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Wat. Res. Vol. 34, No. 5, pp. 17051713, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter

THE ROLE OF SLUDGE RETENTION TIME IN THE HYDROLYSIS AND ACIDIFICATION OF LIPIDS, CARBOHYDRATES AND PROTEINS DURING DIGESTION OF PRIMARY SLUDGE IN CSTR SYSTEMS
YEHUDA MIRON*, GRIETJE ZEEMAN{, JULES B. VAN LIERM and GATZE LETTINGAM
Department of Agricultural, Environmental and Systems Technology, Subdepartment of Environmental Technology, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, Netherlands (First received 1 July 1998; accepted in revised form 1 June 1999) AbstractThe eect of the sludge retention time (SRT) between 3 and 15 days, on hydrolysis, acidication and methanogenesis of domestic sewage was researched by simulating a sludge bed segment of an upow anaerobic sludge bed (UASB) system as a completely stirred tank reactor (CSTR). The CSTR systems were fed with primary sludge (settled solids of domestic sewage) as the inuent at 258C. The study revealed that an SRT R 8 days resulted in acidogenic conditions with negligible biogas production, whereas an SRT > 8 days resulted in methanogenic conditions. The hydrolysis of lipids and carbohydrates increased with increasing SRT, whereas protein hydrolysis only occurred under methanogenic conditions. Approximately 20 and 60% of the particulate biopolymers are hydrolysed under acidogenic and methanogenic conditions, respectively. Hydrolysis was found to be the rate-limiting step for the conversion of carbohydrates. Under acidogenic conditions, acidication was the rate-limiting step for conversion of lipids, while both hydrolysis and acidication were limiting for the conversion of proteins. Under methanogenic conditions, hydrolysis was the rate-limiting step in the whole digestion process. None of the main components of primary sludge followed rst order kinetics with respect to hydrolysis. The dewaterability of the sludges from the CSTRs operated under acidogenic conditions deteriorated, whereas the dewaterability of the methanogenic sludge improved in comparison with the dewaterability of the raw primary sludge. # 2000 Elsevier Science Ltd. All rights reserved Key wordsacidication, carbohydrates, dewaterability, domestic sewage, hydrolysis, lipids, primary sludge, proteins, rate limiting step, SRT, two steps, UASB

NOMENCLATURE

INTRODUCTION

Water Research, 1987 Ccarbh total carbohydrates (particulate plus dissolved carbohydrates) g COD/l Clipid= total lipids (neutral lipids plus LCFA) g COD/l Cprotein total proteins (particulate plus dissolved proteins plus amino acids; total proteins are (NkjNH+ 4 N) 1.5/0.16) g COD/l SAA amino acids g COD/l Scarbh dissolved carbohydrates g COD/l SLCFA LCFA (long chain fatty acids) g COD/l volatile fatty acids g COD/l SVFA Xcarbh particulate carbohydrates g COD/l Xlipid neutral lipids g COD/l Xprotein particulate and dissolved proteins g COD/l

*Present address: Kfar-Haim, 42945 Israel. {Author to whom all correspondence should be addressed; e-mail: grietje.zeeman@algemeen.mt.wau.nl

Anaerobic digestion is more and more recognised as an appropriate technique for the treatment of domestic sewage. The feasibility of anaerobic treatment of domestic sewage using an upow anaerobic sludge bed (UASB) reactor was demonstrated in many full scale treatment plants, particularly under warm climate conditions (Van Haandel and Lettinga, 1994). However, so far anaerobic sewage treatment is only applied in countries with high ambient temperatures. Under low temperature conditions, the low degree of hydrolysis may lead to accumulation of suspended solids, resulting in decreased methanogenic capacity and removal eciencies. Under such conditions the application of short hydraulic retention times (HRT) is virtually impossible (Zeeman and Lettinga, 1999). However, the anaerobic treatment of various kinds of complex wastewaters and sludges, such as domestic sewage, may be treated successfully in a two-step

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UASB system as proposed by Wang (1994); Zeeman et al. (1997). The rst step of such a system is mainly aiming at suspended solids removal and partial hydrolysis and acidication. Its euent will be successively treated by a second step methanogenic UASB reactor. With respect to the biological conversion capacity, the solids retention time (SRT) is one of the most important design parameters for both a methanogenic and an acidifying UASB system treating sewage (Zeeman and Lettinga, 1999). However, for a UASB system operated at short SRTs and thus under acidogenic conditions, no sufcient data with respect to the eect of the SRT on the hydrolysis, acidication and methanogenesis are yet available. Among the main components of primary sludge, viz. carbohydrates, lipids and proteins, carbohydrates, are known to be easily and rapidly converted via hydrolysis to simple sugars and subsequently fermented to volatile fatty acids (VFA) (Cohen, 1982). Protein is hydrolysed to amino acids and further degraded to VFA either through anaerobic oxidation linked to hydrogen production or via fermentation according to the Stickland reaction (McInerney and Zehnder, 1988). The former is dependent on the presence of hydrogen-scavengers while the latter is independent of the methanogenic activity in the reactor (Nagase and Matsuo, 1982). Among the lipids, triglycerides are hydrolysed to long chain fatty acids (LCFA) and further oxidised via b oxidation to acetate or propionate. Accumulation of hydrogen inhibits the b oxidation (Novak and Carlson, 1970) since it is thermodynamically unfavourable under standard conditions. b oxidation only occurs when the hydrogen partial pressure is kept low by the presence of hydrogen-scavengers. It is not clear whether the presence of hydrogen also aects hydrolysis of lipids, although recent research shows that the presence of methanogenic activity enhances hydrolysis of lipids (Palenzuela-Rollon, 1999). In addition, the lipid concentration inuences its own hydrolysis (Palenzuela-Rollon, 1999). LCFA are known to inhibit both their own degradation and the methane production from acetate (Hanaki et al., 1981). Eastman and Ferguson (1981) studied the eect of short SRTs (972 h) on hydrolysis and acidication of primary sludge at 358C. They found that the eect of the SRT on hydrolysis of carbohydrates and proteins can be satisfactorily modelled for the acid phase, by expressing these components in terms of COD equivalents and approximating the hydrolysis by rst order kinetics with respect to degradable particulates. They concluded that hydrolysis is the rate limiting step in the acid phase. Lipids were assumed not to be degraded in the acid phase. The objective of this research was to nd the optimum SRT with respect to hydrolysis, acidication

and methanogenesis, for the treatment of domestic sewage in a single or two-step UASB system. The experiments were performed in CSTR systems at 258C, with primary sludge as an inuent, simulating a sludge bed segment of a UASB reactor (Van der Meer, 1979). In addition, the dewaterability of the sludges, was determined in relation to the SRT. The data are also applicable to sludge digestion in one or two step CSTR systems (Ghosh, 1987).

MATERIALS AND METHODS

Experimental set-up
Five CSTRs with an eective volume of 5 l were operated in order to maintain SRTs of 3, 5, 8, 10 and 15 days. The process temperature was controlled at 25 2 18C, by applying recirculation of temperature controlled water through the double wall of the reactors. The reactors were inoculated with diluted digested primary sludge (20 gTS/l) from the wastewater treatment plant (WWTP) of Ede, The Netherlands. The feeding consisted of diluted primary sludge from the aforementioned WWTP, which was stored during the entire experimental period at 48C. Once a day, a certain volume of digested sludge was withdrawn from the reactor, and an equal volume of the primary sludge was pumped into each reactor. The reactors operated at SRTs of 3 and 5 days were fed twice a day with half of the daily amounts per feeding in order to prevent a shock loading. The reactors were mixed intermittently at 100 rpm for 20 s/20 min. Biogas was continuously collected in 5 l gas bags and was daily measured. The pH was daily measured, whereas suspended solids and VFA concentrations were measured once a week. After two months of continuous operation (more than three SRTs) all the reactors were stable, characterised by a constant biogas production, VFA level, suspended solids concentration and pH. Subsequently, within two weeks, three successive sets of measurements were carried out including duplicate analyses for dissolved COD, total solids and volatile solids (TS and VS, respectively), total carbohydrates, dissolved carbohydrates, considered as simple sugars, Kjeldahl-nitrogen (Nkj), amino acids, total ammonium (NH+ 4 N), neutral lipids, LCFA, VFA, methane (CH4). The average results and standard deviations of each of the three sets as well as calculated hydrolysis rates and constants are presented. Analytical methods TS, VS and Nkj were determined according to standard methods (APHA, 1992). Total COD of the sludge was determined according to NEN 6633 (APHA, 1992), modied for high COD concentrations, by weighing 1.53 g sludge sample and adding water up to 20 g and subsequently 20 ml K2Cr2O7 (1 N) plus 40 ml Ag2SO4/H2SO4 (30 g/l Ag2SO4). The mixture was boiled for 2 h and partly titrated with FeSO4(NH4)2. For determination of dissolved COD (SCOD) the sample was diluted ten times and then subsequently ltered with a 4.4 mm paper lter and a 0.45 mm membrane lter (Schleicher and Schuell). The COD of the ltrate was determined according to the photometric method (Knechtel, 1978). VFA were determined by a HP model 5890A gas chromatograph equipped with a 2 m 4 mm glass column with supelcoport (1120 mesh) coated with 10% Fluorrad FC 431. The temperature of the injector, the column and the FID were 200, 130 and 2808C, respectively. N2 at 208C saturated with formic acid was used as a carrier gas. For determination of dissolved proteins plus amino

Digestion of primary sludge in CSTR system


NH4 N 138

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Table 1. The composition of the inuent primary sludge in mg/l and mg COD/l. The numbers in brackets are the percentages of the VS (rst row) and of the CODtot (second row)

1828 2 173 (11.8) 5344 2 381 (17.3)

Lipid

acids (AA) the sample was centrifuged and the Lowry method (Lowry et al., 1951) was applied on the supernatant. Part of the centrifuged sample was treated according to Brown et al. (1989) in order to remove amino acids and the remaining proteins were subsequently determined with the Lowry method. The amino acid concentration was subsequently calculated from the dierence between these two measurements. The standard used for the Lowry method was BSA (bovine serum albumine). NH+ 4 N concentrations were determined with an automatic analyser (Skalar 1520). Total protein concentrations were determined based on Nkj and NH+ 4 N measurements (see Calculations). Total lipids (lipids plus LCFA) concentration was determined gravimetrically after extraction of lipids by petroleum ether, according to the Soxlett extraction method (APHA, 1992). The LCFA were eluted from the total lipids according to Kaluzny et al. (1985) and analysed by HP 5890 series II gas chromatograph equipped with CP-WAX 58 CB column and FID. Helium was the carrier gas. For the determination of total carbohydrates (including dissolved carbohydrates) in the sludge, the sample was mixed with an ultra-Turrax (Kika-Werk, Jank and Kunkel) and diluted 100200 times and subsequently the method of Bardley et al. (1971) was carried out with glucose as a standard. For the determination of dissolved carbohydrates the sample was membrane ltered (0.45 mm) (membrane lter, Schleicher and Schuell) and the same procedure as used for the total carbohydrates measurement was applied. Dewaterability of the sludge was detected by the capillary suction time (CST, in seconds) and by the lterability constant which is calculated based on the CST (Vesilind, 1988). The dimensionless apparatus constant, f, is equal to 0.794. Methane in the biogas was detected using a Fisons 8000 gas chromatograph with a Teon column (1.5 m 2 mm, Chromosorb 108, 6080 mesh) connected in parallel to a steel column (1.2 m 2 mm, Mol sieve 5A, 6080 mesh). The carrier gas was helium, the oven and TCD temperatures 40 and 1008C, respectively. Calculations Conversion factors: . 1 g carbohydrates (assumed as C6H12O6) is equivalent to 1.07 g COD. . 1 g lipid is equivalent to 2.91 g COD (Sayed, 1987). . 1 g protein (assumed as (C4 H6.1O1.2 N)x) is equivalent to 1 g amino acids, 0.16 g Nkj, 0.16 g NH+ 4 N and 1.5 g COD (Sanders et al., 1996). . Xprotein Cprotein SAA : (1) (2) . Xlipid Clipid SLCFA : . XLCFA CLCFA SLCFA : (3) . Hlipid SRTR8 days SLCFA : no b oxidation is assumed to occur under acidogenic conditions). (4) . Hlipid SRT > 8 days X0,lipid Xlipid S0,LCFA : (5) (6) . Hprotein Sprotein NH 4 N 1:5=0:16: . Hcarbh X0,carbh Xcarbh S0,carbh S0,VFA NH4 N0 1:5=0:16: (7) . Htot Dissolved CODSCOD CH4 COD (8) SLCFA : (9) . Alipid C0,lipid Clipid : . Acarbh C0,carbh Ccarbh S0,VFA NH 4 N0 1:5=0:16: (10) . Aprotein NH (11) 4 N 1:5=0:16: (12) . Atot SVFA CH4 COD:
The calculated hydrolysis (H, g COD/l) includes hydrolysis products already present in the inuent. Calculated acidication (A, g COD/l) includes acidica-

Simple sugars Carbohydrates LCFA Amino acids Protein

VS=15529 2 252 (81.8% of TS) CODtot=30851 2 210

3290 2 48 (21.2) 4936 2 72 (16.0)

263 2 31 (1.7) 394 2 46 (1.3)

11852 114 (7.6) 3423 2 239 (11.1)

9732 2 229 (62.7) 10441 2 246 (33.8)

128 2 59 (0.9) 137 2 63 (0.4)

1455 2 51 (9.4) 2093 2 86 (6.8)

VFA

17881 (115) 26768 (87)

Total sum

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tion products already present in the inuent. Acidication of lipids, proteins or carbohydrates is referred to as hydrolysis plus the subsequent acidication. It is assumed that VFA present in the inuent are produced by hydrolysis and acidication of proteins and carbohydrates and not of lipids. The index ``0'' means the concentration in the inuent, otherwise euent is referred to. The rst order hydrolysis constant (Kh) is calculated based on the linearised equation (Eastman and Ferguson, 1981): TTRT F0 SRT 1 , DF Kh 13

where F0 the biodegradable substrate, is the slope of the equation and DF is the measured hydrolysed substrate.

RESULTS AND DISCUSSION

Acidogenic vs. methanogenic conditions The composition of the primary sludge used for the feeding is presented in Table 1. Figure 1 depicts the pH, total hydrolysis, total acidication and methanogenesis as a function of SRT. The gure shows that at SRT R 8 days, acidogenic conditions prevail while at SRT r 10 days methanogenic conditions prevail. The distribution of the inuent COD over the dierent digestion products at dierent SRTs is illustrated in Fig. 2. 19 and 39% of the particulate biopolymer COD present in the inuent are hydrolysed at an SRT of 8 and 15 days, respectively. The VFA concentration, as compared to the inuent, increases with 8 and 11% at an SRT of 3 and 8 days, respectively. Hydrolysis and acidication of lipids The percentage hydrolysis and acidication of lipids as a function of the SRT is given in Fig. 3. About 39% of the total lipids in the inuent are

LCFA, demonstrating that a signicant fraction of the total lipids is already present in a hydrolysed form in the primary sludge. As primary sludge is produced by settling domestic sewage, at least a part of these LCFA might already be present in the sewage itself. In total, approximately 30 dierent LCFA peaks were detected, whereas only nine were identied. The total concentrations of the unidentied LCFA were estimated to be 1520% of the total LCFA for each SRT. It is found that the main saturated and unsaturated LCFA in both the raw primary sludge and the digested primary sludge were C16:0, C18:0 and C18:1, similar to the results of Quemeneur and Marty (1994). The percentage of hydrolysis remarkably diers between the inuent primary sludge and the sludge digested at an SRT of 3 days. The limited increase of the hydrolysis between an SRT of 3 and 8 days may result from the high LCFA concentrations (1.61.8 g/l), causing product inhibition (Hanaki et al., 1981) or physical hindrance due to adsorption. At an SRT of 3, 5 and 8 days, accumulation of LCFA occurred. Apparently, under acidogenic conditions, b oxidation of LCFA is the rate limiting step as compared to hydrolysis, and at 15 days SRT, lipids hydrolysis becomes rate limiting. Figure 4 presents the removal eciencies of the LCFA as a function of the SRT. Negative removal eciency means accumulation of LCFA in the CSTR and vice versa. The degree of accumulation of saturated LCFA at SRT R 8 days, increases with increasing SRT. SRT=10 days can be considered as a ``transient state'' between no LCFA conversion and a complete LCFA conversion. At SRT=10 days, a decrease of the removal eciency is shown with the increase of the chain length from C12:0 to C18:0. Actually, no removal but accumulation of C18:0

Fig. 1. pH, percentage total hydrolysis, total acidication, and methanogenesis of the inuent COD, as a function of SRT in the anaerobic digestion of primary sludge at 258C in a CSTR. SRT=0 days stands for the inuent.

Digestion of primary sludge in CSTR system

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Fig. 2. The various fractions of the COD as a function of SRT in the anaerobic digestion of primary sludge at 258C in a CSTR. Particulate biopolymers include proteins+carbohydrates+lipids and hydrolysates include LCFA+amino acids+simple sugars.

occurs under these conditions. The latter is in agreement with the results of Novak and Carlson (1970), as is the higher removal eciency of C18:2 over C18:1 at each applied SRT. Apparently, the degree of unsaturation, accelerates the degree of degradation. C16:1, C18:1, and C18:2 were the only LCFAs that were removed under acidogenic conditions, similar to the results of Heukelekian and Mueller (1958). Possibly this removal did not proceed via b oxidation but by hydrogenation, stimulated by the elevated hydrogen pressure. Consequently, these unsaturated LCFA are converted to saturated LCFA with the same number of C atoms (Kemp et al., 1975) and accumulate due to the high hydrogen pressure. Therefore, unsaturated LCFA can be considered as an electron sink when interspecies hydrogen transfer does not take place.

Proteins and carbohydrates Protein hydrolysis and acidication are given in Fig. 5. The decrease in hydrolysis and acidication under acidogenic conditions, indicated by a decrease in the NH+ 4 N concentration in comparison with the inuent, demonstrates that no hydrolysis and acidication take place, except for a sharp increase between an SRT of 8 and 10 days, stressing the eect of acidogenic/methanogenic conditions. The low values obtained for hydrolysis and acidication can be partially explained by the relatively high NH+ 4 N concentration in the inuent (Table 1), suggesting that easy degradable protein was already hydrolysed before the sludge was used in our research. Also, the low pH and high lipid concentration could aect the hydrolysis (PalenzuelaRollon, 1999). Moreover, precipitation of ammonium as struvite (MgNH4PO46H2O) (Mamals et al., 1994) may contribute to the decrease of the NH+ 4 N levels, which results in a lower calculated hydrolysis. The lower % acidication as compared to hydrolysis, as indicated in Fig. 5, shows that not only the hydrolysis of proteins but also the acidication of amino acids is limited at SRT R 10 days. The amino acid concentration varies between 174 220 mg/l, which could aect the protein hydrolysis. Sanders (personal communication) found inhibition of gelatine hydrolysis at amino acid concentrations exceeding 200 mg/l. Under methanogenic conditions hydrolysis of proteins is clearly the rate limiting step. The results in Fig. 6 illustrate that hydrolysis of complex carbohydrates continuously increases with increasing SRT, with the largest dierence between inuent primary sludge and sludge digested at SRT=3 days. The results show that the dissolved carbohydrates concentration is relatively constant, between 95192 mg COD/l, which is ca. 1.5% of

Fig. 3. Percentage lipid hydrolysis and acidication of C0,lipid as a function of SRT, at the anaerobic digestion of primary sludge at 258C in a CSTR. SRT=0 days stands for the inuent.

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Fig. 4. Removal eciencies of each LCFA in the inuent, in relation to its concentration in the euent, at dierent SRTs at the anaerobic digestion of primary sludge at 258C in a CSTR.

the total carbohydrates in the inuent at all applied conditions. Therefore, hydrolysis of carbohydrates and not acidication can be considered as the rate limiting step at each SRT. Hydrolysis rst order kinetics? Hydrolysis constants of lipids and carbohydrates (Table 2) were calculated according to Eq. (13), following the method used by Eastman and Ferguson (1981). However, a poor correlation was observed (R 2 < 0.78). Since no hydrolysis of proteins was calculated under acidogenic conditions (Fig. 5), no hydrolysis constant could be calculated. Our results reveal that there is no sense to calculate the hydrolysis constant for total hydrolysis of primary sludge, as is often made in the literature (Pavlostathis and Giraldo-Gomez, 1991). Hydrolysis of none of the components of the primary sludge follows rst order kinetics neither under acidogenic, nor under methanogenic conditions. Eastman and Ferguson (1981) proposed that under acidogenic conditions, rst order kinetics is the most appropriate empirical hydrolysis function for complex heterogeneous substrates such as primary sludge whereas other hydrolysis functions

may be more appropriate for single homogeneous substrates. However, the calculation of the hydrolysis constant in their study was only made for the combined fraction of carbohydrates and proteins, omitting the lipid fraction. Even when Kh could be calculated with a good correlation, it can not serve as a ``universal'' constant, since it is not more than a specic calculation for a given substrate under certain conditions. Considering the various steps in the anaerobic digestion, hydrolysis is still the less dened step. It is already known that factors such as specic surface area, particle shape, and particle size distribution all strongly aect hydrolysis (Hills and Nakano, 1984; Hobson, 1987). According to Hobson (1987) it would be more appropriate to describe hydrolysis based on available surface area of the substrate instead of the concentration of the substrate. Dewaterability Figure 7 shows the lterability constants and capillary suction time (CST) of the inuent and the digested sludges, as a function of applied SRT in the CSTRs. The lterability constant decreases towards a minimum between an SRT of 3 and

Fig. 5. Percentage protein hydrolysis and acidication of the Nkj 1.5/0.16 as a function of SRT, at digestion of primary sludge at 258C in a CSTR. SRT=0 days stands for the inuent.

Fig. 6. Percentage carbohydrates hydrolysis of the fraction (C0,carbh+VFA0 NH+ 4 N 1.5/0.16) as a function of SRT, at digestion of primary sludge at 258C in a CSTR. SRT=0 days stands for the inuent.

Digestion of primary sludge in CSTR system


Table 2. Hydrolysis constants calculated according to the linear equation for SRT 38 and 315 days SRT (days) Kh Carbohydrates Lipids 0.428 0.806 38 R2 0.6523 0.7846 Kh 0.153 0.842 315 R2 0.7276 0.401

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5 days and increases at SRT > 5 days. Only at an SRT of 15 days, a higher lterability constant is achieved in comparison with the inuent. The lterability constants were tted with a polynomial curve y = 0.0002x 20.0029x + 0.0227, R 2=0.9859. Since the lterability constants are calculated from the reciprocal CST value (Vesilind, 1988), the opposite trend was shown for the CST as a function of the SRT. The conclusion from both lterability constants and CSTs is that, compared to the dewaterability of the inuent, the dewaterability of the sludges worsens under acidogenic conditions, while it improves under methanogenic conditions. No signicant dierence in dewaterability is shown between an SRT of 3 and 8 days. These results are in agreement with Lawler et al. (1986) who found that under acidogenic conditions the total surface area increases as a result of the decrease in the mean particle size, with concomitant worsened dewaterability. However, under methanogenic conditions, the total surface area decreases substantially and the dewaterability improves, probably due to decrease of interstitial and vicinal water in the sludge (Vesilind, 1994).
FINAL DISCUSSION

This study shows that the SRT strongly aects the type and rate of bioconversion processes under anaerobic conditions. The results can be applied to one- or two-step sludge digestion in CSTR systems (Ghosh, 1987) and to occulent sludge UASB reactors treating domestic sewage at 258C, considering its sludge-bed or a sludge bed segment as a CSTR.

For a UASB maintained at SRT R 8 days, acidogenic conditions prevail resulting mainly in degradation of carbohydrates. LCFA, when not removed with the excess sludge, and amino acids will thus have to be degraded in a successive methanogenic reactor. Although VFA were found to accumulate and lower the pH in the acidogenic CSTRs, this will not occur in a UASB due to dilution of the sludge-bed by the inuent with a high buer capacity (Wang, 1994). Excess sludge of such a UASB is not stabilised, but the results show that poststabilisation of the sludge in a CSTR at 258C and an SRT of at least 15 days will stabilise the sludge, recover the potential energy and improve the sludge dewaterability. Under tropical conditions, with a more or less constant high ambient and sewage temperature, a two step UASB system is less attractive in comparison with a single step system since both methanogenesis and hydrolysis occur at a relatively short SRT and therefore a short HRT can be applied. For a UASB operated at an SRT of 15 days at 258C, methanogenic conditions will develop, and carbohydrate as well as lipid and protein hydrolysis will take place resulting in more than 50% of the inuent COD to be converted to methane gas. Under these conditions, the ultimate choice for the HRT will depend on the inuent SS concentration, removal and biodegradability (Zeeman and Lettinga, 1999). At temperatures of <158C, long sludge retention times (>100 days) and therefore longer HRTs are necessary in order to provide sucient methanogenesis and hydrolysis (Zeeman and Lettinga, 1999). Therefore, when low ambient and process temperatures prevail during the whole year or in part of it, e.g. under climatic conditions with high summer and low winter temperatures, such as in the Middle East region, a twostep UASB-system is much more attractive. The latter guideline is especially indicated when high SS concentrations occur in the inuent, since a better SS removal is achieved in the rst step of a twostep as compared to a one-step UASB-system (Wang, 1994). As reactors should be dimensioned

Fig. 7. Filterability constants (Vesilind, 1988) and CST of the inuent and digested sludges as a function of SRT in the anaerobic digestion of primary sludge at 258C. SRT=0 days stands for the inuent.

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Yehuda Miron et al. Cohen, A. (1982) Optimisation of anaerobic digestion of soluble carbohydrates containing wastewater by phase separation. Ph.D. thesis. University of Amsterdam, Amsterdam. Eastman J. A. and Ferguson J. F. (1981) Solubilisation of particulate organic carbon during the acid phase anaerobic digestion. JWPCF 53(3), 352366. Ghosh S. (1987) Improved sludge gasication by twophase anaerobic digestion. J. Environ. Eng. 113(6), 12651284. Hanaki H., Matsuo T. and Nagase M. (1981) Mechanism of inhibition caused by long chain fatty acids in anaerobic digestion processes. Biotechnol. Bioeng. 23, 1591 1610. Heukelekian H. and Mueller P. (1958) Transformation of some lipids in anaerobic sludgedigestion. Sew. Ind. Waste 30(9), 11081120. Hills D. J. and Nakano K. (1984) Eects of particle size on anaerobic digestion of tomato waste. Agric. Waste, 285295. Hobson P. N. (1987) A model of some aspects of microbial degradation of particulate substrates. J. Ferm. Technol. 65(4), 431439. Kaluzny M. A., Duncan L. A., Merrit M. V. and Epps D. E. (1985) Rapid separation of lipids classes in high yield and purity using bonded phase columns. J. Lipid Res. 26, 135140. Kemp P., White R. W. and Landler D. J. (1975) The hydrogenation of unsaturated Fatty Acids by ve bacterial isolates from the sheep rumen, including a new species. J. Gen. Microbiol. 90, 100114. Knechtel J. R. (1978) A more economical method for the determination of chemical oxygen demand. JWPCF 166, 2529. Lawler D. F., Chung Y. J., Hwang S. J. and Hull B. A. (1986) Anaerobic digestion: eect on particle size and dewaterability. JWPC 58(12), 11071117. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265275. Mamals D., Pitt P. A., Cheng Y. W., Loianco J. and Jenkins D. (1994) Determination of ferric chloride dose to control struvite precipitation in anaerobic sludge digesters. Water Environ. Res. 66, 912918. McInerney M. J. (1988) Anaerobic hydrolysis of fats and proteins. In Biology of Anaerobic Micro-Organisms, ed. A. J. B. Zehnder, pp. 373415. John Wiley and Sons, New York. Nagase M. and Matsuo T. (1982) Interactions between amino acid-degrading bacteria and methanogenic bacteria in anaerobic digestion. Biotechnol. Bioeng. 24, 22272239. Novak J. T. and Carlson D. (1970) The kinetics of anaerobic long chain fatty acids degradation. JWPCF 42(2), 19321943. Palenzuela-Rollon, A. (1999) Anaerobic digestion of sh wastewater with special emphasis on hydrolysis of suspended solids. Ph.D. thesis, Agricultural University, Wageningen. Pavlostathis S. G. and Giraldo-Gomez E. (1991) kinetic of anaerobic treatment. Water Sci. Technol. 24(8), 3559. Quemeneur M. and Marty Y. (1994) Fatty acids and sterols in domestic wastewater. Water Res. 28(5), 1217 1226. Sanders W. T. M., Van Bergen D., Buijs S., Corstanje R., Gerrits M., Hoogerwerf T., Kanwar S., Zeeman G., Van Groenestijn J. and Lettinga G. (1996) Treatment of waste activated sludge in an anaerobic hydrolysis upow sludge bed reactor. In EWPCA Symposium ``Sludge Treatment and Reuse''. 79 May, Mu nchen, Germany. Sayed S. K. I. (1987) Anaerobic treatment of slaughterhouse wastewater using the UASB process. Ph.D. thesis,

on the lowest temperature, a two step system is to be advised as it can be operated at short total HRTs. Our results can also be applied to predict the conversions in the rst step of a two-step UASB-system under summer conditions. In Jordan, the rst two-step UASB pilot reactor (100 m3) is under research.
CONCLUSIONS

. At digestion of primary sludge in a CSTR at 258C, protein hydrolysis is aected by acidogenic and methanogenic conditions, whereas carbohydrate and lipid hydrolysis are not. . Nor lipid and protein hydrolysis, neither carbohydrate hydrolysis follow rst order kinetics at digestion of primary sludge in a CSTR at 258C. . At digestion of primary sludge in a CSTR at 258C and SRTR8 days, acidogenic conditions prevail and hardly any biogas is produced:
*

about 20% of the COD is present in a hydrolysed form; the strongest increase in hydrolysis and acidication of total COD occurs between the inuent primary sludge (SRT=0 days) and SRT=3 days; hydrolysis is the rate limiting step for conversion of carbohydrates; acidication is the rate limiting step for conversion of lipids; both hydrolysis and acidication are limiting for the conversion of proteins.

. During digestion of primary sludge in a CSTR at 258C and SRT=10 days, LCFAs are only partly acidied. . During digestion of primary sludge in a CSTR at 258C, at SRT>10 days, methanogenic conditions prevail: * Hydrolysis is rate limiting for all particulate substrates.

Acknowledgements We thank Wendy Sanders for critical review and discussions. The research was carried out within the framework of the AVICENNE project AVICT0009, nanced by the EU. We acknowledge NUFFIC, The Netherlands, for the scholarship given to the rst author.
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