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Journal of Bioscience and Bioengineering VOL. 114 No. 3, 243e250, 2012 www.elsevier.com/locate/jbiosc

REVIEW

Microorganisms in landﬁll bioreactors for accelerated stabilization of solid wastes Nguyen Nhu Sang,1, * Satoshi Soda,2 Tomonori Ishigaki,3 and Michihiko Ike2 Institute for Environment and Resources, Vietnam National University e Ho Chi Minh City, 142 To Hien Thanh street, District 10, Ho Chi Minh City, Viet Nam,1 Division of Sustainable Energy and Environmental Engineering, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan,2 and Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan3 Received 24 January 2012; accepted 6 April 2012 Available online 17 May 2012

Landﬁll bioreactors (LBRs) with management of leachate and biogas have presented numerous advantages such as accelerated stabilization of solid wastes, reduced amount of leachate, and in situ leachate treatment. Such advantages have minimized environmental risks, have allowed extension of the useful life of the landﬁll site, and have fostered cost reduction. LBRs of three types have been developed using both anaerobic and aerobic modes: anaerobic, aerobic, and hybrid. Microorganisms in landﬁlls cause various reactions related with organic fractions and heavy metals. Such functions have been stimulated in LBRs by recirculation of leachate with or without aeration. To date, most studies of microorganisms in LBRs have analyzed bacteria and archaea based on 16S rRNA genes and have analyzed fungi based on 18S rRNA genes from a taxonomical viewpoint. Indicator genes for speciﬁc functions in LBRs such as nitriﬁcation, denitriﬁcation, and methane production have also been monitored. The population dynamics of microorganisms in LBRs have been partially clariﬁed, but the obtained data remain limited because of highly heterogeneous features of solid wastes inside LBRs. Systematic monitoring of microorganisms should be established to improve LBR performance. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Landﬁll bioreactor; Solid waste; Stabilization; Microorganisms; Bacteria; Archaea]

Disposal of municipal solid wastes is of growing importance throughout the world. Landﬁll use is the most common method for their disposal at present. In some countries such as Japan, Taiwan and Switzerland, solid wastes are classiﬁed and buried into landﬁlls after incineration (1,2). By contrast, in most countries including Asian developing countries, unclassiﬁed solid wastes containing high organic fractions are buried directly in landﬁlls (3,4). Consequently, waste disposal landﬁlls generate leachate with high concentrations of organic matter, nutrients, toxic chemicals, and heavy metals. They can also emit harmful and greenhouse-effect gases, such as NH3, H2S, N2O, and CH4, because of unfavorable biochemical reactions that occur in the landﬁlls. Because conventional landﬁlls, especially those in developing countries, can heavily pollute the environment and exacerbate severe human health problems, they should be operated in a manner that can minimize environmental pollution. Conventional landﬁlls have been operated anaerobically without control of the amounts of moisture entering and retained in the waste. In such operations, buried wastes decompose slowly, thereby necessitating long periods for stabilization, which engenders long-term risks (5,6). A common technique to enhance biodegradation is to add supplemental water and/or recirculate leachate to the waste to stimulate microbial activity by providing better contact between substrates and microorganisms. Nowadays, such landﬁlls are often designated as bioreactor landﬁlls and * Corresponding author. Tel.: þ84 8 3865 1132; fax: þ84 8 3865 5670. E-mail address: [email protected] (N.N. Sang).

landﬁll bioreactors (LBRs) (7e9). Furthermore, recently developed aerobic LBRs with aeration provide more rapid biodegradation of organic matter, reduced methane production, and enhanced ammoniaenitrogen removal (10,11). Such advantages minimize the health and environmental risks and allow for greater free space availability, thereby extending the landﬁll site life and reducing costs (12,13). Landﬁll stabilization might require 30 years or longer for conventional landﬁlls, although 5 years or less might be necessary to achieve stabilization using LBRs (14). To optimize the design and control of LBRs, it is important to understand the behavior of microbial populations in LBRs completely, especially bacteria possessing speciﬁc functions, because microbial metabolisms play a pivotal role in the stabilization of solid wastes through their complicated interactions with various physical and chemical reactions. The microbial community structure in LBRs might reﬂect their performance, and might be useful as indicators of the degree of waste stabilization. This review article summarizes the known results of microbial monitoring in LBRs of various types for elucidating the waste decomposition and stabilization inside, although a few studies have addressed this topic (2,15e17). The summarized knowledge might yield some insight into the microbial ecology in LBRs, which can engender improvement of the performance of LBRs. FUNDAMENTAL ROLES OF MICROORGANISMS IN LANDFILLS Microorganisms in landﬁlls can cause various reactions depending on the environmental conditions and their substrate-

1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2012.04.007

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Hydrolysis

Acidogenesis

Methanogenesis

Acetogenesis

Stabilization

(methylotrophs)

Sugars, amino acids, fatty acids

CO2 (acidogens)

Complex organic waste

Organic acids, CO2, H2

(acetogens)

(Acetate-utilizing methanogens)

Acetate

(acetate-oxidizing bacteria) (Hydrogen-utilizing CO2 + H2 methanogens) (acetogens)

Ammonification (ammonifying bacteria)

NH4

Sulfate reduction (sulfur-reducing bacteria)

(C, H, O, N, S, etc.)

(denitrifiers)

NH4+

Nitrification

NO2−

(nitrifiers)

Nitrification (nitrifiers)

(aerobes, fungi)

N2, N2O Humic substances NO3− SO42−

Sulfur oxidation (sulfur-oxidizing bacteria)

Depolymerization

Stabilization waste

Lower molecular weight compounds

CO2 (aerobes, fungi)

Stabilization waste

Aerobic digestion

Ammonification

+

HS−, H2S

Denitrification

(ammonifying bacteria)

CH4

Anaerobic digestion

(hydrolytic bacteria)

FIG. 1. Schematic diagram of solid waste decomposition with the role of microorganisms in landﬁll bioreactors. Symbols: continuous line, carbon decomposition; dotted line, sulfur conversion; and dashed line, nitrogen conversion.

speciﬁcity. An overall scheme for anaerobic and aerobic bioconversions of complex organic wastes is presented in Fig. 1. Balanced utilization of both anaerobic and aerobic metabolic pathways is effective for accelerated stabilization in LBRs. Metabolisms on organic fractions of solid waste The anaerobic process for organic waste decomposition generally takes place in ﬁve stages: hydrolysis, acidogenesis, acetogenesis, methanogenesis, and stabilization (11). Complex organic wastes such as carbohydrates, proteins, and lipids are hydrolyzed to monosaccharides, amino acids, and fatty acids. Acidogens break down these hydrolysis products into H2, CO2, and organic acids such as lactate, butyrate, and propionate. Acetogens break down these organic acids into acetate. The acetogen products are converted anaerobically to CH4 via acetate and H2 þ CO2 during methanogenesis. As a result of the biodegradation of dead plant components such as lignin, humic substances are also formed. Humic substances such as humic acids and fulvic acids, which are dark brown to black, are highly resistant to additional biodegradation. Eventually, the reactions will be limited by substrate availability. Fungi, especially Phanerochaete, Phlebia, Trametes belonging to white-rot fungi, and bacteria such as Streptomyces, Nocardia, Pseudomonas are the most important group of lignin degradation (18). Some complex wastes containing organic nitrogen can be digested to ammonia. Sulﬁde is formed by the anaerobic decomposition of sulfur-containing amino acids and the reduction of sulfate in the waste by sulfatereducing bacteria. Although sulﬁde partly escapes in the biogas (H2S), most remains dissolved as HS. During the aerobic process, bacteria and fungi convert complex organic wastes rapidly to lower molecular weight compounds: mostly to CO2 and H2O with stabilized humic substances instead of methane. The higher energy yields of aerobic processes engender faster microbial growth and reproduction. Through the nitriﬁcation process, ammonia is converted to nitrite by ammoniaoxidizing bacteria and further into nitrate by nitrite-oxidizing bacteria. Hydrogen sulﬁde and ammonia are diminished in aerobic conditions, decreasing noxious odors produced in the landﬁll. In anoxic conditions, denitrifying bacteria convert NO2 =NO3 into N2 via N2O. Without the combination of these aerobic and anoxic reactions, nitrogen cannot be removed efﬁciently from landﬁlls.

Metabolisms on metals in solid waste Microorganisms contribute directly and indirectly to the stabilization of heavy metals in landﬁlls. Iron, cadmium, copper, zinc, and nickel are heavy metals that are commonly found in landﬁlls (19). The major sources of these metals are industrial wastes, incinerator ash, and hazardous substances in unclassiﬁed wastes such as batteries, paints, and dyes. The highest metal concentrations of leachate are observed in the acid formation stage, when pH values are low. Metals and metalloids exert toxicity to microorganisms affecting their biochemical activities, cell morphology, and growth. In methanogenic and neutral pH conditions, metals are removed by precipitation as insoluble sulﬁdes, carbonates, hydroxides, and phosphates. In the presence of sulﬁdes produced by sulfatereducing bacteria, most heavy metals form extremely insoluble sulﬁde salts as the major attenuation mechanisms of heavy metals in landﬁlls (20). Under aerobic conditions, the positive redox potential affects metal speciation and mobility. DESIGN OF LBRS AND THEIR PERFORMANCE Implementation of LBR technology requires modiﬁcation of the design and operational criteria associated with traditional landﬁlling. A schematic diagram depicting an LBR is presented in Fig. 2. The bottom liner system must be designed to accommodate the additional ﬂows contributed by leachate recirculation. The gas management facilities must be operated to control ampliﬁed gas production. Monitoring of leachate and gas quality and quantity becomes crucially important for operational decision making. Even pretreatment such as shredding or screening for reduction of waste blocks might be desirable to promote stabilization. Shredding improves water distribution and more equitable settlement because more waste is exposed to microbial activity. In addition, seeding or inoculating of microorganisms to the LBRs has been investigated typically through the addition of excess sludge of wastewater treatment plants (21,22). Composted waste offers an alternative seed source. However, there is still lack of information regarding to microbial augmentation in LBRs. Although widely various leachate recirculation rates exist depending on the solid waste quality, moisture contents should be monitored to ensure uniform distribution. The optimal moisture contents are 40e70% on

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FIG. 2. Schematic diagram of landﬁll bioreactors.

a wet-weight basis (23). Table 1 presents characteristics of leachate of LBRs. Leachate with high concentrations ammonia and volatile fatty acids with low pH might cause a toxic effect on the sensitive methanogenic community (24). Anaerobic LBRs The traditional method of LBR operation involves enhancing anaerobic waste stabilization with leachate recirculation. Initially, the leachate pH might be neutral; but after the onset of anaerobic conditions, it generally shows a noticeable drop, especially during the acidogenesis stage. It eventually increases to neutral conditions in the methanogenesis stage (Table 1). However, surplus recirculation rates of leachate create an imbalance of growth rates between those of fast-growing acidogens and slow-growing methanogens. In anaerobic landﬁlls, CH4 concentrations in biogas are generally 30e70% v/v, although the CO2 concentration varies between 20% and 50% v/v (25,26). Aerobic LBRs In aerobic LBRs, air is injected actively or passively into the landﬁll using the same devices used for extracting gas or injecting leachate: vertical and horizontal wells (Fig. 2). Settlement in aerobic LBR is generally higher than that in the conventional landﬁlls (12,27). The leachate volume can be reduced in aerobic LBRs by evaporation attributed to the elevated temperature of the waste. In addition, aerobic LBRs can reduce methane production and improve the leachate quality (5,6,13,27e33).

In LBRs with forced aeration, the airﬂow rate is a key design and operation parameter. The optimal aeration rate that is suitable for the growth of microorganisms is 0.06e0.94 l/min/kg (34). The general consensus is that an airﬂow that avoids an outlet CO2 concentration of about 15% is sufﬁcient for the aerobic decomposition of solid waste (10). A reduction of CH4 from 80% to 90% at the start of the aerobic process and remaining consistently below 15% were reported from results of previous studies (30,35). Although uniform distribution of oxygen throughout waste is difﬁcult, forced aeration into LBRs must not be allowed to form an explosive gas mixture of methane and oxygen. The costs of forced aeration of an existing landﬁll site have been estimated at 1e3 Euro/m3. Higher costs of 2e3 Euro/m3 are expected under unfavorable conditions, such as small sites or without suitable infrastructure. Under optimized conditions, costs are expected to be 0.5e1 Euro/m3 (11). The aeration costs are offset by savings derived in several ways, including reduced requirements for a surface cup, lower gas and leachate treatment costs, reduction of the aftercare period by at least several decades, and earlier after-use. Passive and semi-aerobic LBRs remove the leachate and gas continuously from the waste using leachate collection and gas venting systems. With proper engineering design, ambient air ﬂows into the waste body because of the temperature differential between the inside of the waste and the ambient air. It subsequently improves the waste stabilization process and elevates

TABLE 1. Leachate compositions of LBRs. LBRs

Anaerobic Semiaerobic

Hybrid

Aerobic

Operations

Leachate recirculation at 0.2 l/d Without leachate recirculation Leachate recirculation at 41 ml/min Without leachate recirculation Leachate recirculation 41 ml/min Cyclic aeration for 6 h at 2 l/min and non-aeration for 6 h; leachate recirculation at 0.2 l/d Continuous aeration at 2 l/min; leachate recirculation at 0.2 l/d

Weight of waste

Operation (days)

1.3 kg of synthetic organic waste 8470 kg of municipal solid waste 8470 kg of municipal solid waste 10300 kg of industrial solid waste 10300 kg of industrial solid waste 1.3 kg of synthetic organic waste

90 1000 1000 1000 1000 90

1.3 kg of synthetic organic waste

90

Values at the end of operation (maximum values) pH 5.76 8.2 7.4 7.9 9.3 8.7

(6.9) (8.9) (10) (8.9) (10) (9.0)

8.5 (8.8)

COD (mg/l) n.a 210 (850) 50 (350) 180 (410) 20 (380) n.a

n.a

TOC (mg/l) 13710 100 30 30 20 600

Ref.

TeN (mg/l)

(30800) 1450 (2590) 28 (3500) 30 (115) 1 (480) 10 (78) (300) 21 (95) (200) 8 (80) (27000) 400 (2300) 28

500 (9000)

200 (1600) 28

BOD: biochemical oxygen demand; COD: chemical oxygen demand; TOC: total organic carbon; TeN: total nitrogen; n.a: not available; numbers in blankets are maximum values in operational time.

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temperatures further. The passive characteristics of the process are beneﬁcial in terms of energy costs. Some data have been projected for new semi-aerobic LBRs in Malaysia, recorded the positive revenue to near 14% the cost of operation and closure care to compare with that of anaerobic sites (3). This is important information for development and management of landﬁll sites in developing countries. Hybrid LBRs Surplus aeration can decrease microbial activity and increase energy consumption. As a hybrid aerobic and anaerobic technique, intermittent aeration is practical to create cyclically aerobic and anaerobic conditions and to reduce energy consumption in LBRs. Rapid stabilization of solid waste is possible with intermittent aeration at various oxidationereduction potentials. Furthermore, neither an aerobic nor an anaerobic condition alone degrades chlorinated organic contaminants such as perchloroethene and trichloroethene (36). Reportedly hybrid bioreactors can exhibit higher settlement than either aerobic or anaerobic bioreactors (13,28). Few studies have examined biogas compositions from hybrid LBRs such as CH4 below 3% v/v, CO2 of 2e12% v/v, and O2 of 8e19% v/v (27,28,37). METHODOLOGIES FOR MICROBIAL MONITORING IN LBRS Analytical techniques for monitoring of microorganisms in LBRs are shown in Table 2. In early studies of landﬁll microbiology, researchers studied hydrolytic, acidogenic, and acetogenic bacteria using culture-based methods such as plate counts and mostprobable-number (MPN) techniques. Various microorganisms

using speciﬁc substrates as the sole carbon source are enumerated using plates with key substrates such as volatile fatty acids and alcohols in LBRs (2). Acetate-using and H2 þ CO2-using methanogens have also been enumerated using MPN techniques (38). Culture-based methods are not always satisfactory for estimating the total number of viable microorganisms. Molecular techniques based on PCR have been used to overcome the limitations of culture-based methods. The DNA extraction method can also bias diversity studies. Harsh extraction methods such as bead beating can shear the nucleic acids, leading to problems in subsequent PCR detection. Different methods of nucleic acid extraction will result in different yields of products. With environmental samples, it is necessary to remove inhibitory substances such as humic acids, which can interfere with subsequent PCR. Depending on characteristics of the sample and following analytical methods, the adequate waste sample processing and extraction methods should be selected and varied with the goals of the study (39). To date, most studies of microorganisms in LBRs have analyzed bacterial and archaeal communities based on 16S rRNA genes using T-RFLP, DGGE, or random cloning techniques from a taxonomical viewpoint (Table 2). Some indicator genes for speciﬁc functions in landﬁlls are also applied, such as amoA gene for ammonia-oxidizing bacteria, nirK and nirS genes for denitrifying bacteria, and mcrA gene for methanogens (Table 2). The amoA gene coding for a subunit of ammonia monooxygenase is a marker reﬂecting phylogeny on ammonia-oxidizing bacteria. The nirK encoding a cytochrome cd1 nitrite reductase and nirS genes encoding a Cu containing nitrite reductase are markers reﬂecting phylogeny of

TABLE 2. Analysis techniques applied for microorganism monitoring regarding to landﬁlls and solid waste treatment. Analysis techniques Adenosine triphosphate (ATP) assay

Plate counts Luminescent immunoassay Most probable number (MPN)

MPN-PCR

Real-time PCR Terminal restriction fragmentlength polymorphism (T-RFLP) Fluorescent in situ hybridization (FISH)

Denaturing gradient gel electrophoresis (DGGE) Microarray Cloning and sequencing of DNA

a b c d e

16Sr RNA gene. amoA gene. nirK, nirS genes. 18S rRNA gene. mcrA gene.

Methods Quantiﬁcation of viable bacterial cells using a luciferin luciferase system (e.g., 1.0 nmol of ATP corresponds to 200 cells). Quantiﬁcation of colonies formed on the plate (colony forming units) Quantiﬁcation of target cells based on the speciﬁcity of the antibody-antigen reaction. Quantiﬁcation of viable cells based on the principles of MPN statistics Quantiﬁcation of PCR products of target DNA by combining the principles of MPN statistics and PCR technique

Ampliﬁcation and simultaneous quantiﬁcation of a targeted DNA molecule Quantiﬁcation of relative abundance of DNA fragments with different sequences of PCR products. Visualization and quantiﬁcation of target genes in microorganisms

Separation of PCR products using electrophoresis based on differences in DNA sequences or mutations Identiﬁcation of DNA by hybridization on a collection of microscopic DNA spots attached to a solid surface Taxonomical identiﬁcation of rRNA genes

Target microorganisms

Ref.

Viable bacteria

2

Heterotrophs Heterotrophs, methanogens, actinomycetes, fungi Ammoinia-oxidizing bacteria

2,60 31 31

Hemicellulolytic bacteria, cellulolytic bacteria, H2-producing acetogens, acetate methanogens, H2þCO2-utilizing methanogens Eubacteria,a ammonia-oxidizing bacteira,b denitrifying bacteriac Eubacteria,a fungi,d ammonia-oxidizing bacteira,b denitrifying bacteriac Eubacteria,a fungi,d methanogense Eubacteria,a Archaeaa

38

Eubacteriaa Eubacteria,a Archaeaa Eubacteria,a Archaea,a Methanosaetaceae, Firmicutes Eubacteria,a Archaeaa Eubacteria,a Archaea,a Methanosetaceae, Methanosarcinaceae, Methanococcales, Methanobacteriales, Methanomicrobiales Eubacteria,a Archaeaa Methanotrophs Methanotrophsa

1,2 28 67 16 68

Eubacteriaa Eubacteria,a Archaeaa Fungi, Neocallimastigales Eubacteria,a Clostridium Eubacteria,a ammonia-oxidizing bacteriab Archaeaa Methanogens Methanotrophs

15,60e62,67 40,41,69 57 42 46 16,17,54 71 70

1 27 28 2

69 70 70

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TABLE 3. Microorganisms in waste samples of LBRs. LBRs

Operations

Anaerobic

Synthetic organic waste

Semi-aerobic

Municipal solid waste without leachate recirculation Municipal solid waste with leachate recirculation Industrial solid waste without leachate recirculation

Hybrid

Aerobic

Industrial solid waste with leachate recirculation Synthetic organic waste with intermittent aeration and leachate recirculation Synthetic organic waste with continuous aeration and leachate recirculation

denitrifying bacteria which transform NO2eN into gaseous nitrogen. The mcrA encoding for the alpha-subunit of the methyl coenzyme-M reductase is a methanogen-speciﬁc molecular marker. MICROORGANISMS IN LBRS Variations in reactor design, operating conditions, and the substrate composition have a strong impact on the development of microbial communities. As portrayed in Table 3, predominant microbial species in LBRs have been identiﬁed based on the 16S rRNA genes. Few studies of dynamism of microbial populations in lab-scale and pilot-scale LBRs have been reported, as shown in Table 4. MPN values of the 16S rRNA gene were not so sensitive to leachate recirculation and aeration. The MPN values of fungal the 18S rRNA gene, amoA, and mcrA genes can increase sharply in the aerobic LBRs (27,28). The MPN values of nirK and nirS genes in the anaerobic LBRs were equivalent to those in the conventional and aerobic LBRs (Table 4). Although those two reductases are found to be mutually exclusive among denitriﬁers, very little is known about their environmental preferences. Bacteria Various bacteria have been found in anaerobic LBRs (Table 3), such as Firmicutes and Proteobacteria (28,40), Bacteroidetes (40,41), Actinobacteria (28) and Chloroﬂexi (41). Clostridium,

Dominant species of bacteria/archaea

Ref.

Bacillus, Clostridium, Enterobacter, Eschericia, Empedobacter, Flavobacterium, Klebsiella, Lactobacillus, Leuconostoc, Providentia, Schineria, Sporanaerobacter, Thermacetogenium Methanoculleus bourgensis, Methanofollis formosanus, Methanosphaera stadtmanae Clostridium ghoni, C. cluyveri, Shigella, Thermodesulfovibrio yellowstonii, Yersinia pestis Listonella anguillarum, Pseudomonas Clostridium ghoni, Collinsella aeofaciens, Hydrogenobacter acidophilus, Rhodopseudomonas, Thrmotoga thermarum, Yersinia pestis Clostridium josui, Pseudomonas stutzeri, Rhodospirillum rubrum, Thiomicrospira Arthrobacter, Bacteroidetes, Cycloclasticus, Clostridium cellulovorans, C. estertheticum, Desulfovibrio giganteus, Thermoactinomyces dichotomicus, Methanoculleus palmolei, Methanosarcina, Symbiont of Trimyema compress Azospirillum lipoferum, Bacteroidetes, Chlorobiaceae bacterium, Desulforhopalus vacuolatus, Natronoanaerobium salstagnum, Thermoactinomyces dichotomicus Methanosarcina, Methanosphaera, Methanoculleus palmolei

40

1

28

Sporanaerobacter, Thermacetogenium, Leuconostoc, Bacillus and Lactobacillus, Pseudomonas, Listonella, Rhodospirillum, Thiomicrospira, Providentia, Klebsiella, Schineria, Escherichia, Enterobacter, Empedobacter, and Flavobacterium were found to be predominant in anaerobic LBRs. Genera that are capable of degrading various substrates, such as Pseudomonas, are present in large numbers in LBRs (1,2,15,28). Flavobacterium and Rhodospirillum degrade mostly proteins and are expected to be present in proteinaceous wastes (28). Clostridium and Bacillus can degrade amino acids, fatty acids and sugars (1,2,28,41,42). Clostridium species are strictly anaerobic and typical H2-producing bacteria. Reportedly, anaerobic cellulolytic Clostridium genus and aerobic bacteria are often detected simultaneously at various sites where cellulose degradation occurs (43). Sulfate-reducing bacteria such as Desulfobacter, Desulfococcus, and Desulfonema belonging to d-Proteobacteria are the ﬁnal decomposers of organic matter in solid waste, as are methanogens. Sulfate-reducing bacteria can grow syntrophically with either H2-producing or acetateproducing bacteria and can compete with methanogens for electron donors such as acetate and H2/CO2 (44). In aerobic LBRs related to highly effective nitrogen removal of organic solid waste and leachate, the number of amoA genes well reﬂects the effects of aeration in the aerobic LBR (27). Ammoniaoxidizing bacteria are also expected to contribute to nitrous oxide production (45). Speciﬁcally, nitrifying bacteria, speciﬁcally

TABLE 4. Populations of microorganisms in LBRs. LBRs (monitoring period) Semi-aerobic for municipal solid wastes (280 days)

Semi-aerobic for industrial solid wastes (280 days)

Anaerobic and aerobic for synthetic organic waste (140 days)

Aerobic for synthetic organic waste (90 days)

Indicator genes

Populations (MPN-DNA/g-dry)

Ref.

With leachate recirculation 105e107 102e104 <8 101 <8 101 105e106 <2 102 <103 <103

1

16S rRNA gene amoA nirK nirS 16S rRNA gene amoA nirK nirS

Without leachate recirculation 105e107 <8 101 <8 101 <2 102 106e107 <2 102 <8 101 <8 101 Anaerobic 105e109 104e108 103e104 103e105 104e105

Aerobic 106e109 105e109 104e105 103e105 104e105

27

16S rRNA gene 18S rRNA gene amoA nirK nirS

With intermittent aeration 109e1010 105e107 104e106

With continuous aeration 109e1010 105e107 104e107

28

16S rRNA gene 18S rRNA gene mcrA

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Nitrosospira and Nitrobacter species, were found in aerobic LBRs (46). In aerobic LBRs, a-Proteobacteria (Rhodopseudomonas, Rhodospirillum), g-Proteobacteria (Pseudomonas), and d-Proteobacteria (Desulforhopalus) were found (Table 3). Firmicutes and Bacteroidetes were also common in these LBRs. Bacteria of the genus Azospirillum, which degrade various organic and nitrogen, were detected. Uncultured species Bacteroidetes bacterium and Thermoactinomyces dichotomicus can degrade amino acids, fatty acids, and sugars. Desulforhopalus, Chlorobiaceae bacterium, and Thermoactinomyces dichotomicus were predominant in aerobic LBRs (Table 3). Sulfuroxidizing bacteria (Thermoactinomyces) and sulfur-reducing bacteria (Desulforhopalus) were found in an aerobic LBR (28). The results show that aerobes and anaerobes can occur simultaneously because of the heterogeneity of the LBR. Perhaps the most extensively studied functional group of microorganisms in aerobic LBRs is methanotrophs belonging to a-Proteobacteria and g-Proteobacteria (47). Methanotrophs have attracted a great deal of attention because they consume 10e90% of methane, a greenhouse gas, before it is emitted to the atmosphere (48,49). It is noteworthy that methanotrophs help reduce not only methane but also another stronger greenhouse gas, nitrous oxide (50,51). Few studies have speciﬁcally examined microorganisms in hybrid LBRs. Bacterial communities similar to those in the aerobic LBR have been found in the hybrid LBR (Table 3). Gene analyses indicated the presence of Arthrobacter, Bacteroidetes, Clostridium cellulovorans, C. estertheticum, Cycloclasticus, Desulfovibrio giganteus, and Thermoactinomyces dichotomicus (28). Because of the lack of a database, many bacteria in the LBRs remain unidentiﬁed (1,28). Those unidentiﬁed bacteria can achieve overall superior stabilization of LBRs and should be studied further. Archaea Methanogens harboring the mcrA genes can be monitored in LBRs (Tables 3 and 4). Three methanogenesis pathways exist: acetoclastic (acetate to CH4), hydrogenotrophic (e.g., reduction of carbon dioxide to CH4 with H2), and methylotrophic (e.g., methanol, methylamines, and dimethyl sulfoxide to CH4) (52). Methanobacteriales (hydrogenotrophic), Methanomicrobiales (hydrogenotrophic), and Methanosarcinales (hydrogenotrophic, acetoclastic, and methylotrophic) are typically found in LBRs (28,40,53e56). Typically Methanoculleus bourgensis, Methanofollis formosanus, and Methanosphaera stadtmanae in anaerobic LBRs, Methanoculleus palmolei, Symbiont of Trimyema compress and Methanosarcina in hybrid LBRs and Methanosphaera, M. palmolei, and Methanosarcina in anaerobic LBRs were found. Fungi Several studies have revealed the presence of fungi in LBRs (27,28,57). Fungi can degrade cellulose and persistent organic pollutants (58), tolerate low nutrient levels, and grow in the presence of low moisture and low pH conditions. Introduction of oxygen can stimulate the metabolism of fungi, the aerobic microorganisms in hybrid and aeration LBRs (27). Development of the fungal population that can decompose diverse high-molecular organic (58) is inferred as important for initiation of accelerated stabilization of landﬁlls. However, the molecular evidence of members of the obligate anaerobic fungal order Neocallimastigales within landﬁlls has also been found (57). The anaerobic gut fungi have attracted more widespread interest as the most active cellulose degraders. CONCLUSIONS AND FUTURE CONSIDERATIONS Although much is known about fundamental microbial metabolism, little is known about the community structures and functions of microorganisms that exist in actual LBRs. Several recent studies have analyzed the bacterial communities in LBRs, although studies of the eukaryotes are scarce. Through those studies, the population dynamics in LBRs have been partially clariﬁed. However,

J. BIOSCI. BIOENG., the obtained data are still limited and cannot directly engender the improvement of LBR performance. An important reason for the limitation of the data is the difﬁculty in the monitoring of microorganisms in LBRs. Because of highly heterogeneous features of solid wastes inside LBRs, analyses of limited quantities of grab or composite samples cannot always reﬂect the whole shape of the microbial community. Microorganisms exist in and on the surface of waste aggregates. Therefore, the ability to separate these cells from the components is vital for studying biodiversity. Consequently, many samples must be obtained from horizontally and vertically different positions in LBRs by boring to fully grasp the microbial community: very timeconsuming and laborious monitoring is necessary (2,17,59,60). Furthermore, periodical sampling is necessary to understand the microbial population dynamics especially in hybrid LBRs which have periodic changes in the operational/environmental conditions, which is more time-consuming and labor-consuming. Therefore, a simple and systematic sampling scheme or strategy must be established for routine and intensive microbial monitoring. Instead of solid waste samples, analyses of leachate samples are apparently a much easier and simpler means for microbial monitoring (42,54,59,61,62). However, whether the microbial community in the leachate can properly reﬂect that in the LBRs status or not has remained unknown to date. Most studies of microbial monitoring of LBRs have analyzed microbial community structure from the taxonomical viewpoint, based on the 16S rRNA genes. The copy number and the diversity of 16S rRNA genes have proposed as a new index of landﬁll stabilization (2,63). However, fewer studies have focused on the functional microbial populations, although monitoring of their behavior is very important for improving the performance of LBRs. Moreover, most such studies have dealt with bacterial populations responsible for nitrogen cycles (nitriﬁcation/denitriﬁcation) and methane generation. The behaviors of the other functional populations have been rarely clariﬁed. Dissimilatory sulﬁte reductase genes (dsr) and adenosine-50 -phosposulate reductase genes (aps) (64) are important maker genes in the sulfur cycle. Particulate and soluble methane monooxygenase genes (pmo and smo) are important maker genes for controlling the greenhouse gas emission (65). Microbial monitoring using functional gene microarrays (66) targeting various genes related on carbon, nitrogen, sulfur, and metal cycles might be a powerful tool for improving LBR performances in the near future.

ACKNOWLEDGMENTS Nguyen Nhu Sang expresses his appreciation to the Society for Biotechnology, Japan, which awarded him the Young Asian Biotechnologist Prize in 2011. This study was partly supported by Sound Material Cycle Society Promotion Research Grant (K113027), Ministry of Environment, Japan.

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