway at this time. In the fi-nal phase, ac eventually rose to 1.058, 1.057, 1.056, 1.071 and 1.059 in treatments L0, L0.05, L0.1, L0.15, and L0.2, respectively, indicat- ing a shift to dominance of the HM pathway. For 5 g-glucose/L treatments H0 through H0.2, ac also decreased in the initiation per- iod from around 1.04 to 1.020, 1.013, 1.016, 1.030, 1.037, respec- tively. However, the values quickly increased to 1.052, 1.055,1.067, 1.075, and 1.082, respectivel y, without any delay, suggestin gan increasing dominance of HM pathway. At low bicarbonate levels of 0–0.05 mol/L, ac remained below 1.045 before the stationary phase (days 1–17, 91–95% CH4 produced). At the high bicarbonate levels of 0.15 and 0.2 mol/L, ac rose to 1.075 and 1.082 on day 29,characterist ic of exclusive CH4 production via the HM pathway (Fey et al., 2004 ).

The fraction of methane produced from the HM pathway (fmc)had a similar trend as ac (Fig. 2g, h). For 2.5 g-glucose/L treatments,fmc decreased from 0.50 to 0.57 to around 0 in the four bicarbon- ate-amended treatments (days 1–5), while the peak value was 0.11 during this time in treatment L0 lacking bicarbonate. Subse- quently, fmc remained at 0 in treatments L0 to L0.2 for 13, 13, 7,5, and 6 days, respectivel y, and then increased. The final value offmc was 0.30, 0.52, 0.61, 0.86, and 0.69 in the five treatments. Asa result, the CH4 generated via the HM pathway was calculated to account for 4.7%, 18.5%, 15.9%, 34.5%, and 29.8% of the total CH4 in treatments L0, L0.05, L0.1, L0.15, and L0.2, respectively.For 5 g-glucose /L treatments, a similar trend occurred, but fmc

was much higher in treatments H0.15 and H0.2. fmc remainedabove 0 during the entire process and reached 0.89 and 1 at the end of the incubation. The HM pathway was calculated to contrib- ute 3.7%, 10.7%, 17.7%, 39.9%, and 58.7% to the total CH4 productionduring the entire process in treatments H0, H0.05, H0.1, H0.15, and H0.2, respectively .

pH was not controlled in the present study, and relatively large pH differenc es occurred among the bicarbonate levels (up to 1.7).At high bicarbonate levels (0.15 and 0.2 mol/L), the pH rose to8.5–8.6 on day 7 in the 2.5 g-glucose /L treatments (Fig. 3), concom- itant with an obvious deceleration of methanogenes is and acetate degradation . Similar results occurred in the 5 g-glucose/L treat- ments. Lower pH (3.8–5.5) has been observed to promote the SAO-HM pathway (Hao et al., 2012; Kotsyurbenk o et al., 2007 ).However, until now, no research has been done on the effect ofhigh pH values (>7.0) on methano genic pathways. Qu, (2007)maintained pH values between 8.0 and 9.0 when studying metha- nogenesis in solid waste and found that the dominant methano- genic pathway was AM most of the time. In the present study,higher pH values generally occurred in treatments with 2.5 g/L glu- cose compare d to 5 g/L glucose, but a much stronger isotope frac- tionation effect was observed in the latter. In the treatments with 5 g/L glucose, small differences in pH (<0.2) occurred among bicar- bonate concentr ations of 0.1, 0.15 and 0.2 mol/L, but differences inthe isotope fractionation effect were substanti al, suggestin g that the changes in methano genic pathways were more likely induced by different bicarbonate concentratio ns than by pH changes.

Meanwhi le, the pH changes would affect the concentratio n offree ammonia, a key inhibitor for anaerobic digestion. In the pres- ent study, 1 g/L NH4Cl was added in the medium, the maximum concentr ation of free ammonia is estimated to be 90 mg-N/L,which is far below the inhibitory value 230–1500 mg-N/L reported in literature (Bujoczek, 2001; Hansen et al., 1998; Lü et al., 2013 ).Therefore, the ammonia effect caused by increasing pH was negligible.

The addition of bicarbonate affects the CO2 concentratio n. CO2

is the product of glucose fermentati on, acetoclastic methano gene- sis, and syntrophic acetate oxidation, whereas it is a substrate inhydrogen otrophic methano genesis. According to thermodynam icanalysis, higher concentratio ns of bicarbonate would be unfavor- able for AM and SAO and favorable for HM. Since SAO and HMare more sensitive to the H2 partial pressure, increased HM at high- er concentratio ns of bicarbonate should theoretically improve SAO as well. As a result, the tandem reaction of SAO-HM will be en- hanced as a whole at higher concentr ations of bicarbonate . In the present study, this phenomeno n obviously occurred and was animportant component of methane dynamics in the test systems.At the later stage of the experiment, ac at the higher bicarbonate levels rose to 1.07–1.082, and fmc reached 0.7–1.0, which showed that the HM pathway gradually became the dominant methano- genic pathway. These results were consisten t with the thermody- namic considerati ons.

Besides, different isotope fractionation effects occurred in treat- ments with different glucose concentr ations. Treatments with 5 g/L glucose and 0.15 or 0.2 mol/L bicarbonate promoted the HMpathway (lower dCH4 and higher ac and fmc) much more than cor- respondi ng treatments with 2.5 g/L glucose, which could be ex- plained by that more acids were produced by 5 g/L glucose. Hao

Fig. 4. Microbial community structure of the sludge under different levels of glucose and bicarbonate using ARISA (d means time (days)). (a) Archaea 2.5 g-glucose/L, (b)archaea 5 g-glucose/L, (c) bacteria 2.5 g-glucose/L, (d) bacteria 5 g-glucose/L.

Fig. 5. Principal component analysis of the ARISA data. (a) Archaea (component 1 vs. component 2), (b) bacteria (component 1 vs. component 2), (c) bacteria (component 1 vs.component 3). The four time points (day) are connected by lines and form a quadrangle for every treatment.

Y. Lin et al. / Bioresource Technology 137 (2013) 245–253 251

L0 L0.05 L0.1 L0.15 L0.2 H0 H0.05 H0.1 H0.15 H0.2

(b)

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Shan

non-

Wie

ner d

iver

sity

inde

x (H

')

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1 (a)

(b)

seed

seed

Fig. 6. Shannon–Wiener diversity index of microbial community. (a) Archaea, (b)bacteria.

252 Y. Lin et al. / Bioresource Technology 137 (2013) 245–253

et al. (2011a) found the dominant contribution of SAO-HM path- way to thermop hilic methanogenes is at high organic loading rate,Lü et al. (2013) reported a gradual strengthening of SAO-HM path- way along with the increase of acetate concentration. Goevert and Conrad (2009) discovered that carbon isotope fractionati on was affected by the acetate concentratio n, and the values of the isotope enrichment factor of total acetate (eac) and acetate-met hyl (eac-methyl) increased as acetate concentratio ns increased. Qu et al.(2009) found that changes in the concentratio ns of VFAs could lead to different isotope fractionation effects and even a metabolic pathway shift for Methanosarcinac eae .

From these observations, it was inferred that higher bicarbonate concentratio ns promote d the SAO-HM pathway, especially when 5 g/L glucose was added. Since the efficiency of acetate degradation by SAO-HM is generally lower than that by AM (Schnürer et al.,1999), the methane production rate should be lower at higher bicarbonate concentrations , as observed in the present study (Fig. 1).

3.3. Dynamics of microbial community

The archaeal and bacterial community structures along the incubation were analyzed by ARISA. The number, position and den- sity of the bands on the ARISA profile could suggest the microbial dynamics. In the archaeal profiles (Fig. 4a and b), a total of 10bands were detected. Bands at 436-bp and 545-bp were the darkest in the seed sludge. For L0 and H0 treatments, the 327-bp band was the most intense, and the 545-bp band was also rela- tively abundant. The 423-bp band appeared at day 6 and day 7and gradually became dominan t in L0.05 and H0.05 treatments.The 423-bp band was also the most intense in the L0.1 and H0.1 treatments, while the 545-bp band was darker in L0.1 than inH0.1. The 545-bp band was the dominant band for L0.15, L0.2

and H0.2 treatment, while in the H0.15 treatment, the 545-bp was dominant at day 2 but the 423-bp band was dominant onday 7, 17 and 35. Furthermore, the intensity of 658- and 620-bp bands gradually strengthene d as the bicarbonate concentr ation in- creased. Based on the present complete genome database of meth- anogens (National Center of Biotechnolo gy Information, NCBI), the ARISA fragment length for Methanosarci nales is lower than 580 bp(see Section S2 of the Supplement ary materials ). Therefore, the 620-bp and 658-bp bands may represent strictly hydrogen otrophic methano gens, which were favored by the bicarbonate addition. For bacteria, 15 bands were detectable on the profile (Fig. 4c and d).The 180-bp and 300-bp bands were present in every treatment,while the intensity of the 215-bp and 300-bp bands generally went up with the bicarbonate concentration. The intensity of the 260-bp,280-bp, and 443-bp bands fluctuated with bicarbonate concentr ation.

Principal component analysis, which was widely applied to ana- lyze environmental DNA fingerprints (Ramette, 2007 ), was per- formed to compare these ARISA band patterns under different condition s (Fig. 5). For archaeal profiles, component 1 (40.0%)and component 2 (33.3%) together explained 73.3% of the variance.The highest loading for component 1 was from the 545-bp band (0.622), and then followed by the bands 360-bp (0.414), 620-bp (0.388), and 658-bp (0.366), while the 423-bp band influencedcomponent 2 most.

Bicarbonate concentration significantly affected the scores ofcomponent 1. Treatment L0 and H0 were clearly distinct from treatment L0.2 and H0.2 on the axis of component 1, and the scores of component 1 generally went up with increasing bicarbonate concentr ation, consistent with the enrichment of the 545-bp,620-bp and 658 bp bands. The score of component 2 reflectedthe fluctuation of the proportion of the 423-bp band. For bacterial band patterns, component 1 (47.6%), component 2 (19.9%), and component 3 (12%) totally accounted for 79.5% of the variance.The 215-bp band and 280-bp band influenced component 1 most,band at 180-bp affected component 2 most, and component 3 was influenced by the 310-bp mostly. Differences were observed in the scores of component 1 and component 2, demonstrating the change of dominant bacteria induced by glucose and bicarbonate concentr ation.

The Shannon–Wiener index of diversity (H0) (Ye et al., 2007 )

was calculated as well to determine the diversity of microbial com- munity. As indicated in Fig. 6, treatment H0.2 apparent ly had high- er values of H0 for both archaea and bacteria, demonstrat ing greatest archaeal and bacterial diversity.

By comparing the N2 atmosph ere and 30% CO2/70% N2 atmo-sphere, Hansson (1982) found higher CO2 partial pressure inhibited the fermentative bacteria and acetoclastic methano gens, and ex- plained that CO2 was assumed to be dissolved in cell membranes which would increase membran e fluidity and impair its function.As reviewed Cooper et al. (2002), CO2/HCO3

- could cause a sus- tained fall of intraceular pH, i.e. CO2-induced acidification, result- ing in increasing cell lysis. Therefore, bicarbonate could be aninhibitory compound for methanogens. In the present study, bicar- bonate significantly influenced the archaeal community , with much higher value of Shannon–Wiener diversity index (H0) was ob- served for both the bacterial and archaeal communitie s of treat- ment H0.2. Since methanogenic pathways were directly related to archaeal community structure, bicarbonate could influence the prevalen ce of different methanogeni c pathways . Especially, the intensified 620-bp and 658-bp bands could suggest hydrogen o-trophic methanogens were promote d by high concentrated bicarbonate . The ARISA profiles for bacteria showed dynamic changes of dominant bands and more diverse bands at treatment H0.2, which might be induced by the change of quantity of syn- trophic acetate-oxidizing bacteria.

Y. Lin et al. / Bioresource Technology 137 (2013) 245–253 253

4. Conclusions

The hydrogenotr ophic methanogenesi s during thermophili canaerobic digestion was found to be promoted by higher concen- tration of bicarbonate, and strengthene d by higher organic loading rate. Since high organic loading rates usually cause a stronger fluc-tuation in pH, increasing the need for bicarbonate , the effect ofbicarbonate would be more substanti al. The inhibitory effects ofhigh concentratio ns of bicarbonate suggested that bicarbonate should be used cautiousl y as a buffer salt in anaerobic processes,especially when methanogenet ic pathways were studied.

Acknowled gements

This research was partially sponsored by National Basic Re- search Program of China (973 Program, No. 2012CB7 19801), Na- tional Natural Science Foundati on of China (No. 51178327; No.21177096), Innovation Program of Shanghai Municipa l Education Commission (No. 13ZZ030), the Shanghai Pujiang Program (No.11PJ140920 0), Fundame ntal Research Funds for the Central Uni- versities (No. 0400219195), State Key Laboratory of Pollution Con- trol and Resource Reuse Foundation (No. PCRRY11 008).

Appendix A. Supplemen tary data

Supplement ary data associated with this article can be found, inthe online version, at http://dx.doi.o rg/10.1016/ j.biortech.2013.03.093.

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