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DOI: 10.1177/0734242X05049771

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48 Waste Management & Research

Waste Manage Res 2005: 23: 48–56Printed in UK – all right reserved

Copyright © ISWA 2005Waste Management & Research

ISSN 0734–242X

Microbial succession associated with organic matter decomposition during thermophilic composting of organic waste

Using dog food as a model of the organic waste, thermophiliccomposting was carried out for 14 days at a fixed temperatureof 60 C. The relationship between organic matter decompo-sition measured by CO2 evolution during the bio-stabilizationprocess and microbial succession expressed as the changes overtime in the restriction fragment length polymorphism (RFLP)patterns of 16S rDNA sequences, of micro-organisms associ-ated with the composting material was also examined. TheCO2 evolution rate peaked on day 3 and gradually decreaseduntil it became extremely small after day 9 of composting, indi-cating that vigorous organic matter decomposition ceasedaround this time. On the other hand, the RFLP patternchanged drastically from day 0 to day 4 or 5, then remainedstable until day 7 or 8, reaching its final configuration, withlittle variations, after day 9 of composting. RFLP analysistherefore indicates that microbial succession continued intothe later stage of composting. Nevertheless, by day 9, the rateof organic matter decomposition was so low that its influenceon microbial populations could be hardly recognized by con-ventional methods of dilution plating. Moreover, the com-post produced by day 9 showed no inhibitory effect on thegrowth of Komatsuna (Brassica campestris L. var. rapiferafroug),indicating that the maturity of compost is sufficient for plantgrowth when the rate of organic matter decomposition hasbecome extremely low and the RFLP patterns become stable.

Kiyohiko NakasakiKazuki Nag Department of Materials Science and Chemical Engineering,Shizuoka University, Hamamatsu, Japan

Shuichi KaritaDepartment of Sustainable Resource Science, Mie University,Tsu, Japan

Keywords: Composting, organic waste, micro-organisms, microbial succession, 16S rDNA, polymerase chain reaction-restriction fragment length polymorphism: wmr 754-6

Corresponding author: Kiyohiko Nakasaki, Department ofMaterials Science and Chemical Engineering, Shizuoka University, 3-5-1 Johoku, Hamamatsu 432-8561, Japan.Tel/fax: +81 53 478 1172; e-mail: [email protected]

DOI: 10.1177/0734242X05049771

Received 19 April 2004; accepted in revised form 4 October 2004

Introduction

Composting is the aerobic process through which biodegrableorganic materials undergo a partial mineralisation and pro-found transformations due to the metabolism of a complexmicrobial population. The result of such a process is a biolog-ically stable and humified end product, the compost, whichcan be applied in agriculture. It has been thought that themicro-organisms contributing to organic matter decomposi-tion will change as composting progresses, since temperature,pH, moisture content, and the quality and quantity of organic

materials also change during composting (Gray et al. 1971, Gol-ueke 1977, Finstein & Morris 1975, de Bertoldi et al. 1983).

Many researchers, including the authors of this paper,have found a clear incidence for microbial succession frommesophiles to thermophiles and vice versa during the periodof increasing temperature, in the early stages of composting,as well as during the temperature descent phase, in the latestages (Sie et al. 1961, Chang & Hudson 1967, de Bertoldiet al. 1981, Nakasaki et al. 1985). There seems no doubt that

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Microbial succession associated with organic matter decomposition

Waste Management & Research 49

temperature is one of the most important factors in the suc-cession of composting micro-organisms, and it is relativelyeasy to ascertain the microbial succession from mesophiles tothermophiles, or conversely from thermophiles to mesophiles,by using different incubation temperatures with, for example,the dilution plating method.

Of course, the incidence of microbial succession associatedwith changes in other operational conditions of compostingcan be obtained by various incubation conditions for dilutionplating. Indeed, many researchers have succeeded in repre-senting the microbial successions that changes in organicmatter constituents would cause, simply by counting the col-onies of cells formed on the various types of selective agarmedia (Waksman et al. 1939, Chang & Hudson 1967, Stut-zenberger et al. 1970, de Bertoldi et al. 1981, Nakasaki et al.1985). This method, based on the abilities of micro-organ-isms to decompose certain kinds of substrates, is valuable, andprovides important clues for understanding the mechanism oforganic matter decomposition during composting. However,that method also requires much time together with a higherskill. Moreover, it is difficult to figure out microbial succes-sion within a given functional group of micro-organismsgrown on the same selective medium. This is because the col-onies of many micro-organisms look morphologically similarand difficulties exist in distinguishing these colonies fromeach other on an agar plate. Therefore, only certain membersof micro-organisms, namely those whose colonies are easilyidentified, can be recognized and counted. Nevertheless, areferenced study examined the species diversity of compost-ing micro-organisms randomly selected on an agar plate(Strom 1985).

Recently, it has become possible to analyse microbial com-munities also from complex habitats by using molecular biol-ogy methods. In fact, numerous papers analysing microbialcommunities from soil (Knaebel & Crawford 1995, Osbornet al. 2000), water (Van Hannen et al. 1998), and compost(Beffa et al. 1996, Blanc et al. 1997, Peters et al. 2000, Dees &Ghiorse 2001, Alfreider et al. 2002, Michel et al. 2002, Ryck-eboer et al. 2003) have been published. Those reports dealingwith compost stabilization elucidated the microbial succes-sion during the process and referred to the advantage of DNA-based methods for monitoring microbial succession. Michelet al. (2002) carried out a wide analysis of terminal-restrictionfragment length polymorphism (T-RFLP) patterns by com-paring the sizes of the fragments from T-RFLP to those pre-dicted by computer-simulated digestions of bacterial DNA.They obtained more detailed and precise information con-cerning the microbial shift among certain bacterial groupsduring composting. In the present study, we tried to apply oneof the molecular biology methods, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to

composting in order to investigate the succession of the micro-flora throughout the process. We focused here on the effect ofthe quantity of organic matter decomposition (expressed ascumulative CO2 released as a consequence of the activity ofmicro-organisms) on the microbial succession. To the best ofour knowledge no reports have been so far published on therelationship between organic matter decomposition duringcomposting and microbial succession analysed by the PCR-RFLP method.

Materials and methods

Starting raw substrate

A commercial dog food with the trade mark Vita-One SoftTM

(Japan Pet Food Co., Ltd., Tokyo, Japan) was used as a modelof organic waste in place of real organic fractions in the munic-ipal solid-waste, or garbage. In preliminary experiments, thedog food was shown to give good reproducible compostingdata, provided its composition was uniform (Nakasaki et al.1998). The carbon and nitrogen contents of the dry weightbasis of the dog food, as determined by element analysis, were46.0 and 5.29%, respectively. Thus, the carbon-to-nitrogenratio was 8.7 to 1. To ensure the uniformity of the entire solidmaterial subjected to composting during an experimentalrun, the dog food was minced before it was mixed with othermaterials, which included saw dust as a bulking agent andcommercial seeding material (Alles G, Matsumoto Microor-ganism Laboratory Co., Ltd., Matsumoto, Japan). The mixingratio of dog food : sawdust : seeding material in this raw mix-ture was 10 : 9 : 1 on a dry weight basis.

The pH of the raw mixture was 5.29. The dog food, our sub-stitute for garbage, showed a decrease in pH in the early stagesof composting, as does garbage. In raw materials with an ini-tially low pH, microbial activities are inhibited by the tempo-rary drop in pH, and thus the decomposition of organic mate-rials in the composting may come to a stop. The pH of the rawmixture was adjusted to around 8.5 by adding slaked lime. Thiswas done to maintain a weakly alkaline condition even whenthe pH dropped in the early stages of composting. Prior to thestart of the experiment, the moisture content of the raw mix-ture was adjusted to approximately 55% by the addition of dis-tilled water.

Composting trials

Figure 1 shows a schematic representation of the experimentalsystem adopted. A bench-scale composting reactor, 400 mmhigh with a diameter of 300 mm, was used. The air stream fromthe compressor was split into two flow rates, low (30 L h–1)and high (400 L h–1). The low flow rate acted as basal aera-




K. Nakasaki, F. Eguchi, S. Karita


tion throughout the experiment. This rate was operating atthe beginning of the biostabilization, with temperatures inthe range of mesophily. On the other hand, the high flow rateadopted at temperatures exceeding 60°C. When the temper-ature began to decrease, while the basal flow rate was main-tained, an electrical ribbon heater surrounding the reactorwas used to keep the composting temperature at about 60°C.In preliminary experiments, it was ascertained that the increaseof low flow rate did not result in an increase of the organic mat-ter degradation rate. Thus, the aeration rate adopted in thepresent research was reputed to be enough to maintain aero-bic conditions throughout the biostabilization process. Theexhaust gas from the reactor was passed through a conicalflask containing an H2SO4 solution in order to absorb NH3;the gaseous stream was then injected into an infrared analyser(RI-550A; Riken Co., Ltd., Tokyo, Japan) for the monitoringof CO2 concentration in the gas. During all composting exper-iments, the CO2 evolution rate, defined as molar CO2 evolu-tion per hour per gram of dry composting material, was calcu-lated. The NH3 evolution rate was also calculated from themeasurement of NH3 quantity absorbed in the H2SO4 solu-tion by titration at 24 h intervals. Moreover, the carbon min-eralization (MC), corresponding to the extent of organic mat-ter decomposition, was taken as a molar ratio of the carbonloss as CO2 to the initial total carbon contained in the dogfood. The MC value at any time could be estimated throughcumulative quantification of the CO2 evolved up to thatpoint. Details of the composting operation and methods forcalculating CO2 evolution rate and carbon mineralizationhave been described elsewhere (Nakasaki et al. 1998).

The compost was manually turned once a day. At each turn-ing, samples were drawn and analysed for pH, moisture content,protein concentration and associated microbial biomass. ThepH was measured in suspensions of compost samples in waterin the ratio of 1 : 9 on a weight basis. The moisture contentwas determined by drying compost samples at 105°C until

constant weight was reached. The protein concentration wasanalysed by the Kjeldahl method.

Distilled water was sprayed on to the compost that remainedin the reactor after each turning in order to prevent it fromdrying out. Compost stabilization was completed within 14days after the process had started. All experiments were per-formed in triplicate runs.

Microbial analyses

Two kinds of microbial analyses were carried out. The firstwas to measure the number of viable microbial cells. Bothmesophilic and thermophilic bacteria (including actinomyc-etes) as well as fungi were isolated on agarized media by dilu-tion plating. The media used were trypticase-soy (BBL) agarmedium for bacteria (trypticase peptone, 17 g; phyton pep-tone, 3 g; NaCl, 5 g; K2HPO4, 2.5 g; glucose, 2.5 g; agar, 15 g;distilled water, 1 L; pH 7.3) and potato-dextrose (Nissui) agarmedium for fungi (potato-dextrose agar, 39 g; distilled water,1 L; pH 5.6). The incubation temperatures were 30°C for mes-ophiles and 60°C for thermophiles. Plates of thermophileswere incubated for 3 days and those of mesophiles for 7 days.Cell densities were expressed as number of colony-formingunits (CFU) of a given microbe per gram of composting mate-rial, dry weight.

In addition to the total viable microbial cells, protein-degrading micro-organisms were isolated on a 1/20 dilutedtrypticase-soy and casein agar medium (1/20 diluted trypticase-soy medium; casein, 10 g; agar, 15 g; distilled water, 1 L; pH 7.3)for the day 4 sample of compost. The incubation temperatureand the period were 60°C and 3 days, respectively. Themicro-organisms that made a halo around the colonies on theagar plate were determined to be protein-degrading micro-organisms.

The other approach to microbial analysis of compost sam-ples was the molecular biology PCR-RFLP method. Samplesof 3 g compost, fresh weight, were mixed with 27 mL sterilewater and homogenized at 10 000 r.p.m. for 10 min, using arotor/stator type homogenizer. These suspensions were cen-trifuged (120 × g, 1 min, 4°C) to remove the particulatefraction. A further centrifugation (27 000 × g, 10 min, 4°C)was needed in order to recover microbial biomass as a pellet.The pellet was suspended in 2 mL of sterile water. After-wards, lysozyme, ribonuclease, and sodium dodecyl sulphatewere added to induce cell lysis. After incubation for 30 minat 37°C, these lysates were centrifuged (11 000 × g, 5 min,4°C) to remove cell debris. Protein was separated from thesupernatant by extraction with Tris-saturated phenol fol-lowed by a further extraction with chloroform. The aqueousphase was collected and precipitated by ethanol addition.DNA extracted from compost samples was purified using a

Fig. 1: Schematic diagram of composting apparatus.






Micro Spin Column (S-300HR; Pharmacia Biotech, Swe-den). Two sets of PCR primers were used; Set A (26f: 5′-ATTCTAGAGTTTGATCATGGCTCA-3′ and 1393r: 5 ′-ATGGTACCGTGTGACGGGCGGTGTGTA-3′) (Beffaet al. 1996), Set B (530f: 5′-TGACTGACTGAGTGCCAGCMGCCGCGG-3′ and 1494r: 5 ′-TGACTGACTGAGGYTACCTTGTTACGACTT-3′) (Borneman et al. 1996). The16S rDNA sequences of compost-associated micro-organismswere amplified with each set of primers, using a DNA thermo-cycler (TP 240; TaKaRa Shuzo Co., Ltd., Kyoto, Japan) withLA Taq polymerase (TaKaRa). Amplification conditions forboth of Set A and Set B primers were as described by previ-ous authors (Beffa et al. 1996, Borneman et al. 1996).

RFLPs of PCR products were compared through gel elec-trophoresis on agarose with fragments digested by three dif-ferent types of restriction endonucleases, namely HaeIII(Toyobo, Kyoto, Japan), AluI (Gibco, Eggenstein, Germany),and Sau3AI (Gibco). Actually, although this possibility isquite rare, the use of only one endonuclease for enzymaticdigestion could have made it possible to obtain similar RFLPpatterns starting from different DNA mixtures, namely differ-ent initial microbial consortia. Digestion with all endonucle-ases was carried out at 37°C for 60 min. The fragments ofdigested DNA were separated by horizontal electrophoresison 2.5% agarose (Agarose S; Nippon Gene, Toyama, Japan)slab gels using a Mupid Mini-Gel electrophoresis apparatus(Mupid-2; Cosmo Bio Co., Ltd., Tokyo, Japan). Electro-phoresis was performed at 50 V for 210 min; afterwards thegels were stained with ethidium bromide and photographedby using a gel printer (GP-1000i; Taitec Co., Ltd., Saitama,Japan) on an UV transilluminator.

Plant growth assay using soil–compost mixture

A plant growth assay of Komatsuna (Brassica campestris L.var. rapiferafroug) was carried out by the standard methodused in Japan using composts produced by day 0 (raw mate-rial) and day 9 composting. Each of the composts was mixedwith 2.5 kg soil at the standard loading levels equivalentto 1.5 g N pot–1, and the soil–compost mixture was put into a1/5000a standard cultivation pot. As a control experiment,soil mixed with 14 : 14 : 14 N–P–K chemical fertilizer atthree different levels equivalent to a 0, 0.5, and 1.0 g N pot–1

were also prepared. Triplicate pots were used for the assayfor soil–compost and soil–chemical fertilizer mixture. TheKomatsuna was harvested after 28 days cultivation, and thefresh weight of the Komatsuna, excluding the root, was thenmeasured. The relative yield was calculated as the ratio ofthe fresh weight of the Komatsuna obtained using each ofthe composts to that obtained by using chemical fertilizer forthe 0.5 g N pot–1.

Results and discussion

Temperature course, CO2 release, and dynamics of carbon min-eralization expressed as Mc during the composting process areshown in Figure 2. Composting temperature was allowed toreach 60°C and then controlled around this value. The tem-perature declined with the turning of the composting mate-rial, but soon returned to the previous level. Bars representingstandard deviations for CO2 evolution rate and carbon min-eralization are generally very short, with a few exceptions.These small ranges indicate that the composting process usedin the present study was highly reproducible. The rate of CO2

evolution grew very fast with a daily cadence after each turn-ing of the composting material. CO2 release attained thehighest peak at day 3, then gradually decreased as the biosta-bilization process proceeded. This is evidence that the rate oforganic matter decomposition was greater in the early stage ofcomposting than in later stages.

On the other hand, carbon mineralization rapidly peakedduring the early stages of composting. The slope of the curvethen progressively decreased its steepness during the course ofthe biostabilization. Finally, carbon mineralization reached avalue as high as approximately 88% in the late stages of com-

Fig. 2: The courses of temperature, CO2 evolution rate, and the car-bon mineralization during composting. Error bars on the CO2 evolu-tion rate, and the carbon mineralization indicate SD (n = 3).






posting. These results are consistent with the data concern-ing CO2 evolution, which was largely released early in thecomposting process.

The courses of pH, NH3 release and cell densities of mes-ophiles and thermophiles during composting are shown inFigure 3. Even in this case, the error bars for pH values, NH3

evolution rate and cell density measurements range within anarrow span, again confirming that the composting processadopted was highly reproducible. The moisture content fluc-tuated throughout the experiments because of the repeatedaddition of water at any compost turning. Nevertheless, com-post humidity was maintained within the optimum range forthe process (40–60%) throughout the biostabilization (Naka-saki et al. 1994) as was not shown here for the sake of brevity.The pH decreased in the early stages of the process, thenincreased again up to nearly 8.7. The NH3 began to evolveinto exhaust gas at 4 days after the initiation of the compost-ing period, then peaked at 5 days and continued to evolveuntil the end of the composting period, although the concen-tration of NH3 was gradually decreased with time. Theseresults confirmed our previous observation that the pH valuedecreased in the early stages of composting due to the accu-mulation of organic acids caused by the degradation of thedog food, and that the pH value increased again due to both

the production of ammonia associated with protein degrada-tion in the dog food and to the decomposition of organicacids (Ohtaki et al. 1998).

Cell density of mesophilic bacteria did not increase in theinitial temperature rise. Nevertheless, mesophiles were quitelow in comparison with the value of 108 CFU g–1 DW thatwas observed in previous research (Nakasaki et al. 1985). Thisdifference in cell density between the past and the presentexperiments may be due to the faster increase in temperaturewithin the latter trials, which prevented the mesophilic bac-teria from growing. On the other hand, thermophilic bacte-ria (including actinomycetes) showed a sharp increment untilday 3 from the start of the biostabilization, finally reaching acount of approximately 108 CFU g–1 DW. Thermophilic bac-teria are of great importance in organic matter decompositionduring thermophilic composting. No remarkable changeswere observed among colonies on agar plates over the first 3days of the process, suggesting that the thermophilic micro-flora stabilized soon with no significant succession within thistime. Mesophilic fungi, as high as 104 CFU g–1 DW (data notshown), were detected only in the initial stages of compost-ing, before quickly diminishing. Thermophilic fungi were notdetected at any time during the experiments. This is in agree-ment with the finding of Golueke (1977) that compostingtemperatures of 60°C or higher are incompatible with thegrowth of even thermophilic eumycetes.

Figure 4 shows changes in the RFLP pattern of endonuclease-digested 16S rDNA sequences of microbial DNA extractedfrom the organic substrate undergoing thermophilic com-posting. The RFLP patterns of compost microbial DNA werehighly reproducible, thus they are not shown here in detail.The examples given in Figure 4 were obtained with three dif-ferent combinations of PCR primers and endonucleases.Light intensity of DNA fragments was rather weak at the startof the process, suggesting that the quantity of DNA amplifiedfrom compost micro-organisms was relatively small. This isconsistent with the finding that cell densities of micro-organ-isms were quite low in the initial stages of biostabilization(Figure 3).

For all combinations of PCR primers and endonucleases(examples of Figure 4 included), the RFLP patterns changeddrastically from day 0 to day 4 or 5, then they appeared stableuntil day 7 or 8, and ultimately reached their final configura-tion with little variations after day 9. Actually, once the tem-perature had increased to 60°C within the first 12 h of biosta-bilization (Figure 2), it was maintained constant together withoptimal aerobic conditions and moisture content throughoutthe experiments. Although pH stabilized after day 4 (Figure 3),RFLP patterns continued to change, indicating that modifi-cations in the quality and availability of organic materialsstrongly affected the succession of the composting micro-organ-

Fig. 3: The courses of pH value, NH3 evolution rate, and the cell den-sities of mesophiles and thermophiles during composting. Error barsindicate SD (n = 3).






isms. RFLP patterns seldom changed after day 9, when the rateof organic matter decomposition had become extremely low(Figure 2). These data suggest that the end of vigorous reactionsdealing with organic matter decomposition resulted in com-pletion of the succession within the thermophilic compostmicroflora. Interestingly, although the conventional dilutionplating method indicated that microbial succession ceases atday 3 of composting, the molecular biology method adoptedshowed modifications in the microbial consortium until day 9.

Although it is known that the RFLP method can be usedas an advanced analytical tool of microflora in combinationwith a computer-aided gel analysis (Michel et al. 2002), tosimplify the investigation we only analysed characteristicchanges in the patterns of RFLP using Set A primers, and

HaeIII observed in day 4 compost. As shown in Figure 4, aband that had not been observed before, approximately 320 bpin size, began to appear from day 4 of composting. As thiscomposting time coincided with the time when the vigorousNH3 evolution occurred (see Figure 3), it could be deducedthat the band of about 320 bp came from a protein-degradingmicro-organism. The course of protein concentration duringcomposting is shown in Figure 5. Degradation of protein in thecomposting material occurred most vigorously from day 3 today 6 of composting. Furthermore, from the day 4 sample ofcompost, we could isolate three distinct strains of protein-degrading micro-organisms, TC4-1 to TC4-3, the colonies ofwhich had different appearances on the agar plate of selectivemedium for protein-degraders.

In Figure 6, RFLP patterns for the purely cultured strainsfrom TC4-1 to TC4-3 are compared with that for day 4 com-

Fig. 4: Changes in PCR-RFLP pattern of endonuclease digestion for the16S rDNA sequences amplified from micro-organisms in the compost-ing material. (a) Set A primers and HaeIII, (b) Set A primers and AluI,(c) Set B primers and HaeIII. Lanes: M, size marker (100 bp ladder);the numbers, days of composting. These photographs were overexposed in order to show some significant fragments with low lightdensity clearly.

Fig. 5: The course of protein concentration during composting. Errorbars indicate SD (n = 3).

Fig. 6: PCR-RFLP pattern of endonuclease digestion using Set A prim-ers and HaeIII for the 16S rDNA sequences amplified from the purelycultured TC4-1 through TC4-3, and from micro-organisms in the day 4compost.






post. TC4-1 is responsible for the 320 bp band, which is char-acteristically observed for day 4 compost; this result does notcontradict the idea that the growth of TC4-1 in the compostresulted in the appearance of a 320 bp band in the RFLP ofday 4 compost. The TC4-2, and TC4-3, on the other hand,did not show the band near 320 bp, but they contain somebands, which are observed also for the day 4 compost. StrainTC4-1 was isolated on the selective agar medium for protein-degraders when the protein in the composting materialdegraded vigorously, and the RFLP pattern of that strain wasmirrored by the RFLP pattern of micro-organisms in the com-post itself. We can thus conclude that the strain TC4-1 con-tributes to the degradation of protein in the present compost-ing process. TC4-1 was identified as Bacillus thermoglucosidasiusfrom morphological and physiological characteristics of thestrain at NCIMB, Japan.

As the RFLP patterns seldom changed after day 9 (Fig-ure 4), and this phenomenon corresponded to the point atwhich the rate of organic matter decomposition had becomeextremely low, it could thus be thought that the inhibitoryeffect of compost on plant growth may be already small atthis stage of composting. Therefore, we conducted a plantgrowth assay of Komatsuna using day 9 compost. The rela-tive yield of the Komatsuna grown on the soil–compostmixture is shown in Figure 7. The relative yield of Komat-suna using day 9 compost with the MC, at approximately80.8%, was similar to that obtained using chemical fertilizer,whereas a severe inhibition on the Komatsuna growth wasobserved for day 0 compost (raw material). These results sug-gest that compost in which the decomposition of easilydegradable organic material has ceased is mature enough forthe growth of Komatsuna, and that it may be possible todetermine compost maturity from observations of changes inthe RFLP pattern.

In the present study, we kept the composting temperatureconstant, as temperature is one of the main factors influenc-ing the dynamics of microbial populations. Hence, the micro-bial succession that we observed during composting wasaffected by changes in the organic matter content. The presentstudy provides a model and a starting point for elucidatingseparately the effects of various factors on microbial succes-sion; trials that focus on the effects of temperature, moisturecontent, aerobiosis, and pH will be required in order to explainthe influence of each factor on microbial succession. In addi-tion to the experimental benefits, the artefact controllingconstant temperature, namely, isothermal composting, hasbeen used practically, for example in the US as the Belts-ville method, in which the optimum thermophilic temper-ature for composting, 60°C or higher, was maintained by reg-ulating the air feed rate based on a feed-back loop of tem-perature control. The thermophilic temperature can be keptconstant merely by controlling the self-heating for as long aperiod as 30 days in a practical large-scale composting facility(Haug, 1993) even though additional heat was required tomaintain a constant temperature in the small-scale labora-tory composting reactor used in the present study, especiallyat the later stage when the rate of organic matter decomposi-tion became extremely low. However, in practical-scale com-posting, the rate of heat loss is relatively smaller, and the rateof heat accumulation is relatively larger than in laboratory-scale composting. As a result, the constant temperature canbe maintained longer in practical-scale composting than inlaboratory-scale composting, even when the rate of organicmatter decomposition, and thus the heat generation rate,becomes small.

Obviously, the temperature will decrease sooner or later inpractical-scale composting, as well. It is well established thatthe decrease in temperature after consumption of the easilydegradable fractions in common non-isothermal compostingprocesses is associated with an important re-adjustment inthe microbial community (e.g., re-colonization by mesophilesbelonging to different physiological groups) (de Bertoldi et al.1983). For this reasons, microbial succession in non-isother-mal composting usually lasts longer than in the thermophiliccomposting presented in this study. From the viewpoint ofmicrobial ecology, in particular, it will be important to eluci-date the microbial succession during the temperature-descend-ing phase of composting, namely, re-colonization by mes-ophiles. In addition, it would be quite interesting to know themicrobial succession associated with the long-term durationof mesophilic temperature in the composting facility, and alsoin the storage of compost before farmland application, whichsometimes lasts 3 to 6 months.

Our present analysis of microbial succession, from the view-point of determining a threshold of compost maturity that

Fig. 7: Relative yield of Komatsuna harvested from the soil–compostmixtures. Relative yields followed by the same letter were not signifi-cantly (P = 0.05) different according to Duncan’s new multiple rangetest.






will not severely inhibit plant growth, revealed that compostwas sufficiently mature for plant growth when the rate oforganic matter decomposition had become extremely low,and the RFLP patterns had approached the final configura-tion in the thermophilic composting. As it is possible forpractical-scale composting to maintain a constant tempera-ture over 14 days, which was the whole period of compostingin the present study, it will be possible to determine a thresh-old of compost maturity from observations of microbial suc-cession in practical-scale composting, as in the present study,although further research will be required in order to proveour hypothesis.

Conclusions

Thermophilic composting was carried out by using dogfood as a model of organic waste and maintaining the tem-perature near 60°C for 14 days. Intensive organic matterdecomposition was monitored throughout the process,allowing the authors to observe that it ceased by day 9 sincethe compost stabilization had began. The dilution platingmethod, a conventional method of microbial analysis, indi-cated that there was no significant change in the totalcount of thermophilic bacteria (including actinomycetes)after an initial increase in the early stages of composting.Moreover, no remarkable qualitative changes were observedamong the colonies growing on agar plates. These resultsindicate that the relevant variations in the microbialcommunity were restricted to only the early stage. Never-theless, RFLP analysis revealed that microbial successionactually continues until the late stages of compost stabiliza-tion along with the decrease of organic matter decomposi-tion by day 9.

The RFLP is, among different molecular biology protocols,one of the simplest methods to determine the microbial com-munity structure in a complex ecosystem such as the com-posting environment. This method is quite useful especiallyto determine whether the microflora in a progressive succes-sion of compost samples remain constant or not. It is howeverdifficult with RFLP to trace changes concerning singlemicrobes, considering that each band in RFLP pattern doesnot correspond to only one specific micro-organism. RFLPanalysis allowed us to demonstrate that microbial successionproceeds to the late stages of compost stabilization with thedecline of active organic matter decomposition. This phe-nomenon had not been previously shown clearly by conven-tional methods such as dilution plating. A more detailedcharacterization of microbial succession during compostingcan be obtained also by computer-aided gel analysis of T-RFLP profiles, as proposed by Michel et al. (2002) or analysisby other DNA-based methods such as denaturing gradient gelelectrophoresis (DGGE) and temperature gradient gel elec-trophoresis (TGGE), in which each band corresponds to acertain microbe. These analyses may elucidate the role ofindividual micro-organisms in the composting process.

In conclusion, the adoption of molecular biology methodsfor the analysis of micro-organisms in the composting matri-ces is expected to give very precise information on the mech-anisms of organic matter transformation through aerobic bio-waste stabilization.

Acknowledgements

This research was supported partly by a Grant-in-Aid for Sci-entific Research from the Ministry of Education, Scienceand Culture of Japan.

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