Minireview Microbiology of Fed-Batch Composting

Microbes Environ. Vol. 20, No. 1, 1–13, 2005 http://wwwsoc.nii.ac.jp/jsme2/ Minireview

Microbiology of Fed-batch Composting

TAKASHI NARIHIRO1 and AKIRA HIRAISHI1*

1 Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi 441–8580, Japan

(Received December 6, 2004—Accepted January 11, 2005)

Repeated fed-batch composting (FBC) processes, which are modifications of traditional composting technology, have in recent years attracted attention not only in their biotechnological aspects but also from ecological viewpoints. FBC processes differ from the conventional batch system in that the biodegradation of solid organic waste proceeds without definitive thermal stages including the thermophilic phase under self-heating conditions. Mesophilic FBC processes for treating household biowaste are also characterized by low moisture contents, alkaline pH and the presence of high population densities of microorganisms under steady state conditions. Kinetic and microbiological studies of the FBC process have increasingly been conducted using commercially available composters as well as laboratory- and bench-scale reactors. Information from recent studies on FBC processes has provided new insight into our understanding of compost microbiology. This article reviews current knowledge of the FBC process with a special emphasis on microbial diversity, succession and activity in garbage composters. The potential application of FBC technology for bioremediation purpose is also discussed.

Key words: fed-batch composting, compost, microbial community, solid-phase bioreactor

ature is increased by self-heating as a result of vigorous mi- Introduction crobial activity. In the thermophilic phase, the temperature Composting provides a good model of microbial commu- reaches 80LC, which not only stimulates the proliferation of nities to study ecological issues such as diversity, succes- thermophilic microorganisms but also prevents the growth sion and competition during the biodegradation and biocon- or survival of mesophilic microorganisms, including meso- version of organic matter with thermal gradients. The philic pathogens. After the thermophilic progression of typical batch composting process proceeds via four major waste decomposition, the microbial activity lowers due to thermal stages, i.e., the mesophilic, thermophilic, cooling the limited availability of degradable organic substances. and maturation phases, each of which has a particular mi- This cooling phase leads to a decline of temperature and al- crobial community structure developing in response to tem- lows mesophilic microorganisms to predominate again. perature and other environmental conditions. In the first Eventually, solid organic waste turns into stable end com- stage, organic substances are decomposed by mesophilic pounds, including humic-like substances. In addition to the microorganisms at moderate temperature. Then, the temper- traditional batch process, sequencing-batch or semi-continuous composting systems are being widely used for the * Corresponding author; E-mail: [email protected], Tel: 81– treatment of various organic wastes. In sequencing-batch 532–44–6913, Fax: 81–532–44–6929 composting systems with a long type of field-scale reactor, Abbreviations: FBC, fed-batch composting; FUSBIC, flowerpot-using solid biowaste composting; PCDD/Fs, polychlorinated diben- a thermal gradient occurs during the continuous flow of 25) zo-p-dioxins/dibenzofurans; PLFA, phospholipid fatty acids; composted material from the way in to the way out . SCM, solid waste-compost mixture. Details of physicochemical and microbiological features 2 NARIHIRO and HIRAISHI of the conventional batch composting process have been reviewed8,19,20,23). Composting technology has in recent years been modi- fied for fed-batch treatment of solid organic waste using relatively small-scale reactors. In the repeated fed-batch composting (FBC) process, reactors are daily or repeatedly loaded with fresh organic waste and always operated under relatively nutrient-rich conditions, unlike the conventional batch composting system. Therefore, FBC reactors have a much lower and narrower range of temperature, usually less than 50LC, and provide conditions most favorable for growth of mesophilic microorganisms, unless otherwise incubated forcibly. In Japan, various types of electric FBC reactors are commercially available. In particular, personal composters for the treatment of household garbage have come into wide use. A flowerpot-using solid biowaste composting (FUSBIC) system has been studied as a model of 33,35,41,42) Fig. 1. Schematic illustration of a representative of personal garbage the self-heating FBC process . composters available commercially. Common specifications for Since microorganisms fulfill the key role of waste degra- composters: reactor size, 450–760 (H)P400–575 (W)P335–415 dation in the FBC process as well as the conventional com- (D) mm; working volume, 20–43 L; amount of wood chips added posting system, the study of the resident microorganisms is as the solid matrix, 12–20 L; maximum capacity for waste loading, 1.0–1.5 kg (wet wt) day1. Every hour and just after the addi- prerequisite not only for elucidating the microbiological ba- tion of waste, the solid waste-compost mixture (SCM) is stirred sis of the process but also for improving it from a biotech- with an impeller for 1 to 5 min, this mechanical mixing being the nological point of view. Information from recent studies on only way to maintain aerobic conditions. Reactors are equipped FBC processes has provided new insight into our under- with a fan and a heater regulated with a thermistor. This heating system works only when the core temperature declines to 30LC. standing of compost microbiology. This article summarizes current knowledge of the FBC process with a special emphasis on microbial diversity, succession and activity in personal garbage composters. It also deals with the potential application of FBC technology for bioremediation purposes.

General characteristics of FBC process The characteristics and performance of the FBC process have been studied mainly using commercially available composters and laboratory- and bench-scale reactors. A typical commercial composter used for garbage treatment is il- lustrated in Fig. 1. When FBC reactors are operated with household biowaste under self-heating conditions, they ex- hibit common physicochemical characteristics with respect to temperature, pH, moisture content, conductivity and so on. Changes in these parameters commonly found during the start-up operation of FBC reactors are shown in Fig. 2. Fig. 2. Changes in physicochemical characteristics of SCM com- As mentioned above, the biodegradation of organic waste monly found during the start-up operation of personal garbage in traditional batch composting processes is achieved composters at a waste-loading rate of 0.7 kg (wet wt) day1 under through different thermal stages. In general, maximal mi- self-heating conditions. Line a, core temperature; line b, pH; line crobial activity during composting is found at around c, moisture content; line d, conductivity. The figure was made based on information from references 68 and 71. 60LC67,88). On the other hand, waste decomposition in FBC reactors takes place in lower ranges of temperature under Microbiology of Fed-batch Composting 3 self-heating conditions. During start-up, the temperature in FBC reactors rises at the beginning but becomes stable usually between 30 and 45LC at the fully acclimated stage33,35,41,42,68,71). In each batch cycle in the steady state, temperature fluctuates with the degradation of biowaste within the range noted above26,33). In this respect, the FBC process is similar to the mesophilic or cooling phase of the conventional batch composting system. However, the former process is different from the latter in that the resident microorganisms are repeatedly supplied with fresh biowaste while they proceed with the biodegradation. This is the main reason why the FBC process has a narrower and lower Fig. 3. Mass reduction efficiency of personal garbage composters operated at a waste-loading rate of 0.7 kg (wet wt) day1 under range of temperature than the conventional batch system. In self-heating conditions. Left: (A), cumulative amount of biowaste some cases, the temperature in FBC reactors rises over 50LC added; (B), increased mass of SCM. Right: relationship between by self heating26,33). For accelerating the biodegradation, mass reduction and net solid reduction efficiencies. The figure FBC reactors are operated under slightly thermophilic con- was made based on information from references 33, 35 and 68. ditions by on-off control of a heater30,72,75,81). Also, FBC reactors operated forcibly at different temperatures (10–60LC) CO2 and H2O). FBC reactors showing high mass reduction have been reported45). rates can be operated for a long time without the removal of The mesophilic FBC process for garbage treatment is SCM. However, long-term use brings about the accumula- also characterized by low moisture contents and alkaline tion of excess amounts of minerals in the reactor as judged pH. The moisture content of the solid waste-compost mix- from increasing conductivity (see Fig. 2). In the case of ture (SCM) in commercial FBC reactors in the steady state commercial electric composters, it is desirable to remove is kept between 30 and 40% at a waste-loading rate of 0.7 excess SCM from the reactor every 2 months. Since consid- kg (wet wt) day1 68). This content is much lower than values erable amounts of partially degraded materials remain dur- reported for traditional batch composting processes19,20). In ing the FBC process, secondary treatment of the products is the FUSBIC system, the mass reduction efficiency was necessary to obtain matured compost33,35,41). highest at a moisture content of around 40%, and 50% and FBC reactors working under fully acclimated conditions more moisture caused an effluvium problem33). Therefore, a harbor densities of microorganisms in the order of moisture content of 30–40% seems to be most favorable for 1011 cells g1 (dry wt) as measured by epifluorescence FBC reactors to perform well in terms of mass reduction. microscopy26,27,41,63,68,70,71). The culturability of microorgan- Microbial activity in FBC reactors drops sharply at a mois- isms in the reactors is quite high. In mesophilic FBC reac- ture content of less than 30%43). During start-up, SCM has a tors, microorganisms detectable by the plate-counting meth- low pH at the beginning but shows an alkaline pH in the od numbered as many as 1011 g1 (dry wt), accounting for steady state26,41,44,46,47,68,71). However, changes in pH in FBC 24–92% of the total population with the average being reactors may depend upon the chemical composition of 54%71). Plate counts for reactors with different wood waste added. An FBC process that has acidic conditions in matrices reached 1010–1011 CFU g1 (dry wt)44). Similar the steady state has been reported as described below. plate counts were obtained for a conventional compost- In commercial FBC reactors for personal use, high mass- ing process60). However, FBC processes give higher reduction efficiencies of more than 90% can persist under plate counts than traditional composting processes in self-heating conditions when 0.7 kg (wet wt) of household general15,28,65,73,86). The culturability of microorganisms is garbage is supplied per day (Fig. 3). This waste-loading rate much higher in FBC and other composing processes than in corresponds to the average amount of garbage discharged natural environments1). Total viable counts as determined daily from a family in Japan33). The moisture content of the using a LIVE/DEAD BacLight Bacterial viability kit ac- garbage is 76% on average33), while that of SCM under counted for 18–52% of the total counts in a laboratory-scale steady state conditions ranges from 30 to 40% as noted composting system63) and 66–73% in commercial personal above. Therefore, in an FBC reactor for treating household composters for garbage treatment70). These data suggest that garbage, a mass reduction efficiency of 90% corresponds to almost all the living organisms predominating in FBC reac- 75% reduction of the net solid waste (i.e., conversion to tors are culturable. A plausible explanation for this is that 4 NARIHIRO and HIRAISHI repeated loading of organic waste in the FBC process re- counts38), increased via two phases68). The reason why the sults in the exposure of the resident microorganisms to nu- microorganisms increase through two phases during the trient-rich conditions similar to those of the laboratory-cul- start-up period is not known at this time. However, the two- ture system commonly used. step population increase seems to imply competition be- Plate counting of aerobic heterotrophic bacteria in FBC tween the resident or adaptable microorganisms and import- reactors has been performed using non-selective media in- ed microorganisms. This interesting phenomenon is an im- cubated at different temperatures, with incubation at 30LC portant subject awaiting further study. usually giving the highest count71). Plate counts of thermo- Drastic changes in microbial community structures at the philic bacteria, anaerobic bacteria and fungi are much phylum level during the start-up operation of self-heating lower42,71). These observations indicate that mesophilic aero- FBC reactors have been clearly demonstrated by quinone bic heterotrophic bacteria predominate and play primary profiling41,68), which is a promising lipid biomarker roles in the degradation of organic waste in self-heating approach34,36), as well as PLFA profiling. Ubiquinones are FBC reactors. the predominant quinones at the early stage of FBC but their levels decrease gradually with time. In contrast, partially saturated menaquinones gradually accumulate as the opera- Population dynamics and community structure tion proceeds and predominate under steady state condi- To date, microbial communities of composting processes tions. Interestingly, the stages at which ubiquinones and in one batch system have been studied extensively using partially saturated menaquinones predominate correspond both culture-dependent and culture-independent techniques. to phases I and II as noted above, respectively. Among In culture-dependent approaches to this research, aerobic prokaryotes, only members of the classes Alphaproteobac- heterotrophic bacteria have been isolated by the plate- teria, Betaproteobacteria and Gammaproteobacteria con- counting technique and identified based on phenotypic tain ubiquinones, whereas partially saturated menaquinones characteristics (e.g., carbon nutrition)5,86,87) and 16S rRNA are found mostly in the phylum Actinobacteria13,34,98). gene sequence information7,12,28). The culture-independent Therefore, the quinone profile data indicate that a drastic techniques used so far include PCR-denaturing gradient gel population shift from ubiquinone-containing Proteobacteria electrophoresis (DGGE)24,49,50,53,73,96), 16S rRNA gene clon- to Actinobacteria takes place during the start-up operation ing and sequencing9,15), terminal restriction patterns of am- of the FBC process. This community change was also con- plified 16S rRNA genes59,92) and some other molecular firmed by rRNA-targeted FISH and culture-dependent iso- methods74,77,82,94). In addition, chemical biomarker methods lation and 16S rRNA gene sequencing of the predominant using phospholipid fatty acids (PLFA)11,14,29,31,52,55,85,89,95) and bacteria41). About half of the isolates thus obtained from a respiratory quinones90) have been employed for microbial FBC reactor in the steady state were affiliated with mem- community analyses of various composting processes. bers of the actinobacterial genera including Rhodococcus, Microbial community analyses of the FBC process have Jonesia and Ornithinicoccus. Also, Bacillus strains consti- in recent years been conducted using polyphasic approaches tuted a significant proportion of the predominant culturable including cultivation methods. Microbial population dy- bacteria. These findings indicate that members of Actino- namics during the start-up operation of FUSBIC bacteria predominate and play major roles in mesophilic reactors33,35,41,42) and commercial garbage composters have FBC reactors working under fully acclimated conditions. A been reported68,71). A noteworthy observation with these re- schematic model of microbial population dynamics and suc- actors is that total and plate counts of bacteria increase via cession during start-up of the self-heating FBC process for two phases during the start-up period. For example, in com- garbage treatment is shown in Fig. 4. The predominance of mercial composters operated at a waste-loading rate of 0.7 ubiquinone-containing Proteobacteria at the early stage of kg (wet wt) day1, the first increase in bacterial counts oc- the process may result from the sequential supply of gar- curred during 3–4 weeks from the start of waste delivery bage, which contains proteobacterial species as the most (phase I). At the end of this phase, the populations reached a abundant microorganisms. plateau or declined slightly. Then, bacterial numbers in- The microbial community change during the start-up op- creased again during the subsequent 3–4 weeks (phase II), eration of the FBC process has also been demonstrated by reaching a steady state with cell counts of 4.1–6.3P1011 g1 PCR-DGGE. However, one should note that PCR-DGGE (dry wt)68). Similarly, the respiratory quinone content of analysis occasionally fails to detect actinobacterial clones68). SCM, which is highly correlated with total bacterial This may result from possible biases in DNA extraction Microbiology of Fed-batch Composting 5

constituents49,80). In the case of the self-heating FBC process, the microbial community develops under more nutrient-rich conditions. Therefore, factors other than nutrient availability should be taken into account for the reason why actinobacterial species predominate in the FBC process under steady state conditions68,70). In this connection, matric 61) water potential or water activity (aw) may be a critical ecological determinant. As shown in Fig. 2, the conductivity of SCM increases gradually during the FBC process, sug- gesting a possible decrease in aw under acclimated conditions. Research in our laboratory has revealed that actinobacterial strains isolated from FBC reactors are much more tolerant of low aw than proteobacterial isolates (T. Narihiro et al. unpublished data). Therefore, it can be speculated that members of the Proteobacteria are unable to compete with those of the Actinobacteria under steady state conditions with low aw. Further study on the metabolic response of both phylogenetic groups of bacteria to different aw levels would give more useful information to clarify this assump- tion. The effects of moisture contents and temperature on the microbial community in the FBC process were studied with small-scale model reactors45). The predominated microorganisms detected at a fixed temperature of 30LC and differ- Fig. 4. Schematic model of microbial population dynamics during start-up of the self-heating FBC process. The figure was made ent moisture contents were: Enterococcus and yeast strains based on information from references 41 and 68. at a 20% moisture content, Cellulomonas and Xanthomonas at a 30–60% moisture content and Enterobacter, and Xantho- monas at a 70–80% moisture content. Likewise, the pre- and/or PCR amplification from compost samples. Some dif- dominant microorganisms found at a fixed moisture content ficulty in the amplification of actinobacterial 16S rRNA of 60% and different temperatures were: Enterobacter, Pan- genes from mixed populations has been reported32,39). On the toea, and Xanthomonas at 10LC, Cellulomonas and Xantho- other hand, quinone profiling provides rough but less-biased monas at 20–30LC, Bacillus and Cellulomonas at 40LC, information about microbial communities in terms of both and Bacillus at 50LC. These data suggest that there is a re- quantity and phylogenetic composition. This method also verse relationship between the population levels of Proteo- has the advantage that eukaryotic microorganisms in com- bacteria and gram-positive bacteria against changes in plex communities are detectable by monitoring specific moisture content as well as temperature. It has been pointed ubiquinone species such as partially hydrogenated ones54,71). out that the increase in moisture content during composting The combined use of molecular techniques and chemical results in a decrease in the diffusion of oxygen in solid ma- biomarker methods (i.e., quinone profiling and PLFA fin- trices, thereby preventing the growth and survival of aerobic gerprinting) should provide more definitive information microorganisms23). However, the results obtained with a about compost communities. FUSBIC reactor have shown that the proportion of It has been shown that actinobacterial species are com- ubiquinone-containing Proteobacteria, a typical group of mon members of compost communities19,20,58). In typical aerobic bacteria, to the total population increase along with batch-composting processes, temperature and substrate the moisture content of SCM33,35). Therefore, the negative availability are the major determinants of microbial com- effect of a high moisture content on the composting process munity dynamics. The cooling and maturation phases, is due in part to the shift in population to members of the in which nutrient availability becomes quite low, have a Proteobacteria and their rapid consumption of oxygen. more complex bacterial community than the preceding Microbial community changes in an FBC reactor at a rel- phases, with members of Actinobacteria as the major atively high temperature (45–55LC) and level of moisture 6 NARIHIRO and HIRAISHI

(60–70%) were studied using double-gradient DGGE of moisture content on CO2 evolution during FBC were stud- 26) 46,47) PCR-amplified 16S rRNA gene fragments and FISH . ied using a small-scale reactor . Higher CO2 evolution DGGE patterns showed that members of the genus Bacillus was observed at 40LC and a moisture content of 30–60%. predominated during the overall period of operation. A To date, there have been only scattered reports on enzy- moderately thermophilic and alkaliphilic bacillus designat- matic activities in the FBC process. Changes in extracellular ed strain BLx was isolated from the reactor and classified as gelatinase activity and the population of a particular micro- a new genus and species, Cerasibacillus quisquiliarum64). organism, Cerasibacillus quisquiliarum, during a FBC pro- Members of Bacillus and related genera, as well as of cess were studied by zymography with negative activity Thermus7), have been found to be the major constituents of staining63). Early in the process (days 0–16), gelatinase ac- the microbial population in thermophilic FBC processes46,75) tivity and the population of C. quisquiliarum64), increased. as well as in hot composting processes9,15,73,77). Quinone pro- After 20 days of operation, however, the C. quisquiliarum filing of SCM in commercial garbage composters incubated population decreased, while the gelatinase activity was sus- forcibly at 40–60LC has shown that a menaquinone species, tained. The extracellular proteolytic activity of SCM in me- MK-7, which is the primary quinone component of Bacil- sophilic FBC reactors was monitored using azocasein as the lus, accumulates remarkably with increasing temperature, substrate70). The optimal temperature for the activity (70LC) accounting for more than 50 mol% of the total quinone con- was much higher than the range of the core temperature in tent at 50LC and above (A. Hiraishi, unpublished observa- the reactors. On the other hand, the optimal pH (7–10) for tion). It seems that thermophilic FBC reactors provide more the activity matched the pH of SCM in the reactors. Interest- favorable conditions for the proliferation of Bacillus and re- ingly, there were significant negative correlations between lated gram-positive bacteria with a low GC content. the protease activity and moisture content. Aerobic pro- Some investigators reported a unique FBC process that teolytic bacteria predominating in a FUSBIC reactor were worked under acidic conditions in the steady state3,30), un- isolated by the quantitative agar-plating method. 16S rRNA like the standard mesophilic FBC process. PCR-DGGE gene sequence information of these isolates showed that analysis of this process revealed that members of the genus most of the isolates were members of the phyla Actinobac- Lactobacillus predominated in the solid matrices at pH 4 to teria, Bacteroidetes and Frimicutes, especially those of the 6. genera Bacillus, Cellulosimicrobium and Ornithinicoccus, Collectively, these findings indicate that, although self- and those designated as an unidentified Cytophaga-like heating, mesophilic FBC reactors harbor actinobacterial group. The three genera of gram-positive bacteria noted species as the predominant microorganisms, the microbial above were actually found among isolates obtained as the community structure is greatly affected by temperature, pH predominant bacteria in the FUSBIC process41). Compara- and moisture content. Species of the resident microorgan- tive analyses of SCM and proteolytic isolates with inhibitor isms so far detected in FBC processes are listed in Table 1. and zymography experiments suggest that bacteria belong- The information is based on the results of culture-dependent ing to the phyla Actinobacteria or Firmicutes and producing approaches (e.g., phenotypic characterization and 16S an alkaline serine protease play primary roles in protein di- rRNA gene sequencing of culturable isolates) and culture- gestion in the mesophilic FBC process70). independent PCR-DGGE analyses. The bacterial species Little is known about enzymatic activities other than pro- described so far are limited to members of the phyla Actino- tease activity in FBC processes. It is clearly necessary to bacteria, Bacteroidetes, Firmicutes and Proteobacteria. study enzymes involved in the degradation of carbohydrates and lipids as well as proteins during FBC. Degradation activity Potential for application to bioremediation Many researchers have studied the biodegradation of macromolecules during the composting of various organic Traditional composting processes or compost microor- wastes by monitoring oxygen uptake rate56), calorimetric ganisms have also been studied for bioremediation purpos- patterns62) and intra- and extracellular enzymatic es. There have been several review papers concerning the activities4,22,48,51,79,80,91,93). The biodegradation activity during application of composting technology to the bioremediation the operation of an FBC reactor was studied by measuring of soils contaminated with various pollutants includ- 2,57,83) CO2 evolution, and a simple model for describing the pat- ing aliphatic and aromatic hydrocarbons , nitro- tern has been proposed66). The effects of temperature and aromatic compounds16,76,84), chlorinated compounds17,57,78), Microbiology of Fed-batch Composting 7

Table 1. List of microorganisms detected in FBC reactors

Microorganism identified as a Process Method for c Reactor type b Accession number Reference (and most related to): temperature (LC) identification Alphaproteobacteria Agrobacterium sp. C 30 A, Biolog – 45 Agrobacterium tumefaciens C 30 A, Biolog 45 Mesorhizobium sp. (M. loti) F 30–50 A, 16S rRNA AB098586 41 Ochrobactrum anthropi L, C 30 A, Biolog – 44, 45 Paracoccus panthotrophus F 30–50 A, 16S rRNA AB098590 41 Porphyrobacter sp. (P. tepidarius) C 25–30 B, PCR-DGGE – 43 Rhizobium rhizogenes C 30 A, Biolog – 45 Sphingomonas sp. C 25–30 B, PCR-DGGE – 43 C 30 A, Biolog – 44 Sinorhizobium meliloti C 30–40 B, PCR-DGGE AB116962 68 Unidentified (Oligella ureolytica) C 30–40 B, PCR-DGGE AB116966 68 Betaproteobacteria Acidovorax delafieldii C 40 A, Biolog – 45 Alcaligenes latus C 20–30 A, Biolog – 45 Alcaligenes sp. F 30–50 A, 16S rRNA AB098570 41 Alcaligenes xylosoxidans L 30 A, Biolog – 44 Burkholderia cepacia C 30 A, Biolog – 45 Burkholderia gladioli L 30 A, Biolog – 44 Comamonas sp. L 30 A, Biolog – 44 Comamonas testosteroni C 30–40 A, Biolog – 45 Delftia acidovorans C 30 A, Biolog – 45 Kingella kingae L 30 A, Biolog – 44 Variovorax sp. (V. paradoxus) F 30–50 A, 16S rRNA AB098595 41 Gammaproteobacteria Acinetobacter calcoaceticus C 30–40 A, Biolog – 45 Acinetobacter johnsonii C 10–40 A, Biolog – 45 Acinetobacter radioresistens L 30 A, Biolog – 44 Acinetobacter sp. (A. calcoaceticus) F 30–50 A, 16S rRNA AB098569 41 C 30–40 B, PCR-DGGE AB116957 68 Acinetobacter sp. C 30 A, Biolog – 45 Enterobacter asburiae C 30 A, Biolog – 45 Enterobacter cancerogenus C 30 A, Biolog – 45 Enterobacter cloacae L, C 30–40 A, Biolog – 44, 45 Enterobacter sp. (E. aerogenes) F 30–50 A, 16S rRNA AB098582 41 Enterobacter sp. C 20–30 A, Biolog – 45 Klebsiella pneumoniae C 10–20 A, Biolog – 45 Luteimonas sp. (L. mephitis) F 30–50 A, 16S rRNA AB098585 41 C 30–40 A, 16S rRNA AB188220 70 Pantoea agglomerans C 10–30 A, Biolog – 45 Pseudomonas sp. L, C 10–40 A, Biolog – 44, 45 Pseudomonas sp. (P. trivialis) F 30–50 A, 16S rRNA AB098591 41 Rhodanobacter sp. C 25–30 B, PCR-DGGE – 43 Salmonella sp. C 30 A, Biolog – 45 Stenotrophomonas maltophilia C 25–30 B, PCR-DGGE – 43 Stenotrophomonas maltophilia L, C 10–30 A, Biolog – 44, 45 Xanthomonas campestris L, C 10–50 A, Biolog – 44, 45 Firmicutes Bacillus alcalophilus C 30 A, Biolog – 45 Bacillus amyloliquefaciens C 40 A, Biolog – 45 Bacillus azotoformans C 10–30 A, Biolog – 45 Bacillus badius F 30–50 A, 16S rRNA AB098575 41 C 30 A, Biolog – 45 Bacillus coagulans C 40–65 B, PCR-DGGE – 30 C 10–50 A, Biolog – 45 Continued 8 NARIHIRO and HIRAISHI

Table 1. Continued

Microorganism identified as a Process Method for c Reactor type b Accession number Reference (and most related to): temperature (LC) identification Bacillus laevolacticus C 50 A, Biolog – 45 Bacillus licheniformis C 10–50 A, Biolog – 45 Bacillus litoralis C 40–65 B, PCR-DGGE – 30 Bacillus maroccanus C 30 A, Biolog – 45 Bacillus megaterium L, C 10–50 A, Biolog – 44, 45 Bacillus pumilus C 40 A, 16S rRNA – 3 Bacillus pumilus F 30–50 A, 16S rRNA AB098578 41 Bacillus sp. C 40–65 B, PCR-DGGE – 30 Bacillus sp. (B. aminovorans) C 30–50 B, PCR-DGGE AB110655 27 Bacillus sp. (B. clausii) F 30–50 A, 16S rRNA AB098576 41 Bacillus sp. (B. fusiformis) F 30–50 A, 16S rRNA AB098577 41 Bacillus sp. (B. lentus) C 50–60 A, 16S rRNA AB020194 75 Bacillus sp. (B. licheniformis) C 30–40 A, 16S rRNA AB188216 70 C 50–60 A, 16S rRNA AB020195 75 Bacillus sp. (B. subtilis) F 30–50 A, 16S rRNA AB098574 41 C 30–40 A, 16S rRNA AB188212 70 C 50–60 A, 16S rRNA AB020193 75 C 40 A, 16S rRNA – 3 Bacillus sp. (B. thermoamylovorans) C 50–60 A, 16S rRNA AB020196 75 C 46–47 B, PCR-DGGE AB066087 26 Bacillus sp. (B. galactosidilyticus) C 50–60 A, 16S rRNA AB020192 75 Bacillus sphaericus L, C 20–30 A, Biolog – 44, 45 Bacillus subtilis C 40–65 B, PCR-DGGE – 30 C 25–30 B, PCR-DGGE – 43 L, C 10–50 A, Biolog – 44, 45 Bacillus thermoamylovorans C 45–50 A, 16S rRNA AB121094 63 Bacillus thermocloacae C 45 B, PCR-DGGE AB066092 26 Bacillus thermoglucosidasius C 30–50 A, Biolog – 45 Bacillus thermosphaericus C 46–47 B, PCR-DGGE – 26 Brevibacillus brevis C 10–50 A, Biolog – 45 Cerasibacillus quisquiliarum C 45–50 A, 16S rRNA AB107894 63 C 46 B, PCR-DGGE AB066088 26 Enterococcus faecalis C 30–50 B, PCR-DGGE AB110649 27 Enterococcus sp. L, C 30–40 A, Biolog – 44, 45 Enterococcus sp. (E. gallinarum) C 30–50 B, PCR-DGGE AB110652 27 Globicatella sp. (G. sulfidifaciens) C 30–50 B, PCR-DGGE AB110653 27 Lactobacillus alimentarius C 40 B, PCR-DGGE AB042019 3 Lactobacillus fermentum C 30–50 B, PCR-DGGE AB110648 27 Lactobacillus lactis C 40–65 B, PCR-DGGE – 30 Lactobacillus paralimentarius C 40–65 B, PCR-DGGE – 30 Lactobacillus reuteri C 40 B, PCR-DGGE AB042009 3 Lactobacillus sp. C 40–65 B, PCR-DGGE – 30 Lactobacillus sp. (L. fermentum) C 40 B, PCR-DGGE AB042008 3 Lactobacillus sp. (L. pentosus) C 40 A, 16S rRNA – 3 Lactobacillus sp. (L. reuteri) C 40 B, PCR-DGGE AB042017 3 Lactococcus lactis C 40 B, PCR-DGGE AB042007 3 Leuconostoc citreum C 40–65 B, PCR-DGGE – 30 Paenibacillus macerans C 50 A, Biolog – 45 Paenibacillus validus (Bacillus gordonae) C 30 A, Biolog – 45 Pediococcus acidilactici C 40–65 B, PCR-DGGE – 30 Sporosarcina pasteurii (Bacillus pasteurii) C 20–50 A, Biolog – 45 Staphylococcus capitis C 40 B, PCR-DGGE AB042015 3 Staphylococcus hominis C 40 B, PCR-DGGE AB042014 3 Staphylococcus sp. (S. kloosii) C 40 A, 16S rRNA – 3 Staphylococcus sp. (S. hominis) C 40 A, 16S rRNA – 3 Staphylococcus sp. (S. sciuri) C 30–40 A, 16S rRNA AB188210 70 Staphylococcus sp. C 30 A, Biolog – 45 Continued Microbiology of Fed-batch Composting 9

Table 1. Continued

Microorganism identified as a Process Method for c Reactor type b Accession number Reference (and most related to): temperature (LC) identification Staphylococcus warneri C 25–30 B, PCR-DGGE – 43 Unidentified (Clostridium ultunae) C 45–50 B, PCR-DGGE AB066091 26 Unidentified (Lactobacillus plantarum) C 40 B, PCR-DGGE AB042018 3 Unidentified (Lactobacillus kefiri) C 40 B, PCR-DGGE AB042012 3 Unidentified (Staphylococcus pasteuri) C 40 B, PCR-DGGE AB042013 3 Unidentified (Staphylococcus sciuri) C 30–50 B, PCR-DGGE AB110651 27 Weissella paramesenteroides C 40–65 B, PCR-DGGE – 30 Actinobacteria Arthrobacter globiformis F 30–50 A, 16S rRNA AB098573 41 Arthrobacter sp. (A. nicotiniae) F 30–50 A, 16S rRNA AB098571 41 Brachybacterium paraconglomeratum F 30–50 A, 16S rRNA AB098579 41 Brevibacterium sp. (B. lutescens) C 30–40 A, 16S rRNA AB188208 70 Cellulosimicrobium cellulans F 30–50 A, 16S rRNA AB098580 41 Cellulosimicrobium sp. (C. cellulans) C 30–40 A, 16S rRNA AB188217 70 Corynebacterium aquaticum C 30 A, Biolog – 45 Corynebacterium pseudodiphtheriticum L 30 A, Biolog – 44 Corynebacterium sp. C 30–40 A, Biolog – 45 Corynebacterium sp. (C. variabilis) C 40 A, 16S rRNA AB035918 3 Corynebacterium sp. (C. amycolatum) C 30–50 B, PCR-DGGE AB110654 27 Corynebacterium sp. (C. kroppenstedtii) C 30–50 B, PCR-DGGE AB110650 27 Isoptericola sp. (I. halotolerans) C 30–40 A, 16S rRNA AB188223 70 Jonesia sp. (J. denitrificans) F 30–50 A, 16S rRNA AB098583 41 Microbacterium esteraromaticum C 30 A, Biolog – 45 “Micrococcus diversus” C 30 A, Biolog – 45 Micrococcus sp. (M. luteus) C 30–40 A, 16S rRNA AB188213 70 Microoccus sp. L, C 10–30 A, Biolog – 44, 45 Oerskovia enterophila C 50 A, Biolog – 45 Oerskovia turbata L, C 10–40 A, Biolog – 44, 45 Ornithinicoccus hortensis F 30–50 A, 16S rRNA AB098587 41 Ornithinicoccus sp. (O. hortensis) C 30–40 A, 16S rRNA AB188219 70 Ornithinimicrobium sp. (O. humiphilum) C 30–40 A, 16S rRNA AB188211 70 Prauseria sp. (P. hordei) C 30–40 A, 16S rRNA AB188209 70 Rhodococcus equi C 20 A, Biolog – 45 Rhodococcus sp. (R. pyridinivorans) F 30–50 A, 16S rRNA AB08592 41 Rhodococcus sp. C 30 A, Biolog – 45 Rothia dentocariosa C 30–50 A, Biolog – 45 Bacteroidetes Chryseobacterium scophthalmum C 25–30 B, PCR-DGGE – 43 Unidentified (C. fucicola) F 30–50 A, 16S rRNA AB098581 41 C 30–40 A, 16S rRNA AB188214 70 Sphingobacterium multivorum C 25–30 B, PCR-DGGE – 43 Sphingobacterium sp. L, C 30 A, Biolog – 44, 45 Sphingobacterium sp. (S. multivorum) F 30–50 A, 16S rRNA AB098594 41 Unidentified (Gelidibacter mesophilus) C 30–40 B, PCR-DGGE AB116963 68 Unidentified (Flexibacter roseolus) C 30–40 B, PCR-DGGE AB116965 68 Unidentified (Sphingobacterium comitans) C 30–40 B, PCR-DGGE AB116958 68 Sphingobacterium sp. (S. thalpophilum) C 30–40 B, PCR-DGGE AB116961 68 Yeasts Candida entomophia C 30 A, Biolog – 45 Candida etchellsii C 40–65 B, PCR-DGGE – 30 Candida sp. (C. krusei) C 40 A, 16S rRNA – 3 Cryptococcus albidus C 30 A, Biolog – 45 Cryptococcus luteolus C 30 A, Biolog – 45 Pichia farinosa C 40–65 B, PCR-DGGE – 30 Pichia sp. C 30 A, Biolog – 45 Rhodosporidium sp. L 30 A, Biolog – 44 Rhodotorula sp. C 10 A, Biolog – 45 a C, commercial composters; F, FUSBIC reactors; L, laboratory-scale small reactors. b A, culture-based identification; B, culture-independent identification. c Accession numbers for 16S rRNA gene sequences deposited in DDBJ/EMBL/GenBank DNA data banks. 10 NARIHIRO and HIRAISHI pesticide17,20), diesel oil21) and heavy metals6). The informa- the FBC process, such as a lack of different thermal stages, tion accumulated in this field of research suggests that the there is a need to consider a distinct microbiological basis FBC processes as well as conventional systems have poten- for it from that for a conventional composting system. In an tial applications to the bioremediation of polluted soil. effort to study the FBC processes as described herein, sig- The chemical group of polychlorinated dibenzo-p-diox- nificant progress has been made in understanding the biodi- ins/dibenzofurans (PCDD/Fs) is one of the most problemat- versity, ecology and physiology of the microorganisms in- ic environmental pollutants. As a wide variety of dioxin- volved. It is most likely that members of the Actinobacteria degrading microorganisms have been isolated and predominate and fulfill the key roles in the mesophilic FBC characterized, much attention has been paid to the bioreme- process under steady state conditions. An unresolved aspect diation of dioxin-polluted environments using these micro- to this process is why microbial populations increase via organisms or microbial consortia37,97). A preliminary report two phases with a drastic population shift from ubiquinone- has shown that the concentration of PCDD/Fs declined in a containing Proteobacteria to Actinobacteria during the microcosm containing dioxin-polluted soil and compost start-up process. Possibly, matrix water potential is a critical produced from the FUSBIC system40). A FUSBIC reactor determinant of microbial population dynamics, and the met- was shown to be capable of degrading PCDD/Fs spiked or abolic response of compost microbial communities to dif- 69) present as contaminants in household biowaste . In this ferent aw levels should be studied further. Another question case, the concentration of PCDD/Fs present originally in the to be answered is what kinds of microorganisms are respon- biowaste was reduced to approximately 60% after 13 sible for the degradation of polysaccharides, lipids and other months of operation. Interestingly, mono-, di-, and tri-chlo- macromolecules in biowaste during FBC under mesophilic rinated congeners occurred in only trace amounts in the sur- conditions. Moreover, the physical habitat in the composted plus compost produced. These results imply that the trans- material is important in determining the spatial distribution formation of PCDD/Fs by a combination of the reductive and activity of microorganisms. A recent report has intro- dechlorination of highly chlorinated congeners and aerobic duced the interactions and self-organization in the soil-mi- oxidation of the dechlorinated products took place in the crobe complex99). Whether such a microbe-solid complex FUSBIC reactor. exists in compost environments is of great interest. From a Attempts to remedy PCDD/F-contaminated fly ash and biotechnological viewpoint, FBC reactors should be more soil during FBC using a commercial personal composter intensively studied to provide a basis as solid-phase biore- have yielded positive results as well69). Our ongoing studies actors for applications to the bioremediation of polluted soil have indicated that high concentrations of PCDD/Fs and other fields of biotechnology. (5,000 pg-TEQ [toxic equivalent] g1 dry wt) are logarith- mically removed with a half-reduction time of ca. 4 months Acknowledgements during the fed-batch treatment of household garbage using the composter. Real-time PCR assays with specific This study was supported in part by grants K1433 and primers showed that relatively large populations of “Deha- K1522 from the Ministry of the Environment, Japan. Work lococcoides”, a potent dioxin-dechlorinating anaerobic on the application to bioremediation was carried out as a organism10,18), occurred in FBC reactors even under aerobi- part of “The Project for Development of Technologies for cally operated conditions (T. Narihiro et al. unpublished da- Analyzing and Controlling the Mechanism of Biodegrading ta). This observation indicates the possibility that the solid and Processing”, which was entrusted by the New Energy matrices in FBC reactors provide conditions favorable for and Industrial Technology Development Organization the growth and activity of both aerobic and anaerobic mi- (NEDO). This work was also carried out as a part of the 21st croorganisms that are responsible for the composting of gar- Century COE Program “Ecological Engineering for Ho- bage and transformation of PCDD/Fs. 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