a comparative study on the alternating mesophilic and thermophilic two-stage
TRANSCRIPT
Journal of Environmental Sciences 26 (2014) 1–10
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Journal of Environmental Sciences
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A comparative study on the alternating mesophilic and thermophilic two-stageanaerobic digestion of food waste
Jey-R Sabado Ventura∗∗, Jehoon Lee∗∗, Deokjin Jahng∗
Department of Environmental Engineering and Energy, Myongji University, Gyeonggi-Do 449-728, Korea
a r t i c l e i n f o
Article history:Special issue: Sustainable water man-agement for green infrastructure
Keywords:community structurefood wastemethane productionnutrient removaltwo-stage anaerobic digestion
DOI: 10.1016/S1001-0742(13)60599-9
a b s t r a c t
An alternating mesophilic and thermophilic two stage anaerobic digestion (AD) process wasconducted. The temperature of the acidogenic (A) and methanogenic (M) reactors was controlledas follows: System 1 (S1) mesophilic A-mesophilic M; (S2) mesophilic A-thermophilic M; and (S3)thermophilic A-mesophilic M. Initially, the AD reactor was acclimatized and inoculated with digestersludge. Food waste was added with the soluble chemical oxygen demand (SCOD) concentrations of41.4–47.0 g/L and volatile fatty acids of 2.0–3.2 g/L. Based on the results, the highest total chemicaloxygen demand removal (86.6%) was recorded in S2 while S3 exhibited the highest SCOD removal(96.6%). Comparing S1 with S2, total solids removal increased by 0.5%; S3 on the other handdecreased by 0.1 % as compared to S1. However, volatile solids (VS) removal in S1, S2, and S3was 78.5%, 81.7%, and 79.2%, respectively. S2 also exhibited the highest CH4 content, yield, andproduction rate of 70.7%, 0.44 L CH4/g VSadded, and 1.23 L CH4/(L·day), respectively. Bacterialcommunity structure revealed that the richness, diversity, evenness, and dominance of S2 were highexcept for the archaeal community. The terminal restriction fragments dendrogram also revealed thatthe microbial community of the acidogenic and methanogenic reactors in S2 was distinct. Therefore,S2 was the best among the systems for the operation of two-stage AD of food waste in terms of CH4
production, nutrient removal, and microbial communi structure.
Introduction
In the 2007 census by the Ministry of Environment, 14,400tons/day of food waste was generated in Korea (MOE,2007), and more than 50% of the food wastes generatedwere dumped into the sea. However, due to the MarinePollution Act of 2012, ocean dumping of food wastes andother similar types of wastewater is banned. To solve thisproblem, anaerobic digestion (AD) has been developedbecause it can handle waste with high organic contentwith efficient mineralization compared to other processes.Moreover, additional energy in the form of biogas can alsobe harnessed. AD is more practical to use than competing
∗Corresponding author. E-mail: [email protected].∗∗ The authors contribute equally to this work.
processes because of its low maintenance and operationcost, low excess sludge production, and low release ofodor and aerosols (Solera et al., 2002). Other methodssuch as landfilling, composting, and incineration have beensuggested for food waste disposal (Kim et al., 2000; Shin etal., 2001). However, aside from the production of leachate,foul odor, and toxic gases, these processes require landspace and the input of energy to operate; hence, they arenot environmentally friendly and economical.
The advantages of a two-stage system in AD is thepossible enrichment of different bacteria in each digester,which increases stability and allows higher organic loadingrate (OLR) and shorter hydraulic retention time (HRT)than the conventional single-stage digester (Solera et al.,2002). The first stage may act as a metabolic buffer bypreventing pH-shock and build-up of toxic material thatcould be transferred to the methanogenic reactor (Eastman
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and Ferguson, 1981; Solera et al., 2002). Therefore, amore efficient process is established in the two-stage ADsystem. However, disadvantages such as long start-up time,additional reactor cost and operation, and poor granuleformation may be experienced in two-stage AD (Lettingaand Hulshoff, 1991).
Recent studies comparing the advantages of two-stageAD over single stage AD have been reported by Lim etal. (2013), Nathao et al. (2013), and Shen et al. (2013).From their studies, an increase of 7%–15.8% (Shen et al.,2013), 12.8% (Nathao et al., 2013), and 23% (Lim et al.,2013) CH4 yield was obtained in two-stage AD comparedto single stage AD. Other studies on two-stage AD usingfood waste in combination with other substrates such asfruit and vegetable waste (Lin et al., 2012), pulp and papersludge (Lin et al., 2013), and brown water (Lim et al.,2013) have shown improvement in the overall performanceof AD. Aside from this, the pretreatment of food waste(Stabnikova et al., 2008; Shahriari et al., 2013) before two-stage AD further improved its capacity to degrade nutrientsand produce biogas. The role of trace elements was alsoinvestigated thoroughly in AD of food waste (Zhang et al.,2011; Zhang and Jahng, 2012). Moreover, the thermophilicacidogenic reactor (Kim et al., 2013) and thermophilicmethanogenic reactor (Lin et al., 2013) were investigatedin two-stage AD.
The microbial community characterization in AD hasalso been carried out recently to monitor the bacterial orarchaeal microbial activity during operation (Shin et al.,2010; Lim et al., 2013). The study of Shin et al. (2010)suggested that higher diversity of microorganisms wereobserved in the methanogenic reactor of the two-stageAD of food waste-recycling wastewater. Furthermore, themethanogenic community dynamics in an increasing ratioof fruit and vegetable waste to food waste was knownto be dominated by Methanoculleus, Methanosaeta andMethanosarcina. Moreover, Lim et al. (2013) have shownthe predominance of Firmicutes and greater bacterial diver-sity in the two-stage AD of brown water and food waste.This implies that the proper understanding of the microbialbehaviour during an AD operation will further lead tothe improvement of design and digestion process of ADespecially in food waste digestion.
In this study, two-stage AD of food waste was in-vestigated by controlling mainly the temperature ofthe two digesters. Three systems were developed byvarying the temperature profiles of the acidogenic andmethanogenic reactors from both as mesophilic reac-tors to alternating mesophilic and thermophilic reactorsystems. The study were carried out using steady stateconditions, and microbial community structures were alsoanalyzed using 16s ribosomal DNA gene amplificationand terminal-restriction fragment length polymorphism (T-RFLP) analyses. Previous reports have dealt mostly witha thermophilic acidogenic stage followed by a mesophilic
methanogenic stage (Oles et al., 1997; Dinsdale et al.,1997). However, this study was able to extend the in-vestigation to include scenarios of alternating mesophilicor thermophilic two-stage AD and their effect on themicrobial structure of the varying system.
1 Materials and methods
1.1 Food waste source and inocula
The food waste was collected from a food waste recyclingcompany in Yongin Korea. After collection, foreign ma-terials such as bones, plastics, hard ligaments, and otherinorganic materials were removed. The organic loadingconcentration was adjusted using tap water and sieved (No.10) to remove coarse particles larger than 2 mm. Thediluted food wastewater was kept at 4°C to preserve theorganic waste prior to use. The food waste had an initialpH of 3.75 ± 0.15, total solids (TS) of 5.5% ± 0.1%,volatile solids (VS) of 5.25% ± 0.45%, total chemicaloxygen demand (TCOD) of 90.95 ± 4.45 g/L, soluble COD(SCOD) of 44.2 ± 2.8 g/L, and 2.6 ± 0.6 g/L volatile fattyacids (VFAs).
The anaerobic seed sludge was taken from the AD ofthe Yongin Respia wastewater treatment plant, Yongin,Korea. The digester sludge that served as inoculum forthe experiment had an initial pH of 7.3, 16.6 g/L totalsuspended solid (TSS), and 12.0 g/L volatile VSS.
1.2 Two-stage anaerobic digester and operating condi-tions
Two separate bioreactors were designed specifically forthe acidogenic and methanogenic AD. The acidogenicreactor was an acrylic cylindrical reactor with a diameter,height, and effective volume of 22.5 cm, 40 cm, and10 L, respectively (Fig. 1). The CH4 reactor was alsomade of the same material with dimensions of 32 cmdiameter, 48 cm high, and 30 L effective volume. Theacidogenic reactor was operated at the OLR of 4.5 gCOD/(L·day) and operated intermittently (15 min feedpumping and 45 min stop). The HRT was set at 5 and15 days for the acidogenic and methanogenic reactors,respectively. The volumetric flow rate was set at 2 L/day inboth reactors. Both acidogenic and methanogenic reactorswere thermally controlled using a heating jacket with adigital controller (GLTC-DP, GlobalLab, Korea). Initially,mesophilic temperature (36 ± 1°C) was maintained inboth acidogenic and methanogenic reactor for 195 daysfor System 1 (S1); the temperature of the methanogenicreactor was increased to 55 ± 1°C until day 304 in Sys-tem 2 (S2); then, thermophilic acidogenic and mesophilicmethanogenic reactors were set up in System 3 (S3) up today 347. The system in S1 was allowed to stabilize for twoweeks, while one week was allotted for S2 and S3, respec-
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Acidogenic reactor
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Fig. 1 Schematic diagram of the two-stage AD.
tively. A stabilized system was considered to be achievedwhen an increase of VFA concentration in the acidogenicreactor and CH4 production in the methanogenic reactorwere perceived. Also, the subsequent transfer of fermentedfood waste from the thermophilic acidogenic reactor tothe mesophilic methanogenic reactor did not experiencecooling because of the low ratio of feed volume transferredto the succeeding reactor. The thermophilic feed compen-sated the target temperature of the methanogenic reactorwithout cooling in S3.
1.3 Analytical methods
TS, VS, TSS, VSS, TCOD, and SCOD were analyzedaccording to the standard methods (APHA, 2005). The pHof the samples was measured using a pH meter (Orion,Model 370). CH4 and CO2 were analyzed using an HP-6890 gas chromatograph (GC) (Hewlett Packard 6890, PA,USA) with a thermal conductivity detector (TCD) andHP-Plot Q column (30 m × 0.32 mm × 20 µm) (Zhanget al., 2011). For VFA determination, a separate GC-FID (Younglin 6000D, Seoul, Korea) with HP-INOVAXcolumn (30 m × 0.25 mm × 0.25 µm) was used (Zhang etal., 2011).
One-way ANOVA was used to determine the signifi-cance of differences between the different systems whilethe Student t-test was used to compare the nutrient reduc-tion and methane production performance between S1 andS2, S1 and S3, and S2 and S3. The F-value in one-wayANOVA was compared at a critical F-value table for 2d.f. in all systems. The F-test was also performed beforeproceeding to t-test analysis to determine if the variancesbetween two systems were identical or not. The results ofthe F-test determine the proper t-test (equal or unequalvariances) analysis to be used. The t Stat-value of t-test was
compared at a critical t-value table for 87 d.f. between S1-S2 and S1-S3 while 50 d.f. was used for S2-S3 analysis.The following statistical methods were performed usingMicrosoft Office Excel 2010 (Microsoft Corp., Redmond,WA). The confidence interval was set at 95% (α = 0.05)and the total nutrient removal and methane productionprofiles of the different systems were log transformed priorto statistical analysis.
1.4 T-RFLP analysis
The genomic DNA of the samples collected from the aci-dogenic and methanogenic reactors for different systemswas extracted using the Mo Bio Power Soil DNA IsolationKit (MoBio, USA). The genomic DNA extracts wereconfirmed by electrophoresis and stored at –20°C priorto T-RFLP analysis. The obtained DNA extract was PCRamplified using the 16s rDNA universal primers 27F and1392R (Lane, 1991) and archaea universal primers A109fand A934B (Grosskopf et al., 1998). The forward primerswere labelled with 5′-end 6-FAM (phosphoramidite flu-orochrome 5-carboxyfluorescein) (Bionics, Korea). ThePCR was carried in a 50 µL reaction mixture (10X ExTaq buffer, 2.5 mmol/L dNTPs, 0.2 µmol/L primer, 5unit/µL Takara Ex Taq polymerase (Takara, Japan)) con-taining 2 µL of DNA template, according to the followingtemperature cycles: initial 94°C for 5 min, 58°C and55°C annealing for bacteria and archaea respectively; andDNA synthesis (polymerization) at 72°C for 2 min, witha total of 30 cycles at 72°C for 5 min. The restrictionenzyme, HaeIII, was utilized for the digestion of bacterialand archaeal PCR-products.
Four diversity indices, richness, dominance, diversityand evenness, were calculated as described elsewhere(Hurlbert, 1971; Krebs, 1999) using the chromatograms
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obtained from GeneScan Analysis Software v3.7 (AppliedBiosystems, USA). The community similarity analysisbetween acidogenic and methanogenic reactors of S1,S2, and S3 was also estimated through the constructionof a dendrogram using GelCompar II program (AppliedMaths, Belgium) according to the calculation of Pearson’scorrelation coefficients.
2 Results and discussion
2.1 pH and temperature changes
pH is one of the most sensitive operating conditions inan AD because different microorganisms thrive in definedpH ranges. The optimal pH of hydrolytic and acidogenicmicrobes has been found to be between 5.5 and 6.5 (Yuand Fang, 2002; Kim et al., 2003). The acidogenic andmethanogenic stages were controlled at pH 6.6 to 7.8(Lay et al., 1997). The pH and temperature profiles ofthe systems in the two-stage AD are shown in Fig. 2. Tomaintain the optimal pH conditions in the acidogenic andmethanogenic reactor, 1 mol/L HCl and 1 mol/L NaOHwere used to adjust the pH in each reactor. As can be seenin Fig. 2a, the acidogenic and methanogenic pH valueswere 5.0–5.5 and 7.2–8.0, respectively. The pH of thereactors was maintained at these levels to avoid possibleinhibition of the microbial activity. An acidogenic pHbelow pH 5.5 indicated higher VFA concentration in thereactor. The methanogenic reactor also had a pH above8.0, which was possibly due to the initial accumulation ofammonia.
Temperature is also a critical parameter for effective ADimplementation. Increase in operating temperature couldmean higher capital and operating costs (Ferrer et al.,2010). Optimally, AD is operated at 30°C to 40°C. How-ever, it was found that thermophilic conditions could alsobe applied to increase the destruction rate of organic com-pounds, better solid and liquid separation, and pathogen
destruction (Oles et al., 1997). In Fig. 2b, temperatures ofthe different systems were also monitored. As indicatedin section 1.2, S1 was operated at mesophilic conditionfor both reactors. S2 had a mesophilic acidogenic reactorand thermophilic methanogenic reactor, while S3 was thereverse of S2. Comparing closely, the mesophilic conditionin either acidogenic or methanogenic reactor in S1 andS2 maintained an average temperature of 35°C. However,the S3 methanogenic reactor mesophilic condition washigher by 2°C. The absence of cooling of the thermophilicfermented food waste possibly increased the temperatureof the mesophilic methanogenic reactor in S3.
2.2 Nutrient reduction
Figure 3a and e shows the TCOD and TCOD removal ofthe two-stage AD for different systems. The TCOD of S1remained in the range of 10–20 g/L. However, after shiftingto S2, TCOD overloading occurred, which increased theTCOD concentration above 35 g/L for nearly 40 days.After this overloading, the system was stabilized and wentback to its original range of TCOD. In the methanogenicreactor, the TCOD level started at around 40 g/L andgradually increased until the end of S1. The last period(day 155–195) of S1 was shown to have a TCOD level ofat least 80 g/L. This may be due to the effect of nutrientoverloading. However, after the rise in TCOD load, thesystem started to stabilize at a TCOD level around 70 g/Lat S2 to S3.
Comparing the overall TCOD removal (Fig. 3e, Ta-ble 1), S2 had the highest removal of 86.6%, which wasfollowed by S3 (85.1%) and S1 (81.0%). From these obser-vations, it can be inferred that the thermophilic condition ofthe methanogenic reactor was more effective in oxidizingthe available organic nutrients than the thermophilic stateof the acidogenic reactor. Thus, it could be concluded thatmore methanogenic microorganisms were able to thrive athigher temperature than acidogenic microorganisms.
In terms of the SCOD profiles (Fig. 3b and f), the start-up acidogenic SCOD concentration was 20 g/L. A low
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Fig. 2 pH (a) and temperature (b) profiles of the two-stage AD at different systems (S1, S2, and S3).
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level of fluctuations in the SCOD loading was achieved after 3 weeks of incubation. After this period, the SCOD
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Fig. 4 VFA concentrations of (a) acidogenic and (b) methanogenic reactors of S1, S2, and S3 of the two-stage AD.
levels were in the range of 30–40 g/L, starting from themiddle period of S1 up to the final period in S3. The SCODconcentrations of the methanogenic reactor seemed to bebelow 2 g/L in S1 and S2, however, S2 rose higher than20 g/L during start-up. The extreme rise of SCOD level inthe methanogenic reactor of S2 might be due to the shift inthe temperature condition of the reactor from mesophilicto thermophilic state. This might cause the methanogenicmicroorganisms to falter, and hence lower the activity.To cope with the sudden change of temperature, anotheracclimatization stage was experienced by S2. S3, however,it seemed not to be greatly affected by the reversion ofthe thermal conditions of the acidogenic and methanogenicreactors. Although fluctuation was observed in S2, the
total SCOD removal was still 90.5% while S1 and S3were recorded at 92.8% and 96.5%, respectively (Fig. 3f,Table 1).
The TS levels of the acidogenic and methanogenicreactors were also compared (Figs. 3c and g). Around 40–50 g/L of the TS was observed in the incoming TS ofall the reactors. Likewise, TS fluctuations were observedduring the shift of condition from S1 to S2, although morepronounced than the previous SCOD data. The final TSin the methanogenic reactors were maintained at the levelof 20–30 g/L. Comparing the total TS removal of theacidogenic and methanogenic reactors, S2 had the highestremoval 62.7%, followed by S1 (62.2%) (Fig. 3g, Table 1);S3 had TS removal of 62.1%.
Table 1 Acidogenic, methanogenic, and total average nutrient removal and CH4 production of S1, S2, and S3 in the two-stage AD of food waste
Parameters S1 S2 S3
A M Total A M Total A M Total
TCOD removal (%) 26.7 55.0 81.0 a 27.2 59.4 86.6 b,c 32.1 53.0 85.1 a,c
SCOD removal (%) 19.3 73.5 92.8 d 21.5 68.8 90.5 e,f 25.1 71.5 96.5 d,f
TSremoval (%) 22.9 39.3 62.2 g 20.7 42.0 62.7 g 25.3 36.8 62.1 g
VS removal (%) 25.3 53.2 78.5 h 23.1 58.6 81.7 h 27.3 51.9 79.2 h
A: acidogenic reactor; M: methanogenic reactor.Values with the same letter are not significantly different between systems (t Stat 6 t Critical two-tail, α = 0.05).
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Fig. 5 CH4 content (a), CH4 gas production rate (b), and CH4 (c) yield of the methanogenic reactor of S1, S2, and S3.
Journal of Environmental Sciences 26 (2014) 1–10 7
Although TS removal may not give conclusive dataon the reduction of organic matter in the two-stage AD,the gathered data may provide substantial informationon the solid removal in the system. Thus, to investigatethoroughly, the VS content of the food waste was alsomonitored (Fig. 3d).
The VS of the acidogenic reactor was in range of 30–40 g/L while that of the methanogenic reactor was atapproximately 10 g/L (Fig. 3d and h). It took an acclima-tization period of two weeks before the VS concentrationof the acidogenic reactor of S1 attained the VS operatingcondition. Fluctuations in VS content of the food wasteduring the shift to S2 were not as long and abrupt as thefluctuation in SCOD. Much higher VS were observed inS2 and S3 than in S1. However, the methanogenic reactorVS content remained stable during the entire period ofoperation. Comparing closely, the VS removal efficiency ofS1, S2, and S3 was recorded at 78.5%, 81.7%, and 79.2%,respectively (Fig. 3h, Table 1). In contrast to SCODremoval, the TS and VS removal favored S2 compared tothe other systems.
Table 1 shows the summary of the performance andstatistical relationships of the two-stage AD of food wastein the alternating mesophilic and thermophilic condition ofacidogenic and methanogenic reactors. Statistical testingusing one-way ANOVA indicated that the systems are notidentical in terms of TCOD, SCOD, TS, and VS removal(not shown). However, the t-test showed that only S1 andS2 are significantly independent in terms of the TCOD andSCOD reduction capacity. The TS and VS removal of S1,S2, and S3 also did not entail significant differences intheir average solid reduction capacity. The short operatingperiod of S3 as compared to the longer monitoring ofS1 and S2 may have been the source of deviation in theresults of group analysis. Nevertheless, one-way ANOVAsuggests that the systems were not identical.
2.3 VFA production
To investigate the possible occurrence of fluctuations inthe SCOD levels in S2, the change in the VFA profilesof the systems in two-stage AD was determined (Fig.4). As shown in Fig. 4b, the acid concentration of themethanogenic reactor was below 1 g/L, however, when S2was operated, the VFA concentrations were observed tobe above 10 g/L. This provides evidence to support thesudden rise of SCOD level of S2 (Fig. 3b). Therefore, itcan be concluded that the shift to thermophilic conditionof the methanogenic reactor in S2 led to the deteriorationof the performance of the methanogenic microorganisms.Although this did not conform well to the overall per-formance of S2, the start-up fluctuations could be due tothe adjustment period of the anaerobic methanogens to thechange in temperature.
In the acidogenic reactor, acetate was found to be thedominant acid (50%–60%) in all the systems (Fig. 4a). It
was followed by n-butyrate and iso-butyrate at approxi-mately 33%. The other acids measured were propionate,iso-valerate, and n-valerate with concentrations lower than10% of the final acid in all the systems.
2.4 Biogas production
S1 and S2 have been shown not to be related to theCH4 content and yield while the CH4 production ratedid not show significant differences in the methanogenicreactor of all the systems based on t-test analysis. However,based on one-way ANOVA, the CH4 production profiles(CH4 content, production rate, and yield) were significant-ly different between S1, S2, and S3. The CH4 content(Fig. 5a) of S2 was observed to be highest at 70.7%.This was followed by S3 (67.8%), with S1 as the lowestperformer (65.5%). The CH4 production rate was alsohigh in S2 at 1.23 L/(L·day), while S1 and S3 were at0.98 and 1.02 L/(L·day), respectively (Fig. 5b). Comparingfurther, the CH4 yield of S2 was 0.44 L/g VSadded, whichwas 13.6% and 15.9% higher than that in S1 and S3,respectively (Fig. 5c). This indicated that although severefluctuations of nutrient and VS levels were experienced,S2 still managed to overcome the sudden temperaturechange. It should however be noted that the overall CH4production of S2 was the highest. The acidogenic reactor inS3 was also changed to thermophilic condition; however,it was observed that more methanogenic microorganismsare able to dwell in a high-temperature environment thanacidogenic microorganisms (Ferrer et al., 2010). This maybe attributed to the metabolic activity of most acidogenicbacteria, wherein high temperature can be inhibitory.Furthermore, it was stated that acidogenesis is not asso-ciated with thermal hydrolysis, rather on the microbialactivities of the acidogenic reactor (Yu et al., 2013). Thethermophilic conditions might also increase the pH ofthe acidogenic reactor, affecting the microbial activity offermentative microbes.
The temperature-controlled two-stage AD of food wastegave comparable results with other studies dealing with thesame kind of substrate (Table 2). The study had shownthat the mesophilic acidogenic reactor and thermophilicmethanogenic reactor gave the best configuration for boththe organic reduction and biogas production of the two-stage AD of food waste.
2.5 T-RFLP assay results
The T-RFLP data (Table 3) shows that the bacteria havehigher richness, diversity, and dominance in S2 for boththe acidogenic and methanogenic reactors. For archaea, theacidogenic reactor of S2 had the highest richness, diversity,and dominance while the methanogenic reactor of S2 wasthe lowest values (Table 3). The mesophilic archaea in S1were shown to have the highest diversity indices.
Figure 6 shows the T-RFs dendrogram of the bacterialand archaeal community in S1, S2, and S3 of the AD of
8 Journal of Environmental Sciences 26 (2014) 1–10
Table 2 Comparison of the study to others in the literature employing two-stage AD of food waste
Substrate Temp. (°C) HRT (day) Duration TS VS/TS OLR CH4 yield CH4 prod. VS removal Reference
A M A M (day) (%) (%) (kg VS/(L·d)) (L/(kg·VSadded)) (L/(L·day)) (%)
CSTR FW leachate 40.7 37.0 3 27 136 13.8 5.91 2.36 0.55 – 82.6 Kim et al., 2013
Batch FW:PPS (1:1) 37 55 – – – – – – 0.432 – – Lin et al., 2013
CSTR FW:FVW (8:5) 35 35 10 10 – 17.6 0.80 2 0.455 – – Shen et al., 2013
Batch FW (F/M = 7.5) – – – – – – – – 0.094 – – Nathao et al., 2013
Batch (HASL) FW (freeze-thawed) 35 35 – – 12 18.6 94.6 – 0.32 – – Stabnikova et al., 2008
CSTR FW 37 37 – – 120 – – 1.04 0.373 – 90 Cho & Park, 1995
CSTR (HASL) FW 35 35 – – 36 0.46 – – 0.33 – 78 Wang et al., 2005
Semi-cont. FW (microwaved) – – 2 20 – 3.04 94.0 1.39 0.47 1.07 – Shahriari et al., 2013
CSTR FW 35 35 5 20 195 4.5 70.7 3.2 0.38 0.98 78.5 This study
CSTR FW 35 55 5 20 109 5.0 86.7 4.4 0.44 1.23 81.7 This study
CSTR FW 55 37 5 20 43 4.7 85.0 4.0 0.37 1.02 79.2 This study
A: acidogenic reactor; M: methanogenic reactor; CSTR: continuously stirred-tank reactor; FW: food wate; PPS: pulp and paper sludge; FVW: fruit and vegetable waste; F/M:
food to microorganism ratio; HASL: hybrid anaerobic solid-liquid digestion system.
food waste. The bacteria community in the methanogenicreactor in S2 was observed to be 78.7% similar to thatin the acidogenic reactor in S3 (Fig. 6a). It should benoted that under this condition, the methanogenic reac-tor in S2 and acidogenic reactor in S3 were operatedin the thermophilic state. Therefore, a similar group ofdominating bacteria could thrive in this condition. Thissuggested that thermophilic acidogenic and methanogenicbacteria may operate interchangeably between acidogenicor methanogenic states. Some of the thermophilic bacteriain the acidogenic reactor of S3 was also transferred andadapted to the mesophilic methanogenic reactor in S3. Al-ternatively, the bacteria group in the methanogenic reactorof S1 and acidogenic reactor of S2 showed similarities of41.5% while the S1 acidogenic and methanogenic bacteriashowed bacterial community closeness at 36.9%.
Concerning the archaeal community between systems(Fig. 6b), the acidogenic and methanogenic reactor in S2
and S3, respectively, still showed closeness in archaealcommunity structure (98.3%). As hypothesized above, thethermophilic bacteria or archaea might dominate in bothacidogenic and methanogenic reactors in the two-stageAD. Moreover, the archaeal community in S3 acidogenicreactor and S2 and S3 methanogenic reactor were shownto have higher similarities, at 90.6%, compared to the casewith bacteria. The S1 methanogenic reactor and S1 andS2 acidogenic reactors followed the archaeal communitystructure of the S3 acidogenic reactor and S2 and S3methanogenic reactor at 60.4% and 41.9%, respectively.
The microbial diversity indices and similarity patternsshowed that S2 methanogenic microbes have higher ac-tivity than other systems because of the higher diversityindices (Table 3) and distinguishing differences (Fig. 6) ofboth bacteria and archaeal communities. The T-RFs den-drogram revealed that the mesophilic acidogenic reactorwas quite different from the thermophilic methanogenic
Table 3 Diversity indices of the archaea and bacteria samples digested with HaeIII in the acidogenic and methanogenic reactors of the two-stageAD
Acidogenic reactor Methanogenic reactor
S1 S2 S3 S1 S2 S3
Bacteria Richness a 11 13 5 9 16 9Diversity b 2.637 3.366 1.964 2.836 3.392 3.066Evenness c 0.762 0.906 0.846 0.895 0.848 0.967Dominance d 0.779 0.888 0.690 0.833 0.883 0.874
Archaea Richness a 5 14 2 6 5 6Diversity b 1.858 2.854 0.621 1.838 0.934 1.641Evenness c 0.800 0.749 0.621 0.711 0.402 0.635Dominance d 0.662 0.807 0.261 0.654 0.309 0.567
a Richness = number of distinct terminal restriction fragments (T-RFs) peaks;b Shannon-Weaver Diversity = –Σ(PilogPi), where Pi is the proportion for each RFLP pattern;c Evenness = H/Hmax, where H/Hmax = log2S. Hmax is the theoretical maximal Shannon-Weaver diversity index for the clone librariesand S is the total number of RFLP patterns;
d Simpson index = Σ(Pi)2, where Pi is the proportion for each RFLP pattern.
Journal of Environmental Sciences 26 (2014) 1–10 9
b
a
S2-M
S3-A
S3-M
S1-M
S2-A
S1-A
78.7%
23.9%
16.0%
41.5%
36.9%
S2-M
S3-A
S3-M
S1-M
S2-A
S1-A
98.3%
90.6%
60.4%
41.9%
71.2%
Pearson correlation (Opt: 0.80%) (0-100%)
20
20
40 60 80 100 800 500 400 300 250 200 140 120 100 80 70 60 50 40 25 20
Pearson correlation (Opt: 0.80%) (0-100%)
60 80 100 800 500 400 300 250 200 140 120 100 80 70 60 50 40 25 20
Fig. 6 16S rDNA T-RFs dendrogram of the bacteria (a) and archaea (b) in S1, S2, and S3 cut with HaeIII.
reactor, indicating a significant separation of microbialactivity between acidogenic and methanogenic microbes.The separation and increased microbial activity in S2,therefore, supported the initial findings that S2 had thehighest nutrient and solid removal and CH4 productioncapacity.
3 Conclusions
Compared to the other systems, S2 exhibited the high-est TCOD, TS, and VS removal. An SCOD level ofabove 90% and TS removal of 62% were exhibited inall systems. S3 was observed to have the highest SCODremoval, which was 3.9% and 6.6% higher than S1 andS2, respectively. In terms of CH4 content, CH4 productionrate, and CH4 yield, S2 also had the edge over the othersystems. The CH4 content of the biogas in S2 was 70.7%while S1 and S3 were 65.5% and 67.8%, respectively.The CH4 production rate of S2 was also 20.3% and17.1% higher than S1 and S3, respectively. The CH4yield in S2 was 0.44 L/g VSadded while the CH4 yieldin S1 and S2 were almost comparable (0.38 and 0.37L/g VSadded). The bacterial diversity indices of S2 weremore dominant for both the acidogenic and methanogenicreactors compared to the other systems, except for thearchaeal community. The TRFs dendrogram also revealedthe non-similarities of the bacteria and archaea commu-nities in acidogenic and methanogenic reactors in S2.
Overall, the study showed that S2 (mesophilic acidogenicreactor-thermophilic methanogenic reactor) had the bestperformance in terms of solid removal, nutrient removal,CH4 production, and microbial community distinctioncompared to the other systems.
Acknowledgment
This work was supported by the Korean Ministry of Agri-culture, Food and Rural Affairs (313007-03-1-HD020).
r e f e r e n c e s
APHA (American Public Health Association), AWWA (American WaterWorks Association), and WEF (Water Environment Federation),2005. Standard methods for the examination of water and wastew-ater (20st ed.). Washington DC, USA.
Cho, J.K., Park, S.C., 1995. Biochemical methane potential and solid stateanaerobic digestion of Korean food wastes. Bioresour. Technol.52(3), 245–253.
Dinsdale, R.M., Hawkes, F.R., Hawkes, D.L., 1997. Mesophilic and ther-mophilic anaerobic digestion with thermophilic pre-acidificationof instant-coffee-production wastewater. Water Res. 31(8), 1931–1938.
Eastman, J.A, Ferguson, J.F., 1981. Solubilization of particulate organiccarbon during the acid phase of anaerobic digestion. J. WaterPollut. Control. Fed. 53, 352–366.
Ferrer, I., Vazquez, F., Font, X., 2010. Long term operation of athermophilic anaerobic reactor: Process stability and efficiencyat decreasing sludge retention time. Bioresour. Technol. 101(9),2972–2980.
10 Journal of Environmental Sciences 26 (2014) 1–10
Grosskopf, R., Janssen, P.H., Liesack, W., 1998. Diversity and structure ofthe methanogenic community in anoxic rice paddy soil microcosmsas examined by cultivation and direct 16S rRNA gene sequenceretrieval. Appl. Environ. Microbiol. 64(3), 960–969.
Hurlbert, S.H., 1971. The nonconcept of species diversity: a critique andalternative parameters. Ecol. 52(4), 577–586.
Kim, I.S., Kim, D.H., Hyun, S.H., 2000. Effect of particle size and sodiumion concentration on anaerobic thermophilic food waste digestion.Water Sci. Technol. 41, 67–73.
Kim, M., Gomec, C., Ahn Y., Speece, R., 2003. Hydrolysis and acidogen-esis of particulate organic material in mesophilic and thermophilicanaerobic digestion. Environ. Technol. 24(9), 1183–1190.
Kim, S., Bae, J., Choi, O., Ju, D., Lee, J., Sung, H. et al., 2013.A pilot scale two-stage anaerobic digester treating food wasteleachate (FWL): Performance and microbial structure analysisusing pyrosequencing. Proc. Biochem. 49(2), 301–308.
Krebs, C.J., 1999. Ecological Methodology (2nd ed.). Wesley Longman,NY, USA.
Lane, D.J., 1991. 16S/23S rRNA Sequencing. In: Stackebrandt, E.,Goodfellow, M. (Eds.), Nucleic Acid Techniques in BacterialSystematics. John Wiley & Sons Ltd., New York, pp. 115–175.
Lay, J.J., Li, Y.Y., Noike, T., 1997. Influences of pH and moisture contenton the methane production in high-solids sludge digestion. WaterRes. 31(6), 1518–1524.
Lettinga, G., Hulshoff Pol, L.W., 1991. UASB-process design for varioustypes of wastewaters. Water Sci. Technol. 24(8), 87–107.
Lim, J.W., Chen, C.L., Ho, I.J.R., Wang, J.Y., 2013. Study of microbialcommunity and biodegradation efficiency for single- and two-phaseanaerobic co-digestion of brown water and food waste. Bioresour.Technol. 147, 193–201.
Lin, J., Zuo, J.N., Ji, R.F., Chen, X.J., Liu, F.L., Wang, K.J. et al., 2012.Methanogenic community dynamics in anaerobic co-digestion offruit and vegetable waste and food waste. J. Environ. Sci. 24(7),1288–1294.
Lin, Y.Q., Wu, S.B., Wang, D.H., 2013. Hydrogen-methane productionfrom pulp & paper sludge and food waste by mesophilic-thermophilic anaerobic co-digestion. Int. J. Hydrogen Energy38(35), 15055–15062.
Nathao, C., Sirisukpoka, U., Pisutpaisal, N., 2013. Production of hydro-gen and methane by one and two stage fermentation of food waste.
Int. J. Hydrogen Energy 38(35), 15764–15769.Oles, J., Dichtl, N., Niehoff, H., 1997. Full scale experience of two
stage thermophilic- mesophilic sludge digestion. Water Sci. Techol.36(6), 449–456.
Shahriari, H., Warith, M., Hamoda, M., Kenned, K., 2013. Evaluation ofsingle vs. staged mesophilic anaerobic digestion of kitchen wastewith and without microwave pretreatment. J. Environ. Manage.125, 74–84.
Shen, F., Yuan, H., Pang, Y., Chen, S., Zhu, B., Zou, D. et al., 2013.Performances of anaerobic co-digestion of fruit & vegetable waste(FVW) and food waste (FW): Single-phase vs. two-phase. Biore-sour. Technol. 144, 80–85.
Shin, S.G., Han, G., Lim, J., Lee, C., Hwang, S., 2010. A comprehensivemicrobial insight into two-stage anaerobic digestion of food waste-recycling wastewater. Water Res. 44(17), 4838–4849.
Shin, H.S., Han, S.K., Song, Y.C., Lee, C.Y., 2001. Performance ofUASB reactor treating leachate from acidogenic fermenter in thetwo-phase anaerobic digestion of food waste. Water Res. 35(14),3441–3447.
Solera, R., Romero, L., Sales, D., 2002. The evolution of biomass ina two-phase anaerobic treatment process during start-up. Chem.Biochem. Eng. Q. 16(1), 25–30.
Stabnikova, O., Liu, X.Y., Wang, J.Y., 2008. Digestion of frozen/thawedfood waste in the hybrid anaerobic solid-liquid system. WasteManage. 28(9), 1654–1659.
Wang, J.Y., Zhang, H., Stabnikova, O., Tay, J.H., 2005. Comparisonof lab-scale and pilot-scale hybrid anaerobic solid-liquid systemsoperated in batch and semi-continuous modes. Proc. Biochem.40(11), 3580–3586.
Yu, H.G., Fang, H.H., 2002. Acidogenesis of dairy wastewater at variouspH levels. Water Sci. Technol. 45(10), 201–206.
Yu, J., Zheng, M., Tao, T., Zuo, J., Wang, K., 2013. Waste activated sludgetreatment based on temperature staged and biologically phasedanaerobic digestion system. J. Environ. Sci. 25(10), 2056–2064.
Zhang, L., Lee, Y.W., Jahng, D., 2011. Anaerobic co-digestion of foodwaste and piggery wastewater: Focusing on the role of traceelements. Bioresour. Technol. 102(8), 5048–5059.
Zhang, L., Jahng, D., 2012. Long-term anaerobic digestion of food wastestabilized by trace elements. Waste Manage. 32(8), 1509–1515.