BIOENERGYAND BIOFUELS

Anaerobic digestion of swine manure under natural zeoliteaddition: VFA evolution, cation variation, and relatedmicrobial diversity

Lin Lin & Chunli Wan & Xiang Liu & Zhongfang Lei &Duu-Jong Lee & Yi Zhang & Joo Hwa Tay & Zhenya Zhang

Received: 3 August 2013 /Revised: 1 October 2013 /Accepted: 2 October 2013 /Published online: 25 October 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Batch experiments were carried out on anaerobicdigestion of swine manure under 10 % of total solids and60 g/L of zeolite addition at 35 °C. Four distinctive volatilefatty acid (VFAs) evolution stages were observed during theanaerobic process, i.e., VFA accumulation, acetic acid(HAc) and butyric acid (HBu) utilization, propionic acid(HPr) and valeric acid (HVa) degradation, and VFAdepletion. Large decreases in HAc/HBu and HPr/HVaoccurred respectively at the first and second biogas peaks.Biogas yield increased by 20 % after zeolite addition, about356 mL/g VSadded with accelerated soluble chemical oxygendemand degradation and VFA (especially HPr and HBu)consumption in addition to a shortened lag phase betweenthe two biogas peaks. Compared with Ca2+ and Mg2+ (100–300 mg/L) released from zeolite, simultaneous K+ and NH4

+

(580–600 mg/L) adsorptions onto zeolite particlescontributed more to the enhanced biogasification, resultingin alleviated inhibition effects of ammonium on acidogenesisand methanogenesis, respectively. All the identified

anaerobes could be grouped into Bacteroidetes andFirmicutes, and zeolite addition had no significant influenceon the microbial biodiversity in this study.

Keywords Swinemanure . Anaerobic digestion . Naturalzeolite . Volatile fatty acids (VFAs) .Microbial diversity .

Cations

Introduction

According to the statistics released by the Ministry ofEnvironmental Protection of the People’s Republic of China,about 11.86 million tons of chemical oxygen demand (COD)and 0.83 million tons of ammonium nitrogen (NH4–N) weredischarged from agricultural sources, accounting for 47.4 and31.7 % of the total annual COD and NH4–N discharges,respectively in 2011, which was largely contributed by thelivestock industry (MEPPRC 2012). Anaerobic digestion hasbeen widely applied in practice to decompose the organicsubstances of the wastes and wastewaters from livestockfarms and alleviate environmental pollution in addition tothe recovery of combustible methane.

Anaerobic digestion of livestock manure such as swinemanure as the sole substrate, however, sometimes turns outunsuccessful due to the inhibition of high ammonium levels(Hansen et al. 1998). Zeolite addition has been proven to be aneffective way to mitigate ammonium inhibition due to its highadsorption capacity and selectivity for ammonium, resultingin enhanced biogasification performance during the digestionprocess (Ho and Ho 2012; Montalvo et al. 2012). Besides, thisenhancement effect is also noticed and attributable to theincreased biomass due to microorganism immobilization onthe surface of zeolite particles (Adu-Gynamfi et al. 2012;Fernández et al. 2007) and the increase in Ca2+ ions releasedfrom zeolite (Tada et al. 2005). Up to now, however, the

L. Lin :C. Wan :X. Liu : Z. Lei :D.<J. Lee :Y. ZhangDepartment of Environmental Science and Engineering,Fudan University, 220 Handan Road, Shanghai 200433, China

D.<J. Leee-mail: djlee@ntu.edu.tw

Z. Lei (*) : Z. ZhangGraduate School of Life and Environmental Sciences, University ofTsukuba, 1-1-1 Tennoidai, Tsukuba, Ibaraki 305-8572, Japane-mail: lei.zhongfang.gu@u.tsukuba.ac.jp

D.<J. LeeDepartment of Chemical Engineering, National Taiwan University,Taipei 106, Taiwane-mail: djlee@ntu.edu.tw

J. H. TayDepartment of Civil Engineering, Schulich School of Engineering,University of Calgary, 2500 University Drive NW, Calgary, Canada

Appl Microbiol Biotechnol (2013) 97:10575–10583DOI 10.1007/s00253-013-5313-z

enhancement mechanism of zeolite addition to anaerobicdigestion is still not very clear and under investigation becauseof the complexity of the anaerobic process and the variety offeedstocks. In addition, little information could be found onthe relationship between the evolution of volatile fatty acids(VFAs), the most important intermediates produced in thedigestion process, and the cations released from zeolite,especially under the real condition of anaerobic digestion ofswine manure. The related variation of microbial diversityduring this process is also scarce.

The objective of this study was to test the effect of naturalzeolite addition on the anaerobic digestion of swine manure inthe field, in which the total solid (TS) content was about 10%.The variations of biogas production, pH and total alkalinity,ammonium, VFA, and soluble chemical oxygen demand(SCOD) were analyzed based on batch experiments. Inaddition, Ca2+, Mg2+, Na+, and K+ concentrations releasedinto the system along with the biodiversity changes ofmicroorganisms were determined to reveal the relationshipbetweenVFA evolution and cation levels during the anaerobicprocess.

Materials and methods

Swine manure

Raw swine manure was from a swine farm located inKunshan, Jiangsu Province, China. Its characteristics areshown in Table 1. Before dosed into the batch reactors, theTS of the manure was diluted by flushing water to around10 %, in accordance with the operation condition for theanaerobic digester installed in the farm.

Natural zeolite

Natural zeolite, obtained from Shenshi Mine located inJinyun, Zhejiang Province, China, was prepared throughcrushing, sieving (0.5–1.5 mm), washing with deionized

water, and air drying at 105 °C for 6 h. X-ray diffraction(Bruker-D8 Advance, USA) analysis revealed that the mainmineral species in zeolite were heulandite, clinoptilolite–Na,and quartz. Its chemical composition is given in Table 2. Theammonium adsorption capacity of zeolite is about 13.3 mg Nper gram zeolite at an initial ammonium concentration of2000 mg N/L (Lin et al. 2013).

Experimental setup

The batch experiments were carried out in two 1,000-mLground flasks with a working volume of 600 mL, one withnatural zeolite addition and another without zeolite as thecontrol (labeled as Zeo-digester and C-digester, respectively).The two digesters were both fed with swine manure underinitial TS, volatile solids (VS), and total ammonium nitrogen(TAN) of 103.6 g/L, 72.9 g/L and 2,182 mg N/L, respectively.It was reported that TAN>1,500mg/L at pH>7.4 would causeammonium inhibition (Van Velsen 1979), implying that≥48 g/L of zeolite used in this study was necessary foravoiding inhibition. In the present study, 60 g/L of zeolitewas dosed into the Zeo-digester with expectation of mitigatingammonium inhibition during the digestion process.

After dosing, the headspace of each flask was flushed withN2 for 2 min and then closed airtight by rubber caps with twoholes left: one for sampling and another for biogas collection.The flasks were placed in a thermostat controlled at 35±1 °Cand shaken manually twice a day, 2 min for each time. Theanaerobic process lasted for 50 days.

Analysis

Determinations of TS and VS, SCOD, TAN, and totalalkalinity (titrated to pH 4.3) were in accordance with standardmethods (APHA 2005). pH was measured online using a pHmeter (Multi 340i-WTW, Germany). Concentrations ofcations including Na+, Ca2+, K+, and Mg2+ were measuredusing atomic absorption spectroscopy (Hitachi-Z 5000,Japan). Free ammonia nitrogen (NH3–N) concentration wasestimated using the following equation based on the measuredTAN concentration (Anthonisen et al. 1976).

NH3−N½ � mg=Lð Þ= TAN½ � mg=Lð Þ ¼ 10pH= 10pH þ e6;344 273þTð Þ� �

ð1Þwhere T is the temperature in degree Celsius.

Table 1 Characteristics of the swine manure used in the experiment

Parameters Unit Valuea

Total solids (TS) % 17.62

Volatile solids (VS) % of TS 70.36

Alkalinity mg CaCO3/L 4,627

Soluble chemical oxygen demand (SCOD) mg/L 35,421

Total nitrogen (TN) mg N/L 4,259

Total ammonium nitrogen (TAN) mg N/L 3,540

Total phosphorus (TP) mg P/L 70.30

pH – 7.17

a Data are the mean values of duplicate tests

Table 2 Chemical composition of the natural zeolite used in theexperiment

Composition SiO2 Al2O3 Fe2O3 K2O CaO MgO Na2O Others

Weight (%) 69.58 12.20 0.87 1.13 2.59 0.13 2.59 10.91

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Biogas production was daily monitored with a waterdisplacement method using a 1,000-mL graduated cylinderconnected to each reactor. Methane (CH4) content in thebiogas produced and VFAs (C2–C5) including acetic acid(HAc), propionic acid (HPr), n -butyric acid (n -HBu), iso-butyric acid (i-HBu), n -valeric acid (n -HVa), and iso-valericacid (i-HVa) in the digestate were determined using a gaschromatograph (7890A, Agilent, USA) fitted with HP-PLOT 80/100 mesh (2 m×1/8 in.×2.0 mm SS) packedcolumn and TCD detection, and HP-FFAP (30 m×0.25 mm×0.25 μm) capillary column and FID detection,respectively.

As for microbial community analysis, DNA Isolating Kit(MoBio Laboratories, Inc., Carlsbad, CA, USA) was used toextract the genomic DNA according to the manufacturer’sinstructions. Then, the V3 region of the 16S rRNA wasamplified by PCR using universal eubacterial primer pairs(8F, 5′-GAGAGTTTGATCCTGGCTCAG-3′ with a GCclamp and 518R, 5′-ATTACCGCGGCTGCTGG-3′). Asdescribed previously (Wan et al. 2011), the PCR productswere separated with denaturing gradient gel electrophoresis(DGGE) using the Dcode™ universal mutation detectionsystem (Bio-Rad Laboratories, Hercules, CA, USA). Afterband excision, DNA re-amplification, purification, cloning,and sequencing (ABI3730, Sangon Biotech, China), thenucleotide sequences of the dominant DGGE bands wereanalyzed using the BLAST program in GenBank.

Biogas production and pH were daily measured, and theVFAs, SCOD, ammonium, total alkalinity, and cationconcentrations in the supernatant after centrifugation (10,000 rpm for 10 min) were determined once every 5 days.The experimental data were expressed as the mean values ofduplicate tests with deviations <4 %.

Results

Overall performance and digestion characteristics

Figure 1 depicts the daily variation of biogas production andthe corresponding methane content from the two digesters, inwhich both were characterized as double-peak curves. After8 days from the start-up, the daily biogas production increasedquickly and reached the first peaks of 840 and 675 mL in theZeo-digester and the C-digester on day 15, and then a decreasetrend followed and lasted for 4 and 8 days, respectively. Afterthis decline period, the biogas production once againincreased quickly and reached the highest amounts (thesecond peak) of 1,050 and 720 mL, respectively, in the Zeo-digester on day 29 and the C-digester on day 37, with bothmethane contents up to 80 %. After the second peak, the dailybiogas production decreased sharply and the whole digestionprocess almost ceased at around days 34 and 40 for the Zeo-

digester and the C-digester, respectively. During theoccurrence of the two biogas peaks, the methane contentsvaried between 50 and 82 % in the two digesters.

pH and alkalinity profiles

pH is the crucial factor in maintaining good performance of ananaerobic system. The desirable pH ranges between 6.8 and7.2 when methane production ability and microbialcommunities are taken into consideration (Sreekrishmanet al. 2004). The results indicate that the two digestersexperienced obvious pH fluctuations during the digestionprocess, coincidently with the variation of daily biogasproduction (Figs. 1 and 2). At the beginning, pH dropped toaround 6.5 in the first 5 days, mainly due to the rapidconversion of easily degradable organics into fatty acids.Then, the pH rose quickly in the next 10 days and reached7.3 at the first biogas production peak (Figs. 1 and 2). Later,the pH values gradually increased and remained around 7.8and 7.6 in the C-digester and the Zeo-digester, respectively. Aslight difference in pH value from day 35 to day 50 betweenthe two digesters was noticed, probably attributable to theirslight difference in alkalinity (420–820 mg CaCO3 per liter)and buffering capacity during the digestion process (Fig. 2).

Fig. 1 Variations of daily biogas production (a) and methane content (b)in the two digesters during anaerobic digestion of swine manure

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A similar trend was also observed for alkalinity variation inthe two digesters during the anaerobic process (Fig. 2): first,the total alkalinity increased slightly and then advanced fastuntil the appearance of the second biogas peak (about 30–40 days), and finally to 4,847 and 5,468 mg CaCO3/L in theZeo-digester and the C-digester, respectively.

Ammonium and free ammonia profiles

Ammonium is essential for bacterial growth, but it alsoinhibits the anaerobic digestion process at high levels (Sungand Liu 2003). As shown in Fig. 3, the TAN concentrationincreased quickly in the C-digester during the first 10 daysowing to the ammonification of organic nitrogenoussubstances such as proteins and urea in the acidogenesis phase(Kayhanian 1999). In the Zeo-digester, however, TANdecreased during the first 5 days, mainly resulted from therapid ammonium adsorption by zeolite. In the following40 days, the TAN concentration remained around 2,500 and1,900 mg N/L in the C- and the Zeo-digesters, respectively;

that is, about 600 mg N/L of ammonium was adsorbed ontothe zeolite particles.

VFA and SCOD variations

VFAs play an important role in maintaining an efficientanaerobic digestion performance due to its strong effects onpH and alkalinity in the digester, thus the activity ofmethanogens (Buyukkamaci and Filibeli 2005). In addition,VFAs can also be used as a good parameter to signal theprocess imbalance in anaerobic digesters (Ahring et al.1995). Figure 4 shows the variation of volatile fatty acids(C2–C5) including HAc, HPr, i/n -HBu, and i/n -HVa. Theresult indicates that the amounts of HAc and HPr almostaccounted for 70 % during the whole digestion process.

Based on the changes in VFA concentration andcomposition (Fig. 4), the digestion process in both digesterscould be divided into the following four distinct stages insequence. (1) VFA accumulation: the total VFAs was firstlyaccumulated and this accumulation lasted for about 10 days,up to 18,300 and 19,000 mg COD/L in the C- and the Zeo-digesters on day 10, respectively. (2) Utilization of HAc andn /i-HBu: this stage started from day 10 to day 20,correspondingly with the occurrence of the first biogasproduction peak (Fig. 1). This observation is probablyattributed to the highest biodegradability of HAc owing toits lowest Gibbs free energy among the VFAs (Wang et al.1999). The concentrations of HAc and HBu (sum of n -HBuand i-HBu) were decreased from 3,284 and 4,432 mg COD/Lto 376 and 368 mg COD/L in the C-digester and from 3,262and 3,836 mg COD/L to 325 and 88 mg COD/L in the Zeo-digester, respectively. During this stage, HAc and HBu weredecreased by 89 and 92 % in the C-digester and 90 and 98 %in the Zeo-digester, respectively. (3) Degradation of HPr andn /i-HVa: right after most of the HAc and HBu are used up, thedegradation of HPr and n /i-HVa started from day 20 to day 35,which was accompanied by the second peak of biogas

Fig. 2 Profiles of pH and total alkalinity in the two digesters duringanaerobic digestion of swine manure

Fig. 3 Profiles of total ammonium and free ammonia concentrations inthe two digesters during anaerobic digestion of swine manure

Fig. 4 Variation of VFAs (HAc, HPr, i/n -HBu, and i/n -HVa)concentrations in the two digesters during anaerobic digestion of swinemanure

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production (Fig. 1). The variation of HAc and HPrconcentrations agrees with Pind et al. (1997) and Wanget al. (1999) who found that HPr could only be utilizedunder low HAc level and H2 partial pressure, thus alimiting factor for biogas production. During this stage,the concentrations of HPr and HVa (sum of n -HVa and i-HVa) were decreased by 93 and 90 % in the C-digesterand 99 and 98 % in the Zeo-digester, respectively. (4)VFA depletion: this stage ended until day 50 with thefinal VFA concentration ranging between 289 and495 mg COD/L in the two digesters.

In addition, biodegradable organic substances can beremoved through conversion into methane and CO2 duringthe anaerobic process, resulting in COD decrease in thedigester. As shown in Fig. 5, the initial SCOD concentrationand VFAs/SCOD were 22,100 mg/L and 69 %, respectively,in the two digesters. During the first 10 days, SCODconcentrations in the two digesters increased quickly due toVFA accumulation, and the VFAs/SCOD increased toaround 73 %. As the digestion progressed, the SCOD andVFA/SCOD values decreased gradually because of VFAconsumption by methanogens and finally reached 5,200 mg/L and 6 % in the Zeo-digester and 7,800 mg/L and4 % in the C-digester, respectively. It should be noted that onday 30, the VFA/SCOD value and SCOD removal efficiencyreached 18 and 68% in the Zeo-digester, in contrast to 43 and44 % in the C-digester, respectively, indicating that zeoliteaddition could significantly accelerate SCOD degradationand VFA consumption.

Cation release and their effects on anaerobic digestion

Alkali/alkaline-earth metal cations such as Na+, K+, Ca2+, andMg2+ can stimulate microbial growth and are antagonistic toammonium inhibition (Braun et al. 1981), while high levels of

these cations will also cause toxicity (Soto et al. 1993).Zeolites are microporous aluminosilicate minerals withloosely bound cations, like Na+, K+, Ca2+, and Mg2+, whichcan be easily exchanged by other surrounding cations(Tsitsishvili et al. 1992), i.e., ammonium ions in this study.The ionic exchange process between the zeolite frame andaqueous ammonium solution can be expressed by Eq. 2.

Zeo–Mnþ þ nNH4þ→Zeo− NH4

þð Þn þMnþ ð2Þ

where Zeo and M represent zeolite and the loosely heldcations in zeolite, respectively, and n is the number of electriccharge. Namely, a considerable amount of these cations willbe released into the Zeo-digester when ammonium adsorptionhappens.

As shown in Fig. 6, about 300 mg/L of Ca2+, 50 mg/L ofMg2+, 250mg/L ofNa+, and 1,050mg/L ofK+were containedin the initial feeds for the two digesters. The concentrations ofCa2+, Mg2+, and Na+ are among the stimulatory ranges formethanogens: ≤3,000, ≤720, and ≤1,000 mg/L, respectively,for Ca2+, Mg2+, and Na+ (Ahring et al. 1991; Ahn et al. 2006;Lee et al. 2012). The concentration of K+ in the initial feeds,however, was higher than the inhibition level (>400 mg/L) foracidogenic microorganisms (Chen et al. 2008).

The two digesters exhibited similar variation trends ofCa2+ and Mg2+ concentrations during the digestion process(Fig. 6). In the first 5–10 days, the concentrations of Ca2+

and Mg2+ increased two to three times due to the hydrolysisof swine waste (in the C-digester) in addition to the releasefrom zeolite (in the Zeo-digester). The maximum levels ofCa2+ and Mg2+ were 603 and 142 mg/L in the C-digester and698 and 441 mg/L in the Zeo-digester, respectively, duringthis period. The follow-up rapid decrease in these twocations was detected around the occurrence of the firstbiogas peak, possibly resulted from (1) the utilization bymethanogens, which is necessary for the rapid growth of

Fig. 5 Profiles of SCOD concentration and VFA/SCOD ratio in the twodigesters during anaerobic digestion of swine manure

Fig. 6 Variations of Ca2+, Mg2+, Na+, and K+ concentrations in the C-digester (a) and Zeo-digester (b) during anaerobic digestion of swinemanure

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the microbes, and (2) the precipitation with carbonate andphosphate produced in the anaerobic digestion (Keenan et al.1993). The Ca2+ and Mg2+ concentrations decreased rapidlyto 24 and 41 mg/L in the C-digester and to 43 and 81 mg/L inthe Zeo-digester, respectively, on day 25. Finally, theconcentrations of these two cations remained relativelystable (<20 mg/L) until the end of digestion. According tothe variation of Ca2+ andMg2+ concentrations in the process,the Zeo-digester performed better in biogas production witha shortened digestion duration, possibly attributable to theincreased Ca2+ and Mg2+ levels due to zeolite addition.Stated, Ca2+ and Mg2+ may be more advantageous formethanogenesis than acidogenesis.

On the other hand, different variation trends were observedfor the concentrations of Na+ and K+ (Fig. 6). Although somefluctuations happened, in the C-digester, the Na+ and K+

concentrations were relatively stable during the wholedigestion process. In the first 5–10 days, however, asignificant increase in Na+ and a decrease in K+

concentrations were detected in the Zeo-digester; then, thesetwo cations became relatively stable until the end of digestion.Na+ is the dominant cation released into the bulk solution fromthe zeolite used in this study (Lin et al. 2013), which mainlycontributed to the Na+ increase (by about 600 mg/L) in theZeo-digester. K+ was found to be adsorbed onto zeoliteparticles due to the fact that K+ concentration in the Zeo-digester was decreased by 580 mg/L compared to that in theC-digester. The observation is in accordance with Ames(1960) who pointed out that K+ was preferred than NH4

+ inthe ionic exchange sequence; thus, K+ could compete withammonium for adsorption sites. The high level of potassiummay be responsible for the lower activity of anaerobicmicroorganisms due to its passive influx and neutralizationof the membrane potential (Jarrell et al. 1984). In this study,K+ concentration was decreased from 1,240 mg/L (in the C-digester) to 660 mg/L (in the Zeo-digester) during the first5 days because of zeolite addition, which is beneficial forrelieving K+ inhibitory effect on acidogenic organisms(Fernández and Forster 1994). Stated, K+ decrease to someextent might be another explanation for the betterbiogasification performance of the Zeo-digester.

Biodiversity changes of microbial communityduring anaerobic digestion

As shown in Fig. 7 and Table 3, 13 bands (bands 1–13) weredetected in the DGGE, and all the identified anaerobes can begrouped into two families: Bacteroidetes and Firmicutes.

During the anaerobic digestion process, the populationsand numbers of hydrolytic and acidogenic bacteria wereclosely related to the variation of VFAs. Before digestion,Clostridium sp. (bands 2 and 6), a kind of acid formationbacteria (Sousa et al. 2007), was detected in the original fresh

swine manure sample, possibly due to a large amount of VFAsalready existing in the rawmanure (Fig. 4). On day 5, two newdominant bacteria, Proteiniphilum acetatigenes (bands 1 and12) and Clostridium beijerinckii (band 4), appeared in the twodigesters, which respectively generate acetic and propionicacids (Chen and Dong 2005; Chen et al. 2006) and aceticand butyric acids (Ezeji et al. 2007). Then, Clostridiumbutyricum (band 3) was detected on day 15, which wasreported to produce butyric acids (Zigová et al. 1999). Theincrease in the number and diversity of acid generationbacteria definitely contributed to the obvious VFAaccumulation during the acidogenesis period (Fig. 4). As thedigestion progressed, another two new dominant bacteriaincluding Clostridiaceae bacterium (band 7) andClostridium sp. (band 13), which function in acid andhydrogen production, were mainly involved in the digestionprocess from day 15 on and remained constant until the end ofoperation. It was noticed that Alkaliflexus imshenetskii (band5), an alkaliphilic gliding carbohydrate-fermenting bacteriumwith propionate formation, showed up later in the Zeo-digester (day 20) compared with the C-digester, probablyattributable to the slower increase of alkalinity in the Zeo-digester (Fig. 2). In addition, from day 30 on, the diversity ofmicrobial community in the Zeo-digester was relativelysimple, while more bacteria like Petrimonas (band 8),Clostridium paraputrificum (band 9), C. bacterium (band10), and Bacteroidales bacterium (band 11) were found andcoexisted in the C-digester. All these bacteria were identifiedand functioned as producing acetic acid and H2 (Fig. 7 andTable 3). This phenomenon is possibly attributable to the factthat the digestion process in the Zeo-digester almost reachedits VFA depletion stage after day 30, while there was still arelatively high amount of VFAs remaining in the C-digester(Fig. 4), leading to their difference in the activity of acid-degrading bacteria.

Fig. 7 DGGE profiles of bacterial 16S rDNA gene fragments in C-digester and Zeo-digester during anaerobic digestion of swine manure.Numbers at the top of each lane designate the digestion duration

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Furthermore, the archaea dynamic shifts in the twodigesters were also investigated. The PCR-DGGE resultsshowed that only one dominant archaea, Methanosarcinasiciliae with 98 % similarity (data not shown), could beidentified and existed in both digesters during the wholedigestion process. The bacteria can use different substratesto produce methane, such as H2, CO2, methanol, and aceticacid (Bryant and Boone 1987). Restated, no significantinfluence was found on methanogen diversity after zeoliteaddition.

Discussion

The Zeo-digester exhibited better biogas productionperformance than the C-digester in the following threeaspects: (1) the total biogas production was increased by20 %, i.e., 15,600 mL in the Zeo-digester in contrast to 12,950mL in the C-digester, and their biogas yields were 356 and296 mL/g VSadded, respectively. (2) Methane yield wasincreased by 11 %, 204 and 183 mL CH4/g VSadded,respectively, for the Zeo-digester and the C-digester. (3) Theduration of effective digestion process was shortened by 15%,about 34 and 40 days for the Zeo-digester and the C-digester,respectively. These results demonstrated that zeolite additiondid enhance the biogas production efficiency in anaerobicdigestion even when treating swine waste with a higher TScontent (10 % in this study).

A lag phase was noticed between the two biogas peaks(Fig. 1), probably brought about by the inhibitedmicroorganism activity due to the changes of thephysicochemical environment in the digesters during thisperiod. Ahn et al. (2006) attributed this phenomenon to the

different substrate utilization rates for carbohydrate, protein,and lipid. In this study, zeolite addition could significantlyshorten the lag phase from 22 to 14 days, which, to a largeextent, resulted in a shorter duration of effective digestion inthe Zeo-digester.

The increase in alkalinity during anaerobic digestion wasprobably contributed by ammonium production from proteinhydrolysis followed by VFA consumption and the dissolutionof CO2 produced by methanogens. The alkalinity decreaseafter zeolite addition could be attributed to (1) the decrease ofammonium concentration in the bulk solution (due toammonium adsorption onto zeolite; Fig. 3) and (2) theconsumption of CO3

2− possibly caused by precipitation withexcess Ca2+ and Mg2+ ions released from zeolite.

In this study, the ammonium adsorption capacity of zeolitewas estimated as 10 mg N/g, about 25 % lower than itsmaximum adsorption capacity, 13.3 mgN/g when ammoniumsolution is being used as the bulk liquor (Lin et al. 2013). Thedecrease in ammonium adsorption capacity might be broughtabout by the following two reasons. Firstly, other releasedcations such as K+ and H+ coexisting in the digester might becompetitive with NH4

+ ions during adsorption onto zeolitebased on ionic exchange mechanism. Secondly, the masstransfer of ammonium was possibly hindered and retardedby the high TS concentration and the sedimentation of zeoliteparticles covered by solid substrates. On the other hand, freeammonia concentration fluctuated as its concentration wasclosely related with pH variation, in accordance with Eq. 1.Compared with ammonium ion (NH4

+), free ammonia (NH3)is more toxic due to its passive diffusion into cells through thepotassium pathway, leading to proton imbalance (Gallert andWinter 1997). During the methanogenesis period, freeammonia increased very fast with the increase of pH and

Table 3 Sequence analysis of bands excised from DGGE in Fig 7

BandID

Band accessionno.

Closest relative Closest relativeaccession no.

Taxonomicgroup

Function Similarity(%)

1 KF530887 Proteiniphilum acetatigenes NR043154.1 Bacteroidetes Acetic and propionic acid generation 95

2 KF530888 Clostridium sp. AB238899.1 Firmicutes Acid generation 96

3 KF530889 Clostridium butyricum AB647330.1 Firmicutes Acetic and butyric acid generation 97

4 KF530890 Clostridium beijerinckii AB640693.1 Firmicutes Acetic and butyric acid generation 97

5 KF530891 Alkaliflexus imshenetskii FR839023.1 Bacteroidetes Propionic acid generation 90

6 KF530892 Clostridium sp. GU124459.1 Firmicutes Acid generation 98

7 KF530893 Clostridiaceae bacterium AB298753.2 Firmicutes Hydrogen production 85

8 KF530894 Petrimonas sp. JQ988071.1 Bacteroidetes Acetic acid and hydrogen production 86

9 KF530895 Clostridium paraputrificum AB627080.1 Firmicutes Hydrogen production 96

10 KF530896 Clostridiaceae bacterium JQ259413.1 Firmicutes Hydrogen production 99

11 KF530897 Bacteroidales bacterium GU129091.1 Bacteroidetes Acid generation 90

12 KF530898 Proteiniphilum acetatigenes NR043154.1 Bacteroidetes Acetic and propionic acid generation 87

13 KF530899 Clostridium sp. AB238882.1 Firmicutes Acid generation 98

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finally reached 176 and 86 mg N/L in the C- and the Zeo-digesters, respectively. According to previous studies (VanVelsen 1979; Braun et al. 1981), biogas production would beinhibited when total ammonium is >1,500 mg N/L at pH>7.4or free ammonia exceeds 150 mg N/L. In this study, althoughthe ammonium concentration in the Zeo-digester was notdecreased below 1,500 mg N/L in total ammonium or150 mg N/L in free ammonia under zeolite dosage of60 g/L, the ammonium inhibition was alleviated to a largeextent, most probably due to about the 600 mg N/L lowerlevel of ammonium in the Zeo-digester compared to the C-digester.

A high concentration of free ammonia (>200 mg N/L) wasreported to have a more detrimental effect on the growth ofHPr-degrading acetogenic bacteria than that of methanogenicorganisms (Calli et al. 2005). Possibly due to the quickadaptation to VFA variation for HPr-degrading acetogenicbacteria under a lower ammonium level, the Zeo-digester(TAN=1,900mgN/L, NH3–N=86mgN/L) reached its biogaspeaks earlier and achieved a higher biogas yield comparedwith the C-digester (TAN=2,500 mg N/L, NH3–N=176 mg N/L). In addition, the concentration of i-HBu in theC-digester increased and reached the maximum of 1,410 mg COD/L on day 15, which did not occur in the Zeo-digester (68 mg COD/L). This observation implies that zeoliteaddition could also promote the degradation and utilization ofi-HBu during anaerobic digestion. Detailed mechanism isunder further investigation.

The present study showed that zeolite addition allowed afaster start-up and better performance of an anaerobic digesterof swine manure, especially when there was a highconcentration of ammonium. It was mainly contributed bythe decrease in ammonium and K+ concentrations (by 580–600 mg/L) and increases in Ca2+ and Mg2+ concentrations (by100–300 mg/L). Large decreases in HAc/HBu and HPr/HVaoccurred respectively at the first and second biogas productionpeaks, in which the Zeo-digester exhibited an acceleratedSCOD and VFA (especially HPr and HBu) degradation rates.The biogasification performance in the Zeo-digester wasapproximately enhanced by 20 % when treating swinemanure, yielding 356 mL/g VSadded of biogas and 204 mLCH4/g VSadded, respectively. Zeolite addition exerted nosignificant influence on the biodiversity of the microbialcommunity in this study. In the present work, the batchexperiments were carried out with no inoculation of themicroorganisms acclimated to the anaerobic digestion ofswine manure. Thus, the advantage possessed by the Zeo-digester is pending when the two reactors are operated undersemi-continuous and continuous conditions. In addition, it isstill under investigation whether the acclimation ofmicroorganisms to the anaerobic digestion of swine manurewould have impacts on their different biogasificationbehaviors or not.

Acknowledgments The authors thank the partial financial support bythe “Innovation Foundation for Graduate Students of Fudan University,”China, and the Japan Society for the Promotion of Science (JSPS),Grants-in-Aid for Scientific Research (B, no. 25281048). The authorsare also grateful for the field support provided by the Piggery Farm ofBaimi Village and the Zhangpu Town Government of Kunshan County,Jiangsu Province, China.

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