- Email: [email protected]
Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture
ARTICLE IN PRESS WAT E R R E S E A R C H
41 (2007) 826– 834
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture Vel M. Vadivelu, Jurg Keller, Zhiguo Yuan Advanced Wastewater Management Centre, The University of Queensland, St. Lucia QLD 4072, Australia
ar t ic l e i n f o
abs tra ct
Article history:
The inhibitory effect of free ammonia (FA;NH3) on the metabolism of Nitrobacter is
Received 8 March 2006
investigated using a method that allows decoupling energy generation from growth
Received in revised form
processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of
7 September 2006
Nitrobacter. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the
Accepted 22 November 2006
bacterial population in the reactor was Nitrobacter, while no Nitrospira was detected. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the culture at various FA
Keywords: Anabolism Catabolism Free ammonia Inhibition
levels, in the presence (OURwithCO2 ) or absence (OURwithoutCO2 ) of inorganic carbon (CO2,
1 2 in HCO 3 and CO3 ). The FA inhibition on the respiration initiated at below 1 mgNH3–N L
both cases. OURwithoutCO2 gradually decreased by 12% when the FA concentration increased from 0 to approximately 4 mgNH3–N L1 and remained at the same level till an FA level of 9 mgNH3–N L1 (the highest FA concentration applied in this study). This indicates that FA
Nitrobacter
has a limited inhibitory effect on the respiratory capability of Nitrobacter. Starting from a
Respiration
level that is 15% higher than OURwithoutCO2 when no FA was present, OURwithCO2 decreased more rapidly than OURwithoutCO2 reaching the same level as OURwithoutCO2 when FA was between 6–9 mgNH3–N L1. This implies that in this range of FA the presence of inorganic carbon did not cause any increase in the respiration activity of Nitrobacter. The results suggest that, while still oxidizing nitrite at approximately 75% of the non-inhibited rate, Nitrobacter likely ceased to grow at an FA level of above 6 mgNH3–N L1. While the real mechanisms remain to be identified, this study indicates that the FA inhibition on Nitrobacter is likely much more serious than suggested by previous studies where OURwithCO2 (or the equivalent nitrite oxidation rate) was used as the sole measure of the inhibitory effects. & 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Nitrification is a two-step process where the reduced compound of nitrogen, ammonia, is oxidized to nitrite, which is further oxidized to nitrate. These two steps are carried out by two groups of autotrophic microorganisms, namely ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). Despite the phylogenetic distance between them, AOB and NOB are typically in close physical association in sludge flocs (Daims et al., 2000), with a syntrophic interaction. Under Corresponding author. Tel.: +617 3365 4374; fax: +617 3365 4726.
E-mail address: [email protected] (Z. Yuan). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.11.030
normal growth conditions, nitrate is typically the main product of nitrification, and as such ammonia oxidation is often considered the rate limiting step (Randall and Buth, 1984b, Gil and Choi, 2001). However, incomplete nitrification with various levels of nitrite accumulation has also been widely reported in literature (Hanaki et al., 1990, Yang and Alleman, 1992, Villaverde et al., 2000). Traditionally, nitrite build up is considered undesirable in biological wastewater treatment (BWT) systems. In recent years, however, several processes have been developed which
ARTICLE IN PRESS WAT E R R E S E A R C H
Nomenclature
AOB FA FNA m m
0
m0 NOB OTR
ammonia oxidizing bacteria free ammonia (NH3)
41 (20 07) 82 6 – 834
OUR q
free nitrous acid (HNO2) maintenance energy coefficient (mgN mg 1 0 0 0 COD1 biomass h ) m ¼ m +m (1m/mmax), where m , 0 m , m and mmax are defined below a component of m that is independent of m 1 (mgN mg COD1 biomass h ) a component of m that decreases with m 1 (mgN mg COD1 biomass h ) nitrite oxidizing bacteria oxygen transfer rate (mg h1); OTRwithCO2 and OTRwithoutCO2 refer to OUR in the presence and absence of inorganic carbon, respectively
remove nitrogen through nitritation (ammonia oxidation to nitrite) and denitritation (nitrite reduction to di-nitrogen gas). Removing nitrogen via this so-called nitrite pathway, in comparison to full nitrification and denitrification, reduces the oxygen and carbon demand by 25% and 40%, respectively (Fux et al., 2003). Examples of such processes include the wellknown Single reactor High Activity Ammonia Removal Over Nitrite (SHARON) process (Hellinga et al., 1998) and also a novel sequencing batch reactor (SBR) system reported in Lai et al. (2004). All these processes depend on the elimination or inhibition of NOB so that nitrite accumulates as the end product of nitrification. Accumulation of nitrite results from higher activities of AOB than NOB (Smith et al., 1997). Over the past few decades much work has been conducted to understand the mechanisms of nitrite accumulation, in order to either stimulate or avoid nitrite build-up during nitrification. Factors such as pH, temperature, and the concentrations of dissolved oxygen (DO), CO2 and heavy metals were all found to influence the nitrite build-up (Randall and Buth, 1984a, Hanaki et al., 1990, Surmacz-Gorska et al., 1997). However, the main causes are believed to be the inhibitory effects of free ammonia (FA) (Mauret et al., 1996, Villaverde et al., 2000) and free nitrous acid (FNA) (Anthonisen et al., 1976, Philips et al., 2002). The inhibitory effect of FA on NOB has been widely reported (Balmelle et al., 1992, Philips et al., 2002). While it has been speculated that AOB activities may also be inhibited by FA, NOB has been described to be much more sensitive to FA than AOB (Painter, 1970). Anthonisen et al. (1976) reported that FA initiated inhibition on Nitrobacter at about 0.1–1.0 mgNH3 L1, while the threshold value for Nitrosomonas was about 10–150 mgNH3 L1. In a study on nitrogen removal from high strength wastewater via the nitrite pathway, Abeling and Seyfried (1992) found that nitratation (nitrite oxidation to nitrate) was inhibited by FA at a concentration of 1–5 mgNH3 L1, while a similar effect was not observed on nitritation. The literature data summarized above clearly show that FA has a significant inhibitory effect on the metabolism of NOB. However, most studies reported to date relied on the
YG YO
827
oxygen uptake rate (mg h1); OURwithCO2 and OURwithoutCO2 refer to OUR in the presence and absence of inorganic carbon, respectively specific uptake rate of a substrate (mgSubstrate h1) true bacterial growth yield on a substrate (mgCODbiomass mgSubstrate1) true bacterial growth yield with respect to oxygen (mgCODbiomass mgO1) OURwithCO2 –OURwithoutCO2 (mg h1)
DOUR m specific growth rate of Nitrobacter (h1) mmax maximum specific growth rate of Nitrobacter (h1) mwithoutCO2 specific growth rate of Nitrobacter in the absence of externally supplied CO2 (h1)
measurement of the oxygen uptake rates (OUR, or equivalently the nitrite oxidation rates) at different FA levels. The levels of inhibition were assessed by comparing these rates with that measured in the absence or low, thus noninhibitory, level presence of FA. Such studies only revealed the impact of FA on the respiration of NOB. Little information was gained with regard to the impact of FA on the growth of NOB. In this paper, we report additional experimental information on the inhibitory effects of FA on the metabolic activities of NOB. An enriched Nitrobacter culture was used. Batch tests were designed and carried out to measure the oxygen consumption rates of the culture at various FA levels, in the presence and absence of inorganic carbon. The inhibitory effects of FA on the anabolic and catabolic processes of NOB were assessed through comparing the oxygen consumption rates in the two cases and model-based data analysis.
2.
Materials and methods
2.1.
Operation of SBR to enrich NOB
A SBR was operated to selectively grow an enriched culture of NOB. Mixed liquor from a fully nitrifying wastewater treatment plant in Brisbane, Australia, was used as inoculum for the SBR. This reactor had a working volume of 8 L and was fed 1 with nitrite (synthetic wastewater with 1000 mgNO 2 –N L ) as the sole energy source and bicarbonate as the sole carbon source (detailed composition given below). The SBR was operated with a cycle time of 6 h and a hydraulic retention time (HRT) of 1 day. Each cycle consisted of a 270-min aerobic feeding and a 20-min aerobic reaction period, followed by a 60-min settling and a 10-min decanting period. The reactor was operated in a temperature-controlled room (2271 1C). The DO concentration was maintained within the range of 2.75–3.25 mg L1 using an ON/OFF controller. While not controlled, the pH in the reactor varied in a narrow range between 7.2 and 7.4, due to the strong buffer in the wastewater.
ARTICLE IN PRESS 828
WA T E R R E S E A R C H
The synthetic wastewater comprised per liter (adapted from Kuai and Verstraete, 1998): 4.93 g of NaNO2 (1 g NO 2 –N), 0.4 g of NaHCO3, 1 g of each KH2PO4 and K2HPO4 and 2 ml of a stock solution containing trace elements. The trace element stock solution contained (per liter): 1.25 g EDTA, 0.55 g ZnSO4 7H2O, 0.40 g CoCl2 6H2O, 1.275 g MnCl2 4H2O, 0.40 g CuSO4 5H2O, 0.05 g Na2MoO4 2H2O, 1.375 g CaCl2 2H2O, 1.25 g FeCl3 6H2O and 44.4 g MgSO4 7H2O. Cycle studies were performed regularly by measuring the nitrite and nitrate concentrations every 15–30 min over the entire cycle to confirm the performance of the reactor. The effluent nitrite and nitrate concentrations were measured weekly over the entire period to monitor the long-term performance of the reactor. The microbial community composition was measured using the fluorescent in situ hybridization (FISH) technique, as described below.
2.2.
Methods for off-line chemical analysis
Nitrite and nitrate were analyzed using a Lachat QuikChem8000 flow injection analyser (FIA). FIA samples were obtained through filtering mixed liquor from the SBR using 0.22 mm Millex GP syringe-driven filters. The concentration of mixed liquor (volatile) suspended solid ML(V)SS, and the biomass chemical oxygen demand (COD) were measured by standard methods given in APHA (1998).
2.3.
Identification of biomass composition
Samples were prepared according to Daims et al. (2001). FISH was carried out with EUBMix (EUB338, EUB338-II and EUB338III), specific for all Bacteria (Daims et al., 1999), NIT3 specific for Nitrobacter sp., (Wagner et al., 1996) and Ntspa 662 specific for Nitrospira genera (Daims et al., 2001). All probes were commercially synthesized with 50 FITC (fluoroscein isothiocyanate), or one of the sulfoindocyanine dyes, indocarboncyanine (Cy3) or indodicarbocyanine (Cy5) by ThermoHybaid (Interactiva Division, Ulm, Germany). FISH images were collected using a Zeiss LSM 510 Meta Confocal microscope with a 63 Plan-Apochromat oil immersion lens. FISH quantification was performed according to Hall et al. (2003) where the relative abundance of the interested bacteria was determined as mean percentage of all bacteria.
2.4.
Experiments to determine the inhibitory effects of FA
Batch experiments were carried out on the Titration and OffGas Analysis (TOGA) sensor (Pratt et al., 2003) to determine the inhibitory effects of FA on the metabolisms of the enriched NOB culture. The TOGA sensor is a reactor-based instrument for characterizing biological processes. Each batch test in the TOGA reactor lasted several hours. The reactor was filled with 1.3 L of mixed liquor taken from the parent SBR. Various amounts of the synthetic media (see Section 3) used in the SBR were then injected into the reactor, which initiated nitrite oxidation. The ammonium concentration in the reactor was stepwise increased to a concentration of approximately 1600 mgNH+4–NL1. DO and pH were controlled at around 3 mg L1 and 7.3, respectively. These levels are similar to those
41 (2007) 826– 834
applied to the parent SBR. All the batch tests were conducted at room temperature 2271 1C. Measurement of nitrite and nitrate was carried out during the batch tests in the same way as in a normal cycle study. During each test, the O2 transfer rate (OTR) from the gas to the liquid phase, and the CO2 transfer rate (CTR) from the liquid to the gas phase were measured on-line through gas phase mass balance. The proton production/consumption rate (HPR) was monitored by recording the base/acid addition rate for pH control. Details for the methods for the OTR, CTR and HPR determination can be found in Pratt et al. (2003). Each batch experiment consisted of two separate tests involving the use of two sludge samples freshly removed from the parent SBR. One test was carried out in the presence of inorganic carbon in the liquid phase and the other in the absence of inorganic carbon. These conditions were achieved by supplying specifically designed O2-containing gas mixtures, with and without CO2, respectively, as the feeding gas. In the second phase, the non-CO2-containing gas was passed through the reactor for several hours to strip dissolved inorganic carbon (CO2, bicarbonate and carbonate) prior to the addition of nitrite and ammonium (see above). The completion of CO2 stripping was confirmed by both a very low-intensity off-gas CO2 signal (below the detection limit) and a zero HPR signal (the HPR signal should not be zero if CO2 stripping was occurring). The MLSS and MLVSS concentrations were measured at the beginning and the end of each test.
2.5.
Model used for data interpretation
The following model (Pirt, 1982) was used to assist the evaluation of substrate utilization rates due to the growth and maintenance processes of the NOB culture: m m þ m0 þ m0 1 , (1) q¼ YG mmax where q is the substrate (e.g. nitrite or oxygen) utilization rate, YG is the true bacterial growth yield on the substrate concerned. The term m0+m0 (1m/mmax) models the substrate utilization rate due to maintenance processes, where m0 is a constant maintenance term (independent of m), and m0 (1m/ mmax) characterizes the maintenance energy consumption rate dependent on the growth rate. In the above model, maintenance energy refers to the energy consumed for various cell-survival activities other than biomass production. This includes re-synthesis of damaged cellular materials, maintenance of cell motility and the necessary concentration gradients across cell membrane, and also the maintenance of cell growth potential. The last component is normally significant when the microbial growth is limited by nutrients but not energy. Under this condition, cells will be overloaded with reducing equivalents (NADH) and hence ATP, and tend to spill energy through ATP dissipation and/or proton leakage to maintain the catabolic potential such that growth can be reinitiated rapidly when the nutrient limitation is removed (see e.g. Neijssel and Tempest, 1976). This component is described with the term m0 (1km) in the Pirt (1982) model.
ARTICLE IN PRESS WAT E R R E S E A R C H
3.
Results and discussion
3.1.
Reactor performance and microbial community
Long-term monitoring of the NOB reactor performance revealed that complete nitrite oxidation was achieved. The nitrite level in the effluent was mostly undetectable (i.e. o0.05 mgNO2–N L1, data not shown). Cycle studies showed that there was only a low-level (2 mgN L1) accumulation of nitrite during the course of nitrite feeding, and the accumulated nitrite was oxidized almost immediately after feeding stopped (data not shown). The biomass concentration in the reactor was stable and averaged 716 mgCOD L1. The average effluent solids COD concentration was about 54 mgCOD L1, giving an actual sludge age of approximately 13 days (there was no deliberate wastage of sludge). FISH analysis showed that 73% of the microbial population of the NOB-enriched culture was Nitrobacter, while Nitrospira was undetected. The remaining 27% of bacterial community is believed to be heterotrophs surviving by using the lysate of Nitrobacter as the energy and carbon sources (Vadivelu et al., 2006a).
Batch experiments
Fig. 1(a) and (b) show the typical specific OTRwithCO2 and OTRwithoutCO2 profiles (ratio between measured OTRs and biomass concentration) determined during batch tests with a series of successive ammonium additions, in the presence and absence of inorganic carbon, respectively. The MLVSS
Ammonium-N
Nitrite-N (mg.L
1200 1000
90
800
60
600 400
Nitrite-N
30
200 1:00
2:00
3:00
4:00
5:00
6:00
7:00
8:00
9:00
NH+4
120
800
Ammonium-N
90
600 Nitrite-N
400
30 0 0:00
0 10:00 1000
NO-2
60
Ammonium-N (mg.L-1)
2
120
150 -1)
1400
NO-
0 0:00
b OTR (mgO2.gVSS-1h-1)
NH+4
150 Nitrite-N (mg.L-1)
OTR (mgO2.gVSS-1h-1)
a
concentrations measured at the beginning and end of each test were very similar and the averaged value was used in the calculation of the specific activity. The ammonium-N and nitrite-N profiles measured during the same test are also shown. Nitrite was also added in pulses with the maximal nitrite concentration kept below 60–70 mgN L1. Vadivelu et al. (2006b) showed that nitrite in this range (corresponding to an FNA concentration below 0.008 mgN L1 at pH 7.3) would not have any inhibitory effect on either the catabolic or anabolic processes of this Nitrobacter culture. On the other hand, as shown in Fig. 1, both OTRwithCO2 and OTRwithCO2 displayed a plateau after each addition of nitrite, indicating that the maximum bacterial activity was induced following each addition of nitrite. OTR measured in these experiments are equal to the biological OUR when a pseudo steady state of the biological reactions is attained (Pratt et al., 2003). In reference to Fig. 1(a) and (b), the OUR by the biomass is equal to the measured OTR during periods when OTR is approximately constant. The dynamics of the OTR observed immediately after nitrite addition and when nitrite ran out were jointly caused by the biological oxygen uptake and the mass transfer of oxygen between the gas and liquid phases. In this study, we are primarily interested in the maximum OUR achieved after each addition of ammonium; OTR is, therefore, directly used as OUR in the following sections. OUR was caused by the respiration of both the Nitrobacter and the heterotrophic bacteria. The heterotrophs consume oxygen to oxidize the organics available in the reactor resulting from cell lysis to gain energy for growth. The OUR
200
Ammonium-N (mg.L-1)
3.2.
829
41 (20 07) 82 6 – 834
0 1:00
2:00
3:00
4:00
5:00 6:00 Time (hrs)
7:00
8:00
9:00
10:00
Fig. 1 – The specific oxygen transfer rate caused by pulse additions of nitrite and ammonium to the Nitrobacter culture (a) in the presence of CO2 and (b) in the absence of CO2. The nitrite-N ( ) and ammonium-N ( ) concentrations measured off-line are also plotted. In both tests, pH ¼ 7.370.01, temperature ¼ 2271 1C, DO was approximately 3 mg L1.
ARTICLE IN PRESS 830
WA T E R R E S E A R C H
41 (2007) 826– 834
at approximately 85% of the maximum OURwithCO2 , reached its minimum level when the ammonium concentration was approximately 500 mgN L1. Further increase in ammonium concentration upon this level did not decrease OURwithoutCO2 . The minimum OURwithoutCO2 attained was also approximately 75% of the maximum OURwithCO2 . The experiment was repeated six times and similar results were obtained. For each batch test, the ratios between OURwithCO2 and OURwithoutCO2 attained after each addition of nitrite and the overall maximum OURwithCO2 measured in the batch test (OURmax) are calculated. OURmax in each test was always attained in the presence of CO2 and absence of ammonium, and when nitrite was in excess. Referring to Fig. 1, the first three OUR peaks in Fig. 1(a) satisfy these conditions. Their average was used as OURmax for this particular test. The results from the six experiments are plotted as a function of ammonium/FA concentration in Fig. 2. The inhibitory effects of FA displayed by OURwithCO2 and OURwithoutCO2 could not be adequately described by the commonly used non-substrate inhibition model q ¼ qmax KI =ðKI þ SI Þ, where q is the uptake rate of a substrate (e.g. O2), qmax is the maximum substrate uptake rate without inhibition, SI is the concentration of the inhibitory compound and KI is the inhibition constant. The best fit between this model and the experimental data was clearly unsatisfactory (results not shown).
by the heterotrophic bacteria in the culture was assessed in a separate study (Vadivelu et al., 2006a) as the OUR in the absence of nitrite, which showed that it contributed less than 1% to the maximum OUR shown in Fig. 1(a). Theoretically, the OUR measured under the above condition could be partially contributed by the heterotrophic activity of Nitrobacter, which has been shown to be mixotrophs in previous studies (see e.g. Bock, 1976). This capability of Nitrobacter has to date not been demonstrated in mixed culture settings in the presence of ordinary heterotrophs. If it indeed occurred, the rate must have been very small as reflected by the low OUR values measured. For simplicity, the OUR caused by the activity of heterotrophic bacteria or the heterotrophic activities of Nitrobacter is neglected in the data analysis. In the absence of externally supplied CO2 (Fig. 1(b)), the Nitrobacter culture was expected to grow at a very low rate, utilizing the CO2 produced by the heterotrophs through oxidizing cell lysate. The heterotrophic CO2 production rate can be estimated from the heterotrophic OUR, which was measured to be 1.3 mgO2 gCODbiomass h1 in Vadivelu et al. (2006a). Assuming that cell lysate has a degree of reduction of approximately 4, one mole of CO2 should be produced per mole of oxygen consumed by heterotrophs to oxidize cell lysate. The CO2 production rate would be approximately 0.04 mmolCO2 gCODbiomass h1 ( ¼ 1.3 mgO2 gCODbiomass h1/ (32 mgO2/mmolO2)). Even with the assumption that all the CO2 produced was used by Nitrobacter for growth (i.e. ignoring CO2 stripping), the specific growth rate of Nitrobacter ( ¼ 0.04 mmolCO2 gCODbiomass h1 would be 0.0013 h1 32 mgCODbiomass/mmolCO2), or 0.03 day1. Thus, in the absence of externally supplied carbon source, the growth of Nitrobacter is negligible (mwithoutCO2 0). The addition of ammonium immediately decreased both the OURwithCO2 and OURwithoutCO2 plateaus achieved after each addition of nitrite. The minimum OURwithCO2 plateau attained, when the ammonium concentration was above 900 mgN L1, was approximately 75% of the maximum OURwithCO2 plateau incurred in the absence of ammonium. OURwithoutCO2 , starting
3.3. Inhibitory effects of FA on the respiration of Nitrobacter Fig. 2 clearly shows that the addition of ammonium/FA immediately decreased both OURwithCO2 and OURwithoutCO2 , indicating that the respiration of Nitrobacter was inhibited by FA. The inhibition effect started at an FA concentration below 1.0 mgNH3–N L1. This observation agrees well with Anthonisen et al. (1976), who reported that the FA inhibition on Nitrobacter was initiated at 0.1–1.0 mgNH3–N L1. However, the inhibitory effect is limited. The OURwithoutCO2 data, which
0.4
0.9
0.3 OURwithCO /OURmax 2
0.8 0.7
OURwithoutCO /OURmax 2
y = 2E-07x2 - 0.0005x + 0.9834
0.2
y = 2E-07x2 - 0.0003x + 0.8416
0.1
OUR/OURmax
OUR/OURmax
1
ΔOUR/OURmax
0.6
0 0
200
400
600
800
1000
1200
NH+4-N (mg.L-1) 0
1
2
3
4
5 NH3-N
6
7
8
9
10
(mg.L-1)
Fig. 2 – OURwithCO2 /OURmax and OURwithoutCO2 /OURmax as a function of the FA and ammonium-N concentrations (pH ¼ 7.370.01, 2271 1C, DO was approximately 3 mg L1). DOUR/OURmax was calculated as the difference between the polynomial fits of OURwithCO2 /OURmax and OURwithoutCO2 /OURmax, which is proportional to the specific growth rate of the enriched Nitrobacter culture (see text).
ARTICLE IN PRESS WAT E R R E S E A R C H
represent the respiration activity of the Nitrobacter culture when growth was negligible, and would, therefore, not be affected by possible inhibition of FA on the anabolic processes of Nitrobacter, show that at an FA concentration of 4.0 mgNH3–N L1, the respiration of Nitrobacter was only reduced by 12%. A further increase of FA concentration from 4.0 to 9.0 mgNH3–N L1 (the highest level used in this study) did not have further inhibitory effect on the respiration rate. The finding that FA has a limited inhibitory effect on the respiration of Nitrobacter agrees well with Fux et al. (2003), who reported that the nitrite oxidation rate of the biomass from an SBR system treating dewatering liquor decreased by only 10% (in comparison to its maximum value obtained in the absence of FA) when the FA concentration was at 24–80 mgNH3–N L1. The mechanisms responsible for the FA inhibition on the respiration of Nitrobacter are not clear. It may be due to a direct inhibitory effect of FA on the nitrite oxidoreductase, or an enzyme involved in the electron transport or proton translocation. However, an inhibition of FA on the ATP production (with the use of proton motive force) may result in a similar effect. In the latter case, cell membrane would be more energized if the flux of protons entering cells were slowed down due to inhibition, in which case the translocation of protons during respiration would be more difficult. Independent of the detailed mechanisms involved, it can be concluded that FA inhibits, to a limited extent, the catabolic energy production by Nitrobacter.
3.4.
Effects of FA on the anabolic processes of Nitrobacter
Fig. 2 shows that OURwithCO2 and OURwithoutCO2 reached the same lowest level, 75% of the maximum OURwithCO2 , when FA was above 6 mgNH3–N L1. This implies that at an FA concentration of 6 mgNH3–N L1 or above, the presence of CO2 did not cause any difference in the respiration activity of Nitrobacter compared to the case where CO2 was absent. This suggests that the growth of Nitrobacter likely ceased to occur despite of the presence of CO2, as otherwise the additional ATP and reducing power consumption by the carbon transformation processes would result in a higher proton and electron fluxes, causing a higher respiration rate. The experimental data can be further analyzed with the use of the model presented in Eq. (1). With the assumption that mwithoutCO2 0 (see above), the following equation can be derived: DOUR ¼ OURwithCO2 OURwithoutCO2 m m ¼ þ m0 þ m0 1 m0 þ m0 YO mmax 1 m0 ¼ m, Y O mmax
ð2Þ
where YO is the true biomass yield with respect to oxygen, m is specific growth rate of Nitrobacter when CO2 is in excess, other variables are as explained in a previous section. To calculate DOUR, polynomial models were chosen to describe OURwithCO2 and OURwithoutCO2 . It was found that two 2nd-order polynomials could produce good fit to the experimental data (solid lines in Fig. 2). DOUR was then calculated using these two polynomials, which is also shown in Fig. 2 (as DOUR/OURmax).
41 (20 07) 82 6 – 834
831
Equation 2 shows that DOUR is proportional to m. However, DOUR should not be interpreted as the oxygen consumption rate for growth, which should be m/YO instead. The DOUR profile shows that m started decreasing as soon as FA was added and gradually reached a level close to zero when the FA concentration was about 6 mgNH3–N L1. The results suggest a strong negative impact of FA on the growth of Nitrobacter, which will be further discussed below.
3.5.
Comparison with previous methods and results
In previous studies, the effect of an inhibitory compound on nitrifiers has been assessed by measuring the respiration activity in the presence of both energy and carbon sources (i.e. OURwithCO2 as termed in this study). This approach has been applied to both AOB (Anthonisen et al., 1976, Hellinga et al., 1999) and NOB (Balmelle et al., 1992, Fux et al., 2003). The degree of inhibition was determined by comparing the OURwithCO2 data at different levels of the inhibitory compound with that measured in the absence of this inhibitory compound. With the implicit assumption that the bacterial oxygen consumption rate is proportional to their growth rate, which is a common assumption in activated sludge modeling (se e.g. the IWA Activated Sludge Models, Henze et al., 2000), the reduction in respiration activity was assumed to be the inhibitory effect on the bacterial growth processes. If this method of data interpretation were employed in this study, one would have concluded, based on the measured OURwithCO2 data, that FA had a limited inhibitory effect on the growth of NOB. This effect would be about 25% with an FA level of 6.0–9.0 mgNH3–N L1. However, bacteria produce energy to satisfy the energy requirement by both the growth and maintenance processes. Therefore, the bacterial respiration is not always coupled to their growth, and consequently the respiration rate is not necessarily proportional to the bacterial growth rate (see Eq. (1)). Consequently, the method widely employed to date for assessing the inhibitory effects of a potential inhibitor by measuring the respiration activities alone could potential give misleading results. In this paper, the respiration rates of Nitrobacter in the presence and absence of CO2 were measured. OURwithoutCO2 is unrelated to growth, and, therefore, its dependency on the FA concentrations truly reflects the inhibitory effects of FA on the energy production processes. Meanwhile, the difference between OURwithCO2 and OURwithoutCO2 should be directly related to the growth of Nitrobacter, as shown in Eq. (2). While the experimental data obtained in this study strongly suggest that FA has a strong negative effect on the biosynthesis processes of Nitrobacter, the method does not provide an unambiguous proof that such effects exist. Rather, this effect was inferred from indirect measurement and remains a hypothesis until experimental poof is achieved. Also, the experimental data does not allow identifying the fundamental mechanisms involved in the negative effect observed. The effect could be due to direct inhibition of FA on one or more steps in the carbon transformation processes. However, it may also be caused by energy limitation. As discussed above, energy production by Nitrobacter is inhibited by FA, which, if coupled with an increased maintenance energy requirement
ARTICLE IN PRESS 832
WA T E R R E S E A R C H
41 (2007) 826– 834
due to, for example, the high-level presence of FA, could result in a situation where Nitrobacter cells would have to spend all the energy produced for cell maintenance. Dawes and Ribbons (1964) suggested that in energy limiting conditions bacteria have to meet the maintenance energy requirement before spending energy on growth. Further studies are clearly needed to gain a more fundamental understanding of the inhibitory effects of FA on the metabolism of Nitrobacter and indeed on many other microorganisms as well. Such studies should preferably be carried out using pure cultures and by directly measuring the growth progresses. Despite of the above limitations, this study is to our knowledge the first, which shows that FA may have different inhibitory effects on the catabolic and anabolic processes of Nitrobacter.
3.6. Proposed role of FA inhibition on NOB washout in systems treating high-strength wastewater In recent years, the treatment of high-strength wastewater via nitrite to remove nitrogen has been demonstrated in several different processes. The mechanisms involved in the inhibition of NOB in these systems are still unclear. The results obtained in this study, together with the findings in Vadivelu et al. (2006b, c) on the FA and FNA inhibition on the metabolism of AOB and NOB, are summarized in Table 1. Clearly Nitrosomonas has a much higher level of tolerance to FA and FNA in comparison to Nitrobacter, which may have contributed significantly to the elimination of NOB from systems treating high nitrogen-containing wastewater. The typical concentrations of ammonium and nitrite in a system treating sludge digesting liquor through nitritation are in the range of 300–600 mgN L1 (Hellinga et al., 1999, Fux et al., 2003, Lai et al., 2004). Considering the variation of pH (6.5–8.0) in these systems, the FA and the FNA concentrations may vary between 4.0–30 mgNH3–N L1 and 0.04–0.30 mgHNO2–N L1, respectively. Therefore, both FA and FNA could be inhibiting factors of NOB. It is hypothesized that initially in the establishment of a system, the activities of NOB (non-adapted to high levels of FA) would be inhibited by the level of FA present in these systems, which leads to nitrite accumulation. An elevated level of FNA then forms an even stronger inhibitor to NOB growth, leading to their permanent elimination from the system.
An important factor that should be considered is the capability of microorganisms to adapt to inhibitory compounds. There are studies reported in literature showing that NOB could develop resistance towards FA. Villaverde et al. (2000) showed that the threshold value of specific FA inhibition (above which NOB activity ceased almost completely) increased from 0.2 to 0.7 mgNH 3 –N g biomass after a period of 4 months. Wong Chong and Loehr (1978) reported that Nitrobacter adapted to FA could resist an FA concentration of 40 mgNH3 L1, whereas a non-adapted biomass was inhibited at around 3.5 mgNH3 L1. To our best knowledge, no NOB cultures have been reported in literature to grow under high FNA levels or to be able to adapt to high FNA concentrations. It is, therefore, hypothesized that the most effective long-term inhibitor for NOB in systems treating high-strength wastewater is FNA. It should be mentioned that the Nitrobacter culture used in this study was not exposed to ammonium in the parent SBR, in order to avoid the presence of AOB in the enriched NOB culture. Thus, possible adaptation of these microorganisms to high ammonium concentration is not addressed in this study. Therefore, the results obtained may be valid for nonammonia adapted Nitrobacter cultures only. Nevertheless, it is worthwhile to point out that the FA inhibition on the energy production capacity of Nitrobacter is very limited even for the non-adapted biomass.
4.
Conclusions
The effect of FA on the metabolism of Nitrobacter was assessed using an enriched culture of Nitrobacter, not adapted to FA. It was found that the respiration of Nitrobacter was inhibited by FA even at low concentrations (less than 1 mgNH3–N L1). However, this inhibitory effect was limited. The respiration rate of Nitrobacter was reduced by 12% and 25%, respectively, in the absence and presence of inorganic carbon, when the FA concentration was increased from 0 to 9 mgNH3–N L1. FA may have a strong inhibitory effect on the anabolic processes of Nitrobacter. While the study did not provide unambiguous proof of this inhibitory effect, the experimental data strongly indicated that Nitrobacter likely ceased to grow at an FA concentration of 6–9 mgNH3–N L1. The study clearly
Table 1 – Summary of FNA and FA inhibition on Nitrobacter and Nitrosomonas Nitrobacter
Nitrosomonas
Anabolism
Catabolism
Anabolism
Catabolism
FNA
Likely stopped completely at 0.02 mgHNO2–N L1
No inhibition up to 0.04 mgHNO2–N L1
Likely inhibited completely at 0.40 mgHNO2–N L1
50% inhibition at 0.40–0.63 mgHNO2–N L1
FA
Likely inhibited completely at above 6.0 mgNH3–N L1
Inhibited by 12% at 6.0–9.0 mgNH3–N L1
No inhibition at up to 16.0 mgNH3–N L1
No inhibition at up to 16.0 mgNH3–N L1
ARTICLE IN PRESS WAT E R R E S E A R C H
shows the necessity of physiological studies to reveal the mechanisms of FA inhibition on bacterial metabolism. The conventional methods of assessing the inhibitory effects of FA on Nitrobacter (and possibly other bacteria as well), through simply comparing the overall substrate utilizing rates under inhibitory and non-inhibitory conditions, may produce misleading results. It is suggested that the catabolic and anabolic processes should be investigated separately.
Acknowledgments The Australian Research Council is thankfully acknowledged for funding this research through Project DP0342658. The first author would also like to thank Universiti Sains Malaysia, Malaysia for providing scholarships. Dr. Sandra Hall carried out all the FISH work reported in this paper.
Appendix A.
Supplementary data
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.watres.2006.11.030
R E F E R E N C E S
Abeling, U., Seyfried, C.F., 1992. Anaerobic-aerobic treatment of high-strength ammonium waste-water—nitrogen removal via nitrite. Water Sci. Technol. 26 (5 and 6), 1007–1015. Anthonisen, A.C., Loehr, R.C., Prakasam, T.B.S., Srinath, E.G., 1976. Inhibition of nitrication by ammonia and nitrous acid. J. Water Pollut. Control Fed. 48 (5), 835–852. APHA, 1998. Standard Methods for Examination of Water and Wastewater, 20th ed. American Public Health Association. Balmelle, B., Nguyen, K.M., Capdeville, B., Cornier, J.C., Deguin, A., 1992. Study of factors controlling nitrite buildup in biological processes for water nitrification. Water Sci. Technol. 26 (5 and 6), 1017–1025. Bock, E., 1976. Growth of Nitrobacter in presence of organic-matter, 2. Chemo-organotrophic growth of Nitrobacter-agilis. Arch. Microbiol. 108 (3), 305–312. Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., Wagner, M., 1999. The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22 (3), 434–444. Daims, H., Nielsen, P.H., Nielsen, J.L., Juretschko, S., Wagner, M., 2000. Novel Nitrospira-like bacteria as dominant nitrite-oxidizers in biofilms from wastewater treatment plants: diversity and in situ physiology. Water Sci. Technol. 41 (4 and 5), 85–90. Daims, H., Nielsen, J.L., Nielsen, P.H., Schleifer, K.H., Wagner, M., 2001. In situ characterization of Nitrospira-like nitrite oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67 (11), 5273–5284. Dawes, E.A., Ribbons, D.W., 1964. Some aspects of the endogenous metabolism of bacteria. Bacteriol. Rev. 28 (2), 126–149. Fux, C., Lange, K., Faessler, A., Huber, P., Grueniger, B., Siegrist, H., 2003. Nitrogen removal from digester supernatant via nitrite—SBR or SHARON? Water Sci. Technol. 48 (8), 9–18. Gil, K.I., Choi, E.S., 2001. Modelling of inhibition of nitrite oxidation in biological nitritation processes by free ammonia. Biotechnol. Lett. 23 (24), 2021–2026.
41 (20 07) 82 6 – 834
833
Hall, S.J., Keller, J., Blackall, L.L., 2003. Microbial quantification in activated sludge: the hits and misses. Water Sci. Technol. 48 (3), 121–126. Hanaki, K., Wantawin, C., Ohgaki, S., 1990. Nitrification at lowlevels of dissolved-oxygen with and without organic loading in a suspended-growth reactor. Water Res. 24 (3), 297–302. Hellinga, C., Schellen, A., Mulder, J.W., van Loosdrecht, M.C.M., Heijnen, J.J., 1998. The SHARON process: an innovative method for nitrogen removal from ammonium-rich waste water. Water Sci. Technol. 37 (9), 135–142. Hellinga, C., van Loosdrecht, M.C.M., Heijnen, J.J., 1999. Model based design of a novel process for nitrogen removal from concentrated flows. Math. Comput. Model. Dyn. Syst. 5 (4), 351–371. Henze, M., Gujer, W., Mino, T., van Loosdrecht, M. (Eds.), 2000. Activated Sludge Models ASM1, ASM2, ASM2d and ASM3. IWA Publishing, London. Kuai, L.P., Verstraete, W., 1998. Ammonium removal by the oxygen-limited autotrophic nitrification-denitrification system. Appl. Environ. Microbiol. 64 (11), 4500–4506. Lai, E., Senkpiel, S., Solley, D., Keller, J., 2004. Nitrogen removal of high strength wastewater via nitritation/denitritation using a sequencing batch reactor. Water Sci. Technol. 50 (10), 27–33. Mauret, M., Paul, E., PuechCostes, E., Maurette, M.T., Baptiste, P., 1996. Application of experimental research methodology to the study of nitrification in mixed culture. Water Sci. Technol. 34 (1 and 2), 245–252. Neijssel, O.M., Tempest, D.W., 1976. Bioenergetic aspects of aerobic growth of Klebsiella-Aerogenes Nctc 418 in carbonlimited and carbon-sufficient chemostat culture. Arch. Microbiol. 107 (2), 215–221. Painter, H.A., 1970. A review of literature on inorganic nitrogen metabolism in microorganisms. Water Res. 4 (6), 393. Philips, S., Laanbroek, H.J., Verstraete, W., 2002. Origin, causes and effects of increased nitrite concentrations in aquatic environments. Environ. Sci. Biotechnol. 1, 115–141. Pirt, S.J., 1982. Maintenance energy—a general-model for energylimited and energy-sufficient growth. Arch. Microbiol. 133 (4), 300–302. Pratt, S., Yuan, Z., Gapes, D., Dorigo, M., Zeng, R.J., Keller, J., 2003. Development of a novel titration and off-gas analysis (TOGA) sensor for study of biological processes in wastewater treatment systems. Biotechnol. Bioeng. 81 (4), 482–495. Randall, C.W., Buth, D., 1984a. Nitrite buildup in activated-sludge resulting from combined temperature and toxicity effects. J. Water Pollut. Control Fed. 56 (9), 1045–1049. Randall, C.W., Buth, D., 1984b. Nitrite buildup in activated-sludge resulting from temperature effects. J. Water Pollut. Control Fed. 56 (9), 1039–1044. Smith, R.V., Doyle, R.M., Burns, L.C., Stevens, R.J., 1997. A model for nitrite accumulation in soils. Soil Biol. Biochem. 29 (8), 1241–1247. Surmacz-Gorska, J., Cichon, A., Miksch, K., 1997. Nitrogen removal from wastewater with high ammonia nitrogen concentration via shorter nitrification and denitrification. Water Sci. Technol. 36 (10), 73–78. Vadivelu, V.M., Keller, J., Yuan, Z., 2006a. Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture. Biotechnol. Bioeng. 95 (5), 830–839. Vadivelu, V.M., Yuan, Z., Fux, C., Keller, J., 2006b. The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture. Environ. Sci. Technol. 40 (14), 4442–4448. Vadivelu, V.M., Yuan, Z., Fux, C., Keller, J., 2006c. Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes. Biotechnol. Bioeng. 94 (6), 1176–1188.
ARTICLE IN PRESS 834
WA T E R R E S E A R C H
Villaverde, S., Fdz-Polanco, F., Garcia, P.A., 2000. Nitrifying biofilm acclimation to free ammonia in submerged biofilters. Start-up influence. Water Res. 34 (2), 602–610. Wagner, M., Rath, G., Koops, H.P., Flood, J., Amann, R.I., 1996. In situ analysis of nitrifying bacteria in sewage treatment plants. Water Sci. Technol. 34 (1 and 2), 237–244.
41 (2007) 826– 834
Wong Chong, G.M., Loehr, R.C., 1978. Kinetics of microbial nitrification—nitrite-nitrogen oxidation. Water Res. 12 (8), 605–609. Yang, L., Alleman, J.E., 1992. Investigation of batchwise nitrite buildup by an enriched nitrification culture. Water Sci. Technol. 26 (5 and 6), 997–1005.
41 (2007) 826– 834
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Effect of free ammonia on the respiration and growth processes of an enriched Nitrobacter culture Vel M. Vadivelu, Jurg Keller, Zhiguo Yuan Advanced Wastewater Management Centre, The University of Queensland, St. Lucia QLD 4072, Australia
ar t ic l e i n f o
abs tra ct
Article history:
The inhibitory effect of free ammonia (FA;NH3) on the metabolism of Nitrobacter is
Received 8 March 2006
investigated using a method that allows decoupling energy generation from growth
Received in revised form
processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of
7 September 2006
Nitrobacter. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the
Accepted 22 November 2006
bacterial population in the reactor was Nitrobacter, while no Nitrospira was detected. Batch tests were carried out to measure the oxygen uptake rate (OUR) by the culture at various FA
Keywords: Anabolism Catabolism Free ammonia Inhibition
levels, in the presence (OURwithCO2 ) or absence (OURwithoutCO2 ) of inorganic carbon (CO2,
1 2 in HCO 3 and CO3 ). The FA inhibition on the respiration initiated at below 1 mgNH3–N L
both cases. OURwithoutCO2 gradually decreased by 12% when the FA concentration increased from 0 to approximately 4 mgNH3–N L1 and remained at the same level till an FA level of 9 mgNH3–N L1 (the highest FA concentration applied in this study). This indicates that FA
Nitrobacter
has a limited inhibitory effect on the respiratory capability of Nitrobacter. Starting from a
Respiration
level that is 15% higher than OURwithoutCO2 when no FA was present, OURwithCO2 decreased more rapidly than OURwithoutCO2 reaching the same level as OURwithoutCO2 when FA was between 6–9 mgNH3–N L1. This implies that in this range of FA the presence of inorganic carbon did not cause any increase in the respiration activity of Nitrobacter. The results suggest that, while still oxidizing nitrite at approximately 75% of the non-inhibited rate, Nitrobacter likely ceased to grow at an FA level of above 6 mgNH3–N L1. While the real mechanisms remain to be identified, this study indicates that the FA inhibition on Nitrobacter is likely much more serious than suggested by previous studies where OURwithCO2 (or the equivalent nitrite oxidation rate) was used as the sole measure of the inhibitory effects. & 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Nitrification is a two-step process where the reduced compound of nitrogen, ammonia, is oxidized to nitrite, which is further oxidized to nitrate. These two steps are carried out by two groups of autotrophic microorganisms, namely ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB). Despite the phylogenetic distance between them, AOB and NOB are typically in close physical association in sludge flocs (Daims et al., 2000), with a syntrophic interaction. Under Corresponding author. Tel.: +617 3365 4374; fax: +617 3365 4726.
E-mail address: [email protected] (Z. Yuan). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.11.030
normal growth conditions, nitrate is typically the main product of nitrification, and as such ammonia oxidation is often considered the rate limiting step (Randall and Buth, 1984b, Gil and Choi, 2001). However, incomplete nitrification with various levels of nitrite accumulation has also been widely reported in literature (Hanaki et al., 1990, Yang and Alleman, 1992, Villaverde et al., 2000). Traditionally, nitrite build up is considered undesirable in biological wastewater treatment (BWT) systems. In recent years, however, several processes have been developed which
ARTICLE IN PRESS WAT E R R E S E A R C H
Nomenclature
AOB FA FNA m m
0
m0 NOB OTR
ammonia oxidizing bacteria free ammonia (NH3)
41 (20 07) 82 6 – 834
OUR q
free nitrous acid (HNO2) maintenance energy coefficient (mgN mg 1 0 0 0 COD1 biomass h ) m ¼ m +m (1m/mmax), where m , 0 m , m and mmax are defined below a component of m that is independent of m 1 (mgN mg COD1 biomass h ) a component of m that decreases with m 1 (mgN mg COD1 biomass h ) nitrite oxidizing bacteria oxygen transfer rate (mg h1); OTRwithCO2 and OTRwithoutCO2 refer to OUR in the presence and absence of inorganic carbon, respectively
remove nitrogen through nitritation (ammonia oxidation to nitrite) and denitritation (nitrite reduction to di-nitrogen gas). Removing nitrogen via this so-called nitrite pathway, in comparison to full nitrification and denitrification, reduces the oxygen and carbon demand by 25% and 40%, respectively (Fux et al., 2003). Examples of such processes include the wellknown Single reactor High Activity Ammonia Removal Over Nitrite (SHARON) process (Hellinga et al., 1998) and also a novel sequencing batch reactor (SBR) system reported in Lai et al. (2004). All these processes depend on the elimination or inhibition of NOB so that nitrite accumulates as the end product of nitrification. Accumulation of nitrite results from higher activities of AOB than NOB (Smith et al., 1997). Over the past few decades much work has been conducted to understand the mechanisms of nitrite accumulation, in order to either stimulate or avoid nitrite build-up during nitrification. Factors such as pH, temperature, and the concentrations of dissolved oxygen (DO), CO2 and heavy metals were all found to influence the nitrite build-up (Randall and Buth, 1984a, Hanaki et al., 1990, Surmacz-Gorska et al., 1997). However, the main causes are believed to be the inhibitory effects of free ammonia (FA) (Mauret et al., 1996, Villaverde et al., 2000) and free nitrous acid (FNA) (Anthonisen et al., 1976, Philips et al., 2002). The inhibitory effect of FA on NOB has been widely reported (Balmelle et al., 1992, Philips et al., 2002). While it has been speculated that AOB activities may also be inhibited by FA, NOB has been described to be much more sensitive to FA than AOB (Painter, 1970). Anthonisen et al. (1976) reported that FA initiated inhibition on Nitrobacter at about 0.1–1.0 mgNH3 L1, while the threshold value for Nitrosomonas was about 10–150 mgNH3 L1. In a study on nitrogen removal from high strength wastewater via the nitrite pathway, Abeling and Seyfried (1992) found that nitratation (nitrite oxidation to nitrate) was inhibited by FA at a concentration of 1–5 mgNH3 L1, while a similar effect was not observed on nitritation. The literature data summarized above clearly show that FA has a significant inhibitory effect on the metabolism of NOB. However, most studies reported to date relied on the
YG YO
827
oxygen uptake rate (mg h1); OURwithCO2 and OURwithoutCO2 refer to OUR in the presence and absence of inorganic carbon, respectively specific uptake rate of a substrate (mgSubstrate h1) true bacterial growth yield on a substrate (mgCODbiomass mgSubstrate1) true bacterial growth yield with respect to oxygen (mgCODbiomass mgO1) OURwithCO2 –OURwithoutCO2 (mg h1)
DOUR m specific growth rate of Nitrobacter (h1) mmax maximum specific growth rate of Nitrobacter (h1) mwithoutCO2 specific growth rate of Nitrobacter in the absence of externally supplied CO2 (h1)
measurement of the oxygen uptake rates (OUR, or equivalently the nitrite oxidation rates) at different FA levels. The levels of inhibition were assessed by comparing these rates with that measured in the absence or low, thus noninhibitory, level presence of FA. Such studies only revealed the impact of FA on the respiration of NOB. Little information was gained with regard to the impact of FA on the growth of NOB. In this paper, we report additional experimental information on the inhibitory effects of FA on the metabolic activities of NOB. An enriched Nitrobacter culture was used. Batch tests were designed and carried out to measure the oxygen consumption rates of the culture at various FA levels, in the presence and absence of inorganic carbon. The inhibitory effects of FA on the anabolic and catabolic processes of NOB were assessed through comparing the oxygen consumption rates in the two cases and model-based data analysis.
2.
Materials and methods
2.1.
Operation of SBR to enrich NOB
A SBR was operated to selectively grow an enriched culture of NOB. Mixed liquor from a fully nitrifying wastewater treatment plant in Brisbane, Australia, was used as inoculum for the SBR. This reactor had a working volume of 8 L and was fed 1 with nitrite (synthetic wastewater with 1000 mgNO 2 –N L ) as the sole energy source and bicarbonate as the sole carbon source (detailed composition given below). The SBR was operated with a cycle time of 6 h and a hydraulic retention time (HRT) of 1 day. Each cycle consisted of a 270-min aerobic feeding and a 20-min aerobic reaction period, followed by a 60-min settling and a 10-min decanting period. The reactor was operated in a temperature-controlled room (2271 1C). The DO concentration was maintained within the range of 2.75–3.25 mg L1 using an ON/OFF controller. While not controlled, the pH in the reactor varied in a narrow range between 7.2 and 7.4, due to the strong buffer in the wastewater.
ARTICLE IN PRESS 828
WA T E R R E S E A R C H
The synthetic wastewater comprised per liter (adapted from Kuai and Verstraete, 1998): 4.93 g of NaNO2 (1 g NO 2 –N), 0.4 g of NaHCO3, 1 g of each KH2PO4 and K2HPO4 and 2 ml of a stock solution containing trace elements. The trace element stock solution contained (per liter): 1.25 g EDTA, 0.55 g ZnSO4 7H2O, 0.40 g CoCl2 6H2O, 1.275 g MnCl2 4H2O, 0.40 g CuSO4 5H2O, 0.05 g Na2MoO4 2H2O, 1.375 g CaCl2 2H2O, 1.25 g FeCl3 6H2O and 44.4 g MgSO4 7H2O. Cycle studies were performed regularly by measuring the nitrite and nitrate concentrations every 15–30 min over the entire cycle to confirm the performance of the reactor. The effluent nitrite and nitrate concentrations were measured weekly over the entire period to monitor the long-term performance of the reactor. The microbial community composition was measured using the fluorescent in situ hybridization (FISH) technique, as described below.
2.2.
Methods for off-line chemical analysis
Nitrite and nitrate were analyzed using a Lachat QuikChem8000 flow injection analyser (FIA). FIA samples were obtained through filtering mixed liquor from the SBR using 0.22 mm Millex GP syringe-driven filters. The concentration of mixed liquor (volatile) suspended solid ML(V)SS, and the biomass chemical oxygen demand (COD) were measured by standard methods given in APHA (1998).
2.3.
Identification of biomass composition
Samples were prepared according to Daims et al. (2001). FISH was carried out with EUBMix (EUB338, EUB338-II and EUB338III), specific for all Bacteria (Daims et al., 1999), NIT3 specific for Nitrobacter sp., (Wagner et al., 1996) and Ntspa 662 specific for Nitrospira genera (Daims et al., 2001). All probes were commercially synthesized with 50 FITC (fluoroscein isothiocyanate), or one of the sulfoindocyanine dyes, indocarboncyanine (Cy3) or indodicarbocyanine (Cy5) by ThermoHybaid (Interactiva Division, Ulm, Germany). FISH images were collected using a Zeiss LSM 510 Meta Confocal microscope with a 63 Plan-Apochromat oil immersion lens. FISH quantification was performed according to Hall et al. (2003) where the relative abundance of the interested bacteria was determined as mean percentage of all bacteria.
2.4.
Experiments to determine the inhibitory effects of FA
Batch experiments were carried out on the Titration and OffGas Analysis (TOGA) sensor (Pratt et al., 2003) to determine the inhibitory effects of FA on the metabolisms of the enriched NOB culture. The TOGA sensor is a reactor-based instrument for characterizing biological processes. Each batch test in the TOGA reactor lasted several hours. The reactor was filled with 1.3 L of mixed liquor taken from the parent SBR. Various amounts of the synthetic media (see Section 3) used in the SBR were then injected into the reactor, which initiated nitrite oxidation. The ammonium concentration in the reactor was stepwise increased to a concentration of approximately 1600 mgNH+4–NL1. DO and pH were controlled at around 3 mg L1 and 7.3, respectively. These levels are similar to those
41 (2007) 826– 834
applied to the parent SBR. All the batch tests were conducted at room temperature 2271 1C. Measurement of nitrite and nitrate was carried out during the batch tests in the same way as in a normal cycle study. During each test, the O2 transfer rate (OTR) from the gas to the liquid phase, and the CO2 transfer rate (CTR) from the liquid to the gas phase were measured on-line through gas phase mass balance. The proton production/consumption rate (HPR) was monitored by recording the base/acid addition rate for pH control. Details for the methods for the OTR, CTR and HPR determination can be found in Pratt et al. (2003). Each batch experiment consisted of two separate tests involving the use of two sludge samples freshly removed from the parent SBR. One test was carried out in the presence of inorganic carbon in the liquid phase and the other in the absence of inorganic carbon. These conditions were achieved by supplying specifically designed O2-containing gas mixtures, with and without CO2, respectively, as the feeding gas. In the second phase, the non-CO2-containing gas was passed through the reactor for several hours to strip dissolved inorganic carbon (CO2, bicarbonate and carbonate) prior to the addition of nitrite and ammonium (see above). The completion of CO2 stripping was confirmed by both a very low-intensity off-gas CO2 signal (below the detection limit) and a zero HPR signal (the HPR signal should not be zero if CO2 stripping was occurring). The MLSS and MLVSS concentrations were measured at the beginning and the end of each test.
2.5.
Model used for data interpretation
The following model (Pirt, 1982) was used to assist the evaluation of substrate utilization rates due to the growth and maintenance processes of the NOB culture: m m þ m0 þ m0 1 , (1) q¼ YG mmax where q is the substrate (e.g. nitrite or oxygen) utilization rate, YG is the true bacterial growth yield on the substrate concerned. The term m0+m0 (1m/mmax) models the substrate utilization rate due to maintenance processes, where m0 is a constant maintenance term (independent of m), and m0 (1m/ mmax) characterizes the maintenance energy consumption rate dependent on the growth rate. In the above model, maintenance energy refers to the energy consumed for various cell-survival activities other than biomass production. This includes re-synthesis of damaged cellular materials, maintenance of cell motility and the necessary concentration gradients across cell membrane, and also the maintenance of cell growth potential. The last component is normally significant when the microbial growth is limited by nutrients but not energy. Under this condition, cells will be overloaded with reducing equivalents (NADH) and hence ATP, and tend to spill energy through ATP dissipation and/or proton leakage to maintain the catabolic potential such that growth can be reinitiated rapidly when the nutrient limitation is removed (see e.g. Neijssel and Tempest, 1976). This component is described with the term m0 (1km) in the Pirt (1982) model.
ARTICLE IN PRESS WAT E R R E S E A R C H
3.
Results and discussion
3.1.
Reactor performance and microbial community
Long-term monitoring of the NOB reactor performance revealed that complete nitrite oxidation was achieved. The nitrite level in the effluent was mostly undetectable (i.e. o0.05 mgNO2–N L1, data not shown). Cycle studies showed that there was only a low-level (2 mgN L1) accumulation of nitrite during the course of nitrite feeding, and the accumulated nitrite was oxidized almost immediately after feeding stopped (data not shown). The biomass concentration in the reactor was stable and averaged 716 mgCOD L1. The average effluent solids COD concentration was about 54 mgCOD L1, giving an actual sludge age of approximately 13 days (there was no deliberate wastage of sludge). FISH analysis showed that 73% of the microbial population of the NOB-enriched culture was Nitrobacter, while Nitrospira was undetected. The remaining 27% of bacterial community is believed to be heterotrophs surviving by using the lysate of Nitrobacter as the energy and carbon sources (Vadivelu et al., 2006a).
Batch experiments
Fig. 1(a) and (b) show the typical specific OTRwithCO2 and OTRwithoutCO2 profiles (ratio between measured OTRs and biomass concentration) determined during batch tests with a series of successive ammonium additions, in the presence and absence of inorganic carbon, respectively. The MLVSS
Ammonium-N
Nitrite-N (mg.L
1200 1000
90
800
60
600 400
Nitrite-N
30
200 1:00
2:00
3:00
4:00
5:00
6:00
7:00
8:00
9:00
NH+4
120
800
Ammonium-N
90
600 Nitrite-N
400
30 0 0:00
0 10:00 1000
NO-2
60
Ammonium-N (mg.L-1)
2
120
150 -1)
1400
NO-
0 0:00
b OTR (mgO2.gVSS-1h-1)
NH+4
150 Nitrite-N (mg.L-1)
OTR (mgO2.gVSS-1h-1)
a
concentrations measured at the beginning and end of each test were very similar and the averaged value was used in the calculation of the specific activity. The ammonium-N and nitrite-N profiles measured during the same test are also shown. Nitrite was also added in pulses with the maximal nitrite concentration kept below 60–70 mgN L1. Vadivelu et al. (2006b) showed that nitrite in this range (corresponding to an FNA concentration below 0.008 mgN L1 at pH 7.3) would not have any inhibitory effect on either the catabolic or anabolic processes of this Nitrobacter culture. On the other hand, as shown in Fig. 1, both OTRwithCO2 and OTRwithCO2 displayed a plateau after each addition of nitrite, indicating that the maximum bacterial activity was induced following each addition of nitrite. OTR measured in these experiments are equal to the biological OUR when a pseudo steady state of the biological reactions is attained (Pratt et al., 2003). In reference to Fig. 1(a) and (b), the OUR by the biomass is equal to the measured OTR during periods when OTR is approximately constant. The dynamics of the OTR observed immediately after nitrite addition and when nitrite ran out were jointly caused by the biological oxygen uptake and the mass transfer of oxygen between the gas and liquid phases. In this study, we are primarily interested in the maximum OUR achieved after each addition of ammonium; OTR is, therefore, directly used as OUR in the following sections. OUR was caused by the respiration of both the Nitrobacter and the heterotrophic bacteria. The heterotrophs consume oxygen to oxidize the organics available in the reactor resulting from cell lysis to gain energy for growth. The OUR
200
Ammonium-N (mg.L-1)
3.2.
829
41 (20 07) 82 6 – 834
0 1:00
2:00
3:00
4:00
5:00 6:00 Time (hrs)
7:00
8:00
9:00
10:00
Fig. 1 – The specific oxygen transfer rate caused by pulse additions of nitrite and ammonium to the Nitrobacter culture (a) in the presence of CO2 and (b) in the absence of CO2. The nitrite-N ( ) and ammonium-N ( ) concentrations measured off-line are also plotted. In both tests, pH ¼ 7.370.01, temperature ¼ 2271 1C, DO was approximately 3 mg L1.
ARTICLE IN PRESS 830
WA T E R R E S E A R C H
41 (2007) 826– 834
at approximately 85% of the maximum OURwithCO2 , reached its minimum level when the ammonium concentration was approximately 500 mgN L1. Further increase in ammonium concentration upon this level did not decrease OURwithoutCO2 . The minimum OURwithoutCO2 attained was also approximately 75% of the maximum OURwithCO2 . The experiment was repeated six times and similar results were obtained. For each batch test, the ratios between OURwithCO2 and OURwithoutCO2 attained after each addition of nitrite and the overall maximum OURwithCO2 measured in the batch test (OURmax) are calculated. OURmax in each test was always attained in the presence of CO2 and absence of ammonium, and when nitrite was in excess. Referring to Fig. 1, the first three OUR peaks in Fig. 1(a) satisfy these conditions. Their average was used as OURmax for this particular test. The results from the six experiments are plotted as a function of ammonium/FA concentration in Fig. 2. The inhibitory effects of FA displayed by OURwithCO2 and OURwithoutCO2 could not be adequately described by the commonly used non-substrate inhibition model q ¼ qmax KI =ðKI þ SI Þ, where q is the uptake rate of a substrate (e.g. O2), qmax is the maximum substrate uptake rate without inhibition, SI is the concentration of the inhibitory compound and KI is the inhibition constant. The best fit between this model and the experimental data was clearly unsatisfactory (results not shown).
by the heterotrophic bacteria in the culture was assessed in a separate study (Vadivelu et al., 2006a) as the OUR in the absence of nitrite, which showed that it contributed less than 1% to the maximum OUR shown in Fig. 1(a). Theoretically, the OUR measured under the above condition could be partially contributed by the heterotrophic activity of Nitrobacter, which has been shown to be mixotrophs in previous studies (see e.g. Bock, 1976). This capability of Nitrobacter has to date not been demonstrated in mixed culture settings in the presence of ordinary heterotrophs. If it indeed occurred, the rate must have been very small as reflected by the low OUR values measured. For simplicity, the OUR caused by the activity of heterotrophic bacteria or the heterotrophic activities of Nitrobacter is neglected in the data analysis. In the absence of externally supplied CO2 (Fig. 1(b)), the Nitrobacter culture was expected to grow at a very low rate, utilizing the CO2 produced by the heterotrophs through oxidizing cell lysate. The heterotrophic CO2 production rate can be estimated from the heterotrophic OUR, which was measured to be 1.3 mgO2 gCODbiomass h1 in Vadivelu et al. (2006a). Assuming that cell lysate has a degree of reduction of approximately 4, one mole of CO2 should be produced per mole of oxygen consumed by heterotrophs to oxidize cell lysate. The CO2 production rate would be approximately 0.04 mmolCO2 gCODbiomass h1 ( ¼ 1.3 mgO2 gCODbiomass h1/ (32 mgO2/mmolO2)). Even with the assumption that all the CO2 produced was used by Nitrobacter for growth (i.e. ignoring CO2 stripping), the specific growth rate of Nitrobacter ( ¼ 0.04 mmolCO2 gCODbiomass h1 would be 0.0013 h1 32 mgCODbiomass/mmolCO2), or 0.03 day1. Thus, in the absence of externally supplied carbon source, the growth of Nitrobacter is negligible (mwithoutCO2 0). The addition of ammonium immediately decreased both the OURwithCO2 and OURwithoutCO2 plateaus achieved after each addition of nitrite. The minimum OURwithCO2 plateau attained, when the ammonium concentration was above 900 mgN L1, was approximately 75% of the maximum OURwithCO2 plateau incurred in the absence of ammonium. OURwithoutCO2 , starting
3.3. Inhibitory effects of FA on the respiration of Nitrobacter Fig. 2 clearly shows that the addition of ammonium/FA immediately decreased both OURwithCO2 and OURwithoutCO2 , indicating that the respiration of Nitrobacter was inhibited by FA. The inhibition effect started at an FA concentration below 1.0 mgNH3–N L1. This observation agrees well with Anthonisen et al. (1976), who reported that the FA inhibition on Nitrobacter was initiated at 0.1–1.0 mgNH3–N L1. However, the inhibitory effect is limited. The OURwithoutCO2 data, which
0.4
0.9
0.3 OURwithCO /OURmax 2
0.8 0.7
OURwithoutCO /OURmax 2
y = 2E-07x2 - 0.0005x + 0.9834
0.2
y = 2E-07x2 - 0.0003x + 0.8416
0.1
OUR/OURmax
OUR/OURmax
1
ΔOUR/OURmax
0.6
0 0
200
400
600
800
1000
1200
NH+4-N (mg.L-1) 0
1
2
3
4
5 NH3-N
6
7
8
9
10
(mg.L-1)
Fig. 2 – OURwithCO2 /OURmax and OURwithoutCO2 /OURmax as a function of the FA and ammonium-N concentrations (pH ¼ 7.370.01, 2271 1C, DO was approximately 3 mg L1). DOUR/OURmax was calculated as the difference between the polynomial fits of OURwithCO2 /OURmax and OURwithoutCO2 /OURmax, which is proportional to the specific growth rate of the enriched Nitrobacter culture (see text).
ARTICLE IN PRESS WAT E R R E S E A R C H
represent the respiration activity of the Nitrobacter culture when growth was negligible, and would, therefore, not be affected by possible inhibition of FA on the anabolic processes of Nitrobacter, show that at an FA concentration of 4.0 mgNH3–N L1, the respiration of Nitrobacter was only reduced by 12%. A further increase of FA concentration from 4.0 to 9.0 mgNH3–N L1 (the highest level used in this study) did not have further inhibitory effect on the respiration rate. The finding that FA has a limited inhibitory effect on the respiration of Nitrobacter agrees well with Fux et al. (2003), who reported that the nitrite oxidation rate of the biomass from an SBR system treating dewatering liquor decreased by only 10% (in comparison to its maximum value obtained in the absence of FA) when the FA concentration was at 24–80 mgNH3–N L1. The mechanisms responsible for the FA inhibition on the respiration of Nitrobacter are not clear. It may be due to a direct inhibitory effect of FA on the nitrite oxidoreductase, or an enzyme involved in the electron transport or proton translocation. However, an inhibition of FA on the ATP production (with the use of proton motive force) may result in a similar effect. In the latter case, cell membrane would be more energized if the flux of protons entering cells were slowed down due to inhibition, in which case the translocation of protons during respiration would be more difficult. Independent of the detailed mechanisms involved, it can be concluded that FA inhibits, to a limited extent, the catabolic energy production by Nitrobacter.
3.4.
Effects of FA on the anabolic processes of Nitrobacter
Fig. 2 shows that OURwithCO2 and OURwithoutCO2 reached the same lowest level, 75% of the maximum OURwithCO2 , when FA was above 6 mgNH3–N L1. This implies that at an FA concentration of 6 mgNH3–N L1 or above, the presence of CO2 did not cause any difference in the respiration activity of Nitrobacter compared to the case where CO2 was absent. This suggests that the growth of Nitrobacter likely ceased to occur despite of the presence of CO2, as otherwise the additional ATP and reducing power consumption by the carbon transformation processes would result in a higher proton and electron fluxes, causing a higher respiration rate. The experimental data can be further analyzed with the use of the model presented in Eq. (1). With the assumption that mwithoutCO2 0 (see above), the following equation can be derived: DOUR ¼ OURwithCO2 OURwithoutCO2 m m ¼ þ m0 þ m0 1 m0 þ m0 YO mmax 1 m0 ¼ m, Y O mmax
ð2Þ
where YO is the true biomass yield with respect to oxygen, m is specific growth rate of Nitrobacter when CO2 is in excess, other variables are as explained in a previous section. To calculate DOUR, polynomial models were chosen to describe OURwithCO2 and OURwithoutCO2 . It was found that two 2nd-order polynomials could produce good fit to the experimental data (solid lines in Fig. 2). DOUR was then calculated using these two polynomials, which is also shown in Fig. 2 (as DOUR/OURmax).
41 (20 07) 82 6 – 834
831
Equation 2 shows that DOUR is proportional to m. However, DOUR should not be interpreted as the oxygen consumption rate for growth, which should be m/YO instead. The DOUR profile shows that m started decreasing as soon as FA was added and gradually reached a level close to zero when the FA concentration was about 6 mgNH3–N L1. The results suggest a strong negative impact of FA on the growth of Nitrobacter, which will be further discussed below.
3.5.
Comparison with previous methods and results
In previous studies, the effect of an inhibitory compound on nitrifiers has been assessed by measuring the respiration activity in the presence of both energy and carbon sources (i.e. OURwithCO2 as termed in this study). This approach has been applied to both AOB (Anthonisen et al., 1976, Hellinga et al., 1999) and NOB (Balmelle et al., 1992, Fux et al., 2003). The degree of inhibition was determined by comparing the OURwithCO2 data at different levels of the inhibitory compound with that measured in the absence of this inhibitory compound. With the implicit assumption that the bacterial oxygen consumption rate is proportional to their growth rate, which is a common assumption in activated sludge modeling (se e.g. the IWA Activated Sludge Models, Henze et al., 2000), the reduction in respiration activity was assumed to be the inhibitory effect on the bacterial growth processes. If this method of data interpretation were employed in this study, one would have concluded, based on the measured OURwithCO2 data, that FA had a limited inhibitory effect on the growth of NOB. This effect would be about 25% with an FA level of 6.0–9.0 mgNH3–N L1. However, bacteria produce energy to satisfy the energy requirement by both the growth and maintenance processes. Therefore, the bacterial respiration is not always coupled to their growth, and consequently the respiration rate is not necessarily proportional to the bacterial growth rate (see Eq. (1)). Consequently, the method widely employed to date for assessing the inhibitory effects of a potential inhibitor by measuring the respiration activities alone could potential give misleading results. In this paper, the respiration rates of Nitrobacter in the presence and absence of CO2 were measured. OURwithoutCO2 is unrelated to growth, and, therefore, its dependency on the FA concentrations truly reflects the inhibitory effects of FA on the energy production processes. Meanwhile, the difference between OURwithCO2 and OURwithoutCO2 should be directly related to the growth of Nitrobacter, as shown in Eq. (2). While the experimental data obtained in this study strongly suggest that FA has a strong negative effect on the biosynthesis processes of Nitrobacter, the method does not provide an unambiguous proof that such effects exist. Rather, this effect was inferred from indirect measurement and remains a hypothesis until experimental poof is achieved. Also, the experimental data does not allow identifying the fundamental mechanisms involved in the negative effect observed. The effect could be due to direct inhibition of FA on one or more steps in the carbon transformation processes. However, it may also be caused by energy limitation. As discussed above, energy production by Nitrobacter is inhibited by FA, which, if coupled with an increased maintenance energy requirement
ARTICLE IN PRESS 832
WA T E R R E S E A R C H
41 (2007) 826– 834
due to, for example, the high-level presence of FA, could result in a situation where Nitrobacter cells would have to spend all the energy produced for cell maintenance. Dawes and Ribbons (1964) suggested that in energy limiting conditions bacteria have to meet the maintenance energy requirement before spending energy on growth. Further studies are clearly needed to gain a more fundamental understanding of the inhibitory effects of FA on the metabolism of Nitrobacter and indeed on many other microorganisms as well. Such studies should preferably be carried out using pure cultures and by directly measuring the growth progresses. Despite of the above limitations, this study is to our knowledge the first, which shows that FA may have different inhibitory effects on the catabolic and anabolic processes of Nitrobacter.
3.6. Proposed role of FA inhibition on NOB washout in systems treating high-strength wastewater In recent years, the treatment of high-strength wastewater via nitrite to remove nitrogen has been demonstrated in several different processes. The mechanisms involved in the inhibition of NOB in these systems are still unclear. The results obtained in this study, together with the findings in Vadivelu et al. (2006b, c) on the FA and FNA inhibition on the metabolism of AOB and NOB, are summarized in Table 1. Clearly Nitrosomonas has a much higher level of tolerance to FA and FNA in comparison to Nitrobacter, which may have contributed significantly to the elimination of NOB from systems treating high nitrogen-containing wastewater. The typical concentrations of ammonium and nitrite in a system treating sludge digesting liquor through nitritation are in the range of 300–600 mgN L1 (Hellinga et al., 1999, Fux et al., 2003, Lai et al., 2004). Considering the variation of pH (6.5–8.0) in these systems, the FA and the FNA concentrations may vary between 4.0–30 mgNH3–N L1 and 0.04–0.30 mgHNO2–N L1, respectively. Therefore, both FA and FNA could be inhibiting factors of NOB. It is hypothesized that initially in the establishment of a system, the activities of NOB (non-adapted to high levels of FA) would be inhibited by the level of FA present in these systems, which leads to nitrite accumulation. An elevated level of FNA then forms an even stronger inhibitor to NOB growth, leading to their permanent elimination from the system.
An important factor that should be considered is the capability of microorganisms to adapt to inhibitory compounds. There are studies reported in literature showing that NOB could develop resistance towards FA. Villaverde et al. (2000) showed that the threshold value of specific FA inhibition (above which NOB activity ceased almost completely) increased from 0.2 to 0.7 mgNH 3 –N g biomass after a period of 4 months. Wong Chong and Loehr (1978) reported that Nitrobacter adapted to FA could resist an FA concentration of 40 mgNH3 L1, whereas a non-adapted biomass was inhibited at around 3.5 mgNH3 L1. To our best knowledge, no NOB cultures have been reported in literature to grow under high FNA levels or to be able to adapt to high FNA concentrations. It is, therefore, hypothesized that the most effective long-term inhibitor for NOB in systems treating high-strength wastewater is FNA. It should be mentioned that the Nitrobacter culture used in this study was not exposed to ammonium in the parent SBR, in order to avoid the presence of AOB in the enriched NOB culture. Thus, possible adaptation of these microorganisms to high ammonium concentration is not addressed in this study. Therefore, the results obtained may be valid for nonammonia adapted Nitrobacter cultures only. Nevertheless, it is worthwhile to point out that the FA inhibition on the energy production capacity of Nitrobacter is very limited even for the non-adapted biomass.
4.
Conclusions
The effect of FA on the metabolism of Nitrobacter was assessed using an enriched culture of Nitrobacter, not adapted to FA. It was found that the respiration of Nitrobacter was inhibited by FA even at low concentrations (less than 1 mgNH3–N L1). However, this inhibitory effect was limited. The respiration rate of Nitrobacter was reduced by 12% and 25%, respectively, in the absence and presence of inorganic carbon, when the FA concentration was increased from 0 to 9 mgNH3–N L1. FA may have a strong inhibitory effect on the anabolic processes of Nitrobacter. While the study did not provide unambiguous proof of this inhibitory effect, the experimental data strongly indicated that Nitrobacter likely ceased to grow at an FA concentration of 6–9 mgNH3–N L1. The study clearly
Table 1 – Summary of FNA and FA inhibition on Nitrobacter and Nitrosomonas Nitrobacter
Nitrosomonas
Anabolism
Catabolism
Anabolism
Catabolism
FNA
Likely stopped completely at 0.02 mgHNO2–N L1
No inhibition up to 0.04 mgHNO2–N L1
Likely inhibited completely at 0.40 mgHNO2–N L1
50% inhibition at 0.40–0.63 mgHNO2–N L1
FA
Likely inhibited completely at above 6.0 mgNH3–N L1
Inhibited by 12% at 6.0–9.0 mgNH3–N L1
No inhibition at up to 16.0 mgNH3–N L1
No inhibition at up to 16.0 mgNH3–N L1
ARTICLE IN PRESS WAT E R R E S E A R C H
shows the necessity of physiological studies to reveal the mechanisms of FA inhibition on bacterial metabolism. The conventional methods of assessing the inhibitory effects of FA on Nitrobacter (and possibly other bacteria as well), through simply comparing the overall substrate utilizing rates under inhibitory and non-inhibitory conditions, may produce misleading results. It is suggested that the catabolic and anabolic processes should be investigated separately.
Acknowledgments The Australian Research Council is thankfully acknowledged for funding this research through Project DP0342658. The first author would also like to thank Universiti Sains Malaysia, Malaysia for providing scholarships. Dr. Sandra Hall carried out all the FISH work reported in this paper.
Appendix A.
Supplementary data
Supplementary data associated with this article can be found in the online version at doi:10.1016/j.watres.2006.11.030
R E F E R E N C E S
Abeling, U., Seyfried, C.F., 1992. Anaerobic-aerobic treatment of high-strength ammonium waste-water—nitrogen removal via nitrite. Water Sci. Technol. 26 (5 and 6), 1007–1015. Anthonisen, A.C., Loehr, R.C., Prakasam, T.B.S., Srinath, E.G., 1976. Inhibition of nitrication by ammonia and nitrous acid. J. Water Pollut. Control Fed. 48 (5), 835–852. APHA, 1998. Standard Methods for Examination of Water and Wastewater, 20th ed. American Public Health Association. Balmelle, B., Nguyen, K.M., Capdeville, B., Cornier, J.C., Deguin, A., 1992. Study of factors controlling nitrite buildup in biological processes for water nitrification. Water Sci. Technol. 26 (5 and 6), 1017–1025. Bock, E., 1976. Growth of Nitrobacter in presence of organic-matter, 2. Chemo-organotrophic growth of Nitrobacter-agilis. Arch. Microbiol. 108 (3), 305–312. Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., Wagner, M., 1999. The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22 (3), 434–444. Daims, H., Nielsen, P.H., Nielsen, J.L., Juretschko, S., Wagner, M., 2000. Novel Nitrospira-like bacteria as dominant nitrite-oxidizers in biofilms from wastewater treatment plants: diversity and in situ physiology. Water Sci. Technol. 41 (4 and 5), 85–90. Daims, H., Nielsen, J.L., Nielsen, P.H., Schleifer, K.H., Wagner, M., 2001. In situ characterization of Nitrospira-like nitrite oxidizing bacteria active in wastewater treatment plants. Appl. Environ. Microbiol. 67 (11), 5273–5284. Dawes, E.A., Ribbons, D.W., 1964. Some aspects of the endogenous metabolism of bacteria. Bacteriol. Rev. 28 (2), 126–149. Fux, C., Lange, K., Faessler, A., Huber, P., Grueniger, B., Siegrist, H., 2003. Nitrogen removal from digester supernatant via nitrite—SBR or SHARON? Water Sci. Technol. 48 (8), 9–18. Gil, K.I., Choi, E.S., 2001. Modelling of inhibition of nitrite oxidation in biological nitritation processes by free ammonia. Biotechnol. Lett. 23 (24), 2021–2026.
41 (20 07) 82 6 – 834
833
Hall, S.J., Keller, J., Blackall, L.L., 2003. Microbial quantification in activated sludge: the hits and misses. Water Sci. Technol. 48 (3), 121–126. Hanaki, K., Wantawin, C., Ohgaki, S., 1990. Nitrification at lowlevels of dissolved-oxygen with and without organic loading in a suspended-growth reactor. Water Res. 24 (3), 297–302. Hellinga, C., Schellen, A., Mulder, J.W., van Loosdrecht, M.C.M., Heijnen, J.J., 1998. The SHARON process: an innovative method for nitrogen removal from ammonium-rich waste water. Water Sci. Technol. 37 (9), 135–142. Hellinga, C., van Loosdrecht, M.C.M., Heijnen, J.J., 1999. Model based design of a novel process for nitrogen removal from concentrated flows. Math. Comput. Model. Dyn. Syst. 5 (4), 351–371. Henze, M., Gujer, W., Mino, T., van Loosdrecht, M. (Eds.), 2000. Activated Sludge Models ASM1, ASM2, ASM2d and ASM3. IWA Publishing, London. Kuai, L.P., Verstraete, W., 1998. Ammonium removal by the oxygen-limited autotrophic nitrification-denitrification system. Appl. Environ. Microbiol. 64 (11), 4500–4506. Lai, E., Senkpiel, S., Solley, D., Keller, J., 2004. Nitrogen removal of high strength wastewater via nitritation/denitritation using a sequencing batch reactor. Water Sci. Technol. 50 (10), 27–33. Mauret, M., Paul, E., PuechCostes, E., Maurette, M.T., Baptiste, P., 1996. Application of experimental research methodology to the study of nitrification in mixed culture. Water Sci. Technol. 34 (1 and 2), 245–252. Neijssel, O.M., Tempest, D.W., 1976. Bioenergetic aspects of aerobic growth of Klebsiella-Aerogenes Nctc 418 in carbonlimited and carbon-sufficient chemostat culture. Arch. Microbiol. 107 (2), 215–221. Painter, H.A., 1970. A review of literature on inorganic nitrogen metabolism in microorganisms. Water Res. 4 (6), 393. Philips, S., Laanbroek, H.J., Verstraete, W., 2002. Origin, causes and effects of increased nitrite concentrations in aquatic environments. Environ. Sci. Biotechnol. 1, 115–141. Pirt, S.J., 1982. Maintenance energy—a general-model for energylimited and energy-sufficient growth. Arch. Microbiol. 133 (4), 300–302. Pratt, S., Yuan, Z., Gapes, D., Dorigo, M., Zeng, R.J., Keller, J., 2003. Development of a novel titration and off-gas analysis (TOGA) sensor for study of biological processes in wastewater treatment systems. Biotechnol. Bioeng. 81 (4), 482–495. Randall, C.W., Buth, D., 1984a. Nitrite buildup in activated-sludge resulting from combined temperature and toxicity effects. J. Water Pollut. Control Fed. 56 (9), 1045–1049. Randall, C.W., Buth, D., 1984b. Nitrite buildup in activated-sludge resulting from temperature effects. J. Water Pollut. Control Fed. 56 (9), 1039–1044. Smith, R.V., Doyle, R.M., Burns, L.C., Stevens, R.J., 1997. A model for nitrite accumulation in soils. Soil Biol. Biochem. 29 (8), 1241–1247. Surmacz-Gorska, J., Cichon, A., Miksch, K., 1997. Nitrogen removal from wastewater with high ammonia nitrogen concentration via shorter nitrification and denitrification. Water Sci. Technol. 36 (10), 73–78. Vadivelu, V.M., Keller, J., Yuan, Z., 2006a. Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture. Biotechnol. Bioeng. 95 (5), 830–839. Vadivelu, V.M., Yuan, Z., Fux, C., Keller, J., 2006b. The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture. Environ. Sci. Technol. 40 (14), 4442–4448. Vadivelu, V.M., Yuan, Z., Fux, C., Keller, J., 2006c. Stoichiometric and kinetic characterisation of Nitrobacter in mixed culture by decoupling the growth and energy generation processes. Biotechnol. Bioeng. 94 (6), 1176–1188.
ARTICLE IN PRESS 834
WA T E R R E S E A R C H
Villaverde, S., Fdz-Polanco, F., Garcia, P.A., 2000. Nitrifying biofilm acclimation to free ammonia in submerged biofilters. Start-up influence. Water Res. 34 (2), 602–610. Wagner, M., Rath, G., Koops, H.P., Flood, J., Amann, R.I., 1996. In situ analysis of nitrifying bacteria in sewage treatment plants. Water Sci. Technol. 34 (1 and 2), 237–244.
41 (2007) 826– 834
Wong Chong, G.M., Loehr, R.C., 1978. Kinetics of microbial nitrification—nitrite-nitrogen oxidation. Water Res. 12 (8), 605–609. Yang, L., Alleman, J.E., 1992. Investigation of batchwise nitrite buildup by an enriched nitrification culture. Water Sci. Technol. 26 (5 and 6), 997–1005.