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Wat. Res. Vol. 34, No. 9, pp. 24132422, 2000 7 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter

NITROGEN REMOVAL IN A MODIFIED ANAEROBIC BAFFLED REACTOR (ABR): 1, DENITRIFICATION

WILLIAM P. BARBERM and DAVID C. STUCKEY*M
Department of Chemical Engineering and Chemical Technology, Imperial College of Science, Technology, and Medicine, Prince Consort Road, London SW7 2BY, UK (First received 1 July 1999; accepted 1 December 1999) AbstractIn order to achieve nitrogen removal within a single reactor unit, an eight compartment anaerobic baed reactor (ABR) was modied to accommodate an in-situ aerobic stage in the penultimate compartment, which allowed ammonia oxidation to nitrite and nitrate via nitrication. In theory, the nitrite/nitrate may be recycled to the inlet of the ABR and be reduced via denitrication. This paper deals with the anoxic denitrication of a nitrate feed, while a later paper examines nitrication in the aerobic stage. Denitrication occurred almost exclusively in the front two compartments of the anaerobic baed reactor, with rates of 0.335 (82% reduction) and 0.085 kg NO3/ kg VSS.d (96% reduction) in compartments 1 and 2, respectively. Denitrication had several positive eects on overall reactor performance, and this was due to the following factors: the utilisation of an oxidisable electron donor in the form of the feed COD; increased system pH at the reactor inlet thus improving environmental conditions; a high hydrogen demand during dissimilatory nitrate reduction to ammonium, therefore improving conditions for syntrophic bacteria; and, the generation of ammonium from dissimilatory reduction which provided slowly growing bacteria at the front of the ABR with a reduced nitrogen source. The overall eect of these inuences was improved hydrogenotrophic methanogenesis. Denitrication also increased the residual COD in the rst two compartments, and this was attributed to faster growth/decay rates of denitrifying bacteria. Nitrate reduction also inuenced the ratio of volatile fatty acids produced and catabolised, with a signicant reduction in propionate and butyrate, while acetate levels increased. 7 2000 Elsevier Science Ltd. All rights reserved Key wordsanaerobic baed reactor, denitrication, dissimilatory nitrate reduction to ammonium, nitrogen removal, residual COD, VFAs

INTRODUCTION

Nitrogen exists in the environment in several oxidation states, with ammonia, nitrate and diatomic nitrogen gas being the main forms, and the rst two can cause severe environmental problems. Nitrates and phosphates can stimulate eutrophication where pollution is caused in waterways by heavy algal growth, as they are both rate-limiting nutrients for the process (Barnes and Bliss, 1983). Nitrate contaminated water supplies have also been linked to outbreaks of infant disease (Sawyer et al., 1994). Ammonia is extremely toxic to mammals (0.2 mg/l of ammonia is harmful to a variety of aquatic life Sawyer et al., 1994), and can be rapidly oxidised by nitrifying bacteria, thus depleting the aquatic oxygen supply. Nitrogen compounds are found in high concentrations in the wastewaters of many industries, while the average nitrogen composition in domestic sewage is approximately 60% ammonia,
*Author to whom all correspondence should be addressed. Tel.: +44-171-594-5591; fax: +44-171-594-5629; email: d.stuckey@ic.ac.uk

40% organic nitrogen, with trace amounts of nitrite and nitrate (less than 1%Barnes and Bliss, 1983). Traditional methods for removing nitrogen from wastewaters include: dilution of the wastewater; pre-treatment; addition of magnesium and phosphorous to induce struvite precipitation; air stripping; breakpoint chlorination; and ion exchange (Metcalf and Eddy, 1991). However, these methods are costly, require the addition of chemicals, and may release toxic compounds to the environment, and have therefore led to the development of microbiological techniques. Biological removal of nitrogen involves a combination of a two stage aerobic anoxic process which incorporates: (a) the bacterial oxidation (nitrication ) of ammonia to nitrite and then nitrate; and (b) the microbiological reduction (denitrication ) of the nitrite/nitrate to gaseous nitrogen compounds (Cole, 1994). Denitrication may be dened as a process whereby nitrogen oxyanionsnitrates, nitrites, nitrous oxide, nitrogen dioxide and nitric oxide are reduced to gaseous nitrogen. There are three types of microbial nitrate reduction (Tiedje, 1988). These are: (1) denitrication; (2) nitrate assimila-

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tion; and (3) dissimilatory nitrate reduction to ammonium (DNRAStouthamer, 1988), which is the reverse of nitrication (Cole, 1994) shown in Eqs 1 (denitrication/nitrate assimilation) and 2 (DNRA), respectively: 5organic-C 2H2 O 4NO 4 3 2N2 4OH 5CO2 560 kJamol N 1

NO 2H 4H2 4 NH 3H2 O 600 kJamol N 3 4 2 Equation 1 is catalysed by both heterotrophic and autotrophic micro-organisms, which use nitrate in a respiratory mechanism to replace oxygen in conditions of low oxygen availability, thus requiring an oxidisable substrate as an energy source (Winkler, 1981). Denitrication proceeds in four stages with the chemical intermediates being nitrite, nitric oxide and nitrous oxide (Cole, 1994). Accumulation of intermediate products, such as nitrite, may be due to competition during the dierent stages of denitrication, or a lack of appropriate enzymes (Cole, 1994). Oxygen is a strong suppresser of denitrication; aerobic denitrication rates are only 0.33% of the anoxic rate (Tiedje, 1988). Nitrogen removal via nitrication and denitrication is possible by combining aerobic and anoxic units with recycle (Horan, 1996); however, anoxic ammonium oxidation may become a promising alternative (van de Graaf et al., 1996). Potentially, the costs involved with having separate anaerobic and aerobic reactors can be greatly decreased by modifying an anaerobic baed reactor (ABR). The ABR is a high rate bioreactor which has numerous advantages over other reactors and these include; better resilience to hydraulic and organic shock loadings, longer biomass retention times, lower sludge yields, and the ability to partially separate the various phases of anaerobic catabolism (Barber and Stuckey, 1999a). The latter property causes a shift in bacterial populations allowing increased protection against toxic materials (such as ammonia), and a higher resistance to changes in environmental parameters such as pH and temperature. The implications of these advantages mean that nitrogen removal is possible within one reactor unit. Two unit systems generally consist of an initial aerobic stage to reduce ammonia to acceptable levels before entering an anoxic reactor. However, these systems require the addition of an external electron donor to achieve denitrication (Metcalf and Eddy, 1991). In contrast, systems which start with an anoxic reactor are limited by the pH increase caused during proteolysis, and this inherently increases the toxic fraction of ammonia in the reactor. Therefore, these systems can only handle low strength ammo-

nia wastestreams, or require long acclimatisation times. However, this reactor conguration can be easily handled in an ABR due to a lower pH at the reactor inlet caused by the partial biological shift to acidogenesis from methanogenesis. The lower pH would shift equilibrium to the non-toxic ammonium form, thus providing the capacity for higher ammonia loadings. Here, an external electron donor is not required, and denitrication at the reactor inlet would actually improve COD removal by oxidising a fraction of the COD entering the reactor in order to support nitrate reduction. Therefore, the aim of this work was to incorporate existing anoxic/aerobic technologies into a single ABR in order to achieve nitrogen removal. This paper deals with denitrication occurring in the anaerobic region at the front of the reactor, whilst aerobic nitrication within an in-situ aeration compartment at the rear of the reactor is the subject of a subsequent paper (Barber and Stuckey, 2000).
MATERIALS AND METHODS

A mesophilically operated (3520.28C) anaerobic baed reactor with a working volume of 10 l consisting of eight discrete compartments of equal volume was modied to allow nitrogen removal, and the typical experimental setup is shown in Fig. 1. The reactor was initially seeded with 8 l of screened digested sludge containing 26 g/l TSS (18 g/l VSS), and kept at a volumetric loading rate of 4.8 gCOD/l.d (OLR=0.274 gCOD/g VSS.d) with a 20 h hydraulic retention time (HRT). Sodium nitrate (1000 mg/ l) was added along with a basal nutrient solution to a 4000 mg/l COD of synthetic sucrose/protein wastewater. The composition of the nutrients and synthetic wastewater has been previously documented (Nachaiyasit and Stuckey, 1995). Biogas composition, including nitrogen, was measured by GCTCD whilst volatile fatty acids were determined on an HPLC. The details of both these methods are as previously described (Barber and Stuckey, 1998). Conventional parameters such as pH, COD, alkalinity and solids concentrations (total and volatile) were monitored according to Standard Methods (APHA, 1989). Commercial test kits were used for the determination of ammonia (medium rangeNesslerisation method; low rangesalycilate method), nitrate (cadmium reduction method), and nitrite (ferrous sulphate method) according to the procedures provided by Hach (1997). The coecients of variation of these methods were between 4 and 6%.
RESULTS AND DISCUSSION

Initial response Despite the overwhelming thermodynamic advantage that denitrication has over methanogenesis, nitrogen levels in the o-gas did not increase signicantly until one month after the addition of nitrate in the feed, with a peak of >30% nitrogen after 36 days. This delay may have been due to: sludge wastage caused by an observed increase in foaming (Winkler, 1981); inhibition eects (Akunna et al., 1994); and the initial development of a denitrifying population. Foam traps were inserted in the gas

ABR nitrogen removal: denitrication

Fig. 1. Typical experimental layout for modied anaerobic baed reactor. 2415

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Fig. 2. Denitrication intermediates prole and eciency. Nitrate (*), nitrite (w), % denitrication eciency (q).

Fig. 4. Compartment gas production before nitrate addition. Methane (*), carbon dioxide (R), nitrogen (Q).

lines to reduce the solids lost, but the long period of wastage inevitably lengthened the time required for the benets of denitrication to be noticed. However, once a stable micro-community of facultative denitriers had been established, reactor operation and performance were very stable. Denitrication rates Figure 2 shows the average proles of nitrate and nitrite, whilst Fig. 3 shows daily compartmental biogas composition. Other intermediates, such as nitric and nitrous oxides, play a less signicant role during wastewater treatment and therefore were not determined. Virtually all nitrate was removed in the rst two compartments (i.e. within 2/8 20=5 h). Recorded levels were 176 and 37 mg nitrate/l, and 43 and 0.7 mg nitrite/l in compartments 1 and 2, respectively, which corresponded to a denitrication eciency of 82% and 96%. From these data, along with volatile solids information, denitrication rates were calculated to be 0.335, 0.085 and 0.027 kg NO3/kg VSS.d in compartments 1, 2 and 3, respectively. The gure for compartment 1 compared very favourably with rates quoted using methanol, whilst

compartments 2 and 3 compare well for a wastewater carbon source (Metcalf and Eddy, 1991). Amounts of di-nitrogen gas produced were 4.7 l/d (or 3.76 volume nitrogen/volume of compartment), 1.4 l/d (1.12 v/v) and 0.41 l/d (0.33 v/v) in compartments 1, 2 and 3, respectively. Gas production and composition From the stoichiometry of denitrication, 5 moles of organic carbon are required by 4 moles of nitrate to produce 2 moles of nitrogen and 5 moles of carbon dioxide (Winkler, 1981), as shown in equation (1). Figures 4 and 5 and Table 1 show the following trends: nitrogenesis was favoured to methane at the front of the reactor; denitrication caused an increase in levels of carbon dioxide, which was the major contributor to biogas during denitrication; methane composition in the biogas increased after denitrication; the presence of nitrate increased overall gas production from 22.6 l/ d to 37.6 l/d (66% increase); denitrication caused a very slight increase in overall methane production in spite of a fall in the methane composition from 68 to 43%; most gas production occurred at the front of the ABR during denitrication compared

Fig. 3. Average compartmental biogas composition proles before (shaded), and after (hollow), the addition of 1000 mg/l nitrate. Methane (*, w), carbon dioxide (R, r), nitrogen (Q, q).

Fig. 5. Compartment gas production after nitrate addition. Methane (*), carbon dioxide (R), nitrogen (Q).

ABR nitrogen removal: denitrication

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with nitrate-free conditions; denitrication signicantly improved methane production in compartment 1 (from 3 to 5 l/d, increase of 67%). Most of the results can be readily explained from thermodynamics or stoichiometry. Denitrication is thermodynamically more favourable than methanogenesis, thus nitrogen production will increase over methane causing a concomitant rise in carbon dioxide levels according to equation (1), and this was found experimentally (Akunna et al., 1994). Based on McCarty's yield calculations (McCarty, 1966, 1971), bacterial yields from carbohydrate as an electron donor are 0.534 and 0.208 g VSS/g electron donor when nitrate and carbon dioxide are used as electron acceptors, respectively. However, the most signicant advantage comes from the time required for a doubling in bacterial population which is approximately 16 times faster for nitrate as electron acceptor than carbon dioxide (1.1526 h1 compared with 0.0702 h1). In other words, anaerobic processes will be regulated by the presence of nitrate, which is preferentially utilised. Nevertheless, one interesting observation was made during the experimental period, and this concerned the benecial eect that denitrication had on methanogenesis in the rst compartment. From thermodynamics, denitrication should repress methane production; however, methane production increased on average by 67% in the front compartment when nitrate was present. Although dicult to prove, many factors or combination of factors may have contributed to the improved methane levels. Two of these factors may have been as a result of dissimilatory nitrate reduction to ammonium (Stouthamer, 1988), as shown in equation (2). This reaction is as equally appealing to mixed bacterial consortia as nitrate reduction to nitrogen (equation (1)), and as a result will also take place in preference to other reactions. In fact, several researchers have shown the dominance of this reaction over nitrogen production under anoxic conditions, with it accounting for 4070% of nitrogen reduction (Akunna et al., 1994; du Preez and Maree, 1994). The consequences of this reaction are two-fold: rstly, it has a very high hydrogen demand (4 moles of hydrogen required per mole of nitrate reduced); and secondly, ammonium ions are released. The high hydrogen demand enables hydrogen levels to be kept low enough to allow syntrophic reactions to proceed, and this inevitably results in a build-up of methane precursors and ulti-

mately methanogenesis with superior reactor performance. However, hydrogen may also be converted directly to methane at the front of the ABR. Hydrogen levels in the gas phase were occasionally measured and were slightly lower than during non-denitrifying conditions. Ammonium ions released during dissimilatory nitrate reduction may also have improved methane production due to an enhanced availability of reduced nitrogen as a nutrient to methanogens. Although ammonia is often blamed for the cessation of methane production at high concentration and pH (McCarty and McKinney, 1961), at lower pH (in the front of the ABR) and lower concentrations it has been found to encourage methanogenesis. Although environmental conditions at the front of the ABR favour acidogenic bacteria, some methanogens are capable of growth at low pH and high acid concentrations (Speece, 1996), and in the interstices of ocs, conditions are often substantially dierent from the bulk. However, methane production is repressed due to faster growth and assimilation of nutrients by the other bacteria present. It seemed that methanogens were not necessarily restrained by environmental conditions, but instead by nutrient bioavailability at the front of the ABR. An excess of ammonium may have provided methanogens with an enhanced reduced nitrogen source, which under nitrate-free conditions they would have had diculty assimilating due to their slower kinetics and growth rates. This nding was further substantiated during nitrication experiments (Barber and Stuckey, 2000) where methane levels increased by >15% (with no pH dierence) in the front compartment of the ABR with the addition of 500 mg/l ammonia to the feed. Another possible reason for the improved methane levels in compartment 1 was a change in environmental conditions, and this can be seen from equation (1). Denitrication directly increased pH (by 0.25 units) due to the release of hydroxyl ions, and indirectly by consuming acid intermediates. Such an increase in pH, however small, may have increased methane production. Eect of denitrication on COD removal From stoichiometry, 1 kg of nitrate requires 0.645 kg of a carbon source for denitrication to occur; the consequence of this in the ABR is that COD removal will inevitably improve. From the denitrication rates, an additional 529 mg/l COD

Table 1. Eect of denitrication on average gas production Pre-denitrication Total gas (l/d) Nitrogen (l/d) Methane (l/d) Carbon dioxide (l/d) 22.0 0 15.3 6.7 During denitrication 37.6 6.4 16.0 15.2 Post-denitrication 29.9 0 15.6 14.3

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Fig. 6. Eect of denitrication on COD removal eciency. Nitrate-free (w), 1000 mg/l nitrate (*), theoretical values based on experimental denitrication data (r).

(1 g/l nitrate 0.82 (eciency) 645 mg COD required/l=529 mg/l) in compartment 1, and an additional 111 mg/l and 26 mg/l in compartments 2 and 3, respectively, would be removed. Figure 6 shows the COD removal rates for nitrate-free and denitrication conditions; the triangles are based on the calculations shown above for the additional COD removal by using the non-denitrifying case as the starting data. Figure 7 shows the eect of denitrication on residual (non-VFA) COD production. It shows typical exponential decay proles for residual COD for the ABR under normal steady-state conditions (Schiener et al., 1998; Barker and Stuckey, 1998). Residual COD is mainly in the form of Soluble Microbial Products (SMPNamkung and Rittmann, 1986), and is the result of substrate utilisation (UAPUtilisation Associated Products) and biomass decay (BAPBiomass Associated Products, Namkung and Rittmann, 1986). In the ABR, SMPs may act as a microbial diusion barrier to low pH conditions, and between 26 and 48% of the COD are SMP in the rst compartment (Schiener et al., 1998). These gures are signicantly higher than in stirred tank reactors being fed glucose (Namkung

Fig. 7. Eect of denitrication of recalcitrant COD production. 1000 mg/l nitrate (+), nitrate-free (q).

and Rittmann, 1986). The gure for the nitrate free reactor (4 g COD/l, 20 h HRT, OLR=4.8 g COD/ l.d) of approximately 40% is in agreement with the ndings of Schiener et al. and may be due to low pH conditions. The underlying hypothesis behind SMP production in the ABR is that harsh environmental conditions at the front of the reactor will increase production there, and as a result, the nature of the material will be in the form of UAPs (Schiener et al., 1998). SMPs produced at the front of the reactor will slowly be catabolised down the reactor, and converted to volatile fatty acids thus providing substrates for other trophic groups, whilst the fraction of biomass decay products (high molecular weight fractions, e.g. cell walls and cell lysis products) would increase substantially as substrate concentration decreases. Therefore, BAPs are the major contributor to euent COD (Schiener et al., 1998). This hypothesis adequately describes the case for the nitrate-free reactor, which shows typical SMP production. However, SMP production rose from 40 to 60% of the total COD in compartment 1 when nitrate was present. Levels increased from 1285 to 1710 mg/l (increase of 425 mg/l or approximately 35%) and, from 705 to 1060 mg/l (increase of 355 mg/l or 50%) in compartments 1 and 2. Nevertheless, under a denitrication free environment (compartments 37) there was negligible eect on overall SMP production. SMP production is hypothesised to act as a protective measure against harsh conditions, for example the formation of a diusion barrier against low pH (Schiener et al., 1998), and toxic materials (Barker and Stuckey, 1998). However, neither of these conditions existed during denitrication in the ABR. During nitrate reduction, pH levels increased according to stoichiometry by an average of 0.25 units, and denitrication (via dissimilatory reduction to ammonium) caused an increase in bulk ammonium levels (required as a reduced nitrogen source by bacteria). Therefore, the increase observed in SMP production was unlikely to be due to environmental conditions, which in fact improved. The answer may lie in the composition of the SMPs produced; under normal conditions in the ABR, most SMPs at the front of the reactor would be associated with substrate catabolism (i.e. UAP), with only a small fraction of decay products (BAP). As shown earlier, bacterial yields for facultative denitriers using carbohydrate as an electron donor are approximately 55%, compared with 20% for anaerobic bacteria. Using McCarty's technique (1966, 1971), the following yields were calculated for denitrication and anaerobic catabolism for the following substrates (with denitrifying yield rst): acetate, 43% vs 3%; propionate, 61% vs 5%; butyrate, 74% vs 7%; and protein, 64% vs 12%. In addition, the generation times with nitrate as an electron acceptor were all substantially shorter for

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Fig. 8. Nitrate free VFA prole. Formate (w), acetate (*), propionate (Q), butyrate (R).

all substrates. This will inevitably result in substantial changes in the population over time. It is well known that aerobic and facultative bacterial species have substantially higher substrate dependent endogenous decay rates, b, than their anaerobic counterparts as a result of faster growth rates. Decay rates for denitriers have been calculated as 0.45 d1 (Chen et al., 1992), which are several orders of magnitude higher than anaerobes such as methanogens (0.004 d1Pavlostathis and Giraldo-Gomez, 1991). Bearing these considerations in mind, the increase in SMPs due to denitrication was most probably due to endogenous decay of facultative bacteria, and therefore would be in the form of BAPs with high molecular weights. Previous experimental work by Chen et al. (1992) has proved that biomass decay products, or ``secondary substrates'', are produced in large quantities during denitrication within a biolm. The authors examined the nature of the refractory material and discovered that at least 50% of the recalcitrant matter had a molecular weight in excess of 10,000, which is consistent with BAP production, and may explain the data presented in this research.
EFFECT OF DENITRIFICATION ON VFA PROFILES

Figures 8 and 9 show average VFA proles for

Fig. 9. VFA prole on addition of 1000 mg/l nitrate. Formate (w), acetate (*), propionate (Q), butyrate (R).

non-denitrifying and denitrifying environments. The signicant dierences in the two graphs are: superior overall VFA removal when nitrate was present; and an increase in acetate coupled to a signicant decrease in propionate (approximately 200% decrease) and a slight fall in butyrate (approximately 25% decrease) at the front of the nitrate fed reactor compared with the nitrate-free case. Previous ndings have shown similar trends (Akunna et al., 1994) where propionate levels decreased and coincided with an increase in acetate. Our reactor was started-up at a long retention time, and as a result developed strong methanogenic populations of both acetotrophic and hydrogenotrophic genera throughout the length of the reactor. This was manifested by high methane content in the biogas in all the reactor compartments (Barber and Stuckey, 1998). Low hydrogen concentrations were also noted in the gaseous phase, and this indicated a mature environment for syntrophic bacteria, and this is further demonstrated in Fig. 9 by low propionate concentrations. However, during denitrication, hydrogen levels were further reduced due to the high hydrogen demand of dissimilatory reduction of nitrate to ammonium (equation (2)). This ultimately improved environmental conditions for propionate degradation, which is critically inuenced by hydrogen levels. Despite this, denitriers have a low anity for propionate, with butyrate and acetate being preferred substrates (Takai et al., 1997). This suggested that the syntrophic bacteria responsible for propionate degradation before nitrate was added became more ecient at propionate oxidation due to the improved environmental conditions caused by hydrogen utilising denitriers. Subsequently, lower propionate levels were observed at the front of the nitrate fed reactor in Fig. 9. Complete propionate oxidation is only possible under anaerobic conditions by several species of sulphate reducing bacteria which consume propionate irrespective of hydrogen concentration (Widdel, 1988). However, all other bacteria can only incompletely oxidise propionate into simpler molecules, namely acetate and carbon dioxide. Therefore, it is reasonable to assume that the elevated acetate levels found during denitrication in the front-most compartments were a direct result of improved incomplete propionate oxidation. From stoichiometry, 1 mole of propionate oxidised yields 1 mole of acetate, therefore from experimental data the reduction of propionate observed (approximately 260 mg/l) would increase the bulk acetate concentration by about 220 mg/l. However, closer observation of Figs 8 and 9 shows an increase of approximately half the amount expected from stoichiometry. It appears that bacteria which were relatively dormant under nitrate-free conditions were being stimulated by acetate in the presence of nitrate. It is well known from the literature that

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facultative denitriers have a high anity for acetate (Akunna et al., 1993), and will utilise it as an electron donor with nitrate as the terminal electron acceptor. Therefore, improved acetate utilisation during denitrication is likely to be caused by acetotrophic denitriers, thus explaining the lower acetate increase expected from improved propionate oxidation. Improved acetoclastic methanogenesis was ruled out since it could only account for 2% of the increase in methane noted based on stoichiometric calculations. Therefore, increased methane yields were due to improved reductive methanogenesis which increased the methane percentage in the biogas by approximately 20%. According to McCarty and Mosey's (1991) hypothesis, butyrate is produced under stressed conditions to counteract the potential toxicity caused by low pH. Previous work (Nachaiyasit and Stuckey, 1997; Barber and Stuckey, 1998) is in agreement with this hypothesis, and has found high butyrate:acetate ratios in compartment 1 under low pH conditions. These ratios have subsequently reduced sharply as the acid intermediates penetrated the reactor. In the nitrate-free case (Fig. 9) the butyrate:acetate ratio was 0.29 in the rst compartment, whilst the bulk pH was 6.7. This ratio decreased to 0.18 during denitrication due to the increased pH (of 7) caused by the release of hydroxide ions. Therefore, since denitrication reduced the stress level experienced by the microbial community at the front of the ABR by increased pH, butyrate production was not stimulated. Consequently, butyrate concentrations were lower under a denitrifying environment as shown in Fig. 9. Eect of denitrication on ammonia proles As stated earlier, a second denitrication pathway results in the production of ammonium ions as demonstrated in equation (2). Under anaerobic conditions ammonia formation from nitrate is a common occurrence (Akunna et al., 1994). Ammonia proles for nitrate-free and nitrate-rich conditions

Fig. 10. Eect of denitrication on ammonia concentration. Nitrate-free (w), 1000 mg/l nitrate (*), theoretical (r).

are exhibited in Fig. 10. Under nitrate-free conditions, ammonium would be produced from the proteolysis of the proteinaceous material in the feed stream at the front of the ABR. Subsequently, bacteria would assimilate this ammonium in order to synthesise vital amino acids. Excess, or un-bioavailable, ammonium then passes through the reactor un-metabolised subsequently entering the reactor euent. Nevertheless, when nitrate is added to the reactor, ammonium may be formed via dissimilatory reduction. From Fig. 10, the presence of nitrate added on average 177 mg ammonia/l (or 0.177 mg NH3/mg NO3 reduced) to each reactor compartment except for compartment 1 where ammonia levels increased by only 95 mg/l. Both major denitrication routes (Eqs (1) and (2)) have approximately the same free energy of formation per mol of nitrate reduced. Therefore, it would not be unreasonable to assume that there were similar populations of both organisms, and that both reactions proceeded at approximately the same rate. If this assumption was correct, then about half of the nitrate reduced would go through nitrogen production, and the other half through ammonium production. However, the rate of these reactions is critically dependent on substrate type: glucose fermentation resulted in a rate of ammonium production of 2.7 and 5.9 mg N/g VSS.h with nitrate and nitrite as electron acceptors, respectively, while acetate was exclusively converted to nitrogen gas at rates as high as 27.8 and 23.8 mg N/g VSS.h (Akunna et al., 1993). From equation (2), one mole of nitrate is reduced to one mole of ammonium; therefore, 1000 mg/l nitrate should produce 290 mg/ l ammonium. If both reactions were taking place at approximately equal rates, then half of the nitrate available will be converted to ammonium, and therefore dissimilatory nitrate reduction to ammonium (assuming 100% ecient and non-limiting conditions) will add 145 mg ammonia/l. Data on the addition of 145 mg/l ammonia (based on experimental denitrication eciencies and 50% conversion of nitrate to ammonium) on ammonia proles are shown in Fig. 10 by the triangles. From this gure it is clear that this assumption of 50% conversion results in a good t to the experimental data. Nevertheless, these theoretical gures must be viewed with caution due to the complex ammonium requirements in each compartment. Calculated ammonia requirements (during this study) show that bacteria which reduce nitrate to nitrogen gas have a high nitrogen demand which may explain the lower increase in compartment 1. Since the ammonia demand is high for the fermentation of glucose and reduction of protein, it is likely that bacteria with slower growth are being starved of nitrogen in the rst compartment of the ABR. This may partially explain the observed increase in methane production in compartment 1 during deni-

ABR nitrogen removal: denitrication

Table 2. Summary of denitrication experiments Parameter Overall gas production Overall methane production Overall CO2 production Overall VFA production Acetate level Propionate level Butyrate level Hydrogen level Production of refractory COD pH Overall COD removal Bulk ammonia levels

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During denitrication +66% +7% +220% + + +0.25 units Improved by <5% +177 mg/l.comp

Post-denitrication 35% 7% 15% + + + ND 0.27 units Reduced H4% 200 mg/l.comp

Refractory COD levels were signicantly higher due to denitrication in compartments 1 (increase of 35%) and 2 (increase of 50%). In subsequent compartments negligible dierences were observed. Units mg/l.comp=mg ammonia/litre/compartment.

trication as ammonium would be produced from the denitrication of glucose (Akunna et al., 1993). This nding was further substantiated during nitrication experiments when a signicant increase in methane was noted after the addition of 500 mg/l ammonia to the inuent (Barber and Stuckey, 2000).
CONCLUSIONS

. Denitrication occurred almost exclusively in the front two compartments of the ABR due to its rapid kinetics, and the large variety of the bacteria which catalyse the reaction. Denitrication rates of 0.335 and 0.085 kg NO3/kg VSS.d were achieved in compartments 1 and 2, respectively. . Denitrication had several positive eects on overall reactor performance (Table 2), and this was due to the following factors: requirement of an oxidisable electron donor; increased system pH; the high hydrogen demand of dissimilatory nitrate reduction to ammonium (improving conditions for the syntrophic incomplete oxidation of fatty acids such as propionate ultimately leading to methane precursors); and, the generation of ammonium which provided slow growing bacteria at the front of the ABR with a reduced nitrogen source. These factors appeared to enhance hydrogenotrophic methanogenesis which increased methane percentage in the biogas by approximately 20%. This may lead to competition for hydrogen between methanogenesis and DNRA. . Denitrication caused a signicant increase in residual COD in the rst two chambers, probably due to the substantially higher growth and endogenous decay rates of facultative bacteria. The nature of the residual COD probably altered from mainly substrate utilisation products (UAPs) to biomass associated products (BAPs) in agreement with previous work. . Denitrication also caused a shift in the ratio, and quantity, of volatile fatty acids produced and

catabolised. This was manifested in a signicant reduction in propionate and butyrate concentrations, whilst acetate levels rose. Propionate reduction was probably due to improved conditions for syntrophic bacteria (increased pH and lower [H2]), whilst the excretion of hydroxyl ions decreased the tendency of the bacteria to produce butyrate. Acetate levels increased by only 50% of the stoichiometric level from propionate, and this implied direct acetate utilisation by acetotrophic denitriers which have a high anity for acetate. Improved acetoclastic methanogenesis was ruled out since it could only account for 2% of the increase in methane noted.

AcknowledgementsWPB would like to thank the BBSRC for the award of a studentship.

REFERENCES

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