Journal of Environmental Sciences 26 (2014) 110

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A comparative study on the alternating mesophilic and thermophilic two-stage anaerobic 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

article info
Article history: Special issue: Sustainable water management for green infrastructure Keywords: community structure food waste methane production nutrient removal two-stage anaerobic digestion
DOI: 10.1016/S1001-0742(13)60599-9

abstract
An alternating mesophilic and thermophilic two stage anaerobic digestion (AD) process was conducted. The temperature of the acidogenic (A) and methanogenic (M) reactors was controlled as 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 digester sludge. Food waste was added with the soluble chemical oxygen demand (SCOD) concentrations of 41.447.0 g/L and volatile fatty acids of 2.03.2 g/L. Based on the results, the highest total chemical oxygen 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 hand decreased by 0.1 % as compared to S1. However, volatile solids (VS) removal in S1, S2, and S3 was 78.5%, 81.7%, and 79.2%, respectively. S2 also exhibited the highest CH4 content, yield, and production rate of 70.7%, 0.44 L CH4 /g VSadded , and 1.23 L CH4 /(Lday), respectively. Bacterial community structure revealed that the richness, diversity, evenness, and dominance of S2 were high except for the archaeal community. The terminal restriction fragments dendrogram also revealed that the 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, 14,400 tons/day of food waste was generated in Korea (MOE, 2007), and more than 50% of the food wastes generated were dumped into the sea. However, due to the Marine Pollution Act of 2012, ocean dumping of food wastes and other similar types of wastewater is banned. To solve this problem, anaerobic digestion (AD) has been developed because it can handle waste with high organic content with ecient mineralization compared to other processes. Moreover, additional energy in the form of biogas can also be harnessed. AD is more practical to use than competing
Corresponding

author. E-mail: djahng@mju.ac.kr. The authors contribute equally to this work.

processes because of its low maintenance and operation cost, low excess sludge production, and low release of odor and aerosols (Solera et al., 2002). Other methods such as landlling, composting, and incineration have been suggested for food waste disposal (Kim et al., 2000; Shin et al., 2001). However, aside from the production of leachate, foul odor, and toxic gases, these processes require land space and the input of energy to operate; hence, they are not environmentally friendly and economical. The advantages of a two-stage system in AD is the possible enrichment of dierent bacteria in each digester, which increases stability and allows higher organic loading rate (OLR) and shorter hydraulic retention time (HRT) than the conventional single-stage digester (Solera et al., 2002). The rst stage may act as a metabolic buer by preventing pH-shock and build-up of toxic material that could be transferred to the methanogenic reactor (Eastman

Journal of Environmental Sciences 26 (2014) 110

and Ferguson, 1981; Solera et al., 2002). Therefore, a more ecient process is established in the two-stage AD system. However, disadvantages such as long start-up time, additional reactor cost and operation, and poor granule formation may be experienced in two-stage AD (Lettinga and Hulsho, 1991). Recent studies comparing the advantages of two-stage AD over single stage AD have been reported by Lim et al. (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 compared to single stage AD. Other studies on two-stage AD using food waste in combination with other substrates such as fruit and vegetable waste (Lin et al., 2012), pulp and paper sludge (Lin et al., 2013), and brown water (Lim et al., 2013) have shown improvement in the overall performance of AD. Aside from this, the pretreatment of food waste (Stabnikova et al., 2008; Shahriari et al., 2013) before twostage AD further improved its capacity to degrade nutrients and produce biogas. The role of trace elements was also investigated thoroughly in AD of food waste (Zhang et al., 2011; Zhang and Jahng, 2012). Moreover, the thermophilic acidogenic reactor (Kim et al., 2013) and thermophilic methanogenic reactor (Lin et al., 2013) were investigated in two-stage AD. The microbial community characterization in AD has also been carried out recently to monitor the bacterial or archaeal 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 were observed in the methanogenic reactor of the two-stage AD of food waste-recycling wastewater. Furthermore, the methanogenic community dynamics in an increasing ratio of fruit and vegetable waste to food waste was known to be dominated by Methanoculleus, Methanosaeta and Methanosarcina. Moreover, Lim et al. (2013) have shown the predominance of Firmicutes and greater bacterial diversity in the two-stage AD of brown water and food waste. This implies that the proper understanding of the microbial behaviour during an AD operation will further lead to the improvement of design and digestion process of AD especially in food waste digestion. In this study, two-stage AD of food waste was investigated by controlling mainly the temperature of the two digesters. Three systems were developed by varying the temperature proles of the acidogenic and methanogenic reactors from both as mesophilic reactors to alternating mesophilic and thermophilic reactor systems. The study were carried out using steady state conditions, and microbial community structures were also analyzed using 16s ribosomal DNA gene amplication and terminal-restriction fragment length polymorphism (TRFLP) analyses. Previous reports have dealt mostly with a 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 investigation to include scenarios of alternating mesophilic or thermophilic two-stage AD and their eect on the microbial 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 recycling company in Yongin Korea. After collection, foreign materials such as bones, plastics, hard ligaments, and other inorganic materials were removed. The organic loading concentration was adjusted using tap water and sieved (No. 10) to remove coarse particles larger than 2 mm. The diluted food wastewater was kept at 4C to preserve the organic waste prior to use. The food waste had an initial pH of 3.75 0.15, total solids (TS) of 5.5% 0.1%, volatile solids (VS) of 5.25% 0.45%, total chemical oxygen 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 fatty acids (VFAs). The anaerobic seed sludge was taken from the AD of the Yongin Respia wastewater treatment plant, Yongin, Korea. The digester sludge that served as inoculum for the experiment had an initial pH of 7.3, 16.6 g/L total suspended solid (TSS), and 12.0 g/L volatile VSS. 1.2 Two-stage anaerobic digester and operating conditions Two separate bioreactors were designed specically for the acidogenic and methanogenic AD. The acidogenic reactor was an acrylic cylindrical reactor with a diameter, height, and eective volume of 22.5 cm, 40 cm, and 10 L, respectively (Fig. 1). The CH4 reactor was also made of the same material with dimensions of 32 cm diameter, 48 cm high, and 30 L eective volume. The acidogenic reactor was operated at the OLR of 4.5 g COD/(Lday) and operated intermittently (15 min feed pumping and 45 min stop). The HRT was set at 5 and 15 days for the acidogenic and methanogenic reactors, respectively. The volumetric ow rate was set at 2 L/day in both reactors. Both acidogenic and methanogenic reactors were thermally controlled using a heating jacket with a digital controller (GLTC-DP, GlobalLab, Korea). Initially, mesophilic temperature (36 1C) was maintained in both acidogenic and methanogenic reactor for 195 days for System 1 (S1); the temperature of the methanogenic reactor was increased to 55 1C until day 304 in System 2 (S2); then, thermophilic acidogenic and mesophilic methanogenic reactors were set up in System 3 (S3) up to day 347. The system in S1 was allowed to stabilize for two weeks, while one week was allotted for S2 and S3, respec-

Journal of Environmental Sciences 26 (2014) 110

3
Gas vent

Gas vent pH control pH control Gas tank Pump Food waste Temp. controller Sampling port Sampling port Pump Gas tank Temp. controller

Thermal jacket Acidogenic reactor

Fig. 1

Methanogenic reactor
Schematic diagram of the two-stage AD.

tively. A stabilized system was considered to be achieved when an increase of VFA concentration in the acidogenic reactor and CH4 production in the methanogenic reactor were perceived. Also, the subsequent transfer of fermented food waste from the thermophilic acidogenic reactor to the mesophilic methanogenic reactor did not experience cooling because of the low ratio of feed volume transferred to the succeeding reactor. The thermophilic feed compensated the target temperature of the methanogenic reactor without cooling in S3. 1.3 Analytical methods TS, VS, TSS, VSS, TCOD, and SCOD were analyzed according to the standard methods (APHA, 2005). The pH of the samples was measured using a pH meter (Orion, Model 370). CH4 and CO2 were analyzed using an HP6890 gas chromatograph (GC) (Hewlett Packard 6890, PA, USA) with a thermal conductivity detector (TCD) and HP-Plot Q column (30 m 0.32 mm 20 m) (Zhang et al., 2011). For VFA determination, a separate GCFID (Younglin 6000D, Seoul, Korea) with HP-INOVAX column (30 m 0.25 mm 0.25 m) was used (Zhang et al., 2011). One-way ANOVA was used to determine the signicance of dierences between the dierent systems while the Student t-test was used to compare the nutrient reduction and methane production performance between S1 and S2, S1 and S3, and S2 and S3. The F -value in one-way ANOVA was compared at a critical F-value table for 2 d.f. in all systems. The F -test was also performed before proceeding to t-test analysis to determine if the variances between two systems were identical or not. The results of the F -test determine the proper t-test (equal or unequal variances) analysis to be used. The t Stat-value of t-test was

compared at a critical t-value table for 87 d.f. between S1S2 and S1-S3 while 50 d.f. was used for S2-S3 analysis. The following statistical methods were performed using Microsoft Oce Excel 2010 (Microsoft Corp., Redmond, WA). The condence interval was set at 95% ( = 0.05) and the total nutrient removal and methane production proles of the dierent systems were log transformed prior to statistical analysis. 1.4 T-RFLP analysis The genomic DNA of the samples collected from the acidogenic and methanogenic reactors for dierent systems was extracted using the Mo Bio Power Soil DNA Isolation Kit (MoBio, USA). The genomic DNA extracts were conrmed by electrophoresis and stored at 20C prior to T-RFLP analysis. The obtained DNA extract was PCR amplied using the 16s rDNA universal primers 27F and 1392R (Lane, 1991) and archaea universal primers A109f and A934B (Grosskopf et al., 1998). The forward primers were labelled with 5 -end 6-FAM (phosphoramidite uorochrome 5-carboxyuorescein) (Bionics, Korea). The PCR was carried in a 50 L reaction mixture (10X Ex Taq buer, 2.5 mmol/L dNTPs, 0.2 mol/L primer, 5 unit/L Takara Ex Taq polymerase (Takara, Japan)) containing 2 L of DNA template, according to the following temperature cycles: initial 94C for 5 min, 58C and 55C annealing for bacteria and archaea respectively; and DNA synthesis (polymerization) at 72C for 2 min, with a total of 30 cycles at 72C for 5 min. The restriction enzyme, HaeIII, was utilized for the digestion of bacterial and archaeal PCR-products. Four diversity indices, richness, dominance, diversity and evenness, were calculated as described elsewhere (Hurlbert, 1971; Krebs, 1999) using the chromatograms

Journal of Environmental Sciences 26 (2014) 110

obtained from GeneScan Analysis Software v3.7 (Applied Biosystems, USA). The community similarity analysis between acidogenic and methanogenic reactors of S1, S2, and S3 was also estimated through the construction of a dendrogram using GelCompar II program (Applied Maths, Belgium) according to the calculation of Pearsons correlation coecients.

2 Results and discussion

2.1 pH and temperature changes pH is one of the most sensitive operating conditions in an AD because dierent microorganisms thrive in dened pH ranges. The optimal pH of hydrolytic and acidogenic microbes has been found to be between 5.5 and 6.5 (Yu and Fang, 2002; Kim et al., 2003). The acidogenic and methanogenic stages were controlled at pH 6.6 to 7.8 (Lay et al., 1997). The pH and temperature proles of the systems in the two-stage AD are shown in Fig. 2. To maintain the optimal pH conditions in the acidogenic and methanogenic reactor, 1 mol/L HCl and 1 mol/L NaOH were used to adjust the pH in each reactor. As can be seen in Fig. 2a, the acidogenic and methanogenic pH values were 5.05.5 and 7.28.0, respectively. The pH of the reactors was maintained at these levels to avoid possible inhibition of the microbial activity. An acidogenic pH below pH 5.5 indicated higher VFA concentration in the reactor. The methanogenic reactor also had a pH above 8.0, which was possibly due to the initial accumulation of ammonia. Temperature is also a critical parameter for eective AD implementation. Increase in operating temperature could mean higher capital and operating costs (Ferrer et al., 2010). Optimally, AD is operated at 30C to 40C. However, it was found that thermophilic conditions could also be applied to increase the destruction rate of organic compounds, better solid and liquid separation, and pathogen
9 a 8 S1 S2 S3

destruction (Oles et al., 1997). In Fig. 2b, temperatures of the dierent systems were also monitored. As indicated in section 1.2, S1 was operated at mesophilic condition for both reactors. S2 had a mesophilic acidogenic reactor and thermophilic methanogenic reactor, while S3 was the reverse of S2. Comparing closely, the mesophilic condition in either acidogenic or methanogenic reactor in S1 and S2 maintained an average temperature of 35C. However, the S3 methanogenic reactor mesophilic condition was higher by 2C. The absence of cooling of the thermophilic fermented food waste possibly increased the temperature of the mesophilic methanogenic reactor in S3. 2.2 Nutrient reduction Figure 3a and e shows the TCOD and TCOD removal of the two-stage AD for dierent systems. The TCOD of S1 remained in the range of 1020 g/L. However, after shifting to S2, TCOD overloading occurred, which increased the TCOD concentration above 35 g/L for nearly 40 days. After this overloading, the system was stabilized and went back to its original range of TCOD. In the methanogenic reactor, the TCOD level started at around 40 g/L and gradually increased until the end of S1. The last period (day 155195) of S1 was shown to have a TCOD level of at least 80 g/L. This may be due to the eect of nutrient overloading. However, after the rise in TCOD load, the system started to stabilize at a TCOD level around 70 g/L at S2 to S3. Comparing the overall TCOD removal (Fig. 3e, Table 1), S2 had the highest removal of 86.6%, which was followed by S3 (85.1%) and S1 (81.0%). From these observations, it can be inferred that the thermophilic condition of the methanogenic reactor was more eective in oxidizing the available organic nutrients than the thermophilic state of the acidogenic reactor. Thus, it could be concluded that more methanogenic microorganisms were able to thrive at higher temperature than acidogenic microorganisms. In terms of the SCOD proles (Fig. 3b and f ), the startup acidogenic SCOD concentration was 20 g/L. A low
60 b 55 Temperature (C) 50 45 40 35 Acidogenic reactor Methanogenic reactor S1 S2 S3

pH value

7 Acidogenic reactor Methanogenic reactor 6

5 30 4 0 50 100
Fig. 2

150 200 250 Operating period (day)

300

350

25

50

100

150 200 250 Operating period (day)

300

350

pH (a) and temperature (b) proles of the two-stage AD at dierent systems (S1, S2, and S3).

Journal of Environmental Sciences 26 (2014) 110

5
S3

S1 100 a 80

Acidogenic S3 S2

Methaogenic 120 e 100 TCOD removal (%) 80 60 40 20 0 120

Total removal S1

S2

TCOD (g/L)

60

40

20

100 0 b 80 SCOD removal (%) SCOD (g/L)

100 80 60 40 20 0 100 80 TS removal (%) 60 40 20

f (f)

60

40

20

0 60 50 TS (g/L) 40 30 20 10 0 60 50

0 100 d 80 VS removal (%) 60 40 20 0 h

VS (g/L)

40 30 20 10 0 0 50 100 150 200 250 300 350

50

100

150

200

250

300

350

Operating period (day)

Operating period (day)

Fig. 3 (a) TCOD, (b) SCOD, (c) TS, and (d) VS of the acidogenic and methanogenic reactor euent and their corresponding removal (e, f, g, and h) of the two-stage AD at dierent systems (S1, S2, and S3).

level of uctuations in the SCOD loading was achieved

after 3 weeks of incubation. After this period, the SCOD

6
Acetate 16 14 12 VFA (g/L) a S1 S2 Propionate

Journal of Environmental Sciences 26 (2014) 110

iso-Butyrate S3

n-butyrate 16 14 12 VFA (g/L) 10 8 6 4 2 0

iso-Valerate S1

n-Valerate S2

Total VFA S3

10 8 6 4 2 0 0 50 100 150 200 250 Operating period (day) 300 350

50

100

150 200 250 300 Operating period (day)

350

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 3040 g/L, starting from the middle period of S1 up to the nal period in S3. The SCOD concentrations of the methanogenic reactor seemed to be below 2 g/L in S1 and S2, however, S2 rose higher than 20 g/L during start-up. The extreme rise of SCOD level in the methanogenic reactor of S2 might be due to the shift in the temperature condition of the reactor from mesophilic to thermophilic state. This might cause the methanogenic microorganisms to falter, and hence lower the activity. To cope with the sudden change of temperature, another acclimatization stage was experienced by S2. S3, however, it seemed not to be greatly aected by the reversion of the thermal conditions of the acidogenic and methanogenic reactors. Although uctuation was observed in S2, the

total SCOD removal was still 90.5% while S1 and S3 were recorded at 92.8% and 96.5%, respectively (Fig. 3f, Table 1). The TS levels of the acidogenic and methanogenic reactors were also compared (Figs. 3c and g). Around 40 50 g/L of the TS was observed in the incoming TS of all the reactors. Likewise, TS uctuations were observed during the shift of condition from S1 to S2, although more pronounced than the previous SCOD data. The nal TS in the methanogenic reactors were maintained at the level of 2030 g/L. Comparing the total TS removal of the acidogenic and methanogenic reactors, S2 had the highest removal 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 A TCOD removal (%) SCOD removal (%) TSremoval (%) VS removal (%) 26.7 19.3 22.9 25.3 S1 M 55.0 73.5 39.3 53.2 Total 81.0 a 92.8 d 62.2 g 78.5 h A 27.2 21.5 20.7 23.1 M 59.4 68.8 42.0 58.6 S2 Total 86.6 b,c 90.5 e,f 62.7 g 81.7 h A 32.1 25.1 25.3 27.3 S3 M 53.0 71.5 36.8 51.9 Total 85.1 a,c 96.5 d,f 62.1 g 79.2 h

A: acidogenic reactor; M: methanogenic reactor. Values with the same letter are not signicantly dierent between systems (t Stat

t Critical two-tail, = 0.05).

CH4 production rate (L/(L.day))

a CH4 content (%) 80 60 40 20 0 0

CH4 yield (L/g VS added)

100 S1 S2 S3

50 100 150 200 250 300 350 Operating period (day)

1.6 S2 S3 S1 1.4 b 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 50 100 150 200 250 300 350 Operating period (day)

0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 c S1

S2

S3

50 100 150 200 250 300 350 Operating period (day)

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) 110

Although TS removal may not give conclusive data on the reduction of organic matter in the two-stage AD, the gathered data may provide substantial information on the solid removal in the system. Thus, to investigate thoroughly, the VS content of the food waste was also monitored (Fig. 3d). The VS of the acidogenic reactor was in range of 30 40 g/L while that of the methanogenic reactor was at approximately 10 g/L (Fig. 3d and h). It took an acclimatization period of two weeks before the VS concentration of the acidogenic reactor of S1 attained the VS operating condition. Fluctuations in VS content of the food waste during the shift to S2 were not as long and abrupt as the uctuation in SCOD. Much higher VS were observed in S2 and S3 than in S1. However, the methanogenic reactor VS content remained stable during the entire period of operation. Comparing closely, the VS removal eciency of S1, S2, and S3 was recorded at 78.5%, 81.7%, and 79.2%, respectively (Fig. 3h, Table 1). In contrast to SCOD removal, the TS and VS removal favored S2 compared to the other systems. Table 1 shows the summary of the performance and statistical relationships of the two-stage AD of food waste in the alternating mesophilic and thermophilic condition of acidogenic and methanogenic reactors. Statistical testing using one-way ANOVA indicated that the systems are not identical in terms of TCOD, SCOD, TS, and VS removal (not shown). However, the t-test showed that only S1 and S2 are signicantly independent in terms of the TCOD and SCOD reduction capacity. The TS and VS removal of S1, S2, and S3 also did not entail signicant dierences in their average solid reduction capacity. The short operating period of S3 as compared to the longer monitoring of S1 and S2 may have been the source of deviation in the results of group analysis. Nevertheless, one-way ANOVA suggests that the systems were not identical. 2.3 VFA production To investigate the possible occurrence of uctuations in the SCOD levels in S2, the change in the VFA proles of the systems in two-stage AD was determined (Fig. 4). As shown in Fig. 4b, the acid concentration of the methanogenic reactor was below 1 g/L, however, when S2 was operated, the VFA concentrations were observed to be above 10 g/L. This provides evidence to support the sudden rise of SCOD level of S2 (Fig. 3b). Therefore, it can be concluded that the shift to thermophilic condition of the methanogenic reactor in S2 led to the deterioration of the performance of the methanogenic microorganisms. Although this did not conform well to the overall performance of S2, the start-up uctuations could be due to the adjustment period of the anaerobic methanogens to the change in temperature. In the acidogenic reactor, acetate was found to be the dominant acid (50%60%) in all the systems (Fig. 4a). It

was followed by n-butyrate and iso-butyrate at approximately 33%. The other acids measured were propionate, iso-valerate, and n-valerate with concentrations lower than 10% of the nal acid in all the systems. 2.4 Biogas production S1 and S2 have been shown not to be related to the CH4 content and yield while the CH4 production rate did not show signicant dierences in the methanogenic reactor of all the systems based on t-test analysis. However, based on one-way ANOVA, the CH4 production proles (CH4 content, production rate, and yield) were signicantly dierent 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 lowest performer (65.5%). The CH4 production rate was also high in S2 at 1.23 L/(Lday), while S1 and S3 were at 0.98 and 1.02 L/(Lday), respectively (Fig. 5b). Comparing further, the CH4 yield of S2 was 0.44 L/g VSadded , which was 13.6% and 15.9% higher than that in S1 and S3, respectively (Fig. 5c). This indicated that although severe uctuations of nutrient and VS levels were experienced, S2 still managed to overcome the sudden temperature change. It should however be noted that the overall CH4 production of S2 was the highest. The acidogenic reactor in S3 was also changed to thermophilic condition; however, it was observed that more methanogenic microorganisms are able to dwell in a high-temperature environment than acidogenic microorganisms (Ferrer et al., 2010). This may be attributed to the metabolic activity of most acidogenic bacteria, wherein high temperature can be inhibitory. Furthermore, it was stated that acidogenesis is not associated with thermal hydrolysis, rather on the microbial activities of the acidogenic reactor (Yu et al., 2013). The thermophilic conditions might also increase the pH of the acidogenic reactor, aecting the microbial activity of fermentative microbes. The temperature-controlled two-stage AD of food waste gave comparable results with other studies dealing with the same kind of substrate (Table 2). The study had shown that the mesophilic acidogenic reactor and thermophilic methanogenic reactor gave the best conguration for both the organic reduction and biogas production of the twostage AD of food waste. 2.5 T-RFLP assay results The T-RFLP data (Table 3) shows that the bacteria have higher richness, diversity, and dominance in S2 for both the acidogenic and methanogenic reactors. For archaea, the acidogenic reactor of S2 had the highest richness, diversity, and dominance while the methanogenic reactor of S2 was the lowest values (Table 3). The mesophilic archaea in S1 were shown to have the highest diversity indices. Figure 6 shows the T-RFs dendrogram of the bacterial and archaeal community in S1, S2, and S3 of the AD of

Journal of Environmental Sciences 26 (2014) 110

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 A CSTR Batch CSTR Batch CSTR Semi-cont. CSTR CSTR CSTR FW leachate FW:PPS (1:1) FW:FVW (8:5) FW (F/M = 7.5) FW FW (microwaved) FW FW FW M A M 3 2 5 5 5 27 20 20 20 20 (day) 136 12 120 36 195 109 43 VS/TS OLR CH4 yield CH4 prod. VS removal Reference

(%) (%) 13.8 5.91 17.6 0.80 18.6 94.6 0.46 3.04 94.0 4.5 5.0 4.7 70.7 86.7 85.0

(kg VS/(Ld)) (L/(kgVSadded )) (L/(Lday)) (%) 2.36 2 1.04 1.39 3.2 4.4 4.0 0.55 0.432 0.455 0.094 0.32 0.373 0.33 0.47 0.38 0.44 0.37 1.07 0.98 1.23 1.02 82.6 90 78 78.5 81.7 79.2 Kim et al., 2013 Lin et al., 2013 Shen et al., 2013 Nathao et al., 2013 Stabnikova et al., 2008 Cho & Park, 1995 Wang et al., 2005 Shahriari et al., 2013 This study This study This study

40.7 37.0 37 35 37 35 35 35 55 55 35 35 37 35 35 55 37

10 10

Batch (HASL) FW (freeze-thawed) 35 CSTR (HASL) FW

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 methanogenic reactor in S2 was observed to be 78.7% similar to that in the acidogenic reactor in S3 (Fig. 6a). It should be noted that under this condition, the methanogenic reactor in S2 and acidogenic reactor in S3 were operated in the thermophilic state. Therefore, a similar group of dominating bacteria could thrive in this condition. This suggested that thermophilic acidogenic and methanogenic bacteria may operate interchangeably between acidogenic or methanogenic states. Some of the thermophilic bacteria in the acidogenic reactor of S3 was also transferred and adapted to the mesophilic methanogenic reactor in S3. Alternatively, the bacteria group in the methanogenic reactor of S1 and acidogenic reactor of S2 showed similarities of 41.5% while the S1 acidogenic and methanogenic bacteria showed 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 archaeal community structure (98.3%). As hypothesized above, the thermophilic bacteria or archaea might dominate in both acidogenic and methanogenic reactors in the two-stage AD. Moreover, the archaeal community in S3 acidogenic reactor and S2 and S3 methanogenic reactor were shown to have higher similarities, at 90.6%, compared to the case with bacteria. The S1 methanogenic reactor and S1 and S2 acidogenic reactors followed the archaeal community structure of the S3 acidogenic reactor and S2 and S3 methanogenic reactor at 60.4% and 41.9%, respectively. The microbial diversity indices and similarity patterns showed that S2 methanogenic microbes have higher activity than other systems because of the higher diversity indices (Table 3) and distinguishing dierences (Fig. 6) of both bacteria and archaeal communities. The T-RFs dendrogram revealed that the mesophilic acidogenic reactor was quite dierent from the thermophilic methanogenic

Table 3 AD

Diversity indices of the archaea and bacteria samples digested with HaeIII in the acidogenic and methanogenic reactors of the two-stage

Acidogenic reactor S1 Bacteria Richness Diversity b Evenness c Dominance d Richness a Diversity b Evenness c Dominance d
a

Methanogenic reactor S3 5 1.964 0.846 0.690 2 0.621 0.621 0.261 S1 9 2.836 0.895 0.833 6 1.838 0.711 0.654 S2 16 3.392 0.848 0.883 5 0.934 0.402 0.309 S3 9 3.066 0.967 0.874 6 1.641 0.635 0.567

S2 13 3.366 0.906 0.888 14 2.854 0.749 0.807

Archaea

11 2.637 0.762 0.779 5 1.858 0.800 0.662

Richness = number of distinct terminal restriction fragments (T-RFs) peaks; Shannon-Weaver Diversity = (Pi logPi ), where Pi is the proportion for each RFLP pattern; c Evenness = H/H max , where H/H max = log2 S. Hmax is the theoretical maximal Shannon-Weaver diversity index for the clone libraries and S is the total number of RFLP patterns; d Simpson index = ( P )2 , where P is the proportion for each RFLP pattern. i i
a b

Journal of Environmental Sciences 26 (2014) 110

Pearson correlation (Opt: 0.80%) (0-100%) 20 40 60 78.7% 23.9% 80 100
800 500 400 300 250 200 140 120 100 80 70 60 50 40 25 20

S2-M S3-A S3-M

16.0% 41.5% 36.9% S1-M S2-A S1-A Pearson correlation (Opt: 0.80%) (0-100%) 60 80 98.3% 90.6% 60.4%

20

100

800 500 400 300 250 200

140 120 100 80 70 60 50

40

25 20

S2-M S3-A S3-M S1-M

41.9% 71.2% S2-A S1-A

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 signicant separation of microbial activity between acidogenic and methanogenic microbes. The separation and increased microbial activity in S2, therefore, supported the initial ndings that S2 had the highest nutrient and solid removal and CH4 production capacity.

Overall, the study showed that S2 (mesophilic acidogenic reactor-thermophilic methanogenic reactor) had the best performance in terms of solid removal, nutrient removal, CH4 production, and microbial community distinction compared to the other systems. Acknowledgment This work was supported by the Korean Ministry of Agriculture, Food and Rural Aairs (313007-03-1-HD020).

3 Conclusions
Compared to the other systems, S2 exhibited the highest TCOD, TS, and VS removal. An SCOD level of above 90% and TS removal of 62% were exhibited in all systems. S3 was observed to have the highest SCOD removal, which was 3.9% and 6.6% higher than S1 and S2, respectively. In terms of CH4 content, CH4 production rate, and CH4 yield, S2 also had the edge over the other systems. 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% and 17.1% higher than S1 and S3, respectively. The CH4 yield in S2 was 0.44 L/g VSadded while the CH4 yield in S1 and S2 were almost comparable (0.38 and 0.37 L/g VSadded ). The bacterial diversity indices of S2 were more dominant for both the acidogenic and methanogenic reactors compared to the other systems, except for the archaeal community. The TRFs dendrogram also revealed the non-similarities of the bacteria and archaea communities in acidogenic and methanogenic reactors in S2.

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