Bioresource Technology 96 (2005) 13171322

Eect of prefermentation on denitrifying phosphorus removal in slaughterhouse wastewater

M. Merzouki
a

a,b

, N. Bernet

a,*

` , J.P. Delgenes a, M. Benlemlih

Institut National de la Recherche Agronomique, Laboratoire de Biotechnologie de lEnvironnement, Avenue des Etangs, 11100 Narbonne, France b Laboratoire de Microbiologie de lEnvironnement, Faculte des Sciences Dhar Mehraz, B.P. 1796, Fes, Morocco Received in revised form 3 November 2004; accepted 23 November 2004 Available online 20 January 2005

Abstract An anaerobicanoxic sequencing batch reactor (A2 SBR) coupled with a xed-bed nitrication reactor for simultaneous carbon, nitrogen and phosphorus removal was evaluated using slaughterhouse wastewater. Whereas the treatment could not be successfully carried out on the raw wastewater, the process showed very good nutrient removal performances after prefermentation. The removals of COD, N-NH4 and P-PO4 achieved were 99%, 85% and 99%, respectively. The increase in volatile fatty acid (VFA) and phosphate concentrations in the euent after prefermentation may explain the high levels of biological carbon, nitrogen and phosphorus removal observed. A simple prefermentation is, therefore, necessary but sucient to ensure good performances of the denitrifying enhanced biological phosphorus removal (EBPR) process. 2004 Elsevier Ltd. All rights reserved.
Keywords: Anoxic phosphorus removal; Enhanced biological phosphorus removal (EBPR); Sequencing batch reactor (SBR); Fixed-bed nitrication; Prefermentation; Slaughterhouse wastewater; Volatile fatty acids (VFA)

1. Introduction Enhanced biological phosphorus removal (EBPR) is based on the selective enrichment of bacteria accumulating polyphosphate, obtained in a process with a cycle of alternating anaerobic and aerobic phases. As an alternative to this process for biological phosphorus removal, the anaerobicanoxic sequencing batch reactor (A2 SBR) process has been recently proposed by dierent researchers (Vlekke et al., 1988; Wanner et al., 1992; Kuba et al., 1993; Bortone et al., 1994; Sorm et al., 1996). The A2 SBR is an attractive process because of the saving of organic matter and energy ascribed to aeration and low sludge production for complete phosphorus and nitrogen removal (Kuba et al., 1993, 1994, 1996). DurCorresponding author. Tel.: +33 468 425 151/747; fax: +33 468 425 160. E-mail address: bernet@ensam.inra.fr (N. Bernet). 0960-8524/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.11.017
*

ing the anaerobic period, the polyphosphate-accumulating organisms (PAO) take up carbon sources, preferably volatile fatty acids (VFA), and store them as polyhydroxyalkanoates (PHA). The degradation of polyphosphate causes a release of phosphate into the bulk liquid, and a production of energy necessary for anaerobic uptake of carbon sources and their conversion into PHA. During the subsequent aerobic or anoxic phase, PAO take up phosphate from the bulk liquid and store it in the form of polyphosphate, while PHA is used as a carbon and energy source (Jenkins and Tandoi, 1991). The phosphorus uptake under anoxic conditions is attributed to the activity of denitrifying phosphorus accumulating organisms (DNPAO) capable of accumulating high amounts of polyphosphate (Mino et al., 1995). The phosphorus and nitrogen are, therefore, removed simultaneously by these organisms. This is one of the main advantages of applying the A2 SBR process for biological nutrient removal.

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The concept of biological phosphorus removal under alternating anaerobic and anoxic conditions combined with a xed-bed nitrication reactor was proposed for the rst time by Wanner et al. (1992) and Jenicek et al. (1993). This innovative process, called DEPHANOX by Bortone et al. (1996), showed very good performances for phosphorus and nitrogen removal from municipal wastewater. Many industries, such as food industries including slaughterhouses, produce large quantities of wastewater containing high concentrations of organic matter (COD), suspended solids, nitrogen and phosphorus compounds. In the present work, an integral process for simultaneous carbon, nitrogen and phosphorus removal was proposed, to be applied to industrial wastewater. This process is an A2 SBR coupled with a xedbed nitrication reactor. The feasibility and stability of this combined system were investigated while treating a slaughterhouse wastewater.

(1) 1.5 h anaerobic phase. At the beginning of this phase, 0.75 l wastewater was supplied to the reactor, which contained 0.75 l mixed liquor initially, over 7 min. (2) 0.5 h rst settling and feeding period. After 22 min of settlement, 1 l anaerobic supernatant (NH and 4 PO3 rich) was directly fed into the xed-bed 4 reactor. (3) 3.5 h anoxic phase. One litre nitrate and phosphate rich euent from the xed-bed reactor (stored in an exchange tank, see Section 2.3) was sent to the A2 SBR over 3 h at a ow-rate of 5.6 ml min1 (fedbatch mode). At the end of the anoxic phase, the excess sludge was removed to control the sludge retention time at 15 days. (4) 0.5 h second settling and feeding period. After 22 min of settlement, 0.75 l treated euent was pumped out from the reactor. The process was fed initially with a synthetic wastewater for 8 months. The composition of the synthetic wastewater was adapted from Kuba et al. (1993) with higher acetate, ammonia and phosphate concentrations to simulate an industrial wastewater. Then it was fed with a slaughterhouse wastewater, before and after acidogenesis (Table 1). 2.3. Fixed-bed nitrication reactor The nitrication was carried out in a xed-bed column reactor (total volume: 6.8 l, active volume: 4.8 l, total height: 75 cm, bed height: 53 cm) containing pozzolana as a medium. The grains of carrier had an average diameter of 3.6 mm, a density of 2000 kg m3 and a porosity of 0.52. The temperature of the reactor was kept constant at 20 C by a water jacket. pH was controlled at 7.4 by the addition of 1 N NaOH. The hydraulic retention time was 0.96 day. The oxygen necessary

2. Methods 2.1. Experimental set-up The treatment process used in the present work is schematised in Fig. 1. A SBR was operated for simultaneous denitrication and enhanced biological phosphorus removal (anaerobicanoxic SBR, A2 SBR). The SBR was combined with a xed-bed reactor which was operated under aerobic conditions for nitrication. 2.2. Anaerobicanoxic SBR (A2 SBR) Phosphorus removal was carried out in the A2 SBR inoculated with sludge from an activated sludge treatment plant (Roanne, France). The working volume of the SBR was 1.5 l. pH value in the SBR was strictly controlled to 7 0.1 by the addition of 0.5 N H2SO4 or NaOH. The overall hydraulic retention time was 12 h. The A2 SBR was operated in a cycle of 6 h which consisted of four phases:

Table 1 Composition of the raw slaughterhouse euent used Averagea pH CODt (g l1) CODs (g l1) TOCs (g l1) VFAt (mg l1) Acetate Propionate Isobutyrate Butyrate Valerate N-NH4 (mg l1) NTKs (mg l1) P-PO4 (mg l1) TSS (g l1)
a

Standard deviation 0.08 4.19 3.23 0.77 34 26 9 0 0 10 196 5 0.59

NITRIFICATION NH4
+

Aerobic

7.29 7.78 6.21 1.46 117 48 6 0 0 88 410 18 0.87

Fixed bed

NO3

PHOSPHORUS REMOVAL + DENITRIFICATION

Influent

Anaerobic Anoxic

Effluent

A2SBR

Fig. 1. Scheme of proposed carbon, nitrogen and phosphorus removal process.

On four samples within a period of 3 months.

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Anoxic phase 12 10 8
P-PO4 (mg l )
-1

for nitrication was provided by sparging air at the bottom of the column at a ow rate of 200 l h1. The reactor was continuously fed with a synthetic wastewater before coupling with the SBR, and then with anaerobic supernatant (NH and PO4 rich) from the 4 A2 SBR. The hydraulic retention time (HRT) of the reactor was 0.63 days. The nitrate and phosphate rich euent produced in this reactor was kept refrigerated in an exchange tank to be supplied to the A2 SBR during the anoxic phase. 2.4. Feasibility and stability of the two reactors The A2 SBR and the xed-bed nitrication reactor were operated separately before coupling. The combined process was then operated for 11 months, in order to investigate the feasibility and stability of the system with respect to carbon, phosphorus and nitrogen removal in the synthetic and slaughterhouse wastewater. The process was operated for 231 days with a synthetic wastewater; this was then substituted with slaughterhouse wastewater for 100 days. In this paper, the results obtained during the latter period are presented. 2.5. Analytical methods Phosphate, nitrate, nitrite and ammonium analyses were carried out using an ion exchange chromatography system (Dionex-100). Volatile fatty acids (VFAs) were measured with a ame ionisation detector gas chromatograph, Chrompack CP 9050. Total Kjeldahl nitrogen (TKN) was determined by the titrimetric method after mineralisation and distillation using a Buchi appa ratus. Total and soluble chemical oxygen demand (CODt and CODs, respectively) were measured by the potassium dichromate-ferrous ammonium sulphate method (APHA, 1992). Sludge total phosphorus content was determined after perchloric acid digestion (APHA, 1992). Total suspended solids (TSS) and volatile suspended solids (VSS) were determined after centrifugation (APHA, 1992).

Anaerobic Settling 250

N-NH4 and VFA (mg l )

200 150

-1

6 100 4 50 0 0 1 2 3
Time (hours)

2 0 4 5 6

Fig. 2. Cycle variation of P-PO4 (), N-NH4 (s), acetate (m), propionate () and isobutyrate ( ) concentrations in the A2 SBR fed with slaughterhouse raw euents.

3. Results and discussion 3.1. Treatment of raw slaughterhouse euents The treatment of synthetic industrial wastewater in the A2 SBR combined with a xed-bed reactor showed good performance. Acetate, N-NH4 and P-PO4 removal eciencies achieved in the A2 SBR combined were up to 99%, 90% and 98%, respectively (not shown). Thereafter, the process was fed with a slaughterhouse wastewater whose characteristics are presented in Table 1.

An example of variations of VFA, N-NH4 and P-PO4 concentrations in the A2 SBR during a 6 h cycle is shown in Fig. 2. It can be seen that neither phosphorus release nor VFA uptake were observed during the anaerobic phase. Therefore, luxury P uptake could not be observed during the anoxic phase (2.3 mg P g VSS1). As a consequence, the phosphorus content of the sludge (Ps) came down to 1.4% TSS. The absence of P release can be explained by the absence of VFA consumption by PAO. VFA were completely consumed in the anoxic phase by denitrication and in the aerobic lter. Ammonia was mainly consumed by assimilation but also by nitrication. During the one month period of raw euent treatment, ammonia and phosphorus removal performances were low, 48.5% and 47%, respectively (Table 2). The absence of anaerobic VFA uptake due to the low VFA (Table 1) concentration in the slaughterhouse euent caused the failure of enhanced biological phosphorus removal. VFA production during the anaerobic phase seemed to have an inhibiting eect or to compete with P release. Previous workers found that biological nitrogen and phosphorus removal eciencies in A/O SBR were higher when the wastewater contained relatively high nitrogen and phosphorus concentrations and a high readily biodegradable chemical oxygen demand (RBCOD) such as VFA (Keller et al., 1997; Randall et al., 1997; Mazlum and Mazlum, 2000). In order to recover EBPR activity in the A2 SBR, the slaughterhouse wastewater was stored at ambient temperature in a 60 l tank to be fermented. The aim of this prefermentation was to increase the amount of RBCOD as VFA and to increase the phosphate concentration by releasing phosphorus from organic compounds in the euent. This prefermentation has been shown to enhance aerobic EBPR using synthetic substrate (Randall et al., 1997), septic domestic wastewater (McCue et al., 2003) or slaughterhouse wastewater (Subramaniam et al., 1994). No results have been reported in the case of anoxic EBPR.

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Table 2 Average values of phosphorus release and uptake, COD and VFA consumption in the A2 SBR fed with slaughterhouse raw wastewater and prefermented wastewater Raw wastewater Averagea Anaerobic COD consumption (mg C g VSS ) Anaerobic VFA consumption (mg C g VSS1) Anaerobic P release (mg P g VSS1) Anoxic P uptake (mg P g VSS1) Ps (% TSS) COD removal (%) N-NH4 removal (%) P-PO4 removal (%) TSS (g l1) VSS/TSS
a b 1

Prefermented wastewater min.max. 032.7 0 00.15 02.3 11.7 6393.6 1673 1386 4.34.74 0.7900.883 Averageb 104 68.3 4.43 7.74 4 94 77 97 3.45 0.87 min.max. 25193 3589 3.225.90 6.708.80 34.6 9099 6785 8699 3.033.77 0.7710.937

14.46 0 0.06 0.68 1.4 83 48.5 47 4.53 0.835

Calculated on 6 cycle analyses. Calculated on 10 cycle analyses.

3.2. Treatment of slaughterhouse wastewater after prefermentation Evolution of the euent composition during 35 days of anaerobic storage is presented in Fig. 3. After 35 days of storage, about 75% of soluble COD was in the form of VFA, mainly acetate but also propionate, butyrate, isobutyrate and isovalerate. A 30% increase in phosphate concentration was observed as a result of conversion of organic-P. Total and soluble COD removals during the prefermentation period were around 65% and 83%, respectively. This abatement of COD was due to the transformation of a part of organic compounds into VFA and CO2, and to bacterial growth which could explain an increase of TSS from 2 to 2.5 g l1 during the acidication period. The P/CODs ratio increased from 0.0022 to 0.0172. Maximal VFA concentration (3550 mg l1 as COD) was obtained after 14 days of prefermentation at a
900 800
P-PO4, N-NH4 and TKN (mg l )

16000 14000 12000 10000

CODt, CODs and VFA-COD (mg l-1 )

P/CODs ratio of 0.0072. Therefore, it was decided to carry out a 14 day acidication storage of the wastewater as a pretreatment before feeding. The process was operated for 70 days using this prefermented slaughterhouse wastewater. Fig. 4 shows variations of COD, N-NH4 and P-PO4 in the A2 SBR after 26 days. Phosphorus was completely removed in the anoxic phase with an uptake rate of 8 mg P g VSS1. Phosphorus was released in the anaerobic phase with a release rate of 3.7 mg P g VSS1. VFA were consumed in this phase with a consumption rate of 78 mg C g VSS1, or 26 mg acetate g VSS1 h1. This rate was higher than that reported by Bortone et al. (1994), who found that acetate consumption rate varied from 5.5 to 12 mg g VSS1 h1 in the A2 SBR coupled with a batch biolm reactor for nitrication. The CODs, VFA and N-NH4 removal eciencies in the SBR during this cycle were 95%, 97% and 67%, respectively. Prefermentation had the expected eect on the process with the complete restoration of the enhanced biological phosphorus removal in the SBR as shown in Fig. 5. From day 30 and the use of prefermented wastewater, P release concentration increased and varied from 18 to
Anaerobic Settling
N-NH 4, VFA and CODs (mg l )
-1

-1

700 600 500

Anoxic phase 30 25 20 15 10 5 0
P-PO4 (mg l )
-1

1200 1000 800 600 400 200 0 0 1 2 3 Time (hours) 4 5 6

8000 400 300 200 100 0 0 5 10 15 20 Time (days) 25 30 35 6000 4000 2000 0

Fig. 3. Variations of slaughterhouse euent during prefermentation: CODt (j), CODs (h), VFA-COD (), TKN ( ), N-NH4 (s), P-PO4 ().

Fig. 4. Cycle behaviour of CODs (h), VFA (m) N-NH4 (s) and P-PO4 () concentrations in the A2 SBR fed with prefermented slaughterhouse euents.

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35 30 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 100

1321

Raw Fermented wastewater wastewater

tion reactor could be successfully operated, but only after prefermentation of the wastewater to increase VFA concentration and P/COD ratio. Indeed, the conversion of fermentable COD to VFA before utilisation was necessary to maintain enhanced phosphorus removal activity. A complete P removal was observed for 50 days. COD and N-NH4 removal rates reached 99% and 85%, respectively, during the same period. Therefore, prefermentation is a suitable approach in the application of denitrifying EBPR to industrial wastewater containing mainly fermentable organic substrates.

P-PO4 (mg l )

-1

Time (days)

Fig. 5. Daily variations of phosphorus at the end of anaerobic (j) and anoxic (h) phases in the A2 SBR fed with slaughterhouse euent without and with prefermentation.

Acknowledgements The authors thank the International Fund of University Cooperation (FICU) from the University Agency of the French-speaking world (AUF).

30 mg P l1, and P concentration at the end of the anoxic phase decreased and varied from 0 to 3 mg l1. This was due to the increase of VFA and P concentrations in the pre-treated euent and to the consumption of VFA in the anaerobic phase. Prefermentation of the slaughterhouse wastewater increased the P/CODs ratio. Consequently, the P uptake increased, resulting from the increase of the number of PAO in sludge. These results agreed closely with a previous study (Merzouki et al., 2001), and with those found in the literature using A/O systems (Satoh et al., 1992; Liu et al., 1997), suggesting that the EBPR increased with the P/C feeding ratio. The increase of P concentration in the wastewater allowed the increase of P uptake by the DNPAO in the form of poly-P granules. This intracellular stock of polyP in high quantity was used as energy for VFA consumption and P release in high concentrations under anaerobic conditions. Average performances of the system for COD, N-NH4 and P-PO4 removal were 94%, 77% and 97%, respectively (Table 2). Total P removal could be achieved during the last 50 days of steady state operation. The phosphorus sludge content increased from a mean value of 1.44% of TSS. Likewise, the VFA consumption rate increased and varied from 35 to 89 mg C g VSS1, which allowed the P release during the anaerobic phase with an average rate of 4.43 mg P g VSS1 (Table 2). However, the P release decreased between days 65 and 72, and P was incompletely removed at day 72 (92%). This can be explained by a particularly low P concentration in the wastewater (12 mg l1), while this was usually between 20 and 38 mg l1.

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4. Conclusions A process consisting of a A2 SBR fed with slaughterhouse wastewater and coupled to a xed-bed nitrica-

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