World Journal
of Microbiology
and Biotechnology,
9, 59-62
Growth yield of denitrifiers using nitrous oxide as a terminal electron acceptor G.U. Okereke The molar yields (g celbmol) for Alcaligenes faecalis, Pseudomonas stutzeri, Paracoccus denitrificans and Pseudomonas perfecfomarinus batch cultures, under nitrous oxide (N,O) as the electron acceptor, were 11.2, 8.2, 6.1 and 4.4, respectively. Paracoccus denitrificans and Pseudomonasperfectomarinus, which had the slowest growth rates, gave the lowest yields. Large maintenance energy costs may be partially responsible for this. The growth efficiencies of A. faecalis and Ps. perfectomarinus on N,O indicate that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N,O reduction are about 49% and 39% of those in the electron transport system associated with 0, respiration, respectively. Key
words:
Alcaligenes,
denitrification,
electron acceptors, nitrous oxide, Paracoccus,
Denitrification may be defined as the reduction of nitrate, via nitrite and nitrous oxide, to N,, using the pathway as a respiratory alternative to oxygen (Gijskuenen & Robertson 1987). In many bacteria, denitrification only occurs under anaerobic conditions, while other species continue to denitrify at varying levels of dissolved oxygen (Robertson & Kuenen 1984). Stouthamer (1988) reported that, in a number of denitrifying bacteria, N,O is the end product of denitrification. The reduction of NO; to N,O in propionate bacteria is considered to be a detoxication mechanism rather than part of an energy transformation system (Kaspar 1982). Evans et al. (1985) demonstrated that N,O reduction could be blocked by the exposure of soil to air for 24 h, due either to oxygen inhibition or to NO; accumulation from nitrification. Okereke (1984a,b) reported that 59 out of 71 denitrifiers isolated by Gamble et al. (1977) from eight countries utilized N,O as terminal acceptor. Although some literature on N,O utilization and production by some denitrifiers is available, there has not been an extensive study of the growth and energy yield of N,O utilization by denitrifiers in general. Interest in this topic has been stimulated by the hypothesis that N,O released to the atmosphere leads to the partial destruction of the ozone layer which protects the earth from biologically
G.U. Okereke was with the Department of Crop and Soil Science, Michigan State University, East Lansing, MI 48823, USA, and is now with the Biotechnology Research Centre, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Anambra State, Nigeria. @ 1993 Rapid
Communications
of Oxford
Ltd
Pseudomonas
harmful ultraviolet radiation. As far as ecological interpretations are concerned, it is useful to estimate growth yields of denitrifiers on N,O for use in assessing their importance in the ecosystem. In this regard, work with pure cultures can provide useful data, although care has to be taken when using the results to predict what happens in nature. The objective of the study was, therefore, to quantify the growth yields of some denitrifiers that use N,O as the terminal acceptor.
Materials
and Methods
orgunisms suitable strains and media for denitrifiers, isolates which were confirmed as denitrifiers by the protocols of Gamble et al. (1977) were grown on three different media. The strains selected to determine growth yields on N,O and 0, as terminal electron acceptors were Alculigenes fueculis (ATCC X8750), Pseudomonas To select
(ATCC 17588), Purucoccus denitrificuns (ATCC 2008) and Pseudomonas perfectornurinw. The origin of these strains is given by Gamble et u2. (1977).
stufzeri
Compurison of Growth Media Aggregates of sterile soil containing stoch cultures of denitrifiers were either grown on nutrient broth (Difco) or on Tryptic soy broth (Difco) and incubated at 30°C. When turbid, a loopfnl of the culture was transferred into 10 ml of either 0.8% (w/v) nutrient broth (Difco), 3% (w/v) triptic soy broth (TSB) or 3% (w/v) TSB plus 3.5 mM KNO,. Tryptic soy broth contained (g/l): Trypticase peptone, 17 g; phytone peptone, 3 g; NaCl, 5 g; KJ-tpO,, 2.5 g and Bacto dextrose, 2.5 g. Nutrient broth contained (g/l):
G. U. Okereke Bacto beef extract, 3 g and Bacto peptone, 5 g. Growth was scored as visible turbidity after 7 to 14 days. For growth yield experiments, the inocula of the strains were grown anaerobically on TSB and then transferred (4 ml) to side-arm 164-m] Erlenmeyer flasks which contained 100 ml of TSB. The flasks were sealed with rubber stoppers and made anaerobic by evacuating the air and filling with helium (He). Nitrous oxide (0.2 atmosphere) was added with a syringe after removing an equivalent volume of He by syringe. Flasks were incubated at 30°C on a rotary shaker at 150 rev/min. Growth yields, monitored turbidometrically at 640 nm, were converted to cell dry weight by means of a calibration curve for each organism. Justin & Kelly (1978) reported that this was a reliable method for monitoring biomass concentration. At the same time, a gas sample from the headspace of the flask was analysed for N,O by injecting the gas into a gas chromatograph (Model 8515, Carle Instruments, Fullerton, CA) equipped with Propak Q (3 mm x 1.8 m), microthermistor detector and molecular sieve 5A. The column temperature was 45°C and carrier gas was He at a flow rate of 25 ml/min. Peaks were monitored on a chart recorder and quantified by a computing integrator. The total N,O content was determined for the vapour plus solution phase using a coefficient of 0.67, accorrding to the method of Smith et al. (1978). The same procedure was used for growth yield experiments with 0, and nutrient broth as culture components.
Results
and Discussion
To eliminate any experiment was denitrifiers under shows that the
possible toxic effect of N,O, a preliminary conducted to study the growth of the different N,O concentrations. Figure 1 various denitrifiers grew linearly with
Figure 1. Growth 640 nm, in different
of denitrifiers, nitrous oxide
perfectomarinus (A). genes faecalis (0).
60
World ]ouml
measurec concentratil Pseudomonas sfutze
of Microbiology
j as turbidities
at
ons : Pseudomonas ri (m) and Alcali-
and Biotechnology, Vol 9, 1993
Table 1. Comparison of complex 123 isolated denitrifiers.*
media
Isolates
Medium
Nutrient Tryptic Tryptic
for supporting
broth + 0, soy broth + O2 soy broth + 3.5 mM KNO,;
no O2
growth
that %
of
grewt
76 98 89
‘The 123 isolates were from soil and identified as denitrifiers by the protocols of Gamble et al. (1977). t lnoculum was pre-grown on nutrient broth and tryptic soy broth; 110 of the 123 strains taken from the soil stock culture grew on one or both media.
increase in the concentration of N,O. Thus toxicity was not apparent at 20% (v/v) N,O. The adequacy of the procedure used to minimize the influence of contamination by 0, during N,O-dependent growth was demonstrated by Okereke (1984a). The results of another preliminary experiment, to compare the ability of denitrifier isolates to grow aerobically on two media, showed that TSB was more suitable as 98% of the isolates grew in this media, compared with 76% in nutrient broth (Table I). It would appear that the organic substances in nutrient broth were not adequate to satisfy the nutritional demand of many of the denitrifying microflora. A previous study, by dkereke (1984a), &owed that denitrifiers utilized N,O faster in TSB than in nutrient broth. Tryptic soy broth was, therefore, chosen for the studies on growth yields of denitrifiers. Figure 2 shows the growth of denitrifiers corresponding to different N,O consumptions while Table 2 shows the molar growth yields (g cells/mol acceptor) of Alculigenes faecalis, Pseudomonas stutzeri, Paracoccw denitrificans and Ps. perfectomarinus. Koike & Hattori (1975a) reported that Pa. denittificans showed similar cell yields per electron transferred when NO; and NO, were electron acceptors. Their data are also summarized in Table 2. These data do not include maintenance energy, which becomes more significant as growth decreases. The A. fuecalis strains, which grew very quickly, had lower values. The two with the lowest yield also had the slowest growth rates; thus, a large maintenance energy cost may be at least partially responsible for the lower yield. Comparison of the growth rates of A. fuecalis and Ps. perfectomarintts when 0, or N,O was used as a terminal acceptor, showed that the growth rate was higher under aerobic than under anaerobic condition (Table 3). The growth efficiencies of N,O reduction by A. fuecalis and Ps. perfectomarinus were about 49% and 39%, respectively, of those when 0, was used as the terminal electron acceptor. This is to be expected from the greater energy available from aerobic oxidation. Koike & Hattori (1975b) reported that, in a batch culture, the energy yield of Pseudomonas
Growth of denifrifiers on N,O denifrificuns associated with denitrification is approximately half that with aerobic respiration on an electronic basis. Komer & Zumft (1989) demonstrated that the expression of nitrate reductase and N,O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration and of that available for enhanced expression of the N,O reductase. One can conclude that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N,O reduction in this study were about 39% and 49%, for P. perferfomarinus and A. faeculis respectively, of those in the electron transport system associated with 0, respiration. The comparatively higher growth yield reported for A. fuecalisindicates that a more efficient energy coupling system may exist in this denitrifier.
Acknowledgement I
5
1
1.0 Nitrous
1
1.5
oxide
2.0
The research was supported
(mol)
consumed
by a grant from Prof. J. Tiedje,
Department of Crop and Soil Science, Michigan State University, East Lansing, MI, USA.
Figure 2. Growth yields of denitrifier strains with nitrous oxide as terminal electron acceptor: Pseudomonas perfectomarinus (A) ; Pseudomonas sfufzeri ( n ) and Alcaligenes faecalis (0).
Table
2. Growth
yleids
of denitrifier
Strain
Alcaligenes Pseudomonas Paracoccus Pseudomonas Pseudomonas
TSB-tryptic t Data from
l
strains
grown
in batch
Electron acceptor
faecalis stutzeri denitrificans perfectomarinus denitrificanst
soy broth. Koike 8 Hattori
culture
Carbon source
N,O W W NL’ NO, NO,
TSB’ TSB TSB TSB
W
Glutamate
Glutamate Glutamate
with
N20 as electron
Molar growth yield (g ceii/moi acceptor)
acceptor. Yield per electron transferred (g ceils/e)
11.2 8.2 6.1 4.4 28.6 16.9 0.8
Generation time (h)
5.6 4.1 3.0 2.2 5.7 5.6 4.4
1.0 1.2 2.2 2.0 -
(1975a).
References Table 3. Comparison peffecfomerlnus using electron acceptors.
of growth rate of A. faecalls oxygen or nitrous oxide as
Strain
Growth in air
Alcaligenes Pseudomonas
faecalis perfectomarinus
6.6 1.8
rate
and P. terminal
(mglh)
in nitrous 2.8 0.7
oxide
Evans, D.G., Beauchamp, E. & Trevors, J.T. 1985 Sulpbide alleviation of acetylene inhibition of nitrous oxide reduction in soil. Applied and Environmental Microbiology 49, 217-220. Gamble, T.N., Betlach, N.R. & Tiedje, TJ. 1977 Numerically dominant denitrifying bacteria from world soils. Applied and Environmentul Microbiology 33, 926939. Gijskuenen, J. & Robertson, L.A. 1987 Ecology of nit&cation and denitrification in the nitrogen and sulfur cycles, In Society for General Microbiology Symposium 42, eds Cole, J.A. & Ferguson, S. pp. 161-217. Cambridge: Cambridge University Press.
Worki ]ournal of Minobidogy
and Biotechnology, Vol 9, 1993
61
G. U. Okereke Justin, P. & Kelly, D.P. 1978 Growth kinetics of Thiobucillw denifrijicuns in anaerobic and aerobic chemostat culture. Journal of General Microbiology 107, 123-130. Kaspar, H.F. I982 Nitrite reduction to nitrous oxide by Propionibacteria: Detoxication mechanism. Archives of Microbiology 133, 126-130. Kioke, I. & Hattori, A. 1975a Energy yield of denitrification: An estimate from growth yield in continuous cultures of Pseudomonus denitrificuns under nitrate, nitrite and nitrous oxide limited conditions. journal of General Microbiology 88, 11-19. Kioke, I. & Hattori, A. 1975b Growth yield of a denihifying bacterium, Pseudomonas denitrificiuns, under aerobic and denitrifying conditions. ]ournul of General Microbiology 88, 10. Komer, H. & Zumft, W.G. 1989 Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri. Applied and Environmenful Microbiology 55, 167CS1676.
62
World Journal of Microbiology and Biotechnology, Vol 9, 1993
Okereke, G.U. 1984a capability in denitrifiers
Prevalence of from a variety
nitrous oxide reducing of habitats. Plant and Soil
81,421-428. Okereke, G.U. 1984b Possible use of N,O in MPN tubes for enumeration of denitrifiers. Plant and Soil 80, 295-296. Robertson, L.A. & Kuenen, J.G. 1984 Aerobic denitrification-old wine in new bottles. Antonie van Leeuwenhoek, ~ournul of Microbiology and Serology 50, 525-544. Smith, M.S., Firestone, J.K. & Tiedje, J.M. 1978. The acetylene inhibition method for short-term measurement of soil denitrification and its evaluation using Nitrogen-13. Soil Science Society of American ]ournul 42, 611-615. Stouthamer, A.H. 1988 Dissimilatory reduction of oxidation nitrogen compounds. In Biology of Anaerobic Microorganisms, ed Zehunder, A.J.B. pp. 245-303. New York: John Wiley and Sons. (Received
in revised
form
16Jme
1992;
accepted
26]une
1992)
of Microbiology
and Biotechnology,
9, 59-62
Growth yield of denitrifiers using nitrous oxide as a terminal electron acceptor G.U. Okereke The molar yields (g celbmol) for Alcaligenes faecalis, Pseudomonas stutzeri, Paracoccus denitrificans and Pseudomonas perfecfomarinus batch cultures, under nitrous oxide (N,O) as the electron acceptor, were 11.2, 8.2, 6.1 and 4.4, respectively. Paracoccus denitrificans and Pseudomonasperfectomarinus, which had the slowest growth rates, gave the lowest yields. Large maintenance energy costs may be partially responsible for this. The growth efficiencies of A. faecalis and Ps. perfectomarinus on N,O indicate that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N,O reduction are about 49% and 39% of those in the electron transport system associated with 0, respiration, respectively. Key
words:
Alcaligenes,
denitrification,
electron acceptors, nitrous oxide, Paracoccus,
Denitrification may be defined as the reduction of nitrate, via nitrite and nitrous oxide, to N,, using the pathway as a respiratory alternative to oxygen (Gijskuenen & Robertson 1987). In many bacteria, denitrification only occurs under anaerobic conditions, while other species continue to denitrify at varying levels of dissolved oxygen (Robertson & Kuenen 1984). Stouthamer (1988) reported that, in a number of denitrifying bacteria, N,O is the end product of denitrification. The reduction of NO; to N,O in propionate bacteria is considered to be a detoxication mechanism rather than part of an energy transformation system (Kaspar 1982). Evans et al. (1985) demonstrated that N,O reduction could be blocked by the exposure of soil to air for 24 h, due either to oxygen inhibition or to NO; accumulation from nitrification. Okereke (1984a,b) reported that 59 out of 71 denitrifiers isolated by Gamble et al. (1977) from eight countries utilized N,O as terminal acceptor. Although some literature on N,O utilization and production by some denitrifiers is available, there has not been an extensive study of the growth and energy yield of N,O utilization by denitrifiers in general. Interest in this topic has been stimulated by the hypothesis that N,O released to the atmosphere leads to the partial destruction of the ozone layer which protects the earth from biologically
G.U. Okereke was with the Department of Crop and Soil Science, Michigan State University, East Lansing, MI 48823, USA, and is now with the Biotechnology Research Centre, Nnamdi Azikiwe University, P.M.B. 5025, Awka, Anambra State, Nigeria. @ 1993 Rapid
Communications
of Oxford
Ltd
Pseudomonas
harmful ultraviolet radiation. As far as ecological interpretations are concerned, it is useful to estimate growth yields of denitrifiers on N,O for use in assessing their importance in the ecosystem. In this regard, work with pure cultures can provide useful data, although care has to be taken when using the results to predict what happens in nature. The objective of the study was, therefore, to quantify the growth yields of some denitrifiers that use N,O as the terminal acceptor.
Materials
and Methods
orgunisms suitable strains and media for denitrifiers, isolates which were confirmed as denitrifiers by the protocols of Gamble et al. (1977) were grown on three different media. The strains selected to determine growth yields on N,O and 0, as terminal electron acceptors were Alculigenes fueculis (ATCC X8750), Pseudomonas To select
(ATCC 17588), Purucoccus denitrificuns (ATCC 2008) and Pseudomonas perfectornurinw. The origin of these strains is given by Gamble et u2. (1977).
stufzeri
Compurison of Growth Media Aggregates of sterile soil containing stoch cultures of denitrifiers were either grown on nutrient broth (Difco) or on Tryptic soy broth (Difco) and incubated at 30°C. When turbid, a loopfnl of the culture was transferred into 10 ml of either 0.8% (w/v) nutrient broth (Difco), 3% (w/v) triptic soy broth (TSB) or 3% (w/v) TSB plus 3.5 mM KNO,. Tryptic soy broth contained (g/l): Trypticase peptone, 17 g; phytone peptone, 3 g; NaCl, 5 g; KJ-tpO,, 2.5 g and Bacto dextrose, 2.5 g. Nutrient broth contained (g/l):
G. U. Okereke Bacto beef extract, 3 g and Bacto peptone, 5 g. Growth was scored as visible turbidity after 7 to 14 days. For growth yield experiments, the inocula of the strains were grown anaerobically on TSB and then transferred (4 ml) to side-arm 164-m] Erlenmeyer flasks which contained 100 ml of TSB. The flasks were sealed with rubber stoppers and made anaerobic by evacuating the air and filling with helium (He). Nitrous oxide (0.2 atmosphere) was added with a syringe after removing an equivalent volume of He by syringe. Flasks were incubated at 30°C on a rotary shaker at 150 rev/min. Growth yields, monitored turbidometrically at 640 nm, were converted to cell dry weight by means of a calibration curve for each organism. Justin & Kelly (1978) reported that this was a reliable method for monitoring biomass concentration. At the same time, a gas sample from the headspace of the flask was analysed for N,O by injecting the gas into a gas chromatograph (Model 8515, Carle Instruments, Fullerton, CA) equipped with Propak Q (3 mm x 1.8 m), microthermistor detector and molecular sieve 5A. The column temperature was 45°C and carrier gas was He at a flow rate of 25 ml/min. Peaks were monitored on a chart recorder and quantified by a computing integrator. The total N,O content was determined for the vapour plus solution phase using a coefficient of 0.67, accorrding to the method of Smith et al. (1978). The same procedure was used for growth yield experiments with 0, and nutrient broth as culture components.
Results
and Discussion
To eliminate any experiment was denitrifiers under shows that the
possible toxic effect of N,O, a preliminary conducted to study the growth of the different N,O concentrations. Figure 1 various denitrifiers grew linearly with
Figure 1. Growth 640 nm, in different
of denitrifiers, nitrous oxide
perfectomarinus (A). genes faecalis (0).
60
World ]ouml
measurec concentratil Pseudomonas sfutze
of Microbiology
j as turbidities
at
ons : Pseudomonas ri (m) and Alcali-
and Biotechnology, Vol 9, 1993
Table 1. Comparison of complex 123 isolated denitrifiers.*
media
Isolates
Medium
Nutrient Tryptic Tryptic
for supporting
broth + 0, soy broth + O2 soy broth + 3.5 mM KNO,;
no O2
growth
that %
of
grewt
76 98 89
‘The 123 isolates were from soil and identified as denitrifiers by the protocols of Gamble et al. (1977). t lnoculum was pre-grown on nutrient broth and tryptic soy broth; 110 of the 123 strains taken from the soil stock culture grew on one or both media.
increase in the concentration of N,O. Thus toxicity was not apparent at 20% (v/v) N,O. The adequacy of the procedure used to minimize the influence of contamination by 0, during N,O-dependent growth was demonstrated by Okereke (1984a). The results of another preliminary experiment, to compare the ability of denitrifier isolates to grow aerobically on two media, showed that TSB was more suitable as 98% of the isolates grew in this media, compared with 76% in nutrient broth (Table I). It would appear that the organic substances in nutrient broth were not adequate to satisfy the nutritional demand of many of the denitrifying microflora. A previous study, by dkereke (1984a), &owed that denitrifiers utilized N,O faster in TSB than in nutrient broth. Tryptic soy broth was, therefore, chosen for the studies on growth yields of denitrifiers. Figure 2 shows the growth of denitrifiers corresponding to different N,O consumptions while Table 2 shows the molar growth yields (g cells/mol acceptor) of Alculigenes faecalis, Pseudomonas stutzeri, Paracoccw denitrificans and Ps. perfectomarinus. Koike & Hattori (1975a) reported that Pa. denittificans showed similar cell yields per electron transferred when NO; and NO, were electron acceptors. Their data are also summarized in Table 2. These data do not include maintenance energy, which becomes more significant as growth decreases. The A. fuecalis strains, which grew very quickly, had lower values. The two with the lowest yield also had the slowest growth rates; thus, a large maintenance energy cost may be at least partially responsible for the lower yield. Comparison of the growth rates of A. fuecalis and Ps. perfectomarintts when 0, or N,O was used as a terminal acceptor, showed that the growth rate was higher under aerobic than under anaerobic condition (Table 3). The growth efficiencies of N,O reduction by A. fuecalis and Ps. perfectomarinus were about 49% and 39%, respectively, of those when 0, was used as the terminal electron acceptor. This is to be expected from the greater energy available from aerobic oxidation. Koike & Hattori (1975b) reported that, in a batch culture, the energy yield of Pseudomonas
Growth of denifrifiers on N,O denifrificuns associated with denitrification is approximately half that with aerobic respiration on an electronic basis. Komer & Zumft (1989) demonstrated that the expression of nitrate reductase and N,O reductase was controlled by discrete oxygen levels and by the nature of the nitrogenous oxide available for respiration and of that available for enhanced expression of the N,O reductase. One can conclude that the numbers of sites for oxidative phosphorylation in the electron transport system associated with N,O reduction in this study were about 39% and 49%, for P. perferfomarinus and A. faeculis respectively, of those in the electron transport system associated with 0, respiration. The comparatively higher growth yield reported for A. fuecalisindicates that a more efficient energy coupling system may exist in this denitrifier.
Acknowledgement I
5
1
1.0 Nitrous
1
1.5
oxide
2.0
The research was supported
(mol)
consumed
by a grant from Prof. J. Tiedje,
Department of Crop and Soil Science, Michigan State University, East Lansing, MI, USA.
Figure 2. Growth yields of denitrifier strains with nitrous oxide as terminal electron acceptor: Pseudomonas perfectomarinus (A) ; Pseudomonas sfufzeri ( n ) and Alcaligenes faecalis (0).
Table
2. Growth
yleids
of denitrifier
Strain
Alcaligenes Pseudomonas Paracoccus Pseudomonas Pseudomonas
TSB-tryptic t Data from
l
strains
grown
in batch
Electron acceptor
faecalis stutzeri denitrificans perfectomarinus denitrificanst
soy broth. Koike 8 Hattori
culture
Carbon source
N,O W W NL’ NO, NO,
TSB’ TSB TSB TSB
W
Glutamate
Glutamate Glutamate
with
N20 as electron
Molar growth yield (g ceii/moi acceptor)
acceptor. Yield per electron transferred (g ceils/e)
11.2 8.2 6.1 4.4 28.6 16.9 0.8
Generation time (h)
5.6 4.1 3.0 2.2 5.7 5.6 4.4
1.0 1.2 2.2 2.0 -
(1975a).
References Table 3. Comparison peffecfomerlnus using electron acceptors.
of growth rate of A. faecalls oxygen or nitrous oxide as
Strain
Growth in air
Alcaligenes Pseudomonas
faecalis perfectomarinus
6.6 1.8
rate
and P. terminal
(mglh)
in nitrous 2.8 0.7
oxide
Evans, D.G., Beauchamp, E. & Trevors, J.T. 1985 Sulpbide alleviation of acetylene inhibition of nitrous oxide reduction in soil. Applied and Environmental Microbiology 49, 217-220. Gamble, T.N., Betlach, N.R. & Tiedje, TJ. 1977 Numerically dominant denitrifying bacteria from world soils. Applied and Environmentul Microbiology 33, 926939. Gijskuenen, J. & Robertson, L.A. 1987 Ecology of nit&cation and denitrification in the nitrogen and sulfur cycles, In Society for General Microbiology Symposium 42, eds Cole, J.A. & Ferguson, S. pp. 161-217. Cambridge: Cambridge University Press.
Worki ]ournal of Minobidogy
and Biotechnology, Vol 9, 1993
61
G. U. Okereke Justin, P. & Kelly, D.P. 1978 Growth kinetics of Thiobucillw denifrijicuns in anaerobic and aerobic chemostat culture. Journal of General Microbiology 107, 123-130. Kaspar, H.F. I982 Nitrite reduction to nitrous oxide by Propionibacteria: Detoxication mechanism. Archives of Microbiology 133, 126-130. Kioke, I. & Hattori, A. 1975a Energy yield of denitrification: An estimate from growth yield in continuous cultures of Pseudomonus denitrificuns under nitrate, nitrite and nitrous oxide limited conditions. journal of General Microbiology 88, 11-19. Kioke, I. & Hattori, A. 1975b Growth yield of a denihifying bacterium, Pseudomonas denitrificiuns, under aerobic and denitrifying conditions. ]ournul of General Microbiology 88, 10. Komer, H. & Zumft, W.G. 1989 Expression of denitrification enzymes in response to the dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri. Applied and Environmenful Microbiology 55, 167CS1676.
62
World Journal of Microbiology and Biotechnology, Vol 9, 1993
Okereke, G.U. 1984a capability in denitrifiers
Prevalence of from a variety
nitrous oxide reducing of habitats. Plant and Soil
81,421-428. Okereke, G.U. 1984b Possible use of N,O in MPN tubes for enumeration of denitrifiers. Plant and Soil 80, 295-296. Robertson, L.A. & Kuenen, J.G. 1984 Aerobic denitrification-old wine in new bottles. Antonie van Leeuwenhoek, ~ournul of Microbiology and Serology 50, 525-544. Smith, M.S., Firestone, J.K. & Tiedje, J.M. 1978. The acetylene inhibition method for short-term measurement of soil denitrification and its evaluation using Nitrogen-13. Soil Science Society of American ]ournul 42, 611-615. Stouthamer, A.H. 1988 Dissimilatory reduction of oxidation nitrogen compounds. In Biology of Anaerobic Microorganisms, ed Zehunder, A.J.B. pp. 245-303. New York: John Wiley and Sons. (Received
in revised
form
16Jme
1992;
accepted
26]une
1992)
