Journal of Bioscience and Bioengineering VOL. 108 No. 4, 336 – 343, 2009 www.elsevier.com/locate/jbiosc
Effects of intermittent and continuous aeration on accelerative stabilization and microbial population dynamics in landfill bioreactors Nguyen Nhu Sang,1,2 Satoshi Soda,1,⁎ Daisuke Inoue,1 Kazunari Sei,1 and Michihiko Ike1 Division of Sustainable Energy and Environmental Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan 1 and Institute for Environment and Resources, Vietnam National University-Ho Chi Minh City, 142 To Hien Thanh Street, District 10, Ho Chi Minh City, Vietnam 2 Received 23 March 2009; accepted 21 April 2009
Performance and microbial population dynamics in landfill bioreactors were investigated in laboratory experiments. Three reactors were operated without aeration (control reactor, CR), with cyclic 6-h aeration and 6-h non-aeration (intermittently aerated reactor, IAR), and with continuous aeration (continuously aerated reactor, CAR). Each reactor was loaded with highorganic solid waste. The performance of IAR was highest among the reactors up to day 90. The respective solid weight, organic matter content, and waste volume on day 90 in the CR, IAR, and CAR were 50.9, 39.1, and 47.5%; 46.5, 29.3 and 35.0%; and 69, 38, and 53% of the initial values. Organic carbon and nitrogen compounds in leachate in the IAR and the CAR showed significant decreases in comparison to those in the CR. The most probable number (MPN) values of fungal 18S rDNA in the CAR and the IAR were higher than those in the CR. Terminal restriction fragment length polymorphism analysis showed that unique and diverse eubacterial and archaeal communities were formed in the IAR. The intermittent aeration strategy was favorable for initiation of solubilization of organic matter by the aerobic fungal populations and the reduction of the acid formation phase. Then the anaerobic H2-producing bacteria Clostridium became dominant in the IAR. Sulfate-reducing bacteria, which cannot use acetate/sulfate but which instead use various organics/sulfate as the electron donor/acceptor were also dominant in the IAR. Consequently, Methanosarcinales, which are acetate-utilizing methanogens, became the dominant archaea in the IAR, where high methane production was observed. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Accelerative stabilization; Intermittent aeration; Landfill bioreactors; Bacteria; Methanogens]
Many cities in developing countries face serious problems related to disposal of high-organic wastes. Generation of leachate and gases with various contaminants can heavily pollute the environment while causing and exacerbating serious health problems if landfills for waste disposal are not well managed (1, 2). For that reason, landfills should be operated in a mode which minimizes environmental pollution. In conventional landfills, a complex sequence of physically, chemically, and biologically mediated events occurs simultaneously (3). First, high-organic wastes are decomposed mainly into organic acids in anaerobic conditions. Subsequently, the conversion of the acids and the alcohols to acetate, CO2, and H2 occurs. Finally, the products of acetogens are converted anaerobically to CH4 via acetate (acetotrophy) and H2 and CO2 (hydrogenotrophy). Aeration of landfills can activate microorganisms, which can execute important functions for biodegradation and accelerate landfill stabilization (4, 5). Our research has documented the increase in populations of ammonia-oxidizing bacteria and fungi in an aerated landfill bioreactor, where carbon and nitrogen were effectively removed from the solid waste and leachate (5). However, surplus aeration can decrease microbial activity and increase energy con⁎ Corresponding author. Tel.: +81 6 6879 7673; fax: +81 6 6879 7675. E-mail address:
[email protected] (S. Soda).
sumption. A balanced utilization of both aerobic and anaerobic metabolic pathways of the microorganisms might be effective for accelerated stabilization. Intermittent aeration is apparently the most practical strategy to create cyclically aerobic and anaerobic conditions and to reduce energy consumption attributable to the air supply in landfill bioreactors. Rapid stabilization of solid waste is possible with intermittent aeration at various oxygen and oxidation–reduction potential levels (6). Reportedly, an intermittent aeration strategy is favorable for separation of the acid formation phase and the methane fermentation phase, reducing the acid production time (7, 8). Furthermore, intermittent aeration can stimulate nitrifiers and denitrifiers in landfill bioreactors (9, 10). Although much is known about the basic metabolism in landfills, little is known about the microorganisms responsible for those processes, especially in intermittently aerated conditions. Monitoring and characterization of aerobic and anaerobic microorganisms are needed for theoretical and practical operations for landfill bioreactors. Few studies have specifically examined the intermittent aeration of landfill bioreactors and its impacts on microorganisms, except the methanogenic community (6). Furthermore, the performance of landfill bioreactors with intermittent aeration has not been fully compared to that with full aeration. In this study, intermittent aeration for accelerated stabilization of high-organic waste was
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.04.019
VOL. 108, 2009
MICROBIAL POPULATION DYNAMICS IN INTERMITTENT LABDFILL BIOREACTOR
337
FIG. 1. Characteristics of solid waste in landfill reactors. (A) organic matter content, (B) T-N content, and (C) waste volume. Symbols for reactors: diamonds, CR; squares, IAR; circles, CAR. Error bars in (A) show the standard deviation.
evaluated according to the solid waste composition, landfill gas, and leachate. In addition, microbial population dynamics are exhibited as main functions in landfill bioreactors. MATERIALS AND METHODS Landfill bioreactors Three acrylic cylindrical bioreactors of 10 cm diameter, 30 cm height, and 2.85 l in overall volume were arranged in a room maintained at 28 °C. The reactors' configurations included three separate ports on the top for gas measurement, the addition of water, and leachate recirculation (5). The sludge cake collected from a wastewater treatment plant in Osaka, Japan was minced and mixed with wood chips and dry dog food at a dry weight ratio of 2:2:1. The syntehic solid waste used in this study was a model of organic solid waste such as excess activated sludge and the high-organic fraction of municipal solid waste. The sludge cake contains seed microorganisms for the landfill reactors. The synthetic solid waste was compacted 1.3 kg into 2.35 l of each reactor (density of 553 kg/m3). Deionized water was added respectively to all reactors at 300 ml/d on days 1, 5, 10, 17, and 31, and 150 ml/d on days 39, 45, 52, 65, 73, 79, and 87. The leachate was recirculated at 200 ml/d in all reactors. The first reactor was operated without aeration (control reactor, CR). The second reactor was aerated intermittently at 2 l/min (1.5 l/min/kg waste) for 6 h, with nonaeration for 6 h (intermittently aerated reactor, IAR). The third reactor was operated with continuous aeration at 2 l/min (continuously aerated reactor, CAR). Analytical procedures General characteristics of landfill bioreactors A portion of the 10-g wet solid waste was collected from the sampling port of the reactor for measurement of the dry mass, the organic matter and total nitrogen (T-N) contents, and microorganisms for every sampling. The organic matter content was measured according to standard methods (11). The T-N content was determined using Kjeldahl digestion method according to Japanese Industrial Standard K0102. Fresh leachate of 15 ml was collected in the storage container for every sampling. The pH values of leachate were determined using a Navi pH meter (F-52; Horiba Ltd., Kyoto). Leachate samples were centrifuged at 20,000 ×g for 10 min for chemical analysis. Total organic carbon (TOC) was measured using an analyzer (TOC-5000A; Shimadzu Corp., Kyoto). Methane produced from the reactors was sampled daily from the port on the top of the reactors and was examined using a Landfill Gas Analyzer (GA 2000A; Geotechnical Instruments Ltd., Warwickshire). The methane concentrations were measured in the non-aeration phase (0.5 h before aeration). The concentrations of CO2 that passed through a NaOH 5N solution were measured periodically by titration (12).
MPN-PCR analysis DNA in the solid waste samples was extracted using a Fast DNA SPIN Kit for Soil (Qbiogene Inc., CA) for polymerase chain reaction (PCR). The extracted DNA was serially diluted ten-fold at each step; three samples of each dilution were subjected to most probable number (MPN)-PCR (13). The MPN-PCR quantification was performed for eubacterial 16S rDNA with the primers EUBf-933 and EUBr-1387 (14), for fungal 18S rDNA with primers EF4 and Fung5 (15), and for the alpha-subunit for the methyl coenzyme-M reductase (mcrA) with the primers MCRf and MCRr (16). The mcrA gene is a methanogen-specific molecular marker. Terminal restriction fragment length polymorphism (T-RFLP) analysis PCR targeting eubacterial 16S rDNA genes was performed using the 27f and 1392r bacterial primer pair (17). The 5′-end of the forward primer was labeled with 6-carboxyfluorescein (FAM) for T-RFLP analysis. Actually, PCR was performed using one denaturation step at 95 °C for 10 min, followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 57 °C for 1 min, and extension at 72 °C for 3 min, with final extension at 72 °C for 10 min. PCR targeting archaeal 16S rDNA genes was performed using the A109f and A912r archaeal primer pair (18). The 5′ end of the reverse primer was labeled with 6carboxyfluorescein (FAM) for T-RFLP analysis. Actually, PCR was performed using one denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 90 s, with final extension at 72 °C for 6 min. Amplicons from duplicate PCR reaction mixtures were filtered using Montage PCR (Genomics; Millipore Corp., Massachusetts). The filtrate was digested for 5 h separately using HhaI, HaeIII, or MspI at 37 °C (for eubacterial 16S rDNA) or with TaqI at 65 °C or HhaI at 37 °C (for archaeal 16S rDNA). A mixture of digested products with HiDi formamide and GeneScan 2500 TAMRA Size Standard was denatured at 95 °C for 3 min. The fluorescently labeled T-RFs obtained in this manner were separated using capillary electrophoresis (ABI Prism 310; Genetic Analyzer, Applied Biosystems, California) to determine the number and size of T-RFs obtained from each sample. Fragment analyses were conducted using software (GeneScan™ Ver. 3.7; Applied Biosystems, California). The 16S rDNA sequences of the domains Bacteria and Archaea used for the in silico analysis were downloaded from http://mica.ibest.uidaho.edu (19). Statistical analysis Principal component analysis (PCA) and dendrogram analysis of T-RFLP profiles were performed using a software package (PAST ver. 1.34, PAlaeontological Statistics; Hammer). The PCA mathematical technique is used to reduce multidimensional datasets to lower dimensions for analysis. The dendrograms were created using the neighbor-joining clustering algorithm using Bray–Curtis' coefficient of similarity. The Shannon–Weaver index of diversity (H′) (20) was
FIG. 2. Characteristics of leachate generated from landfill reactors. (A) pH, (B) TOC concentration, and (C) T-N concentration. Symbols for reactors: diamonds, CR; squares, IAR; circles, CAR.
338
SANG ET AL.
J. BIOSCI. BIOENG.,
FIG. 3. Characteristics of landfill gas generated from landfill reactors. (A) cumulative CO2 generation and (B) methane concentration. Symbols for reactors: diamonds, CR; squares, IAR; circles, CAR.
calculated as H′ = − Σ Pi log Pi and Pi = ni/N, where ni is the area of peak ith and N is the total areas of peaks. The Simpson index of dominance (D) (21) was calculated using the Pi value in the H′ equation shown above: D = Σ P2i .
RESULTS Accelerative stabilization in landfill bioreactors Fig. 1 shows characteristics of the high-organic waste in the landfill reactors. The organic matter contents in the IAR and the CAR were markedly lower than that of the CR. The increase in the T-N content in the CR was probably attributable to the greater removal of organic carbon than nitrogenous components. The T-N contents fell rapidly in the IAR and the CAR, respectively, after days 50 and 30, indicating effective nitrogen removal by aeration, but at different rates. Furthermore, the waste volume in the IAR on day 90 was lowest in the reactors. Fig. 2 shows characteristics of the leachate generated by the reactors. The pH values of leachate from CR were less than 6.0 throughout the experiment, which reflects the accumulation of fermented acids (acidogenesis phase). The IAR showed lower pH values than the CAR until day 50. Leachate from the IAR and the CAR was characterized by the rapid reduction of the TOC and the T-N concentrations, although temporal increases were observed. Fig. 3 shows cumulative CO2 emissions and methane concentrations from the reactors. The high CO2 emission from the IAR and the CAR proved the high activity of the
TABLE 1. Mass balance of landfill bioreactors during 90-day experiments.
Water balance Input, g (%) - Water in solid waste on day 0 - Supplemental water Output, g (%) - Remaining in solid waste on day 90 - Sampling - Leachate discharge/sampling - Other (evaporation) Solid balance Input, g-dry (%) Output, g-dry (%) - Remaining on day 90 - Sampling - Other (Decomposition, etc.) Carbon balance Input, g (%) Output, g (%) - Remaining on day 90 - Sampling - CO2 emission - Leachate discharge/sampling - Other (CH4, SS in leachate, etc.)
CR
IAR
CAR
664 (18.9) 2850 (81.1)
664 (18.9) 2850 (81.1)
664 (18.9) 2850 (81.1)
1436 (40.9) 107 (3.0) 1420 (40.4) 551 (15.7)
939 (26.7) 101 (2.9) 1400 (39.8) 1074 (30.6)
973 (27.7) 96 (2.7) 995 (28.3) 1450 (41.3)
636 (100)
636 (100)
636 (100)
324 (50.9) 26 (4.1) 286 (45.0)
249 (39.1) 28 (4.4) 359 (56.5)
302 (47.5) 29 (4.6) 305 (47.9)
290 (100)
290 (100)
290 (100)
85.0 (29.3) 10.4 (3.6) 12.3 (4.2) 9.9 (3.4) 172.2 (59.4)
101.3 (35.0) 10.4 (3.6) 15.5 (5.4) 2.5 (0.9) 160.0 (55.2)
134.7 11.0 1.6 22.6 119.9
(46.5) (3.8) (0.6) (7.8) (41.4)
microorganisms. The maximum methane concentration was recorded as 7.7% on day 39 in the IAR in the non-aeration phase. Methane emissions from the CR were not observed, probably because the reactor was in the acidogenesis phase before the methanogenesis phase (Fig. 2A). The methane concentrations in the IAR in the aeration phase and the CAR were less than the detection limit (0.1%) throughout the experiment. Table 1 presents a summary of the mass balance of the landfill bioreactors in 90-day operation. In the water balance, high evaporation and low leachate generation occurred in the aerated bioreactors, especially in the CAR. The solid and carbon contents in the CR, the IAR, and the CAR at the end of operation were, respectively, 50.9, 39.1 and 47.5%, which values were 46.5, 29.3 and 35.0% on the first day. The smallest values of the remaining solid waste and organic matter indicated that the highest decomposition occurred in the IAR. The temporal increase in the TOC concentration in leachate from the IAR suggests that the intermittent aeration accelerated solubilization/ depolimerization of the solid wastes into leachate. High CO2 emissions also occurred in the IAR and the CAR. Microbial population dynamics in landfill bioreactors Fig. 4 shows MPN values of DNA as indicators of microbial populations in the landfill reactors. Eubacterial 16S rDNA increased from about 1.0 × 109 MPN-copies/g-dry on days 0–5.0 × 109 MPN-copies/g-dry on day 90, but no remarkable difference was apparent in the reactors. This result suggests that these values were not so sensitive to aeration. The MPN values of the fungal 18S rDNA in the aerated reactors, especially the IAR, increased sharply during the first 20–30 days and remained higher than that in the CR during the experiment. The mcrA gene increased drastically in the IAR, where high methane production was observed (Fig. 3B). In fact, the CAR was aerated continuously, but the MPN values of the mcrA gene in the CAR were higher than those in the CR. T-RFLP analysis of microorganisms in landfill bioreactors Phylogenetic analysis using T-RFLP analysis showed five known taxa of bacteria (Table 2). At the phylum level, the majority of the 16S rDNA sequences derived from the feed waste were assigned to Bacteroidetes, followed by unknown groups (HhaI fragment 56–57 bp), and Proteobacteria. The library derived from the CR, the IAR and the CAR showed a great difference from the feed waste with the levels of the phylum Actinobacteria, Firmicutes, and Proteobacteria. The anaerobic phylum Firmicutes, e.g. Clostridium, increased in the CR and the IAR in comparison with that in the CAR. Furthermore, uncultured Bacteroidetes (HhaI fragment 96 bp) and sulfate-reducing bacteria of the Desulfovibrio and Desulfohalobium genera in phylum Proteobacteria (HhaI fragment 95 bp) were dominant in the IAR. Results of archaeal communities obtained using T-RFLP analysis are presented in Table 3. Methanogens belonging to the H2-utilizing groups (Methanobacteriales order, HhaI fragment 157 bp) and the acetate-utilizing groups (Methanosarcinales order, HhaI fragment 91,
VOL. 108, 2009
MICROBIAL POPULATION DYNAMICS IN INTERMITTENT LABDFILL BIOREACTOR
339
FIG. 4. Population dynamics of microorganisms in landfill reactors: (A) eubacterial 16S rDNA, (B) fungal 18S rDNA, and (C) archaeal mcrA in landfill reactors. Symbols for reactors: diamonds, CR; squares, IAR; circles, CAR. Error bars show the 95% confidence interval.
534 bp) were found as the major methanogenic archaea during the operational period in the three landfill bioreactors. Methanobacteriales increased gradually from 30% of the archaeal population at the start of operation and became completely dominant up to 55.8% at the end of operation in the CR. Methanobacteriales were also dominant in the CAR on days 20 and 70. In the IAR, Methanobacteriales were gradually replaced by Methanosarcinales, consisting of Methanosarcina and Methanosaeta genera, which favor high acetate concentrations. Methanosarcinales became dominant to approximately 56% on day 20 and continued to dominate for 70 days in the IAR. The increased Methanosarcinales population suggests that this population came to produce methane actively via acetate. Community structure of microorganisms in landfill bioreactors The T-RFLP profiles of eubacterial 16S rDNA were used for PCA, dendrogram analysis, and evaluation of diversity and dominance indices, as presented in Fig. 5. The bacterial communities in the reactors formed different clusters from each other in the twodimensional plot of PC1 and PC2 scores and the dendrogram (Figs. 5A and B). The H′ value was lower and the D value was higher in the IAR than in the other bioreactors on day 70, but all bioreactors showed a similar value on day 90 (Figs. 5C and D). The T-RFLP profiles of archaeal 16S rDNA were also used for PCA, dendrogram analysis, and evaluation of diversity and dominance indices, as depicted in Fig. 6. The archaeal communities in the IAR showed a unique behavior in the two-dimensional plot of PC1 and PC2 scores in comparison to the other reactors. In the dendrogram, the archaeal communities in the two aerated reactors, the IAR and the CAR, in the late experimental phase formed a different cluster from that in the CR (Figs. 6A and B). The H′ values were markedly lower on day 20 in all reactors, but they were higher in the IAR and the CAR on days 70 and 90 (Fig. 6C). The increase of the D values of archaeal communities in the CR and the IAR from day 0 through day 70 depended mainly on the respective developments of Methanobacteriales and Methanosarcinales populations (Fig. 6D and Table 3). DISCUSSION Positive effects of aeration were apparent in the decreases in the organic matter and the nitrogen concentrations in the solid wastes and leachate in the IAR and the CAR (Figs. 1 and 2). The rapid settling of the solid wastes in the IAR and the CAR, especially in the IAR, is an important means to lengthen the life of landfills. The final remaining amount of solid and carbon in the bioreactors also indicated that active solubilization and degradation occurred in the IAR. Those results verified that intermittent aeration is practical for accelerative stabilization of landfills with less energy consumption. Any specific disadvantage of IAR was not observed within the results of this study. It has already been reported that the intermittent aeration stimulated
nitrifiers and denitrifiers in landfill bioreactors, resulting in effective nitrogen removal (9, 10). However, the general performance of landfill bioreactors with intermittent aeration has not been fully compared to that with full aeration. In addition, the mechanisms of the effective removal of organic matter in the IAR have not been fully discussed. In this study, development of the fungal population that can decompose diverse high-molecular organics (22) is inferred to be important for initiation of accelerated stabilization of landfills. Introduction of oxygen stimulated the metabolism of fungi, the aerobic microorganisms in the IAR (Fig. 4B). In addition, the low pH conditions in the IAR, especially during the first 40 days (Fig. 2A), might support the fungal growth rather than the bacterial growth. The gradually rising pH of leachate from the IAR suggests that intermittent aeration reduced the acidogenesis phase through activation of aerobic microorganisms (Fig. 2A). Methane production from acetate can also increase pH. Anaerobic bacteria, namely Clostridium and sulfate-reducing bacteria were found in IAR. Clostridium species are strictly anaerobic and typical H2-producing bacteria (23). Reportedly, anaerobic cellulolytic Clostridium genus and aerobic bacteria are often detected simultaneously at various sites where cellulose degradation occurs (24). Sulfate-reducing bacteria are the final decomposer of organic matter in the solid waste as well as methanogens. Sulfate-reducing bacteria can grow syntrophically with either H2-producing or acetateproducing bacteria and compete with methanogens for electron donors such as acetate and H2/CO2 (25). The high number of mcrA genes that are specific for methane production well reflected the high methane concentration in the IAR. Methane is a greenhouse gas with a high global warming potential but it can be utilized as an energy source if it is recovered. On the contrary, this is a strong greenhouse gas if it was emitted to ambient air. Analyses of the archaeal communities in landfill bioreactors presented differences attributable to intermittent aeration in relation to dominant groups and phylogenetic distribution (Table 3, Fig. 6). The increase in Methanosarcinales was consistent with the concurrent rise in methane production in the IAR (Figs. 3B and 4C). These results suggest that the main degradation pathway was acetotrophy for methanogenesis in the IAR, although the growth rate of the acetate-utilizing methanogens is generally smaller than that of the H2-utilizing methanogens (25). Contrarily, Mertoglu et al. (6) reported that the main degradation pathway for methanogenesis in an intermittently aerated landfill bioreactor was hydrogenotrophy. The H2-utilizing methanogens in the IAR might be outcompeted by sulfate-reducing bacteria, which are generally more efficient at hydrogen utilization than the methanogens (25). Both Desulfovibrio giganteus (26) and Desulfohalobium retbaense (27), which are presumably sulfate-reducing bacteria in the IAR (Table 2), cannot use acetate/sulfate as the electron donor and the electron acceptor. However, other various combinations of the electron donor and the electron acceptor, such as H2/sulfate, lactate/sulfate,
340
SANG ET AL.
J. BIOSCI. BIOENG., TABLE 2. Relative abundance (%) of major phyla of eubacteria in landfill bioreactors.
T-RF size (bp)
Best match in GenBank
Relative abundance (%) Feed waste
HhaI 56–57 123 286 300 991 90 96
HaeIII MspI
258
91
286
93 156 126 133
98 88 95 95 95
202
97
209
97 97 98 190 200
147 273 204
227
218
227 230
221
230 230 230 237
191
135 135 297
320 516 513
224 153 279 525
240
135
240 240
158 230
240
310
240 578
331 231
125 201
228
362
257
164
365
224
159
165
370
163
370
165
370 682
230
974
228
163
CR Day 20 Day 70 Day 90
Not included in database Not included in database Not included in database Not included in database Not included in database Chlorobiaceae bacterium LA53 uncultured Bacteroidetes bacterium nodule ECS04 CFB 6. Flexibacter sp. IUB42 Azospirillum lipoferum (T) Desulforhopalus vacuolatus (T) ltk10 Desulfovibrio giganteus DSM 4370 Desulfohalobium retbaense (T) DSM 5692 Desulfatibacillum alkenivorans PF2803; Olavius algarvensis sulfatereducing endosymbiont 126I-1; sulfate-reducing bacterium PF2802; Desulfosarcina variabilis (T); D. variabilis (T); Desulfococcus multivorans DSM 2059 Desulfomonile limimaris DCB-F Desulfobacula toluolica DSM 7467 Cycloclasticus sp. E2 Butyrivibrio fibrisolvens L2 Natronoanaerobium salstagnum O-M12SP-2 Clostridium cellulovorans (T) DSM 3052 Anaerobacter polyendosporus Clostridium aurantibutyricum (T) NCIMB10659 Clostridium tetani E88 Massachusetts Bacillus sphaericus (T) Lactobacillus catenaformis (T) Clostridium estertheticum (T) NCIMB12511 Paenibacillus macerans (T) ATCC 8244 and DSM 24 Bacillus sp. MC6B22 Bacillus subtilis 168; B. subtilis subsp. subtilis str. 168 Bacillus subtilis 168; B. subtilis subsp. subtilis str. 168 Bacillus subtilis BSK Thermoactinomyces dichotomicus (T) NCIMB 10211 (T) Streptomyces rimosus R6-554 Mycobacterium bovis; M. tuberculosis NCTC 7416 H37Rv phage lambda E; M. bovis AF2122/97; M. tuberculosis H37Rv uncultured Actinobacteridae bacterium MTG-11 Streptomyces bikiniensis (T) DSM40581; S. b. BA5 Arthrobacter sp. RC100; Arthrobacter sp. CPA2 Agromyces mediolanus CNF186; uncultured Agromyces sp. p6 Leifsonia xyli; Citricoccus sp. 3054 Arthrobacter davidanieli RSX II; Leifsonia rubra (T) CMS 76r = type strain Micrococcus sp. MF-1
Phylum
IAR
CAR
Day 20 Day 70 Day 90
Day 20 Day 70 Day 90
14.2 – – – – 2.9 27.9
16.0 – – – – – –
5.6 4.9 9.6 16.9 – – 3.9
4.4 – – – – – 3.3
8.9 – – – – – 16.5
9.6 – – – – – 41.3
7.2 – – – – – 18.6
16.1 – – – 4.6 2.3 8.3
23.1 – – – – 2.4 10.6
10.0 – – – – 5.4 –
– – – –
– – 2.2 –
– – 2.7 –
– – 5.7 –
– – – 3.1
4.4 – – 14.9
– 2.3 – 4.6
– 6.2 – 3.5
– 14.2 5.0 –
3.1 – 4.8 –
–
–
–
–
–
–
–
–
–
6.1
14.1
–
–
–
–
–
–
–
–
5.3
– – –
– – –
– 9.6 –
– – –
– – –
3.2 – –
6.8 – –
– – –
– – 4.2
– – 3.7
–
7.2
–
–
2.0
2.4
–
–
–
–
–
9.5
20.5
19.0
–
–
–
–
–
–
–
–
–
–
8.0
2.4
–
4.9
–
–
–
–
–
–
–
–
10.3
–
–
–
–
–
–
–
7.5
3.8
2.5
–
2.6
2.1
– –
– –
2.0 –
14.3 –
– –
– –
– –
– –
– –
3.5 13.0
–
11.7
–
–
–
–
–
–
–
–
–
–
3.1
6.4
–
–
–
–
–
–
–
–
–
–
14.3
2.2
2.4
10.4
–
–
–
–
–
–
–
–
19.1
–
–
–
2.3
–
–
–
6.5
–
–
–
–
–
Chlorobi Bacteroidetes
Proteobacteria
Firmicutes
Actinobacteria
–: Not assigned (b2%). Some uncultured bacteria of environmental samples in the CR are not shown in this table.
pyruvate/sulfate, and ethanol/sulfate, are available as the electron donor and the electron acceptor for D. giganteus and D. retbaense. Although the CR remained in the acidogenic phase before the
methanogenic phase, Methanobacteriales, which are H2-utilizing methanogens, were predominant in the bioreactor. Higher methane production from the CR would have been observed than that from the
VOL. 108, 2009
MICROBIAL POPULATION DYNAMICS IN INTERMITTENT LABDFILL BIOREACTOR
341
TABLE 3. Relative abundance (%) of major phyla of archaea in landfill bioreactors. T-RF size (bp)
TaqI
Best match in GenBank Feed waste
HhaI
54 62 71 79 498 90
199
90 90
201 156
91
157
392
157
795
157
185
53
392
53
185
76
185
112
185
91
185 283
534 534
392
534
Relative abundance (%)
Not included in database Not included in database Not included in database Not included in database Not included in database Thermococcus chitonophagus str. GC74 DSM 10152 (T). Thermococcus sp. str. GE20. Methanothermobacter thermoflex str. IDZ VKM B-1963 Methanosphaera stadtmanae str. MCB-3 DSM 3091 (T); M. unidentified methanogen ARC21; Methanothermobacter thermophilus str. M VKM B-1786; M defluvii str. ADZ VKM B-1962; Methanobacterium bryantii str. RiH2; M. b. str. M.o.H. DSM 863 (T); M. thermoautotrophicum; M. t. str. ZH3 DSM 9446; M. t. str. CB12 ATCC 43574; M. t. str. KHT-2; M. t. str. delta H; M. t. str. delta H. Methanosphaera unidentified methanogen ARC29; M. u. m. ARC8; M. u. m. ARC14; M. u. m. ARC30; M. u. m. ARC39; M. u. m. ARC43; M. u. m. ARC18; M. u. m. ARC49; M. u. m. ARC62. Methanobrevibacter arboriphilicus str. DH1 DSM 1536; M. curvatus str. RFM-2 DSM 11111 (T). Methanoculleus palmolei str. INSLUZ DSM 4273 (T) Methanospirillum sp.; M. sp. str. R10; M. hungatei str. JF1 DSM 864 (T); Methanofollis tationis DSM 2702 (T); M. liminatans str. GKZPZ DSM 4140 (T); M. l. DSM 10196; M. tationis DSM 2702 (T). symbiont of Trimyema compressa; s. of archaeobacterial s. of T. sp; s. of arch. s. of Metopus contortus. Methanomicrobium mobile str. BP DSM 1539 (T); Methanoplanus petrolearius SEBR 4847 (T) ; Methanoculleus olentangyi str. RC/ER OCM 52 (T); M. MAB1 str. MAB1; M. MAB2 str. MAB2; M. MAB3 str. MAB3; M. BA1 str. BA1; M. bourgense str. MS2 (T) ATCC 43281 (T); M. bourgensis str. MS2; M. olentangyi str. RC/ER ATCC 35293 (T). Methanosarcina sp. str. WH1 DSM 4659; M. mazei str. S-6 ; M. m. str. Go1; M. m. str. C16 ATCC 43340 (T) ; M. m. str. C16 DSM 2053 (T); M. m. str. SarPi; M. acetivorans str. C2A DSM 2834 (T); M. barkeri str. 227 DSM 1538; M. siciliae str. TA/M DSM 3028 (T); M. S. str. C2 J; M. thermophila str. TM-1 DSM 1825 (T); M. barkeri str. MS DSM 800 (T); M. b. str. Sar; M. b. str. CM1. Methanosarcina semesii str. MD1. Methanosaeta concilii str. Opfikon; M. concilii str. Opfikon DSM 2139; M. uncultured archaeon WCHD3-03. Methanosaeta thermoacetophila str. CALS-1 DSM 3870; M. concilii str. FE DSM 3013.
CR
Phylum
IAR
CAR
Day 20
Day 70
Day 90
Day 20
Day 70
Day 90
Day 20
Day 70
Day 90
21.5 – 7.5 1.7 4.2 0.7
6.1 6.6 – – – 11.3
31.1 – – – – –
33.4 – – – – –
– 8.5 – – – –
8.0 2.1 7.4 8.7 – –
19.5 – 5.0 19.6 – 3.3
3.9 13.9 – – – –
11.3 5.8 6.5 10.6 – –
18.5 – 28.3 1.3 –
Thermococcales
6.1 –
– –
– –
– 1.3
– –
– –
– 2.3
– –
– –
– –
Methanobacteriales
9.6
–
10.3
3.3
5.2
–
–
6.5
–
–
–
–
–
–
–
–
0.5
–
38.3
4.0
21.1
58.1
40.7
52.5
13.3
–
–
30.4
–
2.0
7.1
–
–
0.8
–
2.5
6.2
–
3.3
8.3
0.6
0.5
–
–
–
4.1
6.0
–
–
–
–
–
–
–
–
–
0.4
–
9.5
–
2.6
2.1
–
0.7
55.7
56.9
0.9
22.9
8.7
6.7
12.2
3.5
–
0.4
–
–
17.1
–
–
1.8
Methanomicrobiales
Methanosarcinales
–: Not detected.
IAR if the experimental period had been extended beyond 90 days. Continuous aeration in the CAR is presumed to degrade acetate further into CO2; subsequently, Methanobacteriales were not replaced by the acetate-utilizing methanogens Methanosarcinales. Methanogenic diversity in landfill bioreactors is quite dissimilar among different operation modes and in different stabilization phases. Consequently, the transition of the aerobic and anaerobic microbial populations in the IAR was more dynamic than that in either the CR or
the CAR (Table 2 and Fig. 5). The Shannon–Weaver index of eubacteria in the IAR was lowest at days 20 and 70, but it recovered to the same level as those of the CR and the CAR on day 90 (Fig. 5). Furthermore, the Shannon–Weaver index of archaea in the IAR was lowest at day 20, but highest at day 90 (Fig. 6). These results suggest that the intermittent aeration finally formed a diverse microbial community in the IAR. In conclusion, the performance of the IAR with a half time of aeration was highest among the reactors. Reportedly, the range of the
342
SANG ET AL.
J. BIOSCI. BIOENG.,
FIG. 5. Bacterial 16S rDNA analyses: (A) PCA, (B) dendrogram analysis using Bray–Curtis' coefficient, (C) Shannon–Weaver index of diversity (H′), and (D) Simpson index of dominance (D). Symbols for reactors: diamonds, CR; squares, IAR; circles, CAR. Names of samples in (A) and (B): closed triangle, Waste-0, sampling on day 0; Reactor-sampling day (e.g., IAR-70, sampling in the IAR on day 70).
aeration rate, even for landfill bioreactors with full aeration, was large (0.0002–1.3 l/min/kg waste) (5, 28). Moreover, the effects of the period and cycle of intermittent aeration on the performance of
landfill bioreactors have never been studied systematically. The IAR performance is expected to be enhanced under optimized conditions on the intermittent aeration mode. Further monitoring strategies for
FIG. 6. Archaeal 16S rDNA analyses: (A) PCA, (B) dendrogram analysis using Bray–Curtis' coefficient, (C) Shannon–Weaver index of diversity (H′), and (D) Simpson index of dominance (D). Symbols for reactors: diamonds, CR; squares, IAR; circles, CAR. Names of samples in (A) and (B): closed triangle, Waste-0, sampling on day 0; Reactor-sampling day (e.g., IAR-70, sampling in the IAR on day 70).
VOL. 108, 2009
MICROBIAL POPULATION DYNAMICS IN INTERMITTENT LABDFILL BIOREACTOR
microorganisms are necessary to obtain advanced knowledge of the design and operation of landfill bioreactors with intermittent aeration.
12.
ACKNOWLEDGMENTS 13.
The authors gratefully acknowledge the support of the Core University Program between the Japan Society for the Promotion of Science (JSPS) and the Vietnamese Academy of Science and Technology (VAST). Nguyen Nhu Sang gratefully acknowledges the support of the Vietnamese Overseas Scholarship Program (VOSP). This study was partly supported by the Nippon Life Insurance Foundation.
14.
15.
16.
References 17. 1. Sang, N. N., Soda, S., Sei, K., Ishigaki, T., Triet, L. M., Ike, M., and Fujita, M.: Performance of lab-scale membrane bioreactor for leachate from Go Cat landfill in Ho Chi Minh City, Vietnam, Jpn. J. Wat. Treat. Biol., 43, 43–49 (2007). 2. Sormunen, K., Einola, J., Ettala, M., and Rintala, J.: Leachate and gaseous emissions from initial phases of landfilling mechanically and mechanically–biologically treated municipal solid waste residuals, Bioresour. Technol., 99, 2399–2409 (2008). 3. Barlaz, M., Ham, R., and Schaefer, D.: Methane production from municipal refuse: a review of enhancement techniques and microbial dynamics, Crit. Rev. Environ. Control, 19, 557–584 (1990). 4. Ishigaki, T., Sugano, W., Nakanishi, A., Tateda, M., Ike, M., and Fujita, M.: Application of bioventing to waste landfill for improving waste settlement and leachate quality — a lab-scale model study, J. Solid Waste Technol. Manag., 29, 230–238 (2003). 5. Sang, N. N., Soda, S., Sei, K., and Ike, M.: Effect of aeration on stabilization of organic solid waste and microbial population dynamics in lab-scale landfill bioreactors, J. Biosci. Bioeng., 106, 425–432 (2008). 6. Mertoglu, B., Calli, B., Guler, N., Inanc, B., and Inoue, Y.: Effects of insufficient air injection on methanogenic Archaea in landfill bioreactor, J. Hazard. Mater., 142, 258–265 (2007). 7. O'Keefe, D. M. and Chynoweth, D. P.: Influence of phase separation, leachate recycle and aeration on treatment of municipal solid waste in simulated landfill cell, Bioresour. Technol., 72, 55–66 (2000). 8. Jun, D., Yongsheng, Z., Henry, R. K., and Mei, H.: Impacts of aeration and active sludge addition on leachate recirculation bioreactor, J. Hazard. Mater., 147, 240–248 (2007). 9. He, R., Liu, X.-W., Zhang, Z.-J., and Shen, D.-S.: Characteristics of the bioreactor landfill system using an anaerobic–aerobic process for nitrogen removal, Bioresor. Technol., 98, 2526–2532 (2007). 10. Shao, L.-M., He, P. J., and Li, G.-J.: In situ nitrogen removal from leachate by bioreactor landfill with limited aeration, Waste Manag., 28, 1000–1007 (2008). 11. American Public Health Association (APHA): American Water Works Association and Water Pollution Control Federation: Standard methods for the examination of
18.
19.
20. 21. 22. 23.
24.
25. 26.
27.
28.
343
water and wastewater. (20th ed.), American Public Health Association, Washington, DC 20005-2605, 1998. Komilis, D. P. and Ham, R. K.: Carbon dioxide and ammonia emissions during composting of mixed paper, yard waste and food waste, Waste Manag., 26, 62–70 (2006). Picard, C., Ponsonnet, C., Paget, E., Nesme, X., and Simonet, P.: Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction, Appl. Environ. Microbiol., 58, 2717–2722 (1992). Iwamoto, T., Tani, K., Nakamura, K., Suzuki, Y., Kitagawa, M., Eguchi, M., and Nasu, M.: Monitoring impact of in-situ biostimulation treatment on groundwater bacterial community by DGGE, FEMS Microbiol. Ecol., 32, 129–141 (2000). Marshall, M. N., Cocolin, L., Mills, D. A., and VanderGheynst, J. S.: Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost, J. Appl. Microbiol., 95, 934–948 (2003). Springer, E., Matthew, S. S., Carl, R. W., and David, R. B.: Partial gene sequences for the A subunit of methyl-coenzyme M reductase (mcrI) as a phylogenetic tool for the family Methanosarcinaceae, Inter. J. Sys. Bacteriol., 45, 554–559 (1995). Jeffery, A. M., William, G. M., Ruihong, Z., Yanguo, M., and Frank, M.: Bacterial population dynamics in dairy waste during aerobic and anaerobic treatment and subsequent storage, Appl. Environ. Microbiol., 73, 193–202 (2007). Lueders, T. and Friedrich, M.: Archaeal population dynamics during sequential reduction processes in rice field soil, Appl. Environ. Microbiol., 66, 2732–2742 (2000). Conrad, S., Terry, S., Stephan, J. B., James, A. F., and Larry, J. F.: MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms on 16S and 18S rDNA genes, Microb. Ecol., 53, 562–570 (2007). Shannon, C. E. and Weaver, W.: The mathematical theory of communication5th ed, University of Illinois Press, Urbana, 1963. Simpson, E. H.: Measurement of diversity, Nature, 163, 688 (1949). Sodroski, J., Rosen, C., Goh, C. W., and Haseltine, W.: Oxidation of persistent environmental pollutants by a white rot fungus, Science, 228, 1434–1436 (1985). Lin, P. Y., Whang, L. M., Wu, Y. R., Ren, W. J., Hsiao, C. J., Li, S. L., and Chang, J. S.: Biological hydrogen production of the genus Clostridium: Metabolic study and mathematical model simulation, Int. J. Hydrogen Energy, 32, 1728–1735 (2007). Kato, S., Haruta, S., Cui, Z. J., Ishii, M., and Igarashi, Y.: Effective cellulose degradation by a mixed-culture system composed of a cellulolytic Clostridium and aerobic non-cellulolytic bacteria, FEMS Microbiol. Ecol., 51, 133–142 (2004). Liamleam, W. and Annachhatre, A. P.: Electron donors for biological sulfate reduction, Biotechnol. Adv., 25, 452–463 (2007). Brauman, A., Koenig, J. F., Dutreix, J., and Garcia, J. L.: Characterization of two sulfate-reducing bacteria from the gut of the soil-feeding termite, Cubitermes speciosus, Antonie van Leeuwenhoek, 58, 271–275 (1990). Ollivier, B., Hatchikian, C. E., Prensier, G., Guezennec, J., and Garcia, J.-L.: Desulfohalobium retbaense gen. nov. sp. nov. a halophilic sulfate-reducing bacterium from sediments of a hypersaline lake in Senegal, Inter. J. Sys. Bacteriol., 41, 74–81 (1991). Bilgili, M. S., Demir, A., and Özkaya, B.: Quality and quantity of leachate in aerobic pilot-scale landfills, Environ. Manag., 38, 189–196 (2006).