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Unformatted text preview: JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 99, No. 1, 1–11. 2005 DOI: 10.1263/jbb.99.001 © 2005, The Society for Biotechnology, Japan Microbial Diversity in Biodegradation and Reutilization Processes of Garbage SHIN HARUTA,1 TORU NAKAYAMA,2 KOHEI NAKAMURA,3§ HISASHI HEMMI,2 MASAHARU ISHII,3* YASUO IGARASHI,3 AND TOKUZO NISHINO2 Department of Ecosystem Studies, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-8 Yayoi-cho, Inage-ku, Chiba 263-0022, Japan,1 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai, Miyagi 980-8579, Japan,2 and Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan3 Received 12 July 2004/Accepted 21 September 2004 With particular focus on the microbial diversity in garbage treatment, the current status of garbage treatment in Japan and microbial ecological studies on various bioprocesses for garbage treatment are described in detail. The future direction of research in this field is also discussed. [Key words: garbage treatment, compost, microflora, PCR-denaturing gradient gel electrophoresis (DGGE), fluorescence in situ hybridization (FISH), molecular microbial ecology, unculturable, culture-to-difficult] Currently, a large amount of garbage is produced through various activities. Although microbiological treatment methods are considered to be best, we know very little regarding the microbiology involved in such treatment methods. However, much progress has been made over the past few decades regarding the determination of what types of microorganisms exist in a certain system using molecular microbial ecological approaches. In this context, it is appropriate for us to describe the current status of garbage treatment in Japan, and to summarize current and future research in this area with particular focus on microbial activities. Because garbage treatment differs between countries, a description on the current status is confined only to Japan. waste (see Table 1 for definition) and includes residues generated during industrial food-manufacturing processes. However, it accounts for 34% of total general waste (Table 1) and includes residues generated during cooking in restaurants, feeding facilities, and homes, as well as food beyond its expiry date and food refuse. Garbage is a biodegradable, recyclable material and generally contains a significant amount of water (70–90% [w/w]). Degradation of garbage is achieved either physicochemically or microbially, depending on the nature and scale of the garbage. Statistics shows that 46% of garbage in industrial waste is recycled to compost, animal feeds, etc., whereas only 0.7% of garbage in general waste from homes is recycled (Table 1). As a net result, 12% of total garbage is recycled and the rest (88%) is treated by means of incineration, landfill, or other methods. In this section, we briefly survey the current methods that are employed for the treatment of garbage in Japan to clarify characteristics that distinguish the microbial methods from others. Incineration Incineration is the most widespread method for garbage treatment in Japan; 76% (by wet weight) of total garbage is incinerated (2). Emissions from the incineration of garbage along with other municipal wastes contain air pollutants such as SOx, NOx, CO, CO2, hydrogen chloride, and chlorinated hydrocarbons including dioxins, which are endocrine disruptors (3). Thus, the incineration of garbage may cause air pollution, unless the incinerators are equipped with appropriate pollutant control devices. The incineration of garbage can produce energy in the form of steam or electricity if it is combined with an appropriate energy-recovery system. For this specific purpose, I. CURRENT STATUS OF GARBAGE TREATMENT IN JAPAN An enormous amount of waste of plant, animal, or microbial origin is generated during the course of the production, distribution, and consumption of foods. The annual output of such waste, which we here refer to as garbage, in Japan in 2000 was estimated to be 2.20 ´ 107 t (by wet weight, Table 1) (1). Garbage accounts for 1% (by weight) of total industrial * Corresponding author. e-mail: [email protected] phone: +81-(0)3-5841-5143 fax: +81-(0)3-5841-5272 § Present address: Institute of Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Center 6-10, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan. 1 2 J. BIOSCI. BIOENG., HARUTA ET AL. TABLE 1. Annual output of garbage and its treatment in Japan (2000)a Annual output (in 106 t) Incineration or landfill Treatment (in 106 t) Recycling Animal feeds Others – – 0.17 0.10 – – 0.88 0.07 – – Compost Total General wasteb 17.93 17.13 – 0.80 (4.81) 0.44 (0.71) (from industries) (5.52)c (from homes) (12.41) (12.32) – (0.09) 4.05 2.19 0.91 1.86 Industrial wasteb Total 21.98 19.32 – 2.66 a This table was adapted from that which has been reported in previous literature (1). b Industrial waste refers to 20 types of waste that are stipulated by a domestic law and includes sludge, waste plastics, and other waste generated through industrial activities. General waste refers to those which are produced from homes and industries (other than industrial waste). In 2002, annual outputs of total industrial waste and total general waste in Japan were estimated to be 406.0 ´ 106 t and 52.36 ´ 106 t, respectively. c Figures in parentheses show the itemized amounts of garbage in general waste. garbage is stored as a fuel, which is called refuse-derived fuel (RDF), after dehydration by heating, pulverization, and manipulation into an appropriate shape (4). However, it has been pointed out that the manufacturing of RDF from garbage consumes a considerable amount of energy, and garbage can only serve as a low-quality fuel with very low energy efficiency (4). These considerations, together with the recent explosion during the storage of RDF in Mie, Japan, which claimed two lives, pose a question on the validity of the use of garbage as an RDF (4). Landfill Landfill still remains the second method of choice among the methods of garbage treatment in Japan; 12% (by wet weight) of total garbage is buried in soil (2). However, the degradation of garbage in landfill occurs over a prolonged period of time and may also cause the pollution of groundwater and air (5, 6). The use of landfills is becoming less popular due to the lack of land. Other physicochemical methods Other physicochemical approaches to garbage treatment include charcoalization and dehydration, both of which are, at present, very minor methods for the garbage treatment (1, 2). In the charcoalization method, garbage is charred at high temperatures (for example, 250~1000°C) under oxygen-free or oxygendeficient conditions and the resultant product can be used as fuel, adsorbent, and soil conditioner. However, this method is not cost-effective, and the formation of a by-product, tar, during the process remains problematic. In the dehydration method, garbage is dehydrated at lower temperatures (less than 100°C), and the product is used as fertilizer. The main drawback of this method is that the product can easily be rehydrated due to humidity and rainfall, which may cause putrefaction of the material during storage. Moreover, the dehydrated material is not better as a fertilizer than the compost that is produced through microbial processes. The use of a garbage disposer in the sink of home kitchens might also be another solution for garbage treatment. In this case, shredded garbage has eventually to be biodegraded in wastewater disposal plants. At present, the use of such disposers is not widespread in Japan, and were it to occur, this method will cause an increase in sewage sludge, which has to be appropriately treated. Microbial methods for recycling of garbage These methods, which are the topic of this review, take full advantage of microbial activities to degrade organic materials under specific (or controlled) conditions, producing fertilizer (compost), animal feeds, food products (for example, fish sauces), or lactic acid. Microbial methods also include anaerobic digestion of garbage, producing biogas (methane or hydrogen) that can be used as fuels. Thus far, only a small portion of garbage has been treated by means of these microbial methods (Table 1) (1, 2). However, microbial methods share several desirable features that are in striking contrast to those of physicochemical methods as follows, and are expected to play more important roles in the future recycling of garbage. First, microbial methods are versatile; namely, different types of products may be obtained depending on the method employed and the nature of garbage to be processed. This allows site- or community-specific approaches to the recycling of garbage. Second, microbial methods can be performed with low energy consumption compared with other methods. Third, unlike in the case of incineration and landfill, only negligible amounts of environmental pollutants are produced through microbial methods. Finally, products generated through microbial methods can be treated in accordance with the global cycling of materials, as typified by composting and animal feed production. Thus, recycling of garbage by means of microbial methods is expected to have far less impact upon the natural environment compared with the other methods. Although microbial methods share the promising features outlined above, each has specific problems, some of which will be mentioned below. It is important to note here that the characteristics and problems of the respective methods are closely related to the microbiological characteristics of the process. The types of microorganisms that are involved in the microbial degradation process depend largely upon the methods employed, the nature of the garbage, and other environmental factors. Analyses of microflora and its transition during the course of such processes are of great interest from the viewpoint of microbial ecology. Moreover, the analyses should provide important clues that help to overcome the problems with microbial methods for recycling garbage. Unfortunately, only incomplete information is available regarding such analyses performed using classical culture-based methods, because a great majority (99%<) of microbial inhabitants are only viable in the ecological niche of the processes and appear to be unculturable or cultureto-difficult under laboratory conditions (7). However, recent progress in molecular biological approaches to analyze microflora in natural environments has provided a break- VOL. 99, 2005 through to enhance our understanding of the microbiological aspects of the microbial degradation of garbage. II. MICROBIAL ECOLOGICAL STUDIES ON VARIOUS BIOPROCESS FOR GARBAGE TREATMENT Molecular microbial ecological approach to garbage treatment Over the past decade, microbial ecology has been vigorously studied at the molecular biology level. Molecular biological analyses highlight the microbial diversity in nature, which is difficult to clarify by conventional cultivation methods. Various methods have been developed and applied to study the succession, composition, and function of microbial communities (8, 9). In order to characterize a microbial community or determine its composition, the following methods on nucleic acid analysis are widely used. That is, clone library analysis, amplified ribosomal DNA restriction analysis (ARDRA), terminal-restriction fragment length polymorphism (T-RFLP) analysis, random amplified polymorphic DNA (RAPD) analysis, amplified fragment length polymorphism (AFLP) analysis, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and single-strand conformation polymorphism (SSCP) analysis. Moreover, DNA microarray techniques enable high-throughput analysis (10), and dot blot hybridization and quantitative PCR will help to evaluate microbial population. In addition to these methods, other chemical biomarker analyses, that is, phospholipid fatty acid (PLFA), and quinone profiles (11) are also utilized for characterization of a microbial community. On the other hand, microscopic detection of specific microbial cells is achieved by in situ labeling of microbial cells, such as fluorescence in situ hybridization (FISH). Several modifications have been developed to increase the signal intensity, such as HNPPFISH (12), CARD-FISH (13), PNA-FISH (14), RING-FISH (15), in situ PCR (16), and in situ LAMP (17). Further studies should combine the identification of microorganisms with additional information on their functional state. Characterization of microenvironments (measurement of oxygen, hydrogen sulfide, nitrate, nitrite or pH) using microsensors will provide critical information on the microbial activity in habitats. Stable isotope probing (SIP) (18) or microautoradiography-FISH (MAR-FISH) (19) will reveal metabolically active microorganisms in a community. Functional or physiological characterization of a microbial community can be achieved by community-level physiological polymorphism (CLPP) analysis. These methods can be applied to various environments. However, in the course of experimentation, it should always be considered that the results obtained using these methods may include biases caused by the extraction efficiency of nucleic acids, PCR error, or other unknown factors. In particular, the application of such analytical methods to biodegradation process is sometimes limited by the following properties of garbage or compost: (i) huge amounts of organic compounds, (ii) presence of humic acids, (iii) solid fermentation. These factors will make it difficult to purify nucleic acids or to utilize microscopes. However, protocols are being improved to overcome the limitations of individ- MICROBIAL DIVERSITY IN GARBAGE TREATMENT 3 ual garbage samples. To summarize, a polyphasic approach, including traditional cultivation, is desirable to understand the technical biases and limitation of each method, and the results obtained should be interpreted with extreme caution. Composting Composting is the biological conversion of garbage into stable material such as fertilizer or soil amendment material under self-heating and aerobic conditions. The quality of the end product is defined by several criteria. Composting ranges from the treatment of bulk wastes to the treatment of kitchen refuse. In this section, we will briefly summarize the microbial diversity in batchwise composting with field-scale systems and then will focus on individual composting systems operated under fed-batch conditions. The batchwise composting of bulk waste is carried out not only with nonreactor systems, such as windrow or static pile, but also with several types of reactor-based systems, for example, vertical flow systems with an agitated solids bed, inclined flow systems with rotary drums or agitated bins (20). On the basis of the temperature, the composting process under batchwise operation can be divided into four major microbiologically important phases, that is, mesophilic, thermophilic, cooling and maturation. Optimum conditions have been defined empirically regarding aeration, pH, temperature, moisture, and carbon to nitrogen ratio (20). However, composting sometimes unexpectedly leads to undesirable results, such as odor production and incomplete stabilization. Since the 1900s, numerous microbiological studies have been reported on field-scale composting processes as previously reviewed by Finstein and Morris (21). For detailed investigation, in-vessel reactors (or monitored bins) were also developed and analyzed (22–29). Recently, Ryckeboer et al. summarized the huge number of culture-dependent microbial studies and compiled a list of microorganisms isolated using the source material of the composting and the temperature phase during which they were isolated (30). In the mesophilic (and slightly acidic) phase, the bacteria isolated belonged to diverse families (Fig. 1), Alcaligenaceae, Alteromonadaceae, Bacillaceae, Burkholderiaceae, Bradyrhizobiaceae, Caryophanaceae, Caulobacteraceae, Cellulomonadaceae, Clostridiaceae, Comamonadaceae, Corynebacteriaceae, Enterobacteriaceae, Flavobacteriaceae, Flexibacteraceae, Hyphomicrobiaceae, Intrasporangiaceae, Methylobacteriaceae, Microbacteriaceae, Micrococcaceae, Moraxellaceae, Neisseriaceae, Nitrosomonadaceae, Nocardiaceae, Nocardiopsaceae, Paenibacillaceae, Phyllobacteriaceae, Propionibacteriaceae, Pseudomonadaceae, Pseudonocardiaceae, Rhodobacteraceae, Sphingobacteriaceae, Staphylococcaceae, and Xanthomonadaceae, and diverse fungi have also been isolated. On the other hand, in the thermophilic (and alkaline) phase, thermophilic bacteria were found belonging to genera Micromonosporaceae, Streptomycetaceae, Thermoactinomycetaceae, Thermomonosporaceae and Streptosporangiaceae. Hydrogenobacter spp. and Thermus spp. were also found in processes performed at temperatures above 70°C (31, 32). In addition to these bacteria, a methanogenic archaeon, Methanothermobacter thermautotrophicus was isolated from the thermophilic phase (33). After the thermophilic phase, the taxo- 4 HARUTA ET AL. J. BIOSCI. BIOENG., FIG. 1. The neighbor-joining tree of 16S rDNA sequences of Bacteria and Archaea in composting processes. The Bacteria and Archaea isolated from thermophilic/mesophilic phases (30) are represented with the sequences detected by PCR-based methods or their relative species in nucleotide databases (26, 48–52, 54–56). Underlined microorganisms have not yet been isolated from composting processes. nomic diversity increases, and isolated bacteria included a variety of gram-positive and gram-negative bacteria and fungi. Several microorganisms isolated from the cooling/ maturation phase were involved in ammonium oxidation, nitrite oxidation, nitrogen fixation, or natural polymer degradation, indicating the metabolic diversity (30). These culture-dependent approaches clarified the diversity of culturable microorganisms and estimated their metabo- VOL. 99, 2005 lism in the composting process as summarized by Ryckeboer et al. (30). In addition to isolation-based techniques, metabolic fingerprinting methods, such as CLPP analysis, were performed to characterize composting processes (28, 34). Since the 1990s, molecular microbial ecological approaches have been applied to composting processes. Culture-independent methods have the advantage of facilitating the analysis of multiple samples rapidly, leading to easy assessment of microbial succession (27, 28, 35–47). For example, Klamer and Bååth represented the correlation between bacterial succession and temperature transition during the composting of straw materials using the PLFA method (39). The thermophilic phase was characterized by the iso- and anteisobranched PLFAs, which are common in the genus Bacillus. It was also indicated that gram-negative bacteria and fungi increased in the mesophilic phases, and actinomycetes existed throughout the process. Moreover, PCR-based studies demonstrated the existence of microorganisms which had not yet been isolated from composting processes (Fig. 1) (26, 48–57). Unlike in the case of field-scale composting, an on-site fed-batch operation is required for decomposition of daily kitchen garbage, which will reduce the collection of garbage from each family. In Japan, personal composters are coming into wide use to reduce the amount of garbage and/or to produce compost for garden usage. An inexpensive compost bin is traditionally constructed using a wooden box, garbage can, or barrel, and some are commercially available. Several guidelines have been proposed for the construction and operation of compost bins. Nakasaki and Ohtaki have studied fed-batch composting kinetics using a monitored composting system to propose a simple numerical model (58). To our knowledge, however, few microbiological studies have been reported for the fed-batch operation of compost bins. Hiraishi introduced a flowerpot as a simple composting reactor (flowerpot composting system) (59, 60), and determined the microbial population and succession during the fed-batch operation (59–62). The polypropylene flowerpot (approximately 14–30 l capacity) was packed with garden soil. The reactor was successfully loaded with 0.2–0.3 kg of garbage per day. The pH and the core temperature in the reactor were 7–9 and 30–50°C, respectively. In this system, the structure of the microbial community was examined by chemical biomarker analysis, that is, quinone profiling. It was revealed that the predominant microorganisms changed from Proteobacteria to Actinobacteria during the start-up period (62). Members of the Actinobacteria existed stably as major constituents during a one-year operation, although some fluctuations of other diverse microbial populations were observed depending on the seasonal temperature (61). These microbial successions were also indicated by FISH. It is noteworthy that high culturability (>50% of total bacterial cells) was reported for the composting process (62). Moreover, the microbial succession determined by a culture-independent approach was also supported by the change in the population of isolated bacteria (62). Various types of personal electric-powered garbage decomposer (EPGD) are on the market in Japan and some other countries (63, 64). The EPGD is equipped with stirring wings and an air draft to supply air into the system, and MICROBIAL DIVERSITY IN GARBAGE TREATMENT 5 some systems have a heating module and/or a deodorizing device. Sawdust or woodchip is added as a solid starting material before the operation. The small-scale systems for family use are designed to treat approximately 1 kg per day of kitchen garbage produced by each family. For restaurants or hotels, the medium-scale systems are utilized to treat large amounts of garbage (10–40 kg/d). The temperature of the decomposing material is 40–60°C during the process. Hwang et al. reported the degradation pattern of each component in garbage with an experimental composting system (65). Pedro et al. characterized the bacteria isolated from a medium-scale EPGD treating 10–20 kg/d of restaurant refuse for over two years. The inside temperature was maintained at around 50°C to 60°C with a heating module (66). All isolates belonged to the genus Bacillus, and their 16S rDNA sequences were related to those of B. licheniformis, B. subtilis, or B. thermoamylovorans. These are general microorganisms in the mesophilic and thermophilic phases in fieldscale composters (30, 67). The same species were obtained in similar proportions among the samples taken during a 6-month operation indicating the stability of the bacterial community in the composter. Narihiro et al. reported on the microbial diversity in several types of small-scale EPGDs for fed-batch treatment of garbage (0.7 kg/d) (68). All composters used in their study degraded approximately 90% of garbage during a 1-month operation. The pH ranged from 8 to 9, and the core temperature in the reactors ranged from 30°C to 55°C. Members of the Actinobacteria were predominant, as revealed by quinone profile analysis of the composter. Relatively high culturability ranging from 46% to 60% of total bacterial cells was observed in all composters. The quinone profile generated for all colonies on a nutrient agar plate also showed the predominance of the Actinobacteria. These results were quite similar to those from the flowerpot composting system reported by Hiraishi et al. (59–62). Haruta et al. (69) and Nakamura et al. (70) constructed a monitored personal EPGD to measure temperature transition and the amount of exhaust gases. Under appropriate conditions, the temperature increased up to 58°C and the pH was 8–9. Approximately 85% of the garbage was degraded everyday during a 6-week operation. The culturability varied from 0.01% to 30% of total microbial cell counts. The predominant microorganisms belonged to the family Bacillaceae as determined by DGGE analysis (69), quinone profiling, and assaying on nutrient agar plates (unpublished results). Furthermore, a particular strain, Cerasibacillus quisquiliarum BLx (71) was reproducibly detected during the process, and the 16S rDNA sequence related to this strain was also reported in other garbage treatment systems (49, 72). FISH and quantitative PCR revealed the dominance of this strain, and enzymatic analyses indicated that the gelatinases (proteases) produced by this strain contributed to the protein degradation in the process (70). In addition to the operations under alkaline conditions described above, microbiological studies were reported on personal EPGDs operated at neutral or acidic pH (73, 74). Moisture content and/or solid matrices such as sawdust were assumed to be key factors in determining the pH. It 6 HARUTA ET AL. was indicated that (slightly) anaerobic conditions decrease the pH. In contrast to alkaline operation, these processes proceeded at ambient temperature and cannot be categorized as composting processes. (that is, self-heating and aerobic processes). However, a comparative degradation rate was observed in neutral or acidic pH operation. By DGGE analysis, the neutral pH operation (pH 6–7) was microbiologically characterized by the existence of Lactobacillus sp., Corynebacterium sp., Enterococcus sp. and Staphylococcus sp. as well as uncultured or novel bacteria (74). Under acidic conditions (pH 4–6), lactobacilli and yeasts were found as major constituents by DGGE and isolation (73). Inoue et al. evaluated the performance and microbial succession in an EPGD with particular reference to the accumulation of salt during a two-year fed-batch operation (75). The characteristics of isolated bacteria indicated that the prolonged operation was favorable for the growth of moderate halophilic bacteria, as suggested by Haruta et al. (69, 74). The appearance of bacteria pathogenic to humans should be considered during the fed-batch operation with personal reactors in which it is hard to maintain a sufficiently high temperature to inactivate the pathogens. An appropriate microbial community might function as a biological control agent to suppress the growth of unfavorable microorganisms. The acidic operation would be a possible solution to eliminate pathogenic bacteria. Several factors cause differences in the microbial community in each operation, such as aeration, composting materials, or starting materials. No distinct temperature phases are observed in fed-batch operation, and the maintenance of a thermophilic phase is ideal for efficient degradation. However, some microorganisms common to both batchwise and fed-batch operations were found. One of the microorganisms belongs to the Bacilli, and was identified regardless of the detection method. Actinobacteria are also general microorganisms in composting. However, a thermophilic microorganism, such as Thermus sp. or Hydrogenobacter sp. (31, 32) has not been found in personal fed-batch composting systems degrading kitchen garbage. The high temperature (at least above 60°C) necessary for their growth was not achieved in personal composters. A cooling/maturation phase was not defined in fed-batch operation. These phases in batchwise systems are characterized by an increase in metabolic diversity, including the metabolism of inorganic nitrogen-compounds (30). However, Tiquia et al. isolated ammonium oxidizing or nitrite oxidizing bacteria throughout a composting process (76), and molecular analysis revealed the existence of an ammonium-oxidizer even in the thermophilic phase (40). These results imply that bacteria with metabolic activities against inorganic nitrogen-compounds may exist in fed-batch systems. Fungi are also common microorganisms in composting. Both mesophilic and thermophilic fungi were isolated from field-scale composters (reviewed by Ryckeboer et al. [30]). Within the fedbatch composting systems, Narihiro et al. (68) reported that mesophilic fungi could be major constituents within the fungal community. In addition to the microbiological studies described above, the following aspects of composting have been widely stud- J. BIOSCI. BIOENG., ied (77): (i) plant and human pathogens in compost or during composting, (ii) biodegradation of various chemicals, (iii) airborne microorganisms, (iv) effect of protozoa, and (v) microbial community in vermicomposting (78). Moreover, the effect of compost microorganisms on microbial diversity in the soil is also an important topic to evaluate the composting product as a fertilizer or soil amendment material. Although composting is an old and familiar technology, the composting process is one of the most complex biotechnologies because of the myriad physical and biological states during the process. Garbage degradation could be partly explained by the succession and function of predominant strains detected by culture-independent methods. However, a global understanding of the composting process would require the analyses of the whole microbial community including those microorganisms present in low numbers and barely detectable (<0.1% of total microorganisms). In this century, the microbiology of composting should be studied from various aspects, for example, composition, succession, microhabitat, and function of microorganisms within the community, because composting is one of the most important techniques for the reutilization of abundant organic waste. Methane fermentation Methane fermentation from organic waste is carried out in order to harness the energy within the garbage through biodegradation. In this section, we will briefly describe systems, which have been constructed in Japan for the efficient production of methane, for the treatment of organic waste. Later on, we will introduce several reports concerning the microbial diversity in such systems. When we observe the systems in terms of fermentation, they can be divided into two categories. The first category (i and ii, below) comprises acid fermentation and methane fermentation systems. The second category (iii–vi, below) comprises only methane fermentation systems although, in one case (iii, below), methane fermentation itself comprises two steps. (i) Mebius system (79): Both acid fermentation and methane fermentation are performed at 55°C. (ii) IMC bio-gas collecting system (80): Solubilization is performed under acidic and aerobic conditions, then the methane fermentation is performed at 37°C. (iii) Renaissa system (81): Methane fermentation is performed through two steps. The first fermentation is performed at 37°C, and the second fermentation is performed at 55°C. (iv) REM system (82): The system comprises a specialized fermentor, where mixing is carried out by utilizing the positional energy of the produced gases. The methane fermentation itself is performed at 37°C. (v) Kurita Dranco Process (http://www.kwi.jp/product/ index.html): The system comprises a fermentation system that is driven under low water activity. (vi) Thermophilic methane fermentation system (http://www.kajima.co.jp/tech/ katri/leaf/conte/leaf-e-208.html): The thermophilic methane fermentation system is characterized by containing, within a reactor, a support made of carbon fiber to sustain a microbial community for methane fermentation. The fermentation is carried out at 55°C. Various experiments to obtain physicochemical characteristics have been carried out for the systems mentioned above. VOL. 99, 2005 However, no experiments have been reported in terms of microorganisms present within the systems. However, there are several reports where microbial diversity was examined using a laboratory-scale fermentor. Tang et al. reported that phylogenetic diversity within a thermophilic municipal solid-waste digester showed a similar pattern with or without micro-aeration and that microaeration together with the low H2S concentration did not repress the activity of sulfate-reducing bacteria through analyses of FISH, DGGE, and quantitative real-time PCR (83). In the Bacteria, microorganisms affiliated with the phylum Firmicutes were dominant. Also, in the Archaea, a decrease in the population of Methanosarcina and an increase in the population of Methanoculleus was observed as a result of micro-aeration. Griffin et al. (84) and McMahon et al. (85) reported the microbial population dynamics during digestion by a municipal solid-waste digester. However, in terms of microbial diversity, experiments were performed only regarding the overall microbial population dynamics. Because methane fermentation naturally involves the functioning of various microorganisms, experiments toward the elucidation of the microorganisms present are very important in terms of the stability and functionality of methane fermentation. Therefore, changes in microbial communities within such systems in relation to time and space should be clarified in future studies. Also, the function of each type of microorganism should be focused upon. Hydrogen fermentation Due to its cleanness and high-energy yield (122 kJ g–1), hydrogen is a promising future source of energy. This gas can be produced from organic compounds by chemotrophic anaerobes (for example, Clostridium or Enterobacter) under anaerobic conditions or by photosynthetic bacteria (for example, Rhodobacter) under anaerobic light conditions. The microbial production of hydrogen is called hydrogen fermentation, and is expected to play an important role in bioenergy production (86). Thus far, carbohydrates such as glucose, sucrose, starch, cellulose (87, 88), and xylan have mainly been tested as substrate(s) for hydrogen fermentation, by which the continuous production of hydrogen has been attained. There have also been reports on the batch-wise production of hydrogen from organic wastes. The target wastes include agro-industrial residues (89–93), waste molasses (94), food refuse (95), beancurd refuse, rice bran, and wheat bran (96). Using either bacterial isolates (89–91, 93, 94) or enriched natural microflora (91, 92, 95, 96), the production of H2 up to 7.2 mol of H2/mol-hexose (90) was reported. These studies show that the efficiency of hydrogen production from organic wastes may be affected by a variety of factors. Probable factors include temperature (95), pH (92), solid waste concentration (96), food-to-microorganism ratio (92), contents of carbohydrates and nitrogen sources in the substrate waste (89, 91), CO2 concentration (94), and others (89, 90). It is generally observed that the production of hydrogen diminishes and finally stops through repeated additions of organic waste to the fermentation cultures. Hence, in practice, continuous production of hydrogen from organic waste has not successfully been achieved to date. Although the structures of microbial communities in hydrogen fermentation culture MICROBIAL DIVERSITY IN GARBAGE TREATMENT 7 on organic waste have not been analyzed, those of hydrogen fermentation by microflora obtained from sludge compost on cellulose powder have been analyzed in detail by means of culture-based as well as molecular biological approaches (87, 88). These studies provide important information on the microbial ecosystem during the course of hydrogen fermentation on organic waste. These studies also revealed the co-existence of multiple bacterial species in the fermentation culture, most of which belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G + C gram-positive bacteria. Some of these species were identified on the basis of their 16S rDNA sequences to be Thermoanaerobacterium thermosaccharolyticum, Clostridium thermocellum, and C. cellulosi, among which T. thermosaccharolyticum appeared to be mainly responsible for hydrogen production. The T. thermosaccharolyticum strain isolated from the sludge compost was found to be unable to decompose cellulose and was suggested to grow symbiotically with other cellulose-degrading bacteria including clostridia (88). Lactic acid fermentation Lactic acid fermentation is carried out mainly for the purpose of making lactic acid. However, the process of the lactic acid fermentation itself has the advantage of causing a low degree of odor production and suppressing the growth of putrefactive and food poisoning bacteria. The product, lactic acid, can be converted into poly-L-lactate through technological methodologies. Sakai et al. reported that selective accumulation of lactic acid occurs by intermittent pH adjustment during the treatment of kitchen refuse (97). If the pH adjustment during the treatment is performed continuously, selectivity of lactic acid production decreases, which is reinforced by the fact that the numbers of Coliform bacteria and Clostridia increase around 10-fold compared to those present during the intermittent pH adjustment. They also isolated probable lactic acid bacteria responsible for the lactic acid production from the treatment system. Similarly, Wang et al. reported the importance of the pH adjustment of kitchen waste for lactic acid production (98). Although their work is confined to the preservation period of the waste, the finding naturally correlates to the report by Sakai et al. (97). For the industrial production of poly-L-lactate, the quality of the initial fermentation products matters. In this context, Sakai et al. reported a system for the treatment of food waste, where propionic acid fermentation is carried out first to get rid of the existing D-lactate followed by the lactic acid fermentation (99). Because lactic acid bacteria carry out almost all the steps for the production of lactic acid, studies on the microbial community in lactic acid fermentation may not be given high priority. However, organic waste contains macromolecules that are not degraded by lactic acid bacteria. Therefore, the importance of the overall understanding of the community still holds for the lactic acid fermentation. Acidulocomposting Acidulocomposting is a garbagetreatment process utilizing a small bioreactor similar to other EPGDs, which is equipped with a heating and an agitating apparatus that keeps materials at elevated temperatures 8 J. BIOSCI. BIOENG., HARUTA ET AL. FIG. 2. Profiles of PCR-DGGE analyses of the microbial community structures in processed garbage sampled weekly from an acidulocomposting process. An arrow indicates the direction of electrophoresis. The gel contains linear gradients of denaturants (7 M urea and 40% [v/v] formamide as 100%) and the acrylamide concentration shown on the side. Arrowheads, asterisks, and double asterisks indicate the DNA bands that were confirmed to be derived from yeasts, lactic acid bacteria, and Bacillus sp., respectively. (40°C to 65°C). This process, recently reported by Nishino et al. (100) and named based on the availability of its product as compost, can be distinguished from others by the following practical characteristics; it can be operated continuously for a very long period (over two years) with the minimal generation of foul odor, and the pH of the material is spontaneously maintained at 4–6 throughout the process. In contrast, other conventional composting processes using EPDGs are generally operated under neutral to alkaline conditions. The composting activity of such a conventional process gradually diminishes with a decrease in pH of the processed garbage, and, finally, typically 2 to 6 months after the start of the process, the composting activity diminishes and a putrid odor is emitted (101). The PCR-DGGE analyses revealed that the major microorganisms identified in the acidulocomposting were lactic acid bacteria (Fig. 2), such as Lactobacillus and Pediococcus. These lactic acid bacteria continued to be the major species throughout the process and were generally identified irrespective of the types of garbage to be treated. These observations clearly point to the importance of lactic acid bacteria in the process. The dominance of lactic acid bacteria in the acidulocomposting was further confirmed by FISH analyses using probes specific for 16S rRNAs of all eubacteria and lactobacilli-enterococci (102). The results showed that about half of the bacterial population in the compost consisted of lactobacilli-enterococci, which were considered to be metabolically active even in the thermoacidophilic conditions of the process. The number of lacticacid bacterial cells in the acidulocompost was estimated by quantitative PCR analyses to be 8 ´ 105~2 ´ 107 per 1 g of the dried product (102), and this value was apparently lower than those observed in conventional garbage-composting processes (in general, over 1010 cells per 1 g of dried product) (26, 49). The quantitative PCR analyses also revealed the variation in the number of lactic-acid bacterial cells in response to the addition of garbage during the process, where lactic acid bacteria proliferated upon the addition of garbage and subsequently diminished under the conditions of aciduloocomposting. The observed properties of the acidulocomposting should be closely related to its unique microbial community. The operating conditions of the process may allow the growth of a specific class of lactic acid bacteria. In addition, low pH values, which presumably result from organic acids produced by the inhabitant microorganisms, as well as elevated temperatures of the process, likely prevent the growth of other microorganisms including putrefactive bacteria. Moreover, the inhabitant lactic-acid bacteria should produce bacteriocins, bactericidal proteins (103), which could also play an important role in the maintenance of the specific microbial community. Thus, in the acidulocomposting process, the added garbage is degraded by a relatively low number of microorganisms, and the resultant product is pathogen-free and far less putrefactive than garbage. Therefore, acidulocomposting could be regarded as a biological stabilization process of garbage and should be of practical advantage for the recycling of garbage as fertilizer or feed. Final remarks It should be a basic target in microbiology to describe exactly what types of microorganisms exist in a certain system. Through polyphasic approaches, we are now achieving this aim in terms of garbage treatment. The next stage is, of course, to elucidate which microorganism is expressing a certain activity or to clarify which activity is expressed by a certain microorganism. Although such questions have been answered independently, we should develop methods to answer the questions systematically. Although microbial garbage treatment reflects only a small portion of metabolic activities by microorganisms on the earth, we can safely say that all the possible questions to be answered regarding microbiology are included within. Therefore, to summarize, microbial garbage treatment should facilitate the discovery of new frontiers in microbiology. 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