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

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  • eth

    hai

    ed hyded acid

    a largenate se

    of total CH4 generated from hydrogenotrophic methanogenesis general ly

    of biomass into methane is receiving greater attention. Biomass is

    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

    as anaerobic digesters with high concentratio ns of acid or ammo-

    e anaerobt al., 1997

    et al., 2008 ) is preferable. pH as low as 5.5 would extend tphase of methane production from acetate and change thenant pathway of methanogenes is (Hao et al., 2012 ). pH high8 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

    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. L), tel./fax: +86 21 6598

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

    Bioresource Technology 137 (2013) 245253

    Contents lists available at

    Biore source T

    journal homepage: www.els(P. He).converted 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

    nium (Goberna et al., 2009; Hao et al., 2011a,b ).pH is one of the key paramete rs regulating th

    cess. Usually, a neutral pH range (6.67.8) (Lay e0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.03.093ic pro- ; Ward he lag- domi- er than Available online 21 March 2013

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

    increased. 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 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 Received in revised form 10 March 2013 Accepted 13 March 2013

    5 g/L glucose. The stable increased, the proportionh i g h l i g h t s

    High concentrated bicarbonate promot High concentrate d bicarbonate promot Higher concentration of organics led to The boundary concentration of bicarbo

    a r t i c l e i n f o

    Article history:Received 14 January 2013 rogenotrophic methanogenesis. production, lowered acid methanization.r carbon isotope fractionation effect.rving as buffer was 0.15 mol/L.

    a b s t r a c t

    To investigate the inuence of bicarbonate on the metabolic pathway of methanogene sis, different con- centrations of bicarbonate (00.2 mol/L) were applied during thermophilic anaerobic digestion of 2.5 and

    carbon isotopic results demonstrated that, as the bicarbonate concentr ation 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 Inuence of bicarbonate buffer on the mthermophilic anaerobic digestion

    Yucheng Lin a, Fan L a,, Liming Shao c, Pinjing He b,a State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shangbanogenetic pathway during

    200092, PR China

    SciVerse ScienceDi rect

    echnology

    evier .com/locate /bior tech

  • cially affect the methanogenet ic pathway. In consideration of car-

    Anaerobic granular sludge used in this study was collected from

    method.

    chnoa 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 26H2O, 1 g NH4Cl, 0.1 g CaCl 2, 0.2 g Na2S9H2O, 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-EDTA2H2O, 300 mg CoCl 4,200 mg MnCl 24H2O, 200 mg FeSO 47H2O, 200 mg ZnCl 2, 80 mgAlCl36H2O, 60 mg NaWo 42H2O, 40 mg CuCl 22H2O, 40 mg NiSO 4-6H2O, 20 mg H2SeO4, 200 mg HBO 3 and 200 mg NaMoO 42H20.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 Riboavin, 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 nal concentra- bonates 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 inuence 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 acidication 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 4.05.5) (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 rmus, 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-

    246 Y. Lin et al. / Bioresource Tetion 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 2.4. Data tting to the modied Gompertz model

    In this study, the modied Gompert z three-pa rameter model (Behera et al., 2010 ) was tted 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):

    Mt P exp exp Rmax eP k t 1

    1

    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) at2.3. Analysis of gas and liquid samples

    Gas composition (CH4, CO2, and H2) was analyzed using a gas chromatogr aph (GC112A, Shanghai Precision and Scientic 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 Scientic Instrument Co.,LTD, China). Liquid samples were ltered through 0.22- lm polyes- ter lters 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 L1 p-Toluensulfoni c acid. The buffer phase containe d16 mmol L1 BisTris, 4 mmol L1 p-Toluens ulfonic acid and 100 lmol L1 EDTA. The ow 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 IR11 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 relling 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.

    logy 137 (2013) 245253the end of the incubation; t the time (d); Rmax is the maximum CH4product ion rate (mmol CH4/(g-glucosed)); k is the lag phase (d),and e is 2.71828.

  • chno2.5. Data processing of stable isotope signatures

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

    ac dCO2 103

    dCH4 1032

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

    fmc dCH4 dmadmc dma 3

    where, dCH4 and dCO2 are the 13C isotope signatures of total CH4and 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 at80 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- ed 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 nal 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 nalextension 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 nal 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 ShannonWiener 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 t 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 05, 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-glucosed), respectively, demonstrating slowed methane

    Y. Lin et al. / Bioresource Teproduction at 0.15 and 0.2 mol/L bicarbonate. The ultimate meth- ane yields were 17.4117.74 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 insufcient 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 signicantly ow- ing to the high fractionation factor (about 1.08) for CO2 consump -

    logy 137 (2013) 245253 247tion in HM and the comparative ly low fractionation factor (about1.001.01) for CO2 production via SAO. In contrast, d13CH4 fromAM will usually increase with heavier acetate remaining, whereas

  • chnoativ

    e C

    H4 P

    rodu

    ctio

    nm

    ol/g

    -glu

    cose

    )10

    15

    20

    248 Y. Lin et al. / Bioresource Tethe correspond ing d13CO2 might stay relatively constant owing tothe low fractionation factor in AM (Methanos aeta 1.0071.010, Met-hanosarcina 1.0221.027) (Conrad and Klose, 2011 ).

    For 2.5 g-glucose/L treatments amended with bicarbonate , dur- ing the rst 5 days of incubation when 3854% of CH4 was gener- ated, the dCH4 of the four was initially 65.670.7 andincreased to 17.932.9, 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 (6195% CH4 was

    (a)

    (c)

    Time (d)0 10 20 30 40

    VFAs

    (mm

    ol/L

    )

    0

    5

    10

    15

    20

    25

    30

    Acet

    ate

    (mm

    ol/L

    )

    0

    5

    10

    15

    20

    25

    30

    Cum

    ul (m

    0

    5

    (e)

    (c)

    (a)

    Fig. 1. Temporal change of the cumulative CH4, acetate and VFAs. (a, c, e) 2.5 g-glucose/L,j 0.2. The results are means of two replicates. Error bars represent the data range.

    Table 1Calculated results using the modied Gompertz equation for methanogenesis under differ

    Treatment Lag time k (d) CH4 production rate Rmax [mmol CH4/(g-gluc

    L0 1.41 0.15 2.68 0.08 L0.05 0.93 0.16 2.95 0.09 L0.1 0.25 0.25 2.02 0.09 L0.15 0 1.14 0.11 L0.2 0 1.27 0.11 H0 3.12 0.25 1.44 0.06 H0.05 1.50 0.20 2.50 0.10 H0.1 0.64 0.10 2.28 0.04 H0.15 0 1.31 0.06 H0.2 0 0.68 0.09

    Note: The parameter standard error was estimated according to the weighted regressionlogy 137 (2013) 245253generate 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 (84100% 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,

    (d)

    50

    Time (d)0 10 20 30 40 50

    (f)

    (d)

    (b)

    (b, d, f) 5 g-glucose/L. Bicarbonate concentration (mol/L):d 0,s 0.05,. 0.1, 4 0.15,

    ent conditi ons.

    ose d)] Ultimate CH4 yield P (mmol CH4/g-glucose) Coefcient R2

    17.70 0.13 0.992 17.41 0.15 0.987 17.73 0.20 0.981 17.74 0.37 0.952 15.68 0.31 0.949 16.76 0.20 0.991 17.64 0.17 0.987 17.20 0.09 0.996 17.02 0.20 0.986 18.39 0.91 0.948

    .

  • chno

    (a)

    Y. Lin et al. / Bioresource Tesuggesting 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 to13.6. Subsequentl y, the dCH4 gradually decreased from the peak values to 38.9, 48.9, 64.6, 70.0 and 77.5,

    (c)

    (e)

    (g)

    Fig. 2. Temporal change of stable isotopic indicators dCH4, dCO2, ac and fmc. (a, c, e, g) 2.5 . 0.1, 4 0.15, j 0.2.(b)

    logy 137 (2013) 245253 249respectively , in treatments H0, H0.05, H0.1, H0.15 and H0.2, with- out any delay. The dCH4 values signicantly 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,

    (d)

    (f)

    (h)

    g-glucose/L, (b, d, f, h) 5 g-glucose/L. Bicarbonate concentration (mol/L):d 0,s 0.05,

  • 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 2301500 mg-N/L reported in literature (Bujoczek, 2001; Hansen et al., 1998; L et al., 2013 ).Therefore, the ammonia effect caused by increasing pH was negligible.

    The addition of bicarbonate affects the CO2 concentratio n. CO2is 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 1.071.082, and fmc reached 0.71.0, 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 HM

    Time (d)

    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.

    chno2.0, respectively . It should be noted that the measure d dCO2may 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 acaround 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 1.0351.045 during the initia- tion period (days 13), 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 -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 00.05 mol/L, ac remained below 1.045 before the stationary phase (days 117, 9195% 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 15), 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 nal value offmc was 0.30, 0.52, 0.61, 0.86, and 0.69 in the ve 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 fmcwas 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 to8.58.6 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 (3.85.5) 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 (

  • 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) 245253 251

  • chnoL0 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. ShannonWiener diversity index of microbial community. (a) Archaea, (b)

    252 Y. Lin et al. / Bioresource Teet al. (2011a) found the dominant contribution of SAO-HM path- way to thermop hilic methanogenes is at high organic loading rate,L 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 efciency of acetate degradation by SAO-HM is generally lower than that by AM (Schnrer 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 prole could suggest the microbial dynamics. In the archaeal proles (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

    bacteria.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 prole (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 uctuated with bicarbonate concentr ation.

    Principal component analysis, which was widely applied to ana- lyze environmental DNA ngerprints (Ramette, 2007 ), was per- formed to compare these ARISA band patterns under different condition s (Fig. 5). For archaeal proles, 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 inuencedcomponent 2 most.

    Bicarbonate concentration signicantly 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 reectedthe uctuation 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 inuenced component 1 most,band at 180-bp affected component 2 most, and component 3 was inuenced 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 ShannonWiener 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 uidity 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 acidication, result- ing in increasing cell lysis. Therefore, bicarbonate could be aninhibitory compound for methanogens. In the present study, bicar- bonate signicantly inuenced the archaeal community , with much higher value of ShannonWiener 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 inuence the prevalen ce of different methanogeni c pathways . Especially, the intensied 620-bp and 658-bp bands could suggest hydrogen o-trophic methanogens were promote d by high concentrated bicarbonate . The ARISA proles for bacteria showed dynamic

    logy 137 (2013) 245253changes 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.

  • 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

    and M. acetivorans and in rice eld soil. Applied and Environmental Microbiology 75, 26052612.

    Hammer, ., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4,19.

    Hansen, K.H., Angelidaki, I., Ahring, B., 1998. Anaerobic digestion of swine manure:inhibition by ammonia. Water Research 32, 512.

    Y. Lin et al. / Bioresource Technology 137 (2013) 245253 253rate. Since high organic loading rates usually cause a stronger uc-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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    Influence of bicarbonate buffer on the methanogenetic pathway during thermophilic anaerobic digestion1 Introduction2 Material and Methods2.1 Preparation of thermophilic inoculum2.2 Experimental setup2.3 Analysis of gas and liquid samples2.4 Data fitting to the modified Gompertz model2.5 Data processing of stable isotope signatures2.6 DNA extraction and ARISA analysis of bacterial and archaeal community structure

    3 Results3.1 Methanogenic conversion of glucose3.2 Methanogenic pathway analysis by stable carbon isotope signatures3.3 Dynamics of microbial community

    4 Conclusionsack15Appendix A Supplementary dataReferences

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