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
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
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
1 2 J. BIOSCI. BIOENG., HARUTA ET AL. TABLE 1. Annual output of garbage and its treatment in Japan (2000)a
(in 106 t) Incineration
or landfill Treatment (in 106 t)
This table was adapted from that which has been reported in previous literature (1).
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.
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 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
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
Molecular microbial ecological approach to garbage
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 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
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
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)
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
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
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. ). 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
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
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
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.
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
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
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 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
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.
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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