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Nitrous oxide (N2O) production by Alcaligenes faecalis during feast and famine regimes
PII: S0043-1354(99)00374-7
Wat. Res. Vol. 34, No. 7, pp. 2080±2088, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
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NITROUS OXIDE (N2O) PRODUCTION BY ALCALIGENES FAECALIS DURING FEAST AND FAMINE REGIMES S. SCHALK-OTTE1, R. J. SEVIOUR2M, J. G. KUENEN1M and M. S. M. JETTEN1* 1
Kluyverlaboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands and 2Biotechnology Research Centre, LaTrobe University, P.O. Box 199, Bendigo 3550, Victoria, Australia (First received 1 April 1999; accepted in revised form 1 November 1999)
AbstractÐThe environmentally harmful compound, nitrous oxide (N2O), can accumulate as an intermediate in the process of denitri®cation. One important parameter, which can in¯uence this accumulation, is the feeding regime sensed by the bacteria involved, in which an unbalanced supply of electron donor and acceptor may occur. When pulse additions of C-compounds (acetate, butyrate and malate) were given to denitrifying cultures of Alcaligenes faecalis strain TUD, the production rate of N2O was reduced from 9.9±18.5% to 1.8±10.4% of the total nitrite converted, as long as the Csubstrate was in excess. However, as soon as the availability of carbon compounds became exhausted and the culture entered starvation, N2O was one of the main products of denitri®cation and production increased to 32±64% of the total N-feed. Under dynamic feeding conditions, the culture was able to adapt to the ¯uctuating conditions and the ratio of N2O to nitrite decreased. However, during starvation the ratio of N2O to nitrite was still high (2 27%), indicating that with prolonged starvation, the overall N2O emission will increase. Competition between the enzymes of denitri®cation for electrons from the cytochrome c pool could explain the emission of N2O, if the enzyme N2O-reductase has a lower anity for the electron-donor than the other reductases. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐnitrous oxide, N2O/N2 ratio, available carbon, denitri®cation, Alcaligenes faecalis
INTRODUCTION
In wastewater treatment systems, microbial processes are used to convert N-compounds (Jetten et al., 1997). One of these processes is denitri®cation ÿ in which nitrate (NOÿ 3 ) and nitrite (NO2 ) are converted to the harmless compound dinitrogen gas (N2). Intermediates in this process are the gaseous compounds nitric oxide (NO) and nitrous oxide (N2O), both of which are emitted from wastewater treatment systems (SchoÈn et al., 1994), contributing globally 1.0 Tg N yrÿ1 (Kroeze, 1994), equivalent to 5.9% of the total N2O budget. Emission of these gases can contribute to several environmental problems, such as the greenhouse eect and ozone depletion (Wang et al., 1976), and therefore, prevention of such emissions is desirable. In wastewater treatment systems, mixed populations of mainly heterotrophic organisms carry out denitri®cation. These bacteria will experience ¯uctuations in environmental conditions that may lead to incomÿ plete reduction of NOÿ 3 or NO2 to NO or N2O. One parameter that often ¯uctuates, is the amount *Author to whom all correspondence should be addressed. Fax: +31-15-2782355; e-mail: m.s.m.jetten@stm. tudelft.nl
of available electron donor, since the composition of the wastewater varies and is often limited in readily utilisable BOD. Therefore, in many wastewater treatment systems addition of an external carbon source is required to control nitrogen removal (Isaacs et al., 1994). Fluctuations in the composition of the in¯uent and (pulse) additions of external carbon sources may lead to a 'feast and famine' sensation, in which the bacteria experience alternating starvation or excess of C-availability. This unbalanced feeding regime may in¯uence the process of denitri®cation, since an increase in available electrons from the C-source must be matched by an increase in reducing power. In order to increase the rate of reduction of the N-compounds, the activity of the various enzymes involved in denitri®cation ÿ (NOÿ 3 -reductase, NO2 -reductase, NO-reductase and N2O-reductase) must increase. A dierence in activity or anity between these enzymes may lead to an increase in the concentration of undesirable intermediates, for example, NO or N2O. Unfortunately there are little experimental data available to show whether ¯uctuations in C-availability in¯uences emissions of these intermediates. Such information is important if the production of NO or N2O in wastewater treatment systems is to be con-
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N2O production by A. faecalis
trolled. Therefore, the eect of ¯uctuations in Cavailability on emission of intermediates as NO and N2O was studied in pure cultures by pulse addition of an external C-source. As a model organism, the heterotrophic denitri®er Alcaligenes faecalis was used, since this organism is commonly found in wastewater and produces high amounts of the intermediate N2O (Otte et al., 1996). Pulses of dierent carbon sources, including malate and butyrate, which are relatively more oxidised and reduced than acetate, respectively, were given. The role of internal storage material as a reserve C-source, during the imposed famine, and the eect of its reutilisation on N2O emission was also tested. To study the long term eect of feast and famine, a continuous steady state culture of A. faecalis was submitted to dynamic feeding conditions. MATERIALS AND METHODS
Organisms and cultivation Continuous steady state cultures of Alcaligenes faecalis strain TUD (LMD 89.147) were established in Applikon fermenters, as described previously (Otte et al., 1996). The dilution rate (D) used was 0.05 hÿ1, unless stated otherwise, and the volume was kept constant at 2 l by a level controller. The cultures were sparged with dinitrogen gas or helium at 130 ml minÿ1, which is sucient to strip all gases produced from the liquid. A lag of 10 min was observed between production of a gas in the liquid and detection by gas chromatography. Since this bacterium cannot reduce nitrate, but starts denitrifying from nitrite, the cultures were grown with 35 mM sodium nitrite endconcentration under acetate-limitation (15 mM). The medium was supplied in two equivolumetric parts, using an acid medium A and an alkaline medium B (Otte et al., 1996). For each pulse experiment new continuous steady state cultures were used. In the pulse experiments, the feedpumps were turned o after addition of the pulse, leaving the cultures in batch mode during the remainder of the experiment. Acetate was added as a solution of sodium acetate, butyrate was added as butyric acid (J. T. Baker, The Netherlands) and malate as L-malic acid (Sigma-Alldrich, The Netherlands) to the ®nal concentration mentioned in the Results. Dynamic conditions were applied to a continuous acetate-limited culture of A. faecalis using the computer programme BIODACS (Applikon Dependable Instruments, Schiedam, the Netherlands), which controlled the pumps to or from the following culture vessels: an euent vessel, a vessel containing a two times diluted medium A (Otte et al., 1996), a vessel with 0.5 M sodium acetate and a vessel containing 0.5 M sodium nitrite. A 10 h cycle was applied to the culture. At time zero the culture was given a pulse of about 8 mM acetate (®nal concentration) in 10 min. At t = 4.633 h and 9.633 h, 500 ml spent medium was pumped out in 10 min and at t = 4.8 h and 9.8 h 500 ml medium A (2 diluted) was pumped into the chemostat during 10 min. This resulted in a dilution rate of 0.05 hÿ1. At t = 5 h about 17 mM NOÿ 2 (®nal concentration) was added in 10 min. Temperature, stirrer speed, helium supply and pH were controlled in the same fashion as in steady state continuous cultivation. Analytical procedures Biomass was determined by measuring the optical density at 450 nm or 660 nm and by dry weight determi-
2081
nations using 0.2 mm nitrocellulose ®lters. Protein values were measured spectrophotometrically (Goa, 1953). Acetate and butyric acid were determined by GC-analysis, while malate was measured using an L-malic acid test kit (Boehringer Mannheim GmbH, Mannheim, Germany). The nitrite and ammonia concentrations were measured colorimetrically as described by (Griess-Romijn van Eck, 1996) and (Fawcett and Scott, 1960), respectively. All measurements were performed at least in duplo and standard deviations were calculated accordingly. These standard deviations were also taken into account when reaction rates were calculated from these measurements. An elemental composition of A. faecalis biomass of CH2O0.5N0.23 was used for determining kinetic values, as described previously (Otte et al., 1999). Poly-b-hydroxybutyrate (PHB) was determined by Nile Blue A staining (Ostle and Holt, 1982) or by extraction, hydrolysis and esteri®cation of the poly-acids in a one-step process. In this process, about 20 mg of freeze-dried cell material was transferred to test tubes closed with a screwcap. As an internal standard, 1 mg of benzoic acid in 1-propanol was used. To each tube, 1.5 ml of a mixture of concentrated HCl and 1-propanol (1:4) and 1.5 ml dichloroethane was added. Subsequently, the tubes were incubated at 1008C for 2 h with mixing every 15 min. After cooling, 3 ml distilled water was added, the contents vigorously mixed and subsequently centrifuged for a few min (12,000 rpm) to obtain a good phase separation. About 1 ml of the bottom phase (organic phase) was drawn o and ®ltered over a small column of dried waterfree sodium sulfate. After ®ltration the samples were analysed by a GC (Chrompack model 238 A) equipped with a model 910 autosampler, and an HP Innowax column (30 m0.53 mm, Hewlett Packard) and a ¯ame ionisation detector (2508C). Helium was used as a carrier gas and the data acquisition and processing was performed with the program Maestro 2.3 (Chrompack, Interscience). Gas-analysis CO2, N2O and N2 were analysed by on-line gas chromatography, as described previously (Otte et al., 1996), using a GC equipped with a thermal conductivity detector and an electron capture detector. In some cases, atmospheric air interfered with the detection of N2. This interference will show up as 'spikes' of N2, which are not representative for production peaks. NO was analysed by a chemiluminescence based NOx analyser (NO-NO2-NOx analyser
Fig. 1. Gas analysis of pulse addition of approximately 5 mM acetate to an acetate limited anaerobic culture of A. faecalis. At t = 0 the culture was switched from continuous cultivation to batch mode and the acetate pulse was given.
14.720.4 15.020.4 15.820.5 Maximal rate. A pulse of 4.2 mM butyrate was given to an acetate limited culture of A. faecalis. b
a
Anaerobic, D = 0.02 hÿ1
16622 203218 10928
12628 166235 17323
187211 164212 9327
190211 214215 208215
9.920.1 18.521.8 10.420.8
5.820.4 10.421.6 5.220.1
40.028.9 64.427.1 3228.3
1.220.1 1.120.1 1.220.1
Pulse experiments
Anaerobic, D = 0.05 hÿ1 Anaerobic, butyrateb, D = 0.05 hÿ1
Steady state During PHB-ox After pulse Steady state After pulse Steady state Steady state
After pulse
C-source cons. ratea (nmol C minÿ1 mg dwÿ1)
Ratio N2O: NOÿ 2 (% nmol N (nmol N)ÿ1)
model 42 S, Thermo Environmental Instruments Inc., Franklin MA., USA). Gas-samples (1 ml) were injected into a Sample Mixing Unit, designed for small volume samples (Kester et al., 1994).
RESULTS
a NOÿ 2 reduction rate (nmol N minÿ1 mg dwÿ1)
Culture conditions
Table 1. Pulse addition of various C-sources to acetate limited anaerobic cultures of A. faecalis
Max. after pulse
S. Schalk-Otte et al.
PHB formation (% g (g C biomass)ÿ1)
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Pulses of acetate to acetate limited cultures of A. faecalis. In order to investigate the response of an anaerobic denitrifying culture of A. faecalis to a sudden change in available electron donor, pulse experiments were performed. To a continuously grown acetate limited (15 mM) culture of A. faecalis, a pulse of approximately 5 mM sodium acetate was given at t = 0 h (Fig. 1), the pumps were stopped and the culture was allowed to grow in batch mode. After pulsing, the culture immediately started to deplete the NOÿ 2 in the medium at a maximum rate of 126 2 8 nmol N minÿ1 (mg dw)ÿ1, which was slightly lower than the consumption rate under steady state conditions (Table 1). CO2 and N2 production increased, but production of N2O and NO, which already occurred in continuous steady state culture, did not change signi®cantly after the pulse. Accumulation of the storage polymer poly-b-hydroxybutyrate (PHB) was 1.2% g (g C biomass)ÿ1 in acetate limited continuous cultivation as determined by both Nile Blue A staining (not shown) and chemical analysis (Table 1). After the pulse the amount of PHB increased to 7.9% (not shown). In approximately 2 h, the NOÿ 2 was depleted and production of all gaseous N-compounds ceased. Without denitri®cation, cell growth stopped and thus, CO2 production decreased accordingly. As soon as NOÿ 2 was depleted, acetate uptake no longer occurred, suggesting that active transport is involved in its assimilation. At t = 8 h, a pulse of approximately 15 mM NOÿ 2 was given to the culture, which resulted in an immediate increase in production of all gases. N2 production increased steadily, whilst N2O production increased more rapidly and then began to decrease again. Acetate consumption resumed after the NOÿ 2 pulse, at a rate of 187211 nmol C minÿ1 (mg dw)ÿ1, and cellular PHB accumulation increased from 7.9% g (g C)ÿ1 to a maximum of 14.7% (Table 1). As soon as acetate became exhausted (t = 14 h), the culture entered a starvation period and started to use its PHB. The production of N2 decreased to almost zero and CO2 production also decreased, with a half-life of approximately 2.9 h. However, N2O production increased sharply to a maximum of 258 mmol hÿ1, resulting in an N2O to NOÿ 2 ratio of 40%, which is much higher than recorded in steady state cultures (9.9%) or immediately after the acetate pulse (5.8%; Table 1). In the course of PHButilization the N2O production decreased slowly but remained one of the dominant products of denitri®cation. Apparently, when the cell was in a relatively
N2O production by A. faecalis
oxidised state (i.e. during starvation), the N2O production was signi®cantly higher than in steady state or after pulse addition of acetate. When the same experiment was performed with a continuous steady state culture of A. faecalis, which was kept under continuous conditions after the pulse, the same phenomena were observed (data not shown), although interpretation was complicated since wash-out kinetics had to be included to calculate enzyme activities. However, it was evident from these results that during starvation the N2O production increased relative to measurements in steady state and after a pulse of acetate. To study the eect of growth rate on the behaviour of A. faecalis following changes in feeding regime, a pulse of acetate was given to an acetate limited continuous steady state culture grown at D = 0.02 hÿ1. After the pulse of about 4 mM acetate, the same phenomena were observed as with the culture grown at D = 0.05 hÿ1. The production of N2 and CO2 increased slowly after the pulse, while the N2O production did not seem to change signi®cantly (data not shown). Nitrite was consumed at a rate which was slightly higher than the rate of steady state culture (Table 1) and was depleted after 4 h. At t = 7 h the culture was given a pulse of about 12 mM NOÿ 2 , resulting in an increase in N2O production and PHB accumulation. N2 and CO2 production also increased until acetate became exhausted at t = 10 h. At this time, N2O production increased further, while N2 and CO2 production both decreased, resulting in an increase in the ratio of N2O to NOÿ 2 from 5.2% to 32% (Table 1). A pulse of acetate, given to an acetate limited (10 mM), aerobic continuous steady state culture (D = 0.05 hÿ1) of A. faecalis, did not lead to any signi®cant changes in emission of the gaseous intermediates (data not shown). The organism, grown in the presence of 5 mM NOÿ 2 under 67% air satur-
Fig. 2. Gas analysis of pulse addition of approximately 5 mM butyrate to an acetate limited anaerobic culture of A. faecalis. At t = 0 the culture was switched from continuous cultivation to batch mode and the pulse was given.
2083
ation, slightly increased its CO2 production after a pulse addition of about 5 mM acetate. The existing production of N2 did not change signi®cantly and the very low N2O production rate (0.6 mmol hÿ1) increased slightly to 1.0 mmol hÿ1 over 3 h and then decreased again. Accordingly, nitrite consumption was also very low under continuous aerobic cultivation (about 1.9 nmol N minÿ1 (mg dw)ÿ1) and did not increase signi®cantly after addition of a pulse of acetate. A slight increase in PHB content was observed (from 0.1% g (g C)ÿ1 to 1.4%), and as soon as the acetate became exhausted again, the CO2 production decreased with a half-life of about 5.3 h and the N2O production rate fell to 0.5 mmol hÿ1. Pulses of other C-compounds to anaerobic acetate limited cultures of A. faecalis. In order to test the in¯uence of an increase in available electrons on the emission of N2O, an acetate limited continuous steady state culture of A. faecalis was provided with a pulse of about 4 mM butyrate. This compound is more reduced than acetate and will generate more electrons per C-mol oxidised (5 eÿ vs 4 eÿ). After the pulse, the culture was again switched to batch mode. Production of both N2 and CO2 increased immediately after the pulse, whilst a delayed increase in N2O production was observed (Fig. 2). However, this increase was only temporary and N2O production began to decline again before NOÿ 2 became exhausted. Butyrate was assimilated by the cells at a rate of 214215 nmol C minÿ1 (mg dw)ÿ1 (Table 1) and PHB started to increase. At the same time nitrite was taken up at 166235 nmol N minÿ1 (mg dw)ÿ1, which was almost the same as in steady state conditions. As soon as NOÿ 2 became exhausted (t = 2.5), the production of gaseous N-compounds ceased and the CO2 production rate fell. Uptake of butyrate stopped at this point, suggesting that assimilation of this compound also requires active transport. After addition of NOÿ 2 (15 mM), the production of all gases increased again and the cellular PHB content increased from 1.1% g (g C)ÿ1, in steady state, to a maximum of 15.0% (Table 1). When butyrate became exhausted (t = 11 h), N2 production decreased substantially, whilst the N2O production rate increased rapidly to a maximum of 430 mmol hÿ1. Both PHB content and CO2 production then decreased slowly after this time (t1/2= 3.9 h), indicating oxidation of the cellular PHB reserves. As observed previously, during oxidation of this PHB, the ratio of N2O to NOÿ 2 increased (from 10.4% after the pulse to 64.4% during starvation; Table 1). Malate is a more oxidised compound than acetate, but after pulse addition of this compound (4.1 mM) the same phenomena were observed as with butyrate. The organism could not metabolize this compound immediately and entered starvation after the pulse. During this starvation period, N2 production decreased rapidly, while N2O pro-
S. Schalk-Otte et al.
6.820.4 5.220.3 2.320.1 2.820.1 8.320.2 ± 86.3210.7 27.825.3 26.823.9 56.023.2 ± 2.220.1 1.020.1 1.320.2 1.720.1 Maximal. Average ratio over 10 h (1 cycle). c 10th cycle of a culture grown in batch culture prior to submission to dynamic conditions. d n.p.: not performed.
a
b
During PHB-ox After NOÿ 2 -pulse
Ratio N2O: NOÿ 2 (% mM N (mM)ÿ1)
After Ac-pulse
± 4.720.2 2.720.2 3.420.1 2.120.2 10425 320211 14126 11625 515213 17429 266211 452239 550268 317226
In order to mimic conditions in wastewater treatment systems, a continuous anaerobic culture of A. faecalis was submitted to dynamic conditions, in which an alternating feeding regime was established. In a 10 h period, the culture received an 8 mM acetate pulse at t = 0 and t = 10 h. After about 3 h after the ®rst pulse, NOÿ 2 was depleted and so at t = 5 h a pulse of about 16 mM NOÿ 2 was given. At t = 8 h, the acetate was exhausted and the culture entered a starvation period until t = 10 h, when a new acetate pulse was supplied. At this point, the cycle restarted. The culture was maintained at a dilution rate of
Steady state 1st cycle (0±10 h) 10th cycle (100±110 h) 20th cycle (190±200 h) 10th cyclec (90±100 h)
Dynamic conditions
N2O productiona (mmol hÿ1)
duction only decreased slowly (data not shown). The ratio of N2O to NOÿ 2 was high during this starvation period (75 2 25%), but as soon as malate uptake started (after approximately 8 h), CO2 and N2 production increased, while N2O production decreased further (data not shown).
a NOÿ 2 reduction rate (nmol N minÿ1 mg dwÿ1)
Fig. 3. Gas analysis of a continuous anaerobic culture of A. faecalis under dynamic conditions. (a): Gas analysis of the ®rst cycle. (b): gas analysis of the 10th cycle, when the culture has reached a semi steady state. For control of dynamic conditions, see Materials and methods.
Table 2. Anaerobic continuous steady state cultures of A. faecalis under dynamic conditions
Overallb
2.620.1 14.120.4 10.820.2 10.320.2 n.p.d
PHB formationa (% g (g C biomass)ÿ1)
2084
N2O production by A. faecalis
0.05 hÿ1 by refreshing 500 ml of the culture every 5 h. An acetate limited (15 mM) continuous steady state culture was subjected to this 10 h cycle for 200 h, to study the eect of long-term variations in substrate availability. At the start of the experiment, the culture responded to the acetate pulse as described for the pulse experiments. Immediately after the pulse, the N2O production rate started to decrease, while both the N2 and CO2 production rates increased (Fig. 3a). Nitrite was consumed at a maximum rate of 266 2 11 nmol N minÿ1 (mg dw)ÿ1, which was higher than in steady state (Table 2). As soon as NOÿ 2 was depleted, production of gaseous N-compounds ceased, as did acetate uptake and PHB synthesis. After a pulse of NOÿ 2 , production of all gases increased and the ratio of N2O to NOÿ 2 dropped to 2.2%. PHB accumulated to a maximum level of 14.1% (g (g C)ÿ1) until acetate became exhausted (with a maximal rate of 325 2 13 nmol C minÿ1 (mg dw)ÿ1, not shown). At this time, N2 and CO2 production rates decreased, while the N2O production rate increased to a maximum of 320 2 11 mmol hÿ1, again indicating that during starvation, when the cell is in a relatively oxidised state, the relative N2O production increases substantially. The ratio of N2O to NOÿ 2 increased from 4.7% after the acetate pulse to 86.3% during starvation (Table 2). After 100 h (=10 cycles and 5 volume changes), the culture showed the same pattern of response to the ¯uctuations as during the ®rst cycle. After the acetate pulse, NOÿ 2 was consumed at a rate of 452 2 39 nmol N minÿ1 (mg dw)ÿ1, which is much higher than at the start of the experiment (Table 2). The maximum acetate consumption rate observed was 401 2 33 nmol C minÿ1 (mg dw)ÿ1, which is also higher than the rate observed after the ®rst acetate pulse (not shown). PHB accumulated to a maximum level of 10.8% (g (g C)ÿ1) as shown in Table 2. However, the rate at which N2O was produced had also changed (Fig. 3b). As soon as acetate became exhausted, N2O production rate increased, as seen previously, but only to a maximum of 141 2 6 mmol hÿ1, which is much lower than in the initial cycle. The maximal N2 production rate did not change signi®cantly. The ratios of N2O to NOÿ 2 observed after the pulse and during starvation diered, but not as dramatically as during the ®rst cycle (i.e. increasing from 2.7% to 27.8% as compared to 86.3%; Table 2). The overall ratio over 10 h was, therefore, less than half the value observed in the ®rst cycle (2.3% vs 5.2%; Table 2). Apparently A. faecalis could adjust to the ¯uctuating conditions. After 200 h (10 volume changes and 20 cycles) the culture was responding to the ¯uctuations in a very similar way as observed in the 10th cycle (Table 2), indicating that it had already reached a semi-steady state after approximately 100 h. When the same experiment was performed with a
2085
culture, which was pregrown anaerobically in batch mode instead of in continuous steady state culture, the culture behaved similarly to the continuous culture during the ®rst cycle. However, when the culture, grown in batch, reached a semi-steady state after 10 to 20 cycles, the levels of N2O produced were much higher than when the culture was ®rst grown in continuous steady state culture (Table 2). Apparently, the growth history of the organism in¯uenced its ability to adapt to the ¯uctuating conditions. DISCUSSION
During wastewater treatment, addition of an external carbon source (i.e. methanol or acetate) is often essential for the anoxic removal of NOÿ 3 and NOÿ 2 (Isaacs et al., 1994) during denitri®cation. This process is performed by heterotrophic organisms that use an external carbon source for biomass production and to generate reducing equivalents. These equivalents are subsequently used for anaerobic reduction of nitrogen oxides for energy generation. The intermediates in this process nitric oxide (NO) and nitrous oxide (N2O) may be emitted into the environment when the individual steps in denitri®cation are not optimally balanced. One parameter that might in¯uence this balance and thus in¯uence the levels of emission of these intermediates, is the addition of an external carbon source. Fluctuations in the availability of substrate may result in ¯uctuations in the amount of electrons generated in the form of NADH, which needs to be reoxidized. To test the in¯uence of a sudden increase in C-source on NO and N2O emission, pulses of various substrates were given to acetatelimited cultures of A. faecalis. The results show that with an excess of electron donor, N2 production increased, whereas N2O production did not. Under continuous cultivation the ratio of N2O to NOÿ 2 varied from 9.9 to 18.5%, while after the substrate pulse this percentage ranged from 5.2 to 10.4% (Table 1). As soon as the added C-source became exhausted again, N2 production decreased severely and N2O production increased. The percentage of N2O formed from NOÿ 2 increased under these conditions to 32±64%, showing that when the cell is starving for C-compounds, and thus in a relatively oxidised state, the further reduction of N2O to N2 is signi®cantly decreased. Previous studies on the in¯uence of pulse-addition of C-compounds or of dierent C-sources on denitri®cation failed to show this dierence in end-product formation, since measurements of the gas-composition were neglected. However, low denitri®cation eciency during endogenous respiration was observed in a denitrifying sludge (Her and Huang, 1995). During starvation the sludge produced a gas mixture with a lower percentage of N2-gas than seen in the presence of an external C-source. The authors did not
2086
S. Schalk-Otte et al.
give the composition of the remainder of the gas, but an increase in NO or N2O may well have occurred. In the presented experiments, the NOÿ 2 consumption rate did not change signi®cantly after the pulse addition of C-compounds to the culture, whereas the production rates of N2O or N2 did. Although increases in biomass were detected during these pulse-experiments, the increase in N2 production rate observed after pulse addition, did not exceed the maximum N2O reduction rate measured in steady state (150±500 nmol minÿ1 (mg dw)ÿ1; Otte et al., 1996). Therefore, the increase in N2O emission observed on pulsing may not have been due to any dierence in either the amount or the endogenous activity of the nitrous oxide reductase (N2OR), the nitrite reductase (NiR) and the nitric oxide reductase (NOR). Similar results were obtained when a pulse of acetate was given to a culture grown at D = 0.02 hÿ1 (Table 1). This suggests that dierences in growth rate and, thus, dierences in turnover rates of the enzymes, do not determine the observed ¯uctuations in N2O emission. The data presented here suggest that it is more likely that the availability of electrons may in¯uence the various enzymes involved in denitri®cation, possibly by regulating their performance. This could be explained in terms of competition between the various denitri®cation enzymes for electrons, since this might explain the decrease in N2O emission after excess electrons are supplied. Thus, if the N2OR is competing with the NOR and NiR for electrons, then an extra supply of electrons may increase the electron pool, thereby removing any competition and resulting in an increased activity of N2OR (Fig. 4). As soon as the cells become more oxidised (i.e. when the cultures become C-limited), the compe-
Fig. 4. Possible denitri®cation pathway for A. faecalis, depicting the hypothesized competition for electrons by the reductases involved. The gray lines show the transfer of electrons from the cytochrome c pool to the reductases. The black lines show the reduction pathway of the various nitrogen oxides involved. The thickness of the line is indicative for the reaction rate. NiR: nitrite reductase, NOR: nitric oxide reductase, N2OR: nitrous oxide reductase, Cyt C: cytochrome C pool, eÿ: electron, P: periplasm, C: cytoplasm.
tition for electrons will again increase, resulting in a decrease in N2OR-activity and a concomitant increase in N2O emission. Blaszczyk (1993) has suggested that the energetic quantity of the substrate might in¯uence the accumulation of NOÿ 2 from NOÿ 3 in Paracoccus denitri®cans. He showed that growth on ethanol led to less NOÿ 2 accumulation than during growth on the more oxidised substrate acetate. However, the results obtained with A. faecalis suggest that it is not the energetic status of the substrate alone that may aect the emission of gaseous intermediates, since no dierence in behaviour was observed between acetate pulses and those of the more reduced substrate butyrate. Therefore, the level of reduction of the cytochrome pool and/or the respiratory chain may be important. Pidello et al. (1996) also observed a negative relationship between N2O production and the level of reduction in soil samples, which may explain why pulse addition of acetate to an aerobic culture of A. faecalis did not result in a substantial increase in NO or N2O emission. In the presence of oxygen the cytochrome pool will always be in a relatively oxidised state and, thus, will not lead to emission of these intermediates during denitri®cation. However, the nitrite reduction rate under aerobic conditions was very low and results from previous work with A. faecalis (Otte et al., 1999) indicate that the observed N2O production may not be due to reduction of NOÿ 2 , but may be a byproduct of heterotrophic nitri®cation. Therefore, competition between the enzymes of denitri®cation may not have occurred under these conditions. Another regulatory control on the availability of electrons may be at the transcriptional level. For example, Sears et al. (1997) found that the expression of a periplasmic nitrate reductase (Nap) of Paracoccus denitri®cans Pd1222 was higher in butyrate-limited cultures than in the more oxidised malate-limited cultures. They also showed that expression of the membrane bound nitrate reductase (Nar) under aerobic conditions may have been due to redox sensing of FNR-like (FNR=Fumurate and Nitrate Reduction) regulatory proteins (Spiro, 1994). In Escherichia coli, the function of FNR proteins is controlled by a positive redox potential of >+400 mV (Unden et al., 1990). However, the time-spans over which the pulse experiments were performed in the study presented here, make it most unlikely that the observed ¯uctuations in N2O emission are caused by any transcriptional control. For example, the immediate increase in N2 production and decrease in N2O production cannot be explained by the much slower process of transcription. From previous work (Otte et al., 1996) it is known that induction of NiR in A. faecalis takes approximately 20 h at this dilution rate, so transcriptional control is probably not involved, even in the later stages of the experiments.
N2O production by A. faecalis
To study the long-term in¯uence of ¯uctuations in available electron donor, an acetate limited continuous steady state culture was submitted to dynamic conditions. The response of the culture in the ®rst cycle resembled the response observed in the pulse experiments. However, after 10 cycles the culture seemed to have adapted in that its response to the ¯uctuations were less dramatic. Although NOÿ 2 consumption had increased signi®cantly, the ratio of N2O to NOÿ 2 during starvation did not increase as dramatically as during the ®rst cycle (27.8% instead of 86.3%). Therefore, the overall ratio over 10 h had decreased signi®cantly compared either to steady state conditions or to the ®rst cycle (2.3, 6.8 and 5.2%, respectively). This indicates that the culture is able to adapt to these ¯uctuating conditions, as noticed previously with ¯uctuating oxygen concentrations (Otte et al., 1996). In wastewater treatment systems with continuously changing conditions, therefore, sudden changes in the emission of N2O may be dampened. The denitrifying biomass from wastewater treatment plants may well be adapted to these ¯uctuations, as shown in this study for a pure culture of A. faecalis, and produce few partially reduced intermediates. However, in one experiment, a culture of A. faecalis was pregrown in batch mode and thus had a dierent history than the culture described above. This batch grown culture showed much higher N2O production levels even after 20 cycles. This suggested that the composition and history of a culture can in¯uence the overall N2O emission. Both types of dynamic cultures, however, showed that during starvation the relative N2O production can reach very high values. In the dynamic experiments described here, the starvation period was approximately 2 h (one ®fth of the total cycle) and the overall N2O emission was therefore low. If in wastewater treatment plants the starvation period is signi®cantly longer, then N2O emission may also be much more pronounced. Future work will therefore concentrate on the eect of ¯uctuating C-availability on denitrifying biomass from wastewater treatment systems under dierent dynamic regimes, including long periods of starvation.
CONCLUSIONS
1. Nitrous oxide production is high under carbon starvation in denitrifying cultures of A. faecalis. 2. A culture submitted to continuously changing conditions was able to reduce its overall N2O emission by adaptation. 3. Competition between the enzymes of denitri®cation for electrons may explain the increased N2O production during starvation. 4. If in wastewater treatment plants the starvation
2087
periods are signi®cantly longer than those used in this study, N2O emission can be signi®cant.
AcknowledgementsÐThe authors are grateful to M. Zomerdijk and G. van der Steen for acetate and PHB-analyses, respectively. They would also like to thank ir. Jos Schalk for helpful discussions on enzyme properties.
REFERENCES
Blaszczyk M. (1993) Eect of medium composition on the denitri®cation of nitrate by Paracoccus denitri®cans. Appl. Environ. Microbiol. 59, 3951±3953. Fawcett J. K. and Scott J. E. (1960) A rapid and precise method for the determination of urea. J. Clin. Pathol. 13, 156±159. Goa J. (1953) A micro-biuret method for protein determination: determination of total protein in cerebrospinal ¯uid. J. Clin. Lab. Invest. 5, 218±222. Griess-Romijn van Eck E. (1966) Physiological and chemical tests for drinking water. In NEN 1056, IV-2. Nederlands Normalisatie Instituut, Rijswijk, the Netherlands. Her J. J. and Huang J. S. (1995) In¯uences of carbon surface and C/N ratio on nitrate/nitrite denitri®cation and carbon breakthrough. Bioresource Technol. 54, 45±51. Isaacs S. H., Henze M., Soeberg H. and Kuemmel M. (1994) External carbon source addition as a means to control an activated sludge nutrient removal process. Water. Res. 28, 511±520. Jetten M. S. M., Logemann S., Muyzer G. M., Robertson L. A., DeVries S., van Loosdrecht M. C. M. and Kuenen J. G. (1997) Novel principles in the microbial conversion of nitrogen compounds. Antonie Van Leeuwenhoek 71, 75±93. Kester R. A., Wijlhuizen A. G., Duyts H. and Laanbroek H. J. (1994) Chemiluminescence analysis of nitric oxide in small-volume samples by a modi®ed injection method. Biol. Fertil. Soils 18, 260±262. Kroeze C. (1994). Nitrous oxide (N2O) emission inventory and options for control in the Netherlands. RIVM report 773001004, p.3±9. National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands. Ostle A. G. and Holt J. G. (1982) Nile Blue A as a ¯uorescent stain for poly-b-hydroxybutyrate. Appl. Environ. Microbiol. 44, 238±241. Otte S., Grobben N. G., Robertson L. A., Jetten M. S. M. and Kuenen J. G. (1996) Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions. Appl. Environ. Microbiol. 62, 2421±2426. Otte S., Schalk J., Kuenen J. G. and Jetten M. S. M. (1999) Hydroxylamine oxidation and subsequent nitrous oxide production by the heterotrophic ammonia oxidizer Alcaligenes faecalis. Appl. Microbiol. Biotechnol. 51, 255±261. Pidello A., Menendez L. and Perotti E. B. R. (1996) Saccharidic compounds as soil redox eectors and their in¯uence on potential N2O production. Biol. Fert. Soils 23, 173±176. SchoÈn G., Buûmann M. and Geywitz-Hetz G. (1994) Bildung von Lachgas (N2O) im belebten Schlamm aus KlaÈranlagen. Wasser Abwasser 135, 293±301. Sears H. J., Spiro S. and Richardson D. J. (1997) Eect of carbon substrate and aeration on nitrate reduction and expression of the periplasmic and membrane-bound nitrate reductases in carbon-limited continuous cultures
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of Paracoccus denitri®cans pd1222. Microbiology 143, 3767±3774. Spiro S. (1994) The FNR family of transcriptional regulators. Antonie van Leeuwenhoek 66, 23±36. Unden G., Trageser M. and Duchene A. (1990) Eect of positive redox potentials (>+400 mV) on the ex-
pression of anaerobic respiratory enzymes in Escherichia coli. Molecular Microbiology 4, 315±319. Wang W. C., Yung Y. L., Lacis A. A., Mo T. and Hansen J. E. (1976) Greenhouse eects due to man-made perturbations of trace gases. Science 194, 685±690.
Wat. Res. Vol. 34, No. 7, pp. 2080±2088, 2000 # 2000 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/00/$ - see front matter
www.elsevier.com/locate/watres
NITROUS OXIDE (N2O) PRODUCTION BY ALCALIGENES FAECALIS DURING FEAST AND FAMINE REGIMES S. SCHALK-OTTE1, R. J. SEVIOUR2M, J. G. KUENEN1M and M. S. M. JETTEN1* 1
Kluyverlaboratory for Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands and 2Biotechnology Research Centre, LaTrobe University, P.O. Box 199, Bendigo 3550, Victoria, Australia (First received 1 April 1999; accepted in revised form 1 November 1999)
AbstractÐThe environmentally harmful compound, nitrous oxide (N2O), can accumulate as an intermediate in the process of denitri®cation. One important parameter, which can in¯uence this accumulation, is the feeding regime sensed by the bacteria involved, in which an unbalanced supply of electron donor and acceptor may occur. When pulse additions of C-compounds (acetate, butyrate and malate) were given to denitrifying cultures of Alcaligenes faecalis strain TUD, the production rate of N2O was reduced from 9.9±18.5% to 1.8±10.4% of the total nitrite converted, as long as the Csubstrate was in excess. However, as soon as the availability of carbon compounds became exhausted and the culture entered starvation, N2O was one of the main products of denitri®cation and production increased to 32±64% of the total N-feed. Under dynamic feeding conditions, the culture was able to adapt to the ¯uctuating conditions and the ratio of N2O to nitrite decreased. However, during starvation the ratio of N2O to nitrite was still high (2 27%), indicating that with prolonged starvation, the overall N2O emission will increase. Competition between the enzymes of denitri®cation for electrons from the cytochrome c pool could explain the emission of N2O, if the enzyme N2O-reductase has a lower anity for the electron-donor than the other reductases. # 2000 Elsevier Science Ltd. All rights reserved Key wordsÐnitrous oxide, N2O/N2 ratio, available carbon, denitri®cation, Alcaligenes faecalis
INTRODUCTION
In wastewater treatment systems, microbial processes are used to convert N-compounds (Jetten et al., 1997). One of these processes is denitri®cation ÿ in which nitrate (NOÿ 3 ) and nitrite (NO2 ) are converted to the harmless compound dinitrogen gas (N2). Intermediates in this process are the gaseous compounds nitric oxide (NO) and nitrous oxide (N2O), both of which are emitted from wastewater treatment systems (SchoÈn et al., 1994), contributing globally 1.0 Tg N yrÿ1 (Kroeze, 1994), equivalent to 5.9% of the total N2O budget. Emission of these gases can contribute to several environmental problems, such as the greenhouse eect and ozone depletion (Wang et al., 1976), and therefore, prevention of such emissions is desirable. In wastewater treatment systems, mixed populations of mainly heterotrophic organisms carry out denitri®cation. These bacteria will experience ¯uctuations in environmental conditions that may lead to incomÿ plete reduction of NOÿ 3 or NO2 to NO or N2O. One parameter that often ¯uctuates, is the amount *Author to whom all correspondence should be addressed. Fax: +31-15-2782355; e-mail: m.s.m.jetten@stm. tudelft.nl
of available electron donor, since the composition of the wastewater varies and is often limited in readily utilisable BOD. Therefore, in many wastewater treatment systems addition of an external carbon source is required to control nitrogen removal (Isaacs et al., 1994). Fluctuations in the composition of the in¯uent and (pulse) additions of external carbon sources may lead to a 'feast and famine' sensation, in which the bacteria experience alternating starvation or excess of C-availability. This unbalanced feeding regime may in¯uence the process of denitri®cation, since an increase in available electrons from the C-source must be matched by an increase in reducing power. In order to increase the rate of reduction of the N-compounds, the activity of the various enzymes involved in denitri®cation ÿ (NOÿ 3 -reductase, NO2 -reductase, NO-reductase and N2O-reductase) must increase. A dierence in activity or anity between these enzymes may lead to an increase in the concentration of undesirable intermediates, for example, NO or N2O. Unfortunately there are little experimental data available to show whether ¯uctuations in C-availability in¯uences emissions of these intermediates. Such information is important if the production of NO or N2O in wastewater treatment systems is to be con-
2080
N2O production by A. faecalis
trolled. Therefore, the eect of ¯uctuations in Cavailability on emission of intermediates as NO and N2O was studied in pure cultures by pulse addition of an external C-source. As a model organism, the heterotrophic denitri®er Alcaligenes faecalis was used, since this organism is commonly found in wastewater and produces high amounts of the intermediate N2O (Otte et al., 1996). Pulses of dierent carbon sources, including malate and butyrate, which are relatively more oxidised and reduced than acetate, respectively, were given. The role of internal storage material as a reserve C-source, during the imposed famine, and the eect of its reutilisation on N2O emission was also tested. To study the long term eect of feast and famine, a continuous steady state culture of A. faecalis was submitted to dynamic feeding conditions. MATERIALS AND METHODS
Organisms and cultivation Continuous steady state cultures of Alcaligenes faecalis strain TUD (LMD 89.147) were established in Applikon fermenters, as described previously (Otte et al., 1996). The dilution rate (D) used was 0.05 hÿ1, unless stated otherwise, and the volume was kept constant at 2 l by a level controller. The cultures were sparged with dinitrogen gas or helium at 130 ml minÿ1, which is sucient to strip all gases produced from the liquid. A lag of 10 min was observed between production of a gas in the liquid and detection by gas chromatography. Since this bacterium cannot reduce nitrate, but starts denitrifying from nitrite, the cultures were grown with 35 mM sodium nitrite endconcentration under acetate-limitation (15 mM). The medium was supplied in two equivolumetric parts, using an acid medium A and an alkaline medium B (Otte et al., 1996). For each pulse experiment new continuous steady state cultures were used. In the pulse experiments, the feedpumps were turned o after addition of the pulse, leaving the cultures in batch mode during the remainder of the experiment. Acetate was added as a solution of sodium acetate, butyrate was added as butyric acid (J. T. Baker, The Netherlands) and malate as L-malic acid (Sigma-Alldrich, The Netherlands) to the ®nal concentration mentioned in the Results. Dynamic conditions were applied to a continuous acetate-limited culture of A. faecalis using the computer programme BIODACS (Applikon Dependable Instruments, Schiedam, the Netherlands), which controlled the pumps to or from the following culture vessels: an euent vessel, a vessel containing a two times diluted medium A (Otte et al., 1996), a vessel with 0.5 M sodium acetate and a vessel containing 0.5 M sodium nitrite. A 10 h cycle was applied to the culture. At time zero the culture was given a pulse of about 8 mM acetate (®nal concentration) in 10 min. At t = 4.633 h and 9.633 h, 500 ml spent medium was pumped out in 10 min and at t = 4.8 h and 9.8 h 500 ml medium A (2 diluted) was pumped into the chemostat during 10 min. This resulted in a dilution rate of 0.05 hÿ1. At t = 5 h about 17 mM NOÿ 2 (®nal concentration) was added in 10 min. Temperature, stirrer speed, helium supply and pH were controlled in the same fashion as in steady state continuous cultivation. Analytical procedures Biomass was determined by measuring the optical density at 450 nm or 660 nm and by dry weight determi-
2081
nations using 0.2 mm nitrocellulose ®lters. Protein values were measured spectrophotometrically (Goa, 1953). Acetate and butyric acid were determined by GC-analysis, while malate was measured using an L-malic acid test kit (Boehringer Mannheim GmbH, Mannheim, Germany). The nitrite and ammonia concentrations were measured colorimetrically as described by (Griess-Romijn van Eck, 1996) and (Fawcett and Scott, 1960), respectively. All measurements were performed at least in duplo and standard deviations were calculated accordingly. These standard deviations were also taken into account when reaction rates were calculated from these measurements. An elemental composition of A. faecalis biomass of CH2O0.5N0.23 was used for determining kinetic values, as described previously (Otte et al., 1999). Poly-b-hydroxybutyrate (PHB) was determined by Nile Blue A staining (Ostle and Holt, 1982) or by extraction, hydrolysis and esteri®cation of the poly-acids in a one-step process. In this process, about 20 mg of freeze-dried cell material was transferred to test tubes closed with a screwcap. As an internal standard, 1 mg of benzoic acid in 1-propanol was used. To each tube, 1.5 ml of a mixture of concentrated HCl and 1-propanol (1:4) and 1.5 ml dichloroethane was added. Subsequently, the tubes were incubated at 1008C for 2 h with mixing every 15 min. After cooling, 3 ml distilled water was added, the contents vigorously mixed and subsequently centrifuged for a few min (12,000 rpm) to obtain a good phase separation. About 1 ml of the bottom phase (organic phase) was drawn o and ®ltered over a small column of dried waterfree sodium sulfate. After ®ltration the samples were analysed by a GC (Chrompack model 238 A) equipped with a model 910 autosampler, and an HP Innowax column (30 m0.53 mm, Hewlett Packard) and a ¯ame ionisation detector (2508C). Helium was used as a carrier gas and the data acquisition and processing was performed with the program Maestro 2.3 (Chrompack, Interscience). Gas-analysis CO2, N2O and N2 were analysed by on-line gas chromatography, as described previously (Otte et al., 1996), using a GC equipped with a thermal conductivity detector and an electron capture detector. In some cases, atmospheric air interfered with the detection of N2. This interference will show up as 'spikes' of N2, which are not representative for production peaks. NO was analysed by a chemiluminescence based NOx analyser (NO-NO2-NOx analyser
Fig. 1. Gas analysis of pulse addition of approximately 5 mM acetate to an acetate limited anaerobic culture of A. faecalis. At t = 0 the culture was switched from continuous cultivation to batch mode and the acetate pulse was given.
14.720.4 15.020.4 15.820.5 Maximal rate. A pulse of 4.2 mM butyrate was given to an acetate limited culture of A. faecalis. b
a
Anaerobic, D = 0.02 hÿ1
16622 203218 10928
12628 166235 17323
187211 164212 9327
190211 214215 208215
9.920.1 18.521.8 10.420.8
5.820.4 10.421.6 5.220.1
40.028.9 64.427.1 3228.3
1.220.1 1.120.1 1.220.1
Pulse experiments
Anaerobic, D = 0.05 hÿ1 Anaerobic, butyrateb, D = 0.05 hÿ1
Steady state During PHB-ox After pulse Steady state After pulse Steady state Steady state
After pulse
C-source cons. ratea (nmol C minÿ1 mg dwÿ1)
Ratio N2O: NOÿ 2 (% nmol N (nmol N)ÿ1)
model 42 S, Thermo Environmental Instruments Inc., Franklin MA., USA). Gas-samples (1 ml) were injected into a Sample Mixing Unit, designed for small volume samples (Kester et al., 1994).
RESULTS
a NOÿ 2 reduction rate (nmol N minÿ1 mg dwÿ1)
Culture conditions
Table 1. Pulse addition of various C-sources to acetate limited anaerobic cultures of A. faecalis
Max. after pulse
S. Schalk-Otte et al.
PHB formation (% g (g C biomass)ÿ1)
2082
Pulses of acetate to acetate limited cultures of A. faecalis. In order to investigate the response of an anaerobic denitrifying culture of A. faecalis to a sudden change in available electron donor, pulse experiments were performed. To a continuously grown acetate limited (15 mM) culture of A. faecalis, a pulse of approximately 5 mM sodium acetate was given at t = 0 h (Fig. 1), the pumps were stopped and the culture was allowed to grow in batch mode. After pulsing, the culture immediately started to deplete the NOÿ 2 in the medium at a maximum rate of 126 2 8 nmol N minÿ1 (mg dw)ÿ1, which was slightly lower than the consumption rate under steady state conditions (Table 1). CO2 and N2 production increased, but production of N2O and NO, which already occurred in continuous steady state culture, did not change signi®cantly after the pulse. Accumulation of the storage polymer poly-b-hydroxybutyrate (PHB) was 1.2% g (g C biomass)ÿ1 in acetate limited continuous cultivation as determined by both Nile Blue A staining (not shown) and chemical analysis (Table 1). After the pulse the amount of PHB increased to 7.9% (not shown). In approximately 2 h, the NOÿ 2 was depleted and production of all gaseous N-compounds ceased. Without denitri®cation, cell growth stopped and thus, CO2 production decreased accordingly. As soon as NOÿ 2 was depleted, acetate uptake no longer occurred, suggesting that active transport is involved in its assimilation. At t = 8 h, a pulse of approximately 15 mM NOÿ 2 was given to the culture, which resulted in an immediate increase in production of all gases. N2 production increased steadily, whilst N2O production increased more rapidly and then began to decrease again. Acetate consumption resumed after the NOÿ 2 pulse, at a rate of 187211 nmol C minÿ1 (mg dw)ÿ1, and cellular PHB accumulation increased from 7.9% g (g C)ÿ1 to a maximum of 14.7% (Table 1). As soon as acetate became exhausted (t = 14 h), the culture entered a starvation period and started to use its PHB. The production of N2 decreased to almost zero and CO2 production also decreased, with a half-life of approximately 2.9 h. However, N2O production increased sharply to a maximum of 258 mmol hÿ1, resulting in an N2O to NOÿ 2 ratio of 40%, which is much higher than recorded in steady state cultures (9.9%) or immediately after the acetate pulse (5.8%; Table 1). In the course of PHButilization the N2O production decreased slowly but remained one of the dominant products of denitri®cation. Apparently, when the cell was in a relatively
N2O production by A. faecalis
oxidised state (i.e. during starvation), the N2O production was signi®cantly higher than in steady state or after pulse addition of acetate. When the same experiment was performed with a continuous steady state culture of A. faecalis, which was kept under continuous conditions after the pulse, the same phenomena were observed (data not shown), although interpretation was complicated since wash-out kinetics had to be included to calculate enzyme activities. However, it was evident from these results that during starvation the N2O production increased relative to measurements in steady state and after a pulse of acetate. To study the eect of growth rate on the behaviour of A. faecalis following changes in feeding regime, a pulse of acetate was given to an acetate limited continuous steady state culture grown at D = 0.02 hÿ1. After the pulse of about 4 mM acetate, the same phenomena were observed as with the culture grown at D = 0.05 hÿ1. The production of N2 and CO2 increased slowly after the pulse, while the N2O production did not seem to change signi®cantly (data not shown). Nitrite was consumed at a rate which was slightly higher than the rate of steady state culture (Table 1) and was depleted after 4 h. At t = 7 h the culture was given a pulse of about 12 mM NOÿ 2 , resulting in an increase in N2O production and PHB accumulation. N2 and CO2 production also increased until acetate became exhausted at t = 10 h. At this time, N2O production increased further, while N2 and CO2 production both decreased, resulting in an increase in the ratio of N2O to NOÿ 2 from 5.2% to 32% (Table 1). A pulse of acetate, given to an acetate limited (10 mM), aerobic continuous steady state culture (D = 0.05 hÿ1) of A. faecalis, did not lead to any signi®cant changes in emission of the gaseous intermediates (data not shown). The organism, grown in the presence of 5 mM NOÿ 2 under 67% air satur-
Fig. 2. Gas analysis of pulse addition of approximately 5 mM butyrate to an acetate limited anaerobic culture of A. faecalis. At t = 0 the culture was switched from continuous cultivation to batch mode and the pulse was given.
2083
ation, slightly increased its CO2 production after a pulse addition of about 5 mM acetate. The existing production of N2 did not change signi®cantly and the very low N2O production rate (0.6 mmol hÿ1) increased slightly to 1.0 mmol hÿ1 over 3 h and then decreased again. Accordingly, nitrite consumption was also very low under continuous aerobic cultivation (about 1.9 nmol N minÿ1 (mg dw)ÿ1) and did not increase signi®cantly after addition of a pulse of acetate. A slight increase in PHB content was observed (from 0.1% g (g C)ÿ1 to 1.4%), and as soon as the acetate became exhausted again, the CO2 production decreased with a half-life of about 5.3 h and the N2O production rate fell to 0.5 mmol hÿ1. Pulses of other C-compounds to anaerobic acetate limited cultures of A. faecalis. In order to test the in¯uence of an increase in available electrons on the emission of N2O, an acetate limited continuous steady state culture of A. faecalis was provided with a pulse of about 4 mM butyrate. This compound is more reduced than acetate and will generate more electrons per C-mol oxidised (5 eÿ vs 4 eÿ). After the pulse, the culture was again switched to batch mode. Production of both N2 and CO2 increased immediately after the pulse, whilst a delayed increase in N2O production was observed (Fig. 2). However, this increase was only temporary and N2O production began to decline again before NOÿ 2 became exhausted. Butyrate was assimilated by the cells at a rate of 214215 nmol C minÿ1 (mg dw)ÿ1 (Table 1) and PHB started to increase. At the same time nitrite was taken up at 166235 nmol N minÿ1 (mg dw)ÿ1, which was almost the same as in steady state conditions. As soon as NOÿ 2 became exhausted (t = 2.5), the production of gaseous N-compounds ceased and the CO2 production rate fell. Uptake of butyrate stopped at this point, suggesting that assimilation of this compound also requires active transport. After addition of NOÿ 2 (15 mM), the production of all gases increased again and the cellular PHB content increased from 1.1% g (g C)ÿ1, in steady state, to a maximum of 15.0% (Table 1). When butyrate became exhausted (t = 11 h), N2 production decreased substantially, whilst the N2O production rate increased rapidly to a maximum of 430 mmol hÿ1. Both PHB content and CO2 production then decreased slowly after this time (t1/2= 3.9 h), indicating oxidation of the cellular PHB reserves. As observed previously, during oxidation of this PHB, the ratio of N2O to NOÿ 2 increased (from 10.4% after the pulse to 64.4% during starvation; Table 1). Malate is a more oxidised compound than acetate, but after pulse addition of this compound (4.1 mM) the same phenomena were observed as with butyrate. The organism could not metabolize this compound immediately and entered starvation after the pulse. During this starvation period, N2 production decreased rapidly, while N2O pro-
S. Schalk-Otte et al.
6.820.4 5.220.3 2.320.1 2.820.1 8.320.2 ± 86.3210.7 27.825.3 26.823.9 56.023.2 ± 2.220.1 1.020.1 1.320.2 1.720.1 Maximal. Average ratio over 10 h (1 cycle). c 10th cycle of a culture grown in batch culture prior to submission to dynamic conditions. d n.p.: not performed.
a
b
During PHB-ox After NOÿ 2 -pulse
Ratio N2O: NOÿ 2 (% mM N (mM)ÿ1)
After Ac-pulse
± 4.720.2 2.720.2 3.420.1 2.120.2 10425 320211 14126 11625 515213 17429 266211 452239 550268 317226
In order to mimic conditions in wastewater treatment systems, a continuous anaerobic culture of A. faecalis was submitted to dynamic conditions, in which an alternating feeding regime was established. In a 10 h period, the culture received an 8 mM acetate pulse at t = 0 and t = 10 h. After about 3 h after the ®rst pulse, NOÿ 2 was depleted and so at t = 5 h a pulse of about 16 mM NOÿ 2 was given. At t = 8 h, the acetate was exhausted and the culture entered a starvation period until t = 10 h, when a new acetate pulse was supplied. At this point, the cycle restarted. The culture was maintained at a dilution rate of
Steady state 1st cycle (0±10 h) 10th cycle (100±110 h) 20th cycle (190±200 h) 10th cyclec (90±100 h)
Dynamic conditions
N2O productiona (mmol hÿ1)
duction only decreased slowly (data not shown). The ratio of N2O to NOÿ 2 was high during this starvation period (75 2 25%), but as soon as malate uptake started (after approximately 8 h), CO2 and N2 production increased, while N2O production decreased further (data not shown).
a NOÿ 2 reduction rate (nmol N minÿ1 mg dwÿ1)
Fig. 3. Gas analysis of a continuous anaerobic culture of A. faecalis under dynamic conditions. (a): Gas analysis of the ®rst cycle. (b): gas analysis of the 10th cycle, when the culture has reached a semi steady state. For control of dynamic conditions, see Materials and methods.
Table 2. Anaerobic continuous steady state cultures of A. faecalis under dynamic conditions
Overallb
2.620.1 14.120.4 10.820.2 10.320.2 n.p.d
PHB formationa (% g (g C biomass)ÿ1)
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N2O production by A. faecalis
0.05 hÿ1 by refreshing 500 ml of the culture every 5 h. An acetate limited (15 mM) continuous steady state culture was subjected to this 10 h cycle for 200 h, to study the eect of long-term variations in substrate availability. At the start of the experiment, the culture responded to the acetate pulse as described for the pulse experiments. Immediately after the pulse, the N2O production rate started to decrease, while both the N2 and CO2 production rates increased (Fig. 3a). Nitrite was consumed at a maximum rate of 266 2 11 nmol N minÿ1 (mg dw)ÿ1, which was higher than in steady state (Table 2). As soon as NOÿ 2 was depleted, production of gaseous N-compounds ceased, as did acetate uptake and PHB synthesis. After a pulse of NOÿ 2 , production of all gases increased and the ratio of N2O to NOÿ 2 dropped to 2.2%. PHB accumulated to a maximum level of 14.1% (g (g C)ÿ1) until acetate became exhausted (with a maximal rate of 325 2 13 nmol C minÿ1 (mg dw)ÿ1, not shown). At this time, N2 and CO2 production rates decreased, while the N2O production rate increased to a maximum of 320 2 11 mmol hÿ1, again indicating that during starvation, when the cell is in a relatively oxidised state, the relative N2O production increases substantially. The ratio of N2O to NOÿ 2 increased from 4.7% after the acetate pulse to 86.3% during starvation (Table 2). After 100 h (=10 cycles and 5 volume changes), the culture showed the same pattern of response to the ¯uctuations as during the ®rst cycle. After the acetate pulse, NOÿ 2 was consumed at a rate of 452 2 39 nmol N minÿ1 (mg dw)ÿ1, which is much higher than at the start of the experiment (Table 2). The maximum acetate consumption rate observed was 401 2 33 nmol C minÿ1 (mg dw)ÿ1, which is also higher than the rate observed after the ®rst acetate pulse (not shown). PHB accumulated to a maximum level of 10.8% (g (g C)ÿ1) as shown in Table 2. However, the rate at which N2O was produced had also changed (Fig. 3b). As soon as acetate became exhausted, N2O production rate increased, as seen previously, but only to a maximum of 141 2 6 mmol hÿ1, which is much lower than in the initial cycle. The maximal N2 production rate did not change signi®cantly. The ratios of N2O to NOÿ 2 observed after the pulse and during starvation diered, but not as dramatically as during the ®rst cycle (i.e. increasing from 2.7% to 27.8% as compared to 86.3%; Table 2). The overall ratio over 10 h was, therefore, less than half the value observed in the ®rst cycle (2.3% vs 5.2%; Table 2). Apparently A. faecalis could adjust to the ¯uctuating conditions. After 200 h (10 volume changes and 20 cycles) the culture was responding to the ¯uctuations in a very similar way as observed in the 10th cycle (Table 2), indicating that it had already reached a semi-steady state after approximately 100 h. When the same experiment was performed with a
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culture, which was pregrown anaerobically in batch mode instead of in continuous steady state culture, the culture behaved similarly to the continuous culture during the ®rst cycle. However, when the culture, grown in batch, reached a semi-steady state after 10 to 20 cycles, the levels of N2O produced were much higher than when the culture was ®rst grown in continuous steady state culture (Table 2). Apparently, the growth history of the organism in¯uenced its ability to adapt to the ¯uctuating conditions. DISCUSSION
During wastewater treatment, addition of an external carbon source (i.e. methanol or acetate) is often essential for the anoxic removal of NOÿ 3 and NOÿ 2 (Isaacs et al., 1994) during denitri®cation. This process is performed by heterotrophic organisms that use an external carbon source for biomass production and to generate reducing equivalents. These equivalents are subsequently used for anaerobic reduction of nitrogen oxides for energy generation. The intermediates in this process nitric oxide (NO) and nitrous oxide (N2O) may be emitted into the environment when the individual steps in denitri®cation are not optimally balanced. One parameter that might in¯uence this balance and thus in¯uence the levels of emission of these intermediates, is the addition of an external carbon source. Fluctuations in the availability of substrate may result in ¯uctuations in the amount of electrons generated in the form of NADH, which needs to be reoxidized. To test the in¯uence of a sudden increase in C-source on NO and N2O emission, pulses of various substrates were given to acetatelimited cultures of A. faecalis. The results show that with an excess of electron donor, N2 production increased, whereas N2O production did not. Under continuous cultivation the ratio of N2O to NOÿ 2 varied from 9.9 to 18.5%, while after the substrate pulse this percentage ranged from 5.2 to 10.4% (Table 1). As soon as the added C-source became exhausted again, N2 production decreased severely and N2O production increased. The percentage of N2O formed from NOÿ 2 increased under these conditions to 32±64%, showing that when the cell is starving for C-compounds, and thus in a relatively oxidised state, the further reduction of N2O to N2 is signi®cantly decreased. Previous studies on the in¯uence of pulse-addition of C-compounds or of dierent C-sources on denitri®cation failed to show this dierence in end-product formation, since measurements of the gas-composition were neglected. However, low denitri®cation eciency during endogenous respiration was observed in a denitrifying sludge (Her and Huang, 1995). During starvation the sludge produced a gas mixture with a lower percentage of N2-gas than seen in the presence of an external C-source. The authors did not
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give the composition of the remainder of the gas, but an increase in NO or N2O may well have occurred. In the presented experiments, the NOÿ 2 consumption rate did not change signi®cantly after the pulse addition of C-compounds to the culture, whereas the production rates of N2O or N2 did. Although increases in biomass were detected during these pulse-experiments, the increase in N2 production rate observed after pulse addition, did not exceed the maximum N2O reduction rate measured in steady state (150±500 nmol minÿ1 (mg dw)ÿ1; Otte et al., 1996). Therefore, the increase in N2O emission observed on pulsing may not have been due to any dierence in either the amount or the endogenous activity of the nitrous oxide reductase (N2OR), the nitrite reductase (NiR) and the nitric oxide reductase (NOR). Similar results were obtained when a pulse of acetate was given to a culture grown at D = 0.02 hÿ1 (Table 1). This suggests that dierences in growth rate and, thus, dierences in turnover rates of the enzymes, do not determine the observed ¯uctuations in N2O emission. The data presented here suggest that it is more likely that the availability of electrons may in¯uence the various enzymes involved in denitri®cation, possibly by regulating their performance. This could be explained in terms of competition between the various denitri®cation enzymes for electrons, since this might explain the decrease in N2O emission after excess electrons are supplied. Thus, if the N2OR is competing with the NOR and NiR for electrons, then an extra supply of electrons may increase the electron pool, thereby removing any competition and resulting in an increased activity of N2OR (Fig. 4). As soon as the cells become more oxidised (i.e. when the cultures become C-limited), the compe-
Fig. 4. Possible denitri®cation pathway for A. faecalis, depicting the hypothesized competition for electrons by the reductases involved. The gray lines show the transfer of electrons from the cytochrome c pool to the reductases. The black lines show the reduction pathway of the various nitrogen oxides involved. The thickness of the line is indicative for the reaction rate. NiR: nitrite reductase, NOR: nitric oxide reductase, N2OR: nitrous oxide reductase, Cyt C: cytochrome C pool, eÿ: electron, P: periplasm, C: cytoplasm.
tition for electrons will again increase, resulting in a decrease in N2OR-activity and a concomitant increase in N2O emission. Blaszczyk (1993) has suggested that the energetic quantity of the substrate might in¯uence the accumulation of NOÿ 2 from NOÿ 3 in Paracoccus denitri®cans. He showed that growth on ethanol led to less NOÿ 2 accumulation than during growth on the more oxidised substrate acetate. However, the results obtained with A. faecalis suggest that it is not the energetic status of the substrate alone that may aect the emission of gaseous intermediates, since no dierence in behaviour was observed between acetate pulses and those of the more reduced substrate butyrate. Therefore, the level of reduction of the cytochrome pool and/or the respiratory chain may be important. Pidello et al. (1996) also observed a negative relationship between N2O production and the level of reduction in soil samples, which may explain why pulse addition of acetate to an aerobic culture of A. faecalis did not result in a substantial increase in NO or N2O emission. In the presence of oxygen the cytochrome pool will always be in a relatively oxidised state and, thus, will not lead to emission of these intermediates during denitri®cation. However, the nitrite reduction rate under aerobic conditions was very low and results from previous work with A. faecalis (Otte et al., 1999) indicate that the observed N2O production may not be due to reduction of NOÿ 2 , but may be a byproduct of heterotrophic nitri®cation. Therefore, competition between the enzymes of denitri®cation may not have occurred under these conditions. Another regulatory control on the availability of electrons may be at the transcriptional level. For example, Sears et al. (1997) found that the expression of a periplasmic nitrate reductase (Nap) of Paracoccus denitri®cans Pd1222 was higher in butyrate-limited cultures than in the more oxidised malate-limited cultures. They also showed that expression of the membrane bound nitrate reductase (Nar) under aerobic conditions may have been due to redox sensing of FNR-like (FNR=Fumurate and Nitrate Reduction) regulatory proteins (Spiro, 1994). In Escherichia coli, the function of FNR proteins is controlled by a positive redox potential of >+400 mV (Unden et al., 1990). However, the time-spans over which the pulse experiments were performed in the study presented here, make it most unlikely that the observed ¯uctuations in N2O emission are caused by any transcriptional control. For example, the immediate increase in N2 production and decrease in N2O production cannot be explained by the much slower process of transcription. From previous work (Otte et al., 1996) it is known that induction of NiR in A. faecalis takes approximately 20 h at this dilution rate, so transcriptional control is probably not involved, even in the later stages of the experiments.
N2O production by A. faecalis
To study the long-term in¯uence of ¯uctuations in available electron donor, an acetate limited continuous steady state culture was submitted to dynamic conditions. The response of the culture in the ®rst cycle resembled the response observed in the pulse experiments. However, after 10 cycles the culture seemed to have adapted in that its response to the ¯uctuations were less dramatic. Although NOÿ 2 consumption had increased signi®cantly, the ratio of N2O to NOÿ 2 during starvation did not increase as dramatically as during the ®rst cycle (27.8% instead of 86.3%). Therefore, the overall ratio over 10 h had decreased signi®cantly compared either to steady state conditions or to the ®rst cycle (2.3, 6.8 and 5.2%, respectively). This indicates that the culture is able to adapt to these ¯uctuating conditions, as noticed previously with ¯uctuating oxygen concentrations (Otte et al., 1996). In wastewater treatment systems with continuously changing conditions, therefore, sudden changes in the emission of N2O may be dampened. The denitrifying biomass from wastewater treatment plants may well be adapted to these ¯uctuations, as shown in this study for a pure culture of A. faecalis, and produce few partially reduced intermediates. However, in one experiment, a culture of A. faecalis was pregrown in batch mode and thus had a dierent history than the culture described above. This batch grown culture showed much higher N2O production levels even after 20 cycles. This suggested that the composition and history of a culture can in¯uence the overall N2O emission. Both types of dynamic cultures, however, showed that during starvation the relative N2O production can reach very high values. In the dynamic experiments described here, the starvation period was approximately 2 h (one ®fth of the total cycle) and the overall N2O emission was therefore low. If in wastewater treatment plants the starvation period is signi®cantly longer, then N2O emission may also be much more pronounced. Future work will therefore concentrate on the eect of ¯uctuating C-availability on denitrifying biomass from wastewater treatment systems under dierent dynamic regimes, including long periods of starvation.
CONCLUSIONS
1. Nitrous oxide production is high under carbon starvation in denitrifying cultures of A. faecalis. 2. A culture submitted to continuously changing conditions was able to reduce its overall N2O emission by adaptation. 3. Competition between the enzymes of denitri®cation for electrons may explain the increased N2O production during starvation. 4. If in wastewater treatment plants the starvation
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periods are signi®cantly longer than those used in this study, N2O emission can be signi®cant.
AcknowledgementsÐThe authors are grateful to M. Zomerdijk and G. van der Steen for acetate and PHB-analyses, respectively. They would also like to thank ir. Jos Schalk for helpful discussions on enzyme properties.
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