co-digestion of manure and whey for in situ biogas upgrading by the addition of h2: process...

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BIOENERGY AND BIOFUELS Co-digestion of manure and whey for in situ biogas upgrading by the addition of H 2 : process performance and microbial insights Gang Luo & Irini Angelidaki Received: 15 September 2012 / Revised: 22 October 2012 / Accepted: 23 October 2012 / Published online: 11 November 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract In situ biogas upgrading was conducted by intro- ducing H 2 directly to the anaerobic reactor. As H 2 addition is associated with consumption of the CO 2 in the biogas reactor, pH increased to higher than 8.0 when manure alone was used as substrate. By co-digestion of manure with acidic whey, the pH in the anaerobic reactor with the addition of hydrogen could be maintained below 8.0, which did not have inhibition to the anaerobic process. The H 2 distribution systems (diffus- ers with different pore sizes) and liquid mixing intensities were demonstrated to affect the gas-liquid mass transfer of H 2 and the biogas composition. The best biogas composition (75:6.6:18.4) was obtained at stirring speed 150 rpm and using ceramic diffuser, while the biogas in the control reactor con- sisted of CH 4 and CO 2 at a ratio of 55:45. The consumed hydrogen was almost completely converted to CH 4 , and there was no significant accumulation of VFA in the effluent. The study showed that addition of hydrogen had positive effect on the methanogenesis, but had no obvious effect on the aceto- genesis. Both hydrogenotrophic methanogenic activity and the concentration of coenzyme F 420 involved in methanogen- esis were increased. The archaeal community was also altered with the addition of hydrogen, and a Methanothermobacter thermautotrophicus related band appeared in a denaturing gradient gel electrophoresis gel from the sample of the reactor with hydrogen addition. Though the addition of hydrogen increased the dissolved hydrogen concentration, the degrada- tion of propionate was still thermodynamically feasible at the reactor conditions. Keywords Anaerobic digestion . Co-digestion . Hydrogen . In situ biogas upgrading Introduction Anaerobic digestion is an effective method for organic pollu- tion reduction and bioenergy production and has increasing applications worldwide (Angelidaki et al. 2006); (Boe and Angelidaki 2009). The produced biogas consists of 5070 % CH 4 and 3050 % CO 2 . The most common utilization route of biogas is for electricity production, often combined with uti- lization of the excess heat. Alternatively, biogas is upgraded to natural gas quality (biomethane) and used as autogas, or it is injected into the existing natural gas grid. This widens up the opportunities to utilize biogas in distant energy consumption locations (Holm-Nielsen et al. 2009). The most common methods for biogas upgrading include water washing, pres- sure swing adsorption, polyglycol adsorption, and chemical treatment (Osorio and Torres 2009), which are performed outside the anaerobic reactor and require investments in ex- ternal compressors, pumps, membranes, etc. Therefore, the cost for biogas upgrading is relatively high. In situ biogas upgrading has been investigated previously and several methods have been proposed (Lindberg and Rasmuson 2006; Luo et al. 2012; Nordberg et al. 2012), where CH 4 rich biogas could be obtained directly from the anaerobic reactor. Nordberg et al. (2012) investigated a process for in situ biogas upgrading where CO 2 was removed by circulating the sludge stream through a desorption unit and then back to the digestion. The desorption was achieved by aeration of the sludge. CH 4 content in the biogas between 70 % and 87 % was achieved. However, the main disadvantage of the method is that part of CH 4 (8 %; Nordberg et al. 2012) is removed together with CO 2 . Lindeboom et al. (2012) described an in situ biogas upgrading method by operating the anaerobic Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4547-5) contains supplementary material, which is available to authorized users. G. Luo : I. Angelidaki (*) Department of Environmental Engineering, Technical University of Denmark, 2800 Kgs Lyngby, Denmark e-mail: [email protected] Appl Microbiol Biotechnol (2013) 97:13731381 DOI 10.1007/s00253-012-4547-5

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  • BIOENERGYAND BIOFUELS

    Co-digestion of manure and whey for in situ biogasupgrading by the addition of H2: process performanceand microbial insights

    Gang Luo & Irini Angelidaki

    Received: 15 September 2012 /Revised: 22 October 2012 /Accepted: 23 October 2012 /Published online: 11 November 2012# Springer-Verlag Berlin Heidelberg 2012

    Abstract In situ biogas upgrading was conducted by intro-ducing H2 directly to the anaerobic reactor. As H2 addition isassociated with consumption of the CO2 in the biogas reactor,pH increased to higher than 8.0 when manure alone was usedas substrate. By co-digestion of manure with acidic whey, thepH in the anaerobic reactor with the addition of hydrogencould be maintained below 8.0, which did not have inhibitionto the anaerobic process. The H2 distribution systems (diffus-ers with different pore sizes) and liquid mixing intensitieswere demonstrated to affect the gas-liquid mass transfer ofH2 and the biogas composition. The best biogas composition(75:6.6:18.4) was obtained at stirring speed 150 rpm and usingceramic diffuser, while the biogas in the control reactor con-sisted of CH4 and CO2 at a ratio of 55:45. The consumedhydrogen was almost completely converted to CH4, and therewas no significant accumulation of VFA in the effluent. Thestudy showed that addition of hydrogen had positive effect onthe methanogenesis, but had no obvious effect on the aceto-genesis. Both hydrogenotrophic methanogenic activity andthe concentration of coenzyme F420 involved in methanogen-esis were increased. The archaeal community was also alteredwith the addition of hydrogen, and a Methanothermobacterthermautotrophicus related band appeared in a denaturinggradient gel electrophoresis gel from the sample of the reactorwith hydrogen addition. Though the addition of hydrogenincreased the dissolved hydrogen concentration, the degrada-tion of propionate was still thermodynamically feasible at thereactor conditions.

    Keywords Anaerobic digestion . Co-digestion . Hydrogen .

    In situ biogas upgrading

    Introduction

    Anaerobic digestion is an effective method for organic pollu-tion reduction and bioenergy production and has increasingapplications worldwide (Angelidaki et al. 2006); (Boe andAngelidaki 2009). The produced biogas consists of 5070 %CH4 and 3050%CO2. The most common utilization route ofbiogas is for electricity production, often combined with uti-lization of the excess heat. Alternatively, biogas is upgraded tonatural gas quality (biomethane) and used as autogas, or it isinjected into the existing natural gas grid. This widens up theopportunities to utilize biogas in distant energy consumptionlocations (Holm-Nielsen et al. 2009). The most commonmethods for biogas upgrading include water washing, pres-sure swing adsorption, polyglycol adsorption, and chemicaltreatment (Osorio and Torres 2009), which are performedoutside the anaerobic reactor and require investments in ex-ternal compressors, pumps, membranes, etc. Therefore, thecost for biogas upgrading is relatively high.

    In situ biogas upgrading has been investigated previouslyand several methods have been proposed (Lindberg andRasmuson 2006; Luo et al. 2012; Nordberg et al. 2012), whereCH4 rich biogas could be obtained directly from the anaerobicreactor. Nordberg et al. (2012) investigated a process for insitu biogas upgrading where CO2 was removed by circulatingthe sludge stream through a desorption unit and then back tothe digestion. The desorption was achieved by aeration of thesludge. CH4 content in the biogas between 70% and 87%wasachieved. However, the main disadvantage of the method isthat part of CH4 (8 %; Nordberg et al. 2012) is removedtogether with CO2. Lindeboom et al. (2012) described an insitu biogas upgrading method by operating the anaerobic

    Electronic supplementary material The online version of this article(doi:10.1007/s00253-012-4547-5) contains supplementary material,which is available to authorized users.

    G. Luo : I. Angelidaki (*)Department of Environmental Engineering,Technical University of Denmark,2800 Kgs Lyngby, Denmarke-mail: [email protected]

    Appl Microbiol Biotechnol (2013) 97:13731381DOI 10.1007/s00253-012-4547-5

  • reactor at elevated autogenerated pressure. Higher CH4 con-tent (>90 %) was obtained mainly due to the higher CO2solubilization in the liquid at higher pressure (90 bar).

    We have proposed a novel process for in situ biogasupgrading by the addition of hydrogen (Luo et al. 2012),where H2 and CO2 were biologically converted to CH4.However, the addition of hydrogen to anaerobic reactor treat-ing cattle manure for in situ biogas upgrading led to pHincrease to 8.3 or even higher due to the conversion of CO2,which was inhibitory to the anaerobic digestion (Luo et al.2012). Whey is a waste stream produced from cheese facto-ries, and it has very low pH (4.5 or even lower), alkalinity, andprotein content (Thoma et al. 2006; Gelegenis et al. 2007;Latif et al. 2011). Although it has a very high biodegradability(close to 99 %), it constitutes a difficult substrate to be treateddue to its high organic content and low alkalinity. Co-digestion of whey and cattle manure has been studied previ-ously (Gelegenis et al. 2007; Akassou et al. 2010; Kavacikand Topaloglu 2010), and improved treatment efficiency wasreported due to the necessary nutrients and buffer capacityprovided by manure. We could also expect that pH in theanaerobic reactor will be decreased by co-digestion comparedwith manure alone, considering the low alkalinity and proteincontent of whey. Besides, H2 is a gaseous substrate and it isdifficult to be captured by the microorganisms in the liquidsphase. In our previous study (Luo et al. 2012), the H2 injectedinto the anaerobic reactor was not fully utilized and the pro-duced biogas contained around 20 % H2. It seems that thegasliquid mass transfer is the limiting factor for the process(Pauss et al. 1990; Guiot et al. 2011). An insight into theprocess is necessary since hydrogen is an intermediate productgenerated in the acidogenic and acetogenic phase. The addi-tion of H2 to the anaerobic reactor may increase the H2concentration and thereby lead to the inhibition of VFA (pro-pionate, butyrate etc.) degradation (Siriwongrungson et al.2007). The system could therefore become imbalanced oreven can totally break down due to acidification caused byaccumulation of VFA. However, effective decrease of the H2concentration may be achieved by its consumption by the H2-utilizing methanogens at a rate equal to or greater than itsproduction and injection rate, which may make the VFAdegradation possible again. Thus, it is of outmost importanceto study how the dynamics of this complex process dependupon alterations of H2 concentration.

    The purpose of this study was to apply whey togetherwith manure as means to arrest increase in pH in a process,where in situ upgrading of biogas was desired, by injectionof H2. Different strategies for increasing gasliquid masstransfer were applied in order to increase the hydrogenconsumption rate and CH4 content. Furthermore, the re-sponse of conversion rates, enzymatic activities, microbialcomposition, and its influence on the systems thermody-namics upon H2 addition were studied.

    Material and methods

    Substrate characteristics

    Substrates used in the experiment were cattle manure fromVegger biogas plant and whey from Arla Food, Denmark.The substrates were received in one batch, mixed thoroughly,and distributed in 5 L plastic bottles andwere kept at 20 C forthe entire experimental period. The frozen substrates werethawed and kept at 4 C for 23 days before usage. Substratecharacteristics were analyzed and shown in Table S1.Considering the high organic concentration of whey, it wasdiluted four times and then mixed with manure at a ratio of 2:3.

    Reactor setup and operations

    Two identical 1 L continuously stirred tank reactors (A and B)with working volume of 600mLwere used. The configurationof the reactors was described elsewhere (Liu et al. 2006). Bothreactors were filled with inoculum, which was digested ma-nure from Snertinge biogas plant, in Denmark, operated underthermophilic conditions (55 C). Temperature in the reactorswas controlled at 55 C. The HRTwas controlled at 15 days.The reactors were fed once per day. Hydrogen was continu-ously injected to reactor A, while reactor B was operated ascontrol, without hydrogen injection. The H2 injection flowrate in reactor A was initially set at 1.5 L/(Lday), and thenchanged to 1.7 L/(Lday) after 20 days operation, which wasaround 4 times of CO2 production rate in the control reactor(B), corresponding to the stoichiometric ratio of H2:CO2 forproduction of CH4. The hydrogen gas was injected to thebottom of reactor A either by a column diffuser (pore diame-ters 0.51.0 mm) or ceramic diffuser (1440 m). The reac-tors were mixed by magnetic stirrer, and two different stirringspeeds 150 and 300 rpmwere tested. The operation conditionsfor the reactors could be found in Table 1.

    Pulse load tests

    After the reactors reached steady operation conditions(steady operation condition was defined as a period of 6consecutive days with daily variation of biogas productionrate of

  • milliliter-fresh samples were transferred from the reactors to20-mL-serum bottles. The samples were supplemented withdifferent substratesacetate (20 mM), propionate (10 mM),butyrate (10 mM), and H2/CO2 (80/20, 1 atm). Bottles withfresh samples only, but without substrates, were used ascontrols. The bottles were incubated in a shaker at 55 Cwith shaking speed 300 rpm. All the tests were prepared induplicates.

    Microbial community composition

    Archaeal communities at steady-states were analyzed bypolymerase chain reaction-denaturing gradient gel electro-phoresis (PCR-DGGE). The procedure has been describedin previous publication (Luo et al. 2011). Dominant bandsfrom DGGE were sequenced and identified by comparingthe gene sequences with DNA sequences in the NationalCentre for Biotechnology Information database using theBLAST algorithm. Sequences generated from this workare deposited at GenBank under accession numbersJQ183069-JQ183073.

    Analytical methods

    Total solids (TS), volatile solids (VS), ammonia nitrogen(NH3N), total Kjeldahl nitrogen (TKN), and chemical ox-ygen demand (COD) were analyzed according to APHA(1995). Inorganic carbon was determined by a TOC-5000(Shimadzu, Kyoto, Japan).

    The concentrations of acetate, butyrate and propionatewere determined by gas chromatograph (GC; HewlettPackard, HP5890 series II) equipped with a flame ionizationdetector and HP FFAP column (30 m0.53 mm1.0 m). H2was analyzed by GC-TCD fitted with a 4.5 m3 mms-mstainless column packed with Molsieve SA (10/80). The H2partial pressure was calculated by multiplying the percentageof H2 and the ambient pressure (110

    5Pa). CH4 was analyzedwith GC-TCD fitted with paralleled column of 1.1 m3/16Molsieve 137 and 0.7 m1/4 chromosorb 108. Detailedinformation about the operation conditions of above GC orHPLC was described previously (Kongjan et al. 2009).

    Dissolved H2 in the liquid phase was determined by areduction gas detector (Trace Analytical RGD2) with adetection limit of 0.1 ppmv, after extraction by the methoddescribed by Yu et al. (2006). The calculation of dissolvedH2 was described in the supporting information.

    The acid-forming enzyme acetate kinase (AK) was ana-lyzed as previously described (Ledoux and Lamy 1986)with potassium acetate as substrate. For its determination,25 mL of the fermentation mixture was taken out of theanaerobic fermentation reactors and then washed and re-suspended in 10 mL of 25 mM Tris/HCl buffer (pH 7.4).The suspension was sonicated at 30 kHz and 4 C for30 min to break down the cells of acidogenic bacteria andthen centrifuged at 10,000 rpm and 4 C for 30 min toremove the waste debris. The extracts were used for enzymeactivity assay. Coenzyme F420 was assayed by spectropho-tometric study (Delafontaine et al. 1979).

    Table 1 Summary of steady-state reactor performances

    Phase I Phase II Phase III

    Reactor A B A B A B

    Mixing speed (rpm) 150 150 300 300 150 150

    Gas diffuser Column / Column / Ceramic /

    Biogas production rate (mL/(Lday)) 1,429157 87467 1,235147 89545 1,180130 87355

    Biogas composition (%)

    CH4 533 553.5 682.5 562.8 753.4 56.71.5

    CO2 131.5 453.1 8.81.8 442.9 6.61.2 43.31.6

    H2 343.2 / 23.22.3 / 18.42.6 /

    CH4 production rate (mL/(Lday)) 75743 48031 83931 50125 88537 49422

    CO2 production rate (mL/(Lday)) 18536 39327 10835 39326 7824 37818

    H2 consumption rate (mL/(Lday)) 1,214145 / 1,413128 / 1,482159 /

    H2 mass balance (%)a 95 98 106

    pH 7.740.14 7.280.12 7.840.1 7.330.07 7.890.12 7.310.13

    Acetate (mM) 2.10.1 0.60.2 2.30.2 0.50.3 2.50.4 0.80.3

    Propionate (mM) 0.80.3 0.30.1 0.70.4 0.20.1 0.50.5 0.20.1

    Effluent VS (g/L) 8.90.5 8.50.6 7.80.7 8.30.4 8.40.5 8.20.8

    a H2 mass balance0(4(rCH4ArCH4B)+rbiogasH2)/rH2, where rCH4A is the CH4 production rate from reactor A, rCH4B is the CH4 production ratefrom reactor B, rbiogas is the CH4 production rate from reactor A, H2 is the H2 percentage in the biogas from reactor A, and rH2 is the H2 injectionflow rate

    Appl Microbiol Biotechnol (2013) 97:13731381 1375

  • Results

    Process performance

    Monitored data from the two reactors is shown in Fig. S1and steady operation condition process performance data issummarized in Table 1. The experiments were carried outfor around 150 days. Steady operation condition of eachoperation condition was obtained after operation for approx-imately two to three HRTs, when VFA and biogas produc-tion had reached relatively stable values. During phase I,CH4 content in reactor Awas only 53 %, which was slightlylower than that from reactor B. However, the CO2 content(13 %) was much lower than that (45 %) from reactor B.The CH4 production rate of reactor A was around 58 %higher than that of reactor B due to the conversion of H2 andCO2 to CH4. In order to increase the hydrogen consumptionrate and decrease the H2 content in the biogas, the stirringspeed was increased from 150 to 300 rpm (phase II). The H2consumption rate increased from 1,214 to 1,413 mL/Lday,which resulted in the increase in CH4 content of reactor A to68 % and the decrease in H2 content to 23 %. The increasein methane content of the produced biogas could beexplained by the lower gasliquid mass transfer limitationof hydrogen (Pauss et al. 1990). The intensive stirring mightdecrease the floc size in the reactor and thereby increase theshearing forces. During phase III, we decreased the stirringspeed back to 150 rpm. Instead of intense mixing, wechanged the H2 distribution equipment from column diffuserto ceramic diffuser, which has much smaller pores for betterH2 distribution. The CH4 content further increased to 75 %,which was much higher than that of reactor B. The theoret-ical CH4 production rate was calculated based on the H2consumption rate taking into consideration the basis CH4production rate from reactor B, serving as control reactor,i.e., without hydrogen injection, at each operation condition(Fig. S2). The calculated theoretical values were very closeto the measured values, indicating that the consumed hydro-gen was almost fully converted to CH4. Table 1 also showedthat the addition of hydrogen did not influence the effluentVS concentration. Though there was slightly higher acetateand propionate concentrations in reactor A compared withreactor B, they were still very low (

  • previous study (Luo et al. 2012), but it was not found in thepresent study. It could be attributed to the optimal pH (7.77.8) by co-digestion of manure and whey in the presentstudy which did not inhibit the acetoclastic methanogens.The above results indicated that hydrogenotrophic metha-nogens were enriched in reactor A.

    Effect of hydrogen addition on observed activities of keyenzymes

    Acetogenesis and methanogenesis are two main processesinvolved in anaerobic digestion, and there are several keyenzymes taking part in these processes (Zehnder 1988).During acetogenesis, the organic substrates and intermedi-ates (propionate, butyrate) will all be converted to acetyl-

    CoA, and finally to acetate, H2, and CO2 catalyzed byacetate kinase (Schink 1997). Therefore, acetate kinase is akey enzyme involved in the acetogenesis. It was anticipatedthat the addition of hydrogen would increase the hydrogenpartial pressure and result in inhibition of propionate andbutyrate degradation, thus probably decrease acetate kinaseactivity. However, there was no significant difference inacetate kinase activity in reactors A and B as seen inFig. 2. This indicates that addition of hydrogen had noobvious inhibition of the acetogenesis. This point will bediscussed later by measuring the dissolved hydrogen. It iswell-known that coenzyme F420 plays an important role inthe CH4 production from acetate and H2/CO2 (Baresi andWolfe 1981). The concentrations of coenzyme F420 fromreactor A was at least 20 % higher than that from reactor B,

    136 140 144 1486.5

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    Fig. 1 Time courses of reactor performances under organic loading shock. (A) Biogas production rate and VFA concentration in reactor A. (B) pHand biogas composition in reactor A. (C) Biogas production rate and VFA concentration in reactor B. (D) pH and biogas composition in reactor B

    Table 2 Specific methanogenicactivity on different substrates(mLCH4/gVSd) in reactors Aand B at steady-states

    Phase I Phase II Phase III

    Reactor A B A B A B

    Acetate 11723 12318 13236 13528 12131 11733

    H2/CO2 33643 22825 35021 21530 37838 23135

    Propionate 185 196 256 326 204 186

    Butyrate 347 285 318 257 267 304

    Appl Microbiol Biotechnol (2013) 97:13731381 1377

  • which was consistent with the increased specific methano-genic activities on H2/CO2 in reactor A.

    Effect of hydrogen addition on microbial communitystructures

    The archaeal microbial community structures in both reactorsA and B were analyzed by PCR-DGGE. The archaeal com-munity (Fig. 3) in either reactor A or B displayed no obviousvariation during phase I, II, and III. This implies that thequalitative methanogenic community structure in each reactoris consistent, despite the modification of operating parameters.However, there are differences in DGGE bands between reac-tors A and B. Band 3 was only observed in reactor A.Sequencing results (Table S2) showed that it was closelyrelated to Methanothermobacter thermautotrophicus with98 % similarity. Besides, band 1 in reactor B was brighterthan that in reactor A. It seems the addition of hydrogen alsoaffected the relative abundance of the dominant bands.

    Effect of hydrogen addition on microbial concentration

    The microbial concentration is an important parameter re-lated with the reactor performance. However, it is difficult toestimate the microbial concentration in anaerobic reactorstreating organic wastes with high solids content. It wasreported ATP could be used to represent the microbialconcentration since the decrease or increase in ATP is incorrespondence with microbial concentration (Yu et al.2002). The ATP content in reactor A was higher than thatin reactor B during all three phases (Fig. 4), which indicatedthat higher microbial concentration was grown in reactor A.The higher ATP content in reactor A was also consistentwith the higher methane production rate.

    Effect of hydrogen addition on dissolved hydrogen

    Hydrogen and interspecies hydrogen transfer are key factorsin anaerobic digestion, affecting degradation of intermediates

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    Fig. 2 Comparisons of the enzyme activities in reactors A and B atsteady-states

    Fig. 3 DGGE bands of archaeal communities in reactors A and B atsteady-states (CI, CII, and CIII represent the DGGE result of reactor Bduring phase I, II, and III. HI, HII, and HIII represent the DGGE resultof reactor A during phase I, II, and III)

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    1378 Appl Microbiol Biotechnol (2013) 97:13731381

  • (propionate, butyrate) and thereby the efficiency of the wholebiogas (Majone et al. 2010). In properly operating anaerobicdigesters, the hydrogen concentration is normally low, but animbalance between hydrogen-producing bacteria and hydro-genotrophic methanogens will lead to hydrogen accumula-tion. Addition of hydrogen to anaerobic reactor might resultin increased hydrogen partial pressure, unless high hydrogenconsumption rate can keep the hydrogen content low.Therefore, it is crucial to clarify the effect of hydrogen addi-tion on dissolved hydrogen concentration. Table 3 shows thedissolved and gaseous hydrogen at steady-states in both reac-tors A and B. In reactor A, the addition of hydrogen led to thehigher dissolved hydrogen compared with reactor B.

    Based on the dissolved and gaseous hydrogen concentra-tion, the gasliquid mass transfer (reactor A) or liquidgasmass transfer (reactor B) constants (kLa) could be calculatedbased on previous study (Pauss et al. 1990) and the resultsare shown in Table 3. For reactor A, the increase in mixingspeed (phase II) or change of the gas diffuser (phase III)obviously increased the kLa. For reactor B, the increase inmixing (phase II) increased the kLa from 0.025 (phase I) to0.052 h1 (phase II), and the values were in the range asprevious reported (Pauss et al. 1990; Pauss and Guiot 1993).It is obvious different hydrogen distribution systems ormixing intensity significantly affected the gasliquid masstransfer (reactor A) or liquidgas mass transfer constant.

    Discussion

    By co-digestion of manure and acidic whey, the pH in thebiogas reactor with addition of hydrogen could be main-tained in an optimal range for anaerobic digestion. Duringthe whole operation period, pH in reactor A, with hydrogenaddition, was around 7.77.9, which was higher than thatfrom reactor B (around 7.3). It was consistent with ourprevious study showing that addition of hydrogen to theanaerobic reactor would result in increase in the pH (Luoet al. 2012). Nevertheless, in the present study, where ma-nure and whey were co-digested, pH was always below 8,permitting efficient methane production, despite addition ofhydrogen to the reactor. Alternatively, optimal pH can also

    be ensured by on-line pH control equipment, when there isno availability of acidic waste.

    The results from this study also clearly showed thathigher methane content could be achieved by the increasein reactor mixing or by utilization of efficient H2 distribu-tion. Although the increased mixing (from 150 to 300 rpm)was beneficial for reactor A, it did not result in significantincrease in CH4 production rate of reactor B (Table 1),indicating that 150 rpm was adequate mixing intensity toachieve homogeneous distribution of substrate, enzymes,and microorganisms (Vavilin and Angelidaki 2005;Kaparaju et al. 2008). Previous studies showed that vigorousmixing may even have adverse effect on digestion, as itdisrupts the structure of microbial flocs and the syntrophicrelationships between organisms (Kim et al. 2000; Stroot etal. 2001). Increase in mixing may also significantly increasethe operation cost, though it could increase the hydrogenconsumption rate. Therefore, efficient H2 distribution sys-tems would be a better choice compared to increasing mix-ing intensity. Though the methane content in the presentwork was only 75 %, there is still possibility to furtherincrease the methane content by better H2 distribution sys-tems, such as injection through hollow fiber membrane,which could efficiently distribute the gaseous hydrogen toliquid phase without bubble (Kim et al. 2011).

    The present study also investigated and elucidated howexternal hydrogen addition affects the anaerobic digestionprocess. The study revealed that the addition of hydrogen toanaerobic reactor not only increased the hydrogenotrophicmethanogenic activity, but also changed the archaeal com-munity structure. The appearance of M. thermautotrophicusin reactor A could be related with the addition of hydrogen,since M. thermautotrophicus belongs to microorganismsmediating hydrogenotrophic methanogenesis (Ishii et al.2005). The results from microbial community analysis werealso consistent with the enzyme analysis that the addition ofhydrogen had effects on methanogenesis.

    Although the addition of H2 resulted in the higher dis-solved H2 in the liquid phase (Table 3), we did not observeaccumulation of propionate and butyrate. Propionate is animportant intermediate in anaerobic digestion and accountsfor around a third of methane production in a biogas reactor

    Table 3 Dissolved and gaseous hydrogen in reactors A and B at steady-states

    Phase I Phase II Phase III

    Reactor A B A B A B

    Dissolved hydrogen (Pa) 33045 458 38068 3510 36538 507.8

    Gaseous hydrogen (Pa) 36,0001,150 0.80.2 23,800550 1.20.4 18,400578 0.90.3

    Gpropionate (KJ/mol) 37 54 36 55 35 51kLa (/h) 6.62 0.025 11.78 0.052 16.05 0.026

    Appl Microbiol Biotechnol (2013) 97:13731381 1379

  • (Dong et al. 1994). Generally, the oxidation of propionate toacetate could only proceed at H2 partial pressure lower than10 Pa at standard conditions (Majone et al. 2010). However,both the gaseous and dissolved H2 partial pressures ofreactor A were much higher than 10 Pa. We calculated theGibbs free energy of propionate degradation (Table 3). TheG of reactor A at all steady-states was lower than 30 KJ/mol. Even though the values were higher than those fromreactor B, they were low enough to make the reactionexergonic. Another possible reason for the degradation ofpropionate is that there were microbial flocs in the reactor.Hydrogen was converted to methane at the surface of flocs,and then propionate might still be decarboxylated in deeperlayers and in the center without inhibition by the increase ofH2 partial pressure.

    The in situ biogas upgrading by the addition of H2 aimsto challenge the traditional operational biochemistry of an-aerobic digesters through introduction of external hydrogeninto the anaerobic digester and hence promotion of thehydrogenotrophic methanogenic community. In our process,the conversion of hydrogen into methane has several advan-tages, though hydrogen is thought to be a promising cleanfuel for future. The technologies for compression, transpor-tation, and storage of methane are mature, and methanecould be immediately integrated to the existing infrastruc-ture (Cheng et al. 2009). It should be noted that workingwith H2 in full-scale biogas plants may lift the installation toanother safety requirement. The subsequent application ofthe concept depends on the necessity of biogas upgrading,availability of hydrogen, economical efficiency, and envi-ronmental sustainability.

    Acknowledgments This study was funded by the Danish Council forIndependent Research (12-126632) and Hans Christian rsted PostdocProgram from Technical University of Denmark. The authors wouldlike to thank Hector Garcia for his technical assistance with theexperiments.

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    Appl Microbiol Biotechnol (2013) 97:13731381 1381

    Co-digestion of manure and whey for in situ biogas upgrading by the addition of H2: process performance and microbial insightsAbstractIntroductionMaterial and methodsSubstrate characteristicsReactor setup and operationsPulse load testsSpecific methanogenic activity (SMA) testsMicrobial community compositionAnalytical methods

    ResultsProcess performanceEffect of hydrogen addition on pHEffect of hydrogen addition on specific methanogenic activitiesEffect of hydrogen addition on observed activities of key enzymesEffect of hydrogen addition on microbial community structuresEffect of hydrogen addition on microbial concentrationEffect of hydrogen addition on dissolved hydrogen

    DiscussionReferences