Change in Microbial Numbers during Thermophilic Composting of Sewage Sludge with Reference to CO2 Evolution Rate

Transcription

1 APPLID AD VIROMTAL MICROBIOLOGY, Jan. 1985, p /85/1375$2./ Copyright 1985, Amerian Soiety for Mirobiology Vol. 49, o. 1 Change in Mirobial umbers during Thermophili Composting of Sewage Sludge with Referene to CO2 volution Rate KIYOHIKO AKASAKI, MASAYUKI SASAKI, MAKOTO SHODA,* AD HIROSHI KUBOTA Researh Laboratory of Resoures Utilization, Tokyo Institute of Tehnology, 4259 agatsuta, Midoriku, Yokohama, Japan Reeived 9 August 1984/Aepted 28 September 1984 Dewatered sewage sludge was omposted in a laboratorysale autothermal reator in whih a onstant temperature of 6 C was kept as long as possible by regulating the air feed rate. The hange in CO2 evolution rate was measured ontinuously from the start up through the essation of omposting. The suession of mesophili bateria, thermophili bateria, and thermophili atinomyetes was also observed during the omposting. Speifi CO2 evolution rates of thermophili bateria and atinomyetes in the onstanttemperature region of 6 C were assessed quantitatively. It was found that the CO2 evolution rate was attributed to thermophili bateria at the initial stage of 6 C and to thermophili atinomyetes at the later stage of 6 C. A serious and omplex problem in ities today is the disposal of huge amounts of sewage sludge produed from wastewater treatment failities. Conversion of the sludge into stable organi material by omposting is of great interest reently as one method of sludge treatment. As omposting is a mirobial deomposition proess of organi matter, muh mirobiologial researh has been done in this area. The primary onern, so far, has foused on the pathogeni problems (2, 7, 12, 14) and on mirobial populations in muniipal waste ompost (6, 1) and in agriultural and manure omposting (4, 5, 8, 9, 12, 13). o information was available on mirobial researh of sewage sludge omposting. However, it is diffiult to assess real fators whih affet the mirobial suession unless mirobial analysis is done under arefully ontrolled onditions of omposting. In this paper, isolation of mirobial groups from a sewage sludge ompost was onduted at several stages of the omposting proess in a laboratorysale autothermal reator in whih a onstant temperature of 6 C was maintained as long as possible by ontrolling the air feed rate to the reator. During the omposting, the mirobial ativity was monitored by measuring CO2 evolution rate, and the speifi ativities of isolated groups of miroorganisms were assessed quantitatively. MATRIALS AD MTHODS Apparatus. The omposting reator used is ylindrial (3 mm in diameter, 4 mm in depth) and made of thermoresistant polyvinyl hloride resin with a perforated plate at the bottom to distribute the air supplied from outside. This reator was surrounded with a ubi Styrofoam insulator (1.2 by 1.2 by 1.2 m3) to maintain minimum heat loss from the wall of the reator. Temperature was deteted with three thermoouples whih were plaed at the bottom, enter, and upper parts of the reator. The temperature differene between the upper and bottom parts of the reator was + 2 C at most. Air from a ompressor was split into two streams: one for a lower flow rate, 4 ml/min; and the other for a higher flow rate, 4, ml/min. When the experiment started, the air was supplied at the lower flow rate. One the signal from the thermoouple in the enter of the reator indiated a temperature higher than the set point of 6 C, a temperature * Corresponding author. 37 ontroller hanged the lower flow rate to the higher one and temperature was maintained autothermally at C by the onoff ontrol. A temperature of 6 C has already been found to be optimal for sewage sludge omposting by our researh (1). A shemati diagram of the apparatus is shown in Fig. 1. Composting material and seed. Dewatered sewage sludge ake from the Minamitama Wastewater Treatment Plant (Inagi City, Tokyo, Japan) was used as the omposting material. The sludge was a mixture of raw settled sludge and exess sludge from the ativated sludge proess, ontaining approximately 6% water, 7.5% slaked lime, and 2.5% ferri hloride as dewatering agents. The average omposition of the dried sludge was 5% volatile matter (VM), in whih C, H, and were 25, 4, and 3%, respetively, and 5% ash. Fresh sludge was olleted from the plant just before eah run of the omposting operation. Compost produt, produed in our laboratorysale omposting reator by the proedure desribed herein and ured for 3 to 4 weeks at room temperature, was used as an inoulating seed. It was also asertained that CO2 evolution from the seed was negligible (1). The sludge ake and the seed, whih were ground and sieved to <5 mesh, were mixed in the following ratios of seed/sludge (perent [dry weight]): % (run A), 1% (run B), 24% (run C), 133% (run D). o bulking agent was added to simplify the reation system and delete additional effets derived from suh agents. The initial weight of the sludgeseed mixture paked into the reator was 3,3 to 5, g (wet weight). Operation of the reator. As the sludge ontained lime, the initial ph value of the material was around 11, and a long lag period (about 3 days) was observed before mirobial degradation of the raw material started. To shorten the lag time, CO2 gas from a ylinder was supplied to the reator at the start of eah run at a rate of 5 ml/min to neutralize the lime. The supply of CO2 was ontinued until the ph of the material dereased to around 8. About 1 h later, after neutralization, a sharp rise in temperature and CO2 evolution by mirobial degradation of the sludge were observed. Turning of the ompost material in the reator was onduted by hand every 24 h after the temperature reahed 6 C. The sampling times of the materials during the omposting were as follows: at the time when the CO2 supply was stopped, when the temperature reahed around 43 and Downloaded from on Otober 2, 218 by guest

2 38 AKASAKI T AL. Air _ Flowmeter 6. Styrofoam insulator li.water trap a Co2 gas ylinder 7. Thermoouple 12. Siliagel adsorber 3 Gas meter 8. Temperature ontroller 13. Reorder 4 Perforated plate 9. letromagneti valve 14.CO2 analyzer 5 Reator 1. Pulse transmitter 15. Miroomputer FIG. 1. Shemati diagram of omposting reation system. 6C, when turning was done, and when the operation was stopped. The omposting operation was stopped when temperature ontrol with varying air flow was no longer available and when the temperature dereased to 3C even at a lower air flow rate. The moisture ontent was adjusted to around 6% at the start of the experiments and was not ontrolled during the reation. Isolation of miroorganisms. Bateria, atinomyetes, and fungi, both mesophili and thermophili, were isolated. As preliminary experiments, several media for bateria, atinomyetes, and fungi were tried, and the media in whih the largest number of isolates appeared were adopted as the isolating media. They are as follows: Tryptiase soy agar (BBL Mirobiology Systems) medium for bateria (Tryptiase peptone, 17 g; phytone peptone, 3 g; acl, 5 g; K2HPO4, 2.5 g; gluose, 2.5 g; agar, 2 g; distilled water, 1 liter [ph 7.3]); maltyeast extrat agar medium for atinomyetes (yeast extrat [Difo Laboratories], 4 g; malt extrat [Difo], 8 g; gluose, 4 g; agar, 2 g; distilled water, 1 liter [ph 7.3]); and potatodextrose agar medium for fungi (potatodextrose agar, 39 g; distilled water, 1 liter [ph 5.2]). Other media whih were tried but not used were as follows: for bateria, nutrient medium (Difo), PGY medium (peptone, gluose, yeast extrat), and brain heart infusion medium (Difo); for atinomyetes, ISP no. 4 medium (inorgani saltsstarh agar) and PGY medium; and for fungi, Czapek solution agar (Difo). Several media supplemented with water extrat of the sludge were also tried. However, no remarkable inrease in the number of isolated mirobes was observed. Ten grams (wet weight) of a sample was suspended into 9 ml of sterile water in a sterile homogenizer up and dispersed at 1, rpm for 1 min with a homogenizer (type X3, ihon Seiki Ltd). After serial dilution in sterile water, the suspension was spread onto the three types of medium plates defined above. The inubation temperatures were 3 C for isolation of mesophiles and 6 C for thermophiles. The inubation time was 3 days for mesophili bateria, 2 days for thermophili bateria, and 7 days for atinomyetes and fungi, mesophili and thermophili, respetively. The average number of miroorganisms isolated on three of six plates was onsidered the viable ell number. The deviation of the numbers was <1%. Analysis. The ativity of miroorganisms was ontinuously monitored by measuring the CO2 onentration of the offgas from the reator by means of a CO2 analyzer (type ZFP4, FujiDenki Co., Ltd.). The offgas was passed through both a ondensor and a silia gel olumn to remove moisture before it entered the analyzer (see Fig. 1). The water ontent of the solid sample was determined from the loss of weight after drying at 8 C for 24 h. The dried solids were ground in a mortar and heated at 6 C for 3 min in an eletri oven. After ooling, a few drops of ammonium arbonate solution (25%, wt/vol) were added to the residue followed by heating at 2 C for 1 h. The loss of weight was alulated as VM. As it was found from preliminary experiments that the amount of dryweight redution of the ompost has a linear relationship with the umulative amount of CO2 evolved, the value of VM at a ertain time was estimated from this relationship (see equations 2 and 3). RSULTS Change in mirobial population during omposting proess. The hanges in the number of mesophili bateria, thermophili bateria, and thermophili atinomyetes from runs A, C, and D are shown in Fig. 2 to 4, respetively. As the number of mesophili atinomyetes and fungi were of the order of 12 ells per g of dry ompost, their ontribution was onsidered to be small in this omposting proess. The hange in the number of mesophili bateria was not remarkable ompared with those of thermophili bateria and atinomyetes. It is interesting that appreiable numbers of them existed during the thermophili stage at 6 C. Their I... In +. x n C >~. APPL. VIRO. MICROBIOL Time ( hr) FIG. 2. Time ourses of temperature (T), C2 evolution rate (ro,), the ell number of isolated miroorganisms, and onversion of VM, XVM, during omposting in run A (no seed added). Symbols: *, mesophili bateria (MB);, thermophili bateria (TB); A, thermophili atinomyetes (TA). Arrows on the absissa indiate the points at whih ompost was turned. )2 x I o C n1. a Downloaded from on Otober 2, 218 by guest

3 VOL. 49, 1985 CHAG I MICROBIAL UMBRS DURIG SLUDG COMPOSTIG 39 survival was due to their thermotolerane property, details of whih are explained in the aompanying paper (11). The average numbers of miroorganisms isolated from the raw sludge and the seed are shown in Table 1. The effet of inreasing seed was learly refleted in the inrease in initial numbers of thermophili bateria and thermophili atinomyetes (see Fig. 2 to 4). Pattern of CO2 evolution rate. Time ourses of CO2 evolution rate, ro2, are also shown in Fig. 2 to 4. The ro2 is defined as moles of CO2 evolved per hour per gram (dry weight) of ompost. It seemed that the seeding ratio affeted both the pattern and the values of the CO2 evolution rate. As the seed ratio was inreased, the seond peak of CO2 evolution appeared more losely to the first one and eventually only one peak was seen in run D (Fig. 4). As desribed below, the peak CO2 evolution rate was losely related to speifi groups of miroorganisms. stimation of metaboli ativity for isolated groups. Although a large number of mesophili bateria were isolated from the ompost materials even at the thermophili stage of 6 C, their respiratory ativity at 6 C was found to be negligible, as desribed in the aompanying paper (11). Therefore, the ontribution of both thermophili bateria and thermophili atinomyetes to the overall CO2 evolution rate during a onstant temperature of 6 C was estimated. It was shown by Bah et al. (1) that the CO2 evolution rate is mostly assoiated with degradation of VM of the raw sludge in the omposting material, and the amount of degradable VM in the inoulating seed is negligible ompared with that of the raw sludge. During the thermophili stage, the two groups of miroorganisms (i.e., thermophili bateria and thermophili atinomyetes) were assumed to have their " O *_, o 'IO o. V L ~" U 47 '13..1 :3._. 2, In n v _ x u) a v._ O L yta ~~~~~~~~ ~8 TA~~~~ _n > _ 6 <~~~~~~6 _ <,,,,,j2,, 2.2 C2/,," ~~~~~~~~~~~1 ~~~~~~ Time ( hr) FIG. 4. Time ourses of omposting in run D (133% seed). For symbols and details, see legend to Fig. 2. own speifi CO2 evolution rates whih are the funtion of only the VM onversion of the raw sludge. Based on that assumption, the following linear equation in the thermophili region of 6 C was introdued: nfara + nbrb = rco2 (1) where Ra is the CO2 evolution rate per ell of thermophili atinomyetes (moles of CO2 per hour per ell); Rb is the CO2 evolution rate per ell of thermophili bateria (moles of CO2 per hour per ell); na is the ell number of thermophili atinomyetes (ell per gram [dry solid] of ompost); nb is the ell number of thermophili bateria (ells per gram [dry solid] of ompost); and ro2 is the CO2 evolution rate (moles of CO2 per hour per gram [dry solid] of ompost). The onversion of VM in the raw sludge, XvM, is defined and alulated in the following way: Downloaded from on Otober 2, 218 by guest x () I 6 8 Time ( hr ) FIG. 3. Time ourses of omposting in run C (24% seed). For symbols and details, see legend to Fig. 2. TABL 1. umber of miroorganisms isolated in raw sludge and in seed Cells/g (dry wt) of material Miroorganisms Raw sludge Seed Mesophili Bateria 3.6 x x 18 Atinomyetes < 13 < 13 Fungi 4.2 x x 12 Thermophili Bateria 1.4 x x 17 Atinomyetes 1.4 x x 18 Fungi <1 <1

4 4 AKASAKI T AL. I16 (14 \ KO 12 X r 1 m r 8 \ TB C) 2\ 6 COnVerSiOn Of VM, XVM (%) FIG. 5. stimated speifi CO2 evolution rates of thermophili bateria, Rb, and thermophili atinomyetes, Ra, as a funtion of onversion of VM. Symbols:., thermophili bateria (TB);, thermophili atinomyetes (TA). XVM = (VMOVM,)/VMO (2) 2 R d where VMo is the initial ontent of VM (grams); VM, is the ontent of VM at time t (grams); RCO2 is the CO2 evolution rate at time t (moles of CO2 per hour); and Y is the onversion fator of VM degraded to CO2 produed (grams per mole of GO2). Y was obtained from the experiments by using the following equation: Y = (DSo DSf) RO2 dt (3) where DSo is the weight of dry solid in the initial omposting material (grams); DSf is the weight of dry solid at the time of termination of omposting (grams); and tf is the time when omposting was terminated. ah term in the lefthand side of equation 1 defines the CO2 evolution rate by either thermophili bateria or atinomyetes. If the experimental results of the ell numbers and CO2 evolution rates from four runs are plotted against the onversion of VM in the sludge, four sets of values for na, nb, and rco2 are available for a given onversion of VM. Therefore, Ra and Rb for eah onversion of VM an be alulated by the method of least squares. Figure 5 shows the result. The temperature reahed 6 C after about 7% VM onversion was attained (Fig. 2 to 4). The value for Ra estimated from the leastsquares method was negative between 7 and 1% VM onversion. This is beause the ativity of thermophili atinomyetes is so small ompared with that of thermophili bateria that the values of the first and seond terms in the left side of equation 1 were different in their order. Therefore, the value for Rb in the range of 7 to 1% was estimated by disounting the term of nara in equation 1. By using the alulated values of Ra and Rb in Fig. 5, the ontribution of CO2 evolved by eah group of miroorganisms to the total CO2 evolved was assessed (Fig. 6). The ordinate represents the ratio of CO2 evolution rate attributed to thermophili bateria/overall CO2 evolution rate. It is obvious that CO2 evolution is attributed mainly to thermophili bateria at the initial stage of 6 C onstant temperature and later to thermophili atinomyetes. When the seed inreased, the ontribution of thermophili atinomyetes beame more pronouned. In Fig. 7, the ontribution of the two groups of thermophiles to the CO2 evolution rate for run C is depited by solid lines. ~ It b xcy APPL. VIRO. MICROBIOL. Downloaded from on Otober 2, 218 by guest y L~ 2 L a3 _) Conversion of VM, XVM(%) FIG. 6. stimated CO2 evolution rate attributed to thermophili bateria/overall CO2 evolution rate in the four omposting runs Conversion of VM, XVM ( /) FIG. 7. Contribution of CO2 evolution rates by both thermophili bateria (TB) and thermophili atinomyetes (TA) to the overall CO2 evolution rate measured during omposting, run C.

5 VOL. 49, 1985 CHAG I MICROBIAL UMBRS DURIG SLUDG COMPOSTIG 41 DISCUSSIO Appropriateness of assumption in estimating speifi CO2 evolution rate. When the speifi ativities of thermophili bateria and atinomyetes were estimated by equation 1, the assumption was made that the ativities were only a funtion of VM onversion. If speifi CO2 evolution rate depends on VM onentration as well as VM onversion, Ra and Rb in equation 1 an be replaed by Ra'z and Rb'z, respetively. Here z is an initial VM onentration of raw material and Ra' and Rb' are CO2 evolution rate oeffiients for atinomyetes and bateria, respetively. quation 1 will therefore be modified to: nfara'z + nbrb'z = rco2 (4) The same method of least squares used to assess the values Ra and Rb from equation 1 was applied to obtain the values Ra' and Rb'. The value of z was taken as the weight fration of initial VM onentration in the raw sludge/initial dry solid onentration in the raw material. To assess the adaptabilityof equations 1 and 4, the orrelation fators between the measured ro2 and alulated ro2 from the values Ra and Rb (or Ra' and Rb') were ompared in two ases. They were 88% for equation 1 and 44% for equation 4. This result suggests that the assumption made to use equation 1 is more reasonable. Sine the ompost reation proeeds on a solid surfae whih miroorganisms inhabit, the speifi ativity of CO2 evolution of a miroorganism will depend solely on the availability of VM of the raw sludge. On the other hand, if the growth rate of a miroorganism in the ompost material is onsidered, the speifi ativity of the miroorganism might depend not only on the VM onversion, whih orresponds to quality of the substrate, but also on the initial VM onentration, whih orresponds to quantity of the substrate. In the present study, however, the experimental data obtained were not enough to determine the growth rate of miroorganisms aurately. Speifi ativity of isolated miroorganisms. o quantitative analysis has been made about CO2 evolution rate with referene to ell numbers during omposting. The CO2 evolution rate of ommon mesophili bateria suh as Baillus sp. and Azotobater sp. was estimated to be on the order of 116 to 114 mol of C2/ell per h (3). When only the mesophili bateria were assumed to be responsible for the CO2 evolution at the early stage of the omposting, when the temperature was <4 C, the speifi CO2 evolution rate for the mesophili bateria was estimated to be on the order of 114 mol of C2/ell per h. o data, however, have been reported for orresponding values for thermophili bateria or atinomyetes. The speifi CO2 evolution rate estimated based on equation 1 was on the order of 113 mol of C2/ell per h (Fig. 5). The values seem to be rather high ompared with those of ommon mesophili bateria. However, Pseudomonas sp. was reported to have a high 2 uptake rate (3), orresponding to the order of 113 when onverted to the CO2 evolution rate. Mirobial suession and CO2 evolution rate. At the initial stage of the omposting, mesophili bateria inherent to the raw sludge mainly ontributed CO2 evolution. As temperatures rose, the leading group of miroorganisms whih ontributed to CO2 evolution hanged from mesophili to thermophili miroorganisms. From the result shown in Fig. 7, it is apparent that the first peak in the CO2 evolution rate in Fig. 2 and 3 was also mostly assoiated with the ativity of thermophili bateria, whereas the seond one was assoiated with that of thermophili atinomyetes. Figure 4 shows, however, only one peak in CO2 evolution rate. This an be interpreted as meaning that larger inoulation of seed gave a higher onentration of atinomyetes and the peak of CO2 evolution of atinomyetes oinided with that of thermophili bateria. The ontribution of the atinomyetes depended strongly on the seeding ratio, as shown in Fig. 6 at the region of high onversion of VM. Atinomyetes develop far more slowly than most bateria or fungi and are rather ineffetive ompetitors when nutrient levels are high. Moreover, as they are generally more tolerant to high temperature, they beome more ative in a later period at 6 C, when the level of nutrients beomes lower. The estimated values for speifi CO2 evolution rates in Fig. 5 indiate that the thermophili atinomyetes were more ompetitive when the nutrient level beame lower. The dominane of atinomyetes in the experiments was notied by the white olor on the surfae of the ompost at a later period in the omposting. Fungi ould barely be deteted throughout the experiments. The fat that few fungi were deteted is partly due to the high moisture ontent of the omposts and partly beause the optimal temperature for thermophili fungi is 45 to 5 C; high temperature is thus unfavorable to fungi. Fungi were reported to be essentially absent at 65 C for straw omposting (6). That only restrited groups of miroorganisms appeared in the omposting and the small deviation in the numbers of isolated miroorganisms an be attributed to the high ph value of the limeontaining raw sludge used in the experiments. LITRATUR CITD 1. Bah, P. D., M. Shoda, and H. Kubota Rate of omposting of dewatered sewage sludge in ontinuously mixed isothermal reator. J. Ferment. Tehnol. 62: Bertoldi, M. D., U. Citemesi, and M. Griselli Bulking agents in sludge omposting. Compost Si. 21: Brok, T. D Priniples of mirobial eology. Prentie Hall In., nglewood Cliffs,.J. 4. Chang, Y., and H. J. Hudson The fungi of wheat straw ompost. Trans. Br. Myol. So. 5: Fergus, C. L Thermophili and thermotolerant molds and atinomyetes of mushroom ompost during peak heating. Myologia 56: Finstein, M. S., and M. L. Morris Mirobiology of muniipal solid waste omposting, Adv. Appl. Mirobiol. 19: Golueke, C. G Biologial relamation of solid wastes. Rodale Press, mmaus, Pa. 8. Golueke, C. G., B. J. Card, and P. H. MGauhey A ritial evaluation of inoula in omposting. Appl. Mirobiol. 2: Gray, K. R., K. Sherman, and A. J. Biddlestone A review of omposting. Part 1. Proess Biohem. 6: Kane, B.., and J. T. Mullins Thermophili fungi in a muniipal waste ompost system. Myologia 65: akasaki, K., M. Sasaki, M. Shoda, and H. Kubota Charateristis of mesophili bateria isolated during thermophili omposting of sewage sludge. Appl. nviron. Mirobiol. 49: Savage, J., T. Chase, Jr., and J. D. Mamillan Population hanges in enteri bateria and other miroorganisms during aerobi thermophili windrow omposting. Appl. Mirobiol. 26: Waksman, S. A., T. C. Cordon, and. Hulpoi Influene of temperature upon the mirobiologial population and deomposition proesses in omposts of stable manure. Soil Si. 47: Wiley, B. B., and S. Westerberg Human pathogens in omposted sewage. Appl. Mirobiol. 18: Downloaded from on Otober 2, 218 by guest