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Biochemical Engineering Journal 88 (2014) 19–25

Contents lists available at ScienceDirect

Biochemical Engineering Journal

jo ur nal home page: www.elsev ier .com/ locate /be j

egular Article

half-submerged integrated two-phase anaerobic reactor forgricultural solid waste codigestion

ei Xinga,b, Xiaojie Chenb, Jiane Zuob,∗, Chong Wangc, Jia Linb, Kaijun Wangb

School of Civil Engineering, Beijing Jiaotong University, Beijing 100044, ChinaState Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing 100084, ChinaCollege of Science, Hunan Agricultural University, Changsha 410128, China

r t i c l e i n f o

rticle history:eceived 13 August 2013eceived in revised form 24 March 2014ccepted 27 March 2014vailable online 6 April 2014

eywords:naerobic processeswo-phaseioreactors

a b s t r a c t

Anaerobic digestion is widely used in bioenergy recovery from waste. In this study, a half-submerged,integrated, two-phase anaerobic reactor consisting of a top roller acting as an acidogenic unit and arecycling bottom reactor acting as a methanogenic unit was developed for the codigestion of wheatstraw (WS) and fruit/vegetable waste (FVW). The reactor was operated for 21 batches (nearly 300 d).Anaerobic granular sludge was inoculated into the methanogenic unit. The residence time for the mixedwaste was maintained as 10 d when the operation stabilized, and the temperature was kept at 35 ◦C.The highest organic loading rate was 1.37 kg VS/(m3 d), and the maximum daily biogas production was328 L/d. Volatile solid removal efficiencies exceeded 85%. WS digestion could be confirmed, and effi-ciency was affected by both the ratio of WS to FVW and the loading rate. The dominant bacteria were

aste treatmentodigestioniogas

Bacteroides-like species, which are involved in glycan and cellulose decomposition. Methanogenic com-munity structures, pH levels, and volatile fatty acid concentrations in the acidogenic and methanogenicunits differed, indicating successful phase separation. This novel reactor can improve the mass transferand microbial cooperation between acidogenic and methanogenic units and can efficiently and steadycodigest solid waste.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Anaerobic digestion is an efficient and sustainable technologyhat has been widely used in agricultural waste treatment [1–3].he biogas produced during anaerobic digestion is a renewablenergy, and the liquid and solid residues can be used as green fer-ilizers. However, the anaerobic digestion of agricultural wastesith high solid concentrations is limited by low hydrolysis rates

particularly for lignocellulosic waste such as wheat straw (WS)],ow mass-transfer efficiency, and difficulty in microbial biomasseservation [4]. Anaerobic microecosystem balance is also readilyisrupted because of the different growth rates and optimal con-itions of hydrolytic and fermentative bacteria and methanogens

5].

Aiming to the problem of high solid anaerobic digestion, codi-estion was suggested and investigated. Lignocellulosic waste can

∗ Corresponding author at: School of Environment, Tsinghua University, Beijing00084, China. Tel.: +86 10 6277 2455; fax: +86 10 62772455.

E-mail addresses: weix@bjtu.edu.cn (W. Xing), jiane.zuo@tsinghua.edu.cnJ. Zuo).

ttp://dx.doi.org/10.1016/j.bej.2014.03.016369-703X/© 2014 Elsevier B.V. All rights reserved.

be digested simultaneously with other materials, such as manure,sludge, and vegetable and fruit waste, in order to adjust the C/Nratios of the raw materials and improve mass transfer [6,7]. Twotypes of reactor configurations, namely, single- and two-phasereaction systems, were developed for solid agricultural wastecodigestion. Single-phase reactors can be operated in batch, semi-batch, or continuous modes. The continuously stirred tank reactor(CSTR) is a typical single-phase anaerobic reaction system in whichhydrolytic and fermentative bacteria and methanogens are mixedin a single reaction zone [8]. With the development of high-rateanaerobic reactors, such as upflow anaerobic sludge bed (UASB), forliquid materials, biomass preservation, high methanogenic reactionrates, and high mass-transfer efficiency can be achieved by granu-lar sludge and up-flow. However, hydrolysis limits the applicationof high-rate reactors to high solid anaerobic digestion, particularlyfor agricultural waste such as lignocellulose [9].

Meanwhile, two-phase anaerobic digestion has the advan-tage of operational stability and high-solid digestion efficiency

[10,11]. Two-phase digestion systems spatially separate the acid-ification phase from the methane production phase using twoseparated bioreactors in series. This configuration allows the effi-cient hydrolysis of solid materials while maintaining favorable

20 W. Xing et al. / Biochemical Enginee

Nomenclature and abbreviations

C carbonCSTR continuously stirred tank reactorEGSB expanded granular sludge bedFVW fruit/vegetable wasteHIT half-submerged integrated two-phaseLB Luria-BertaniN nitrogenPCR polymerase chain reactionPMMA polymethyl methacrylateSLBR sequencing leach bed reactorSEM scanning electron microscopyTC total carbonTOC total organic carbonTS total solidUASB upflow anaerobic sludge bedUASS upflow anaerobic solid-stateVFAs volatile fatty acids

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VS volatile solidWS wheat straw

ethane production. Reactor configuration is a major concernn related studies. For example, an upflow anaerobic solid-stateUASS) reactor has been developed for the codigestion of agri-ultural waste [9]. A number of researchers combined an initialigester with a UASB to digest crops or grass [12,13]. The sequenc-

ng leach bed reactor (SLBR) and CSTR coupled with a UASB are bothonsidered promising [14]. Different types of two-phase anaero-ic digesters, including integrated systems, have been effectivelypplied to agricultural waste codigestion [15,16].

In this study, a novel reactor configuration, namely, thealf-submerged, integrated, two-phase anaerobic reactor (HITnaerobic reactor), was developed. The integrated reactor wasased on the principle of combining separated acidogenic andethanogenic units as well as solid and liquid phases in one sin-

le reactor configuration, by using a half-submerged structure.he study aims to accelerate the hydrolysis of solid materials andnhance the mass transfer and syntrophic cooperation betweencidogenic and methanogenic microorganisms. Experiments on WSnd fruit/vegetable waste (FVW) codigestion were performed toest the effectiveness of the HIT anaerobic reactor. The objectivesf the research are as follows: (1) to introduce the design conceptnd operational approach of the proposed HIT anaerobic reactor;2) to evaluate the agricultural solid waste codigestion performancef the proposed reactor in terms of volatile solid (VS) removal andiogas production; and (3) to investigate the phase separation byomparing the pH levels, volatile fatty acid (VFA) concentrations,nd microbial communities in both acidogenic and methanogenicnits.

. Materials and methods

.1. Characteristics of WS and FVW

In this study, WS and FVW served as the main substrates. WSas collected from a farmland near Beijing, China. FVW, includingears, apples, bananas, watermelons, Chinese cabbage, and lettuce,as obtained from the waste dump of a fruit and vegetable mar-

et in Beijing. Both WS and FVW were shredded and homogenized

o small pieces (approximately 5 cm in length) after the fruit coresere removed. Composition characteristics, such as total solid (TS)

nd VS, were analyzed; the results are shown in Table 1. The C/Natio of WS is 49.67, whereas that of FVW is 15.62. These values

ring Journal 88 (2014) 19–25

indicate that nitrogen is insufficient when WS alone is used as theanaerobic digestion feedstock. Certain proportions of WS and FVWare required to improve the C/N balance. In this study, the C/Nratios in the range of 15.62–26.42 were investigated by adjustingthe proportions of WS and FVW.

2.2. HIT anaerobic reactor design

The proposed innovative HIT anaerobic reactor in this studymainly consists of two parts, namely, the acidogenic unit on topand the methanogenic unit at the bottom. The entire apparatusand operation approach (Chinese patent: CN201010576176) areshown in Fig. 1. The reactor was composed of polymethyl methacry-late (PMMA) and stainless steel. A stainless steel roller that couldbe rotated at rates of 3–30 rpm was installed on top. Three innerclapboards separated the roll into six equal parts to ensure thor-ough mixing of the solid waste. Holes with 5 mm diameters wereevenly distributed on the roller surface. The solid materials fed intothe reactor were half-submerged in the liquid zone to allow suffi-cient transfer of the substance. A cuboid reactor with a hot waterjacket composed of PMMA served as the methanogenic unit forthe granular sludge cultivation and anaerobic reaction. A recyclingsystem was installed between the acidogenic and methanogenicunits to pump the hydrolysates from the acidogenic zone intothe methanogenic zone. This process efficiently prevented acidaccumulation and enhanced mass transfer. The biogas releasedfrom the reaction zones was collected from the top of the reactor.After the reaction, the solid residue and the additional water fromraw materials were obtained from the roller and the liquid out-let, respectively, for further analysis. Half-submergence enhancesorganic waste dissolution and hydrolysis, which is rapidly activatedby microorganism retention. The acidogenic and methanogeniczones can be separated in a combined reactor but can also be com-bined via mass transfer to achieve efficient anaerobic digestion.

In the current study, the efficient reaction volume of the reac-tor was 75 L, of which the acidogenic unit roller accounted for25 L, whereas the methanogenic unit cuboid reactor accounted for50 L. However, the volume of the methanogenic unit cuboid reactorcould be reduced to approximately 25 L based on the operationalperformance observed in this study. This volume reduction canfurther improve the utilization efficiency and loading rate of thereactor.

2.3. Experiment design and parameters of the HIT anaerobicreactor

Experiments were conducted on 21 batches throughout anoperation period of nearly 300 d. First, anaerobic granular sludgefrom a full-scale UASB reactor (obtained from Shandong Province,China) that treats starch wastewater was inoculated into themethanogenic unit at 35 ◦C. The sludge concentration in themethanogenic unit was approximately 20 g VS/L. The residencetime for the mixed waste in the acidogenic unit decreased from 24 dto 17 d for the first four batches (considered as start-up periods)and was then maintained at 10 d for the subsequent batches. TheWS/FVW ratios of the different batches ranged from 0.2 to 1.5 (ascalculated form the VS amount), except that of the 15th batch,in which FVW was the sole feedstock. The roller rotating speed,inner recycling flow, and reactor temperature were maintained at5 rpm, 0.45 m3/h, and 35 ◦C, respectively, throughout the experi-mental period. The TS and VS of the raw materials and residues

were respectively determined at the beginning and the end of eachbatch operation. The volume and total organic carbon (TOC) of thewater discharged at the end of each batch operation were also mea-sured. pH levels and VFAs were determined by daily sampling, and

W. Xing et al. / Biochemical Engineering Journal 88 (2014) 19–25 21

Table 1Characteristics of wheat straw (WS) and fruit/vegetable waste (FVW).

Substrate TS (%) VS (%) Element content (%TS) C/N

C H N O

Wheat straw 94.47 83.75 42.22 5.36 0.85 39.68 49.673.26

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Fruit/vegetable waste 7.37 6.49 4

iogas production in each batch was monitored at the same timevery day.

Reactor biogas production was determined using an LML-2-tyle wet gas meter (Changchun Instruments Company, China).he results were then converted to the standard volume. Methaneontents and VFA concentration were determined using gashromatography (N2000, Zhida Company, China; 6890N, Agilentechnologies, USA). TOC and total carbon (TC) were measured using

TOC analyzer (TOC-5000, Shimadzu, Japan). Other parameters,uch as TS and VS, were determined using standard methods (ChinaPA 2002). At the end of operation, microorganisms in the sludgef the methanogenic zone and on the surface of the digested WSere observed by scanning electron microscopy (SEM, Quanta200,

EI Company, USA).

.4. Microbial community analysis using 16S rDNA sequencingechnology

Given the stability of operation in the final three batches underhe same operational parameters, microbial samples were col-ected from the acidogenic and methanogenic units of the reactor

t the end of the 19th batch operation. The samples were gentlyashed and centrifuged for 20 min at 15,000 rpm (CR22G, HITACHIompany, Japan). The samples were then used in DNA extraction,olymerase chain reaction (PCR) amplification, and clone library

ig. 1. Schematic diagram of the innovative half-submerged, integrated, two-phase (HIT4) holes on the roller surface, (5) inner clapboard, (6) rotation axis, (7) sampling orifice, (12) recycling distributor, (13) water outlet, (14) water jacket.

5.18 2.77 38.01 15.62

construction to investigate microbial community structures. TotalDNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomed-icals, LLC, USA). PCR was performed using a PTC-200 system (MJResearch Inc., USA). Primers 63f (5′-CAGGCCTAACACATGCAAGTC-3′) and 1387r (5′-GGG CGG WGT GTA CAA GGC-3′) were usedin PCR to amplify bacterial 16S rDNA genes. The detailed ampli-fication conditions have been reported by Julian et al. [17].Meanwhile, primers 109f (5′-ACKGCTCAGTAACACGT-3′) and 915r(5′-GTGCTCCCCCGCCAATTCCT-3′) were used to amplify archaeal16S rDNA; the amplification conditions are discussed in Großkopf’sreport [18]. Purified PCR products were ligated into the pGEM-Tvector system (Promega Company, USA), transformed into com-petent Escherichia coli DH5� cells, and then incubated at 37 ◦C for18 h. Afterward, clone libraries were generated on Luria-Bertaniplates containing 100 �g/mL of ampicillin, 80 �g/mL of X-Gal, and0.5 mmol/L of isopropyl �-d-1-thiogalactopyranoside. The clonelibrary construction details have been reported in our previouswork [19]. Positive clones were randomly selected for sequenc-ing via the Sanger method of dideoxy or chain termination. Thesequencing was performed by Shenggong Co., Ltd. (Shanghai,China) using a 3730XL DNA Analyzer (ABI, USA). The BLAST search

program (http://www.ncbi.nlm.nih.gov/BLAST/) was used to deter-mine the phylogenetic affiliations of the obtained sequences. Thebeta-diversity index [20] was calculated to determine the differ-ence between two communities.

) anaerobic reactor. (1) Top cover, (2) biogas outlet, (3) acidogenic reaction roller,8) rotation motor, (9) temperature probe, (10) recycling pipe, (11) recycling pump,

22 W. Xing et al. / Biochemical Enginee

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ig. 2. Daily biogas production rate in the first five batches of wheat straw (WS) andruit/vegetable waste (FVS) codigestion.

. Results and discussion

.1. Performance of the HIT anaerobic reactor

The innovative HIT anaerobic reactor was used to codigest 21atches (∼300 days) of WS and FVW. For the first four (start-up)atches, the feed amount was maintained at approximately 0.4 kgS per batch, and the weight ratio of WS to FVW was maintainedt 1:1. The biogas generation rates during the start-up periods arehown in Fig. 2. The maximum biogas generation rate for each batchas initially very low but steadily increased as the operation pro-

ressed. The total biogas production was 0.086 m3 in the first batchnd increased to 0.117 m3 in the fourth batch. In particular, theaximum biogas production rate was observed on the second day

f the fourth and the fifth batch and then on the first day of eachubsequent batch operation. These results indicate that the start-upf the HIT anaerobic reactor was complete.

The residence time for the solid phase in the acidogenic unitas maintained at 10 d from the fifth batch onward, and the reac-

or operation stabilized. The actual residence time for the liquidhase in both acidogenic and methanogenic units was actually lesshan 10 d because the water in the waste gradually released intohe liquid phase and was discharged all at once at the end of eachatch operation. The total biogas production and feed amount forach batch during the stable period are presented in Fig. 3. Dur-ng the operation, the highest organic loading rate in the reactor

as 1.37 kg VS/(m3 d), and the maximum daily biogas generationate was 328 L/d. The maximum total biogas production in thenal batch was 0.75 m3. The biogas production was approximately60 L/kg VS (removed) and was positively and linearly correlatedR2 > 0.9) with the removed WS and FVW. The methane contentsn the biogas generated in each batch ranged from 64.9% to 76.7%.

oreover, the VS removal efficiency exceeded 85%. These resultsre comparable with those obtained from grass silage digestion inertain two-phase anaerobic reactors [21,22].

ig. 3. Total biogas production and feed amount in each stable WS and FVW batchodigestion.

ring Journal 88 (2014) 19–25

Given that the same operational parameters were used in thefinal three batches, a statistical analysis of the stability of the reactorwas performed using batches 19–21. The average value, standarddeviation, and variation coefficient (relative standard deviation) ofVS removal efficiency and total biogas production (Table 2) werecalculated. The variation in the reaction performances of batches19–21 was relatively small. This result indicates that the reactorwas stable under similar operational parameters.

The carbon mass balance was then calculated based on batches19–21. The fed-in TC was calculated as 489.32 g according to theTS and carbon percentage in the feedstock. This value is appli-cable to all three batches. The average biogas production was0.689 ± 0.055 m3, which corresponds to 369.39 ± 29.56 g carbon interms of the molar volume of standard gas. The TC concentrationin the water discharge was 503.67 ± 10.97 mg/L, and the TC thatwas lost in the water was 5.54 ± 0.12 g based on the water dis-charge volume of 11 L. The average TC in the residue dischargewas 82.87 ± 13.44 g. Therefore, the mass balance was determined as93.6% based on the mass ratio of the output TC to the input TC. Thisresult indicates the validity of the monitoring data. The remaining6.4% of the TC may be attributed to detection error and gas leakage.

Given the high codigestion efficiency of the HIT anaerobic reac-tor during the first three days of each batch, the reactor can befurther operated in a sequencing batch mode, in which daily feed-ing is implemented to reduce the residence time. Moreover, theorganic loading rates can be further increased by reducing the inef-fective methanogenic unit volume. This reactor configuration mayeither be applied in localized, small-scale operations or scaled upon the basis of reaction modules.

3.2. WS and FVW codigestion at different ratios

WS is generally difficult to digest because of its high TS andlignocellulose contents. However, WS and FVW codigestion wasachieved using the innovative HIT anaerobic reactor developed inthis study. The results are shown in Table 3. In the 15th batch, inwhich FVS was the sole feedstock, the removal efficiency of totalVS reached 98%. This finding indicates that nearly all of the FVS canbe degraded in the reaction. Therefore, the WS digestion efficiencywas calculated from the total VS reduction and the assumption thatFVS was 98% decomposed. The results show that the amount ofWS removed in typical batches ranged from 27% to 54%, and theefficiency can be affected by both the ratio of WS to FVW and theloading rate of the reactor.

The ratios of “decomposed VS in WS” to the “decomposed VS inFVW” (i.e., Decomposed VSWS:Decomposed VSFVW in Table 3) arecomparable at similar VS loading rates. The ratio was either 0.46or 0.47 when the VS loading rate ranged from 0.5 kg VS/(m3 d) to0.6 kg VS/(m3 d) and ranged from 0.11 to 0.13 when the VS loadingrate increased to 1.20 kg VS/(m3 d). The VSWS and VSFVW exhib-ited proportion reductions. This result indicates successful WS andFVW codigestion. WS cannot be efficiently decomposed becauseof its high C/N ratio. Thus, the addition of FVW can improve theC/N balance in the digestion systems and increase the decomposi-tion efficiency. The loading rate is another key factor in codigestionprocesses. Lehtomaki et al. [7] found that high loading rates lead toinefficient anaerobic codigestion of crop residues and cow manure.In the current study, the Decomposed VSWS:Decomposed VSFVWwas low when the loading rate was high because large amountsof FVW could enhance the preference of microorganisms to thiswaste. However, a comparison of batches 10 and 18 showed thathigher WS proportions at the same loading rate were adverse to

WS decomposition (26.7% at a VSWS:VSFVW of 0.42:1 vs. 54.1% ata VSWS:VSFVW of 0.23:1). These results indicate that appropriateloading rate and WS proportion ranges are necessary for a success-ful WS and FVW codigestion.

W. Xing et al. / Biochemical Engineering Journal 88 (2014) 19–25 23

Table 2Statistical analysis of the VS removal efficiency and the total biogas production in the last three batches.

Batch 19 Batch 20 Batch 21 Average value (A) Standard deviation (D) Coefficient of variation (D/A)

VS removal efficiency 87.4% 89.1% 90.0% 88.8% 1.3% 1.5%Total biogas production 0.641 m3 0.677 m3 0.749 m3 0.689 m3 0.055 m3 8.0%

Table 3Feed amount and efficiency of the reactor in typical batches.

Batch Wheat straw(kgVS)

Fruit/vegetablewaste (kgVS)

VSWS:VSFVW Loading rate(kgVS/(m3 d))

Removal efficiencyof total VS (%)

Removal efficiencyof wheat straw (%)

DecomposedVSWS:decomposedVSFVW

5 0.23 0.22 1.05:1 0.60 65.5 43.1 0.4647.2 30.3 0.4775.3 26.7 0.1189.9 54.1 0.13

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Methanogenic unit

pH v

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Time in batch (d)

6 0.23 0.15 1.52:1 0.5110 0.27 0.64 0.42:1 1.20

18 0.17 0.73 0.23:1 1.20

The daily and cumulative biogas productions of typical batchesre shown in Fig. 4. The daily biogas production in the first threeays of each batch operation was markedly higher than those inther days. This finding reveals that microorganisms acclimatedo the feedstock were enriched in the reactor. Moreover, most ofhe microorganisms could be reserved and maintained in the liquidhase of the HIT anaerobic reactor because of the reactor configura-ion. This characteristic reduces the starting time for each batch ands particularly favorable for high-solid materials such as agricul-ural waste, which normally require a relatively long starting timeor digestion. In particular, biogas production in batch 10 peakedor the second time on the fifth day when both the ratio of WS toVW and the reactor loading rate were relatively high. This findingroves that the codigestion was achieved, and that FVW is moreeadily and rapidly digested than WS. WS and FVW codigestionromoted WS digestion, as visually confirmed by the SEM images,hich showed the presence of a biofilm and fractures on the WS.

.3. pH and VFA concentrations in the acidogenic andethanogenic units

Liquid samples were collected from the acidogenic andethanogenic units of the HIT anaerobic reactor to monitor the

H and VFA concentrations throughout the operation. Data fromll batches showed similar trends. Therefore, only data from batch8 are shown in Figs. 5 and 6 to represent all batches. The pH in the

cidogenic unit decreased from 7.2 to 6.8 (Fig. 5). This value is in theuggested range for optimal hydrolysis [23]. Meanwhile, the pH inhe methanogenic unit remained within the 7.0–7.4 range through-ut the entire period; this result indicates that the methanogens

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Fig. 4. Daily and cumulative biogas

Fig. 5. pH values in the acidogenic and methanogenic units of batch 18.

were optimized according to the suggested conditions [24]. Thefluctuations in the pH in the methanogenic unit were smaller thanthose in the acidogenic unit. It can be deduced that the pH differ-ence between the two units could be further enlarged along withthe loading rate increment. This phenomenon indicates that theinnovative HIT anaerobic reactor provided various reactive condi-tions. Methanogens are more sensitive than hydrolytic bacteria andfermentative bacteria. Therefore, the separation of the acidogenicphase from the methanogenic phase in a single reactor can improvethe stability and efficiency of the digestion process.

Fig. 6 shows the variation in the VFA concentrations in the

acidogenic and methanogenic units. The total VFA concentrationin the acidogenic unit increased to approximately 4000 mg/L inthe first two days. This result indicates effective hydrolysis and

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production in typical batches.

24 W. Xing et al. / Biochemical Enginee

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effect of co-digestion with dairy cow manure, Bioresour. Technol. 99 (2008)8288–8293.

[2] M.F. Demirbas, M. Balat, H. Balat, Biowastes-to-biofuels, Energy Convers. Man-

ig. 6. VFA concentrations in the acidogenic and methanogenic units of batch 18.a) acidogenic unit; (b) methanogenic unit.

cidification. Acetic acid constituted two thirds of the total VFAs.oreover, although propionic acid was present during the entire

rocess, its concentration decreased to 87.9 mg/L at the end ofatch operation. The VFA reduction after the third day was mainlyttributed to VFA consumption in the methanogenic unit as a resultf efficient mass transfer between the two units. The maximum VFAoncentration in the methanogenic unit was lower than 2500 mg/L;his value gradually decreased after the second day. During theeaction, n-butyric acid was degraded into small molecular com-ounds and led to a transitory increase in acetic acid and propioniccid levels. The significant difference in the VFAs of the two unitsnd the lack of VFA accumulation indicates a successful phase sep-ration and an efficient mass transfer.

.4. Microbial community structures in the acidogenic andethanogenic units

Given the stability of the operation in the final three batchesnder the same operational parameters, microorganisms in theeactor were sampled, and microbial community structures werenalyzed at the end of the 19th batch operation using molecu-ar cloning technology. The results indicate that Bacteroides-likepecies related to glycan and cellulose decomposition were theominant bacteria (accounting for 46% of the bacterial community)

n the acidogenic unit. These dominant clones showed 98% similar-ty to the uncultured Bacteroidetes clone L38 (EU887996) obtainedrom a UASB reactor fed with grass silage [25]. These cloneslso exhibited 96% similarity to cultured cellulolytic Bacteroides

p. 22C (AY554420) isolated from a landfill leachate bioreactor.ther clones in the HIT anaerobic reactor were closely related to

xylanolytic anaerobe affiliated with the order Bacteroidales [26].he bacterial community in the reactor included species that belong

ring Journal 88 (2014) 19–25

to the orders Clostridiales and Syntrophobacterales. Previous stud-ies have proven that related species play highly important roles inhydrolysis, fermentation, and acetate producing [27,28]. Thus, thecurrent results also provide important evidence for lignocellulosicand sugary waste codigestion in the HIT anaerobic reactor.

Microbial clone libraries were also constructed for the sludgesamples from the methanogenic unit. The results reveal negligibledifference in the bacterial communities in the two reactor units,as indicated by a calculated beta-diversity index of 0.22. By con-trast, the difference in the archaeal communities of the acidogenicand methanogenic units was significant, as indicated by a beta-diversity index of 0.41. In particular, the genus Methanosaeta, whichhas an important function in granular sludge integration [29],constituted 79% of the archaeal community in the methanogenicunit and only 41% in the acidogenic unit. Meanwhile, the propor-tions of the Methanosarcina, Methanobacterium, Methanoculleus andMethanospirillum genera were higher in the acidogenic unit than inthe methanogenic unit. The differences between the methanogeniccommunity structures further confirm the separation of the aci-dogenic phase from the methanogenic phase in the HIT anaerobicreactor. Hydrolytic and fermentative bacteria in the reactor werenearly similar because of their increased adaptability to a widerange of pH and VFA concentrations. Therefore, the innovative HITanaerobic reactor developed in this study can provide a relativelystable environment for sensitive methanogen and can maintainsyntrophic cooperation between different microorganisms.

4. Conclusions

An innovative, half-submerged, integrated, two-phase anaer-obic reactor was developed to accelerate the hydrolysis of solidmaterials and enhance mass transfer and syntrophic cooperationbetween acidogenic and methanogenic microorganisms. This reac-tor was used to codigest WS and FVW. The average VS removalefficiency exceeded 85% during 21 batch operations that lastedapproximately 300 d. The maximum organic loading rate and max-imum daily biogas production were 1.37 kg VS/(m3 d) and 328 L/d,respectively. The WS digestion efficiency was affected by both theratio of WS to FVW and the loading rate. The microbial commu-nity structures, pH, and VFA concentrations in the acidogenic andmethanogenic units varied and thus indicate successful phase sepa-ration. The innovative HIT anaerobic reactor can provide a relativelystable environment for sensitive methanogens and can maintainbalance in the anaerobic microecosystem.

Acknowledgements

This research was supported by the Major Science and Tech-nology Program for Water Pollution Control and Management ofChina (2012ZX07205-001), and the National Science and Technol-ogy Support Program (2008BADC4B18). We would like to expressour appreciation to the local staff for their kind assistance in wastecollection.

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