Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification
Guocheng Du 1,Jinju Geng 1,Jian Chen 1,2,*and Shiyi Lun 11
School of Biotechnology,Southern Yangtze University,Wuxi 214036,P.R.China 2
Key Laboratory of Industrial Biotechnology,Ministry of Education,Southern Yangtze University,Wuxi 214036,P.R.China
*Author for correspondence:Tel./Fax:+86-510-5888301,E-mail:jchen@sytu.edu
Received 13August 2002;accepted 22January 2003
Keywords:Denitrifying bacteria,mixed culture,nitrifying bacteria,nitrogen compound,simultaneous nitrification
and denitrification
Summary
A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification.A mixture of NaHCO 3and CH 3COONa was lected as the appropriate carbon source for cell growth and nitrogen removal,the concentrations of carbon and nitrogen sources were also examined.Ammonia could be oxidized aerobically to nitrite by the mixed culture,and the intermediate nitrite was then reduced to dinitrogen gas.No nitrite was detected during the process.0.212g of ammonia/l could be removed in 30h and nitrate could not be utilized aerobically by the mixed culture.Nitrite could be degraded aerobically as well as anaerobically.Very little ammonia was degraded anaerobically,but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42h.
Introduction
The adver environmental impacts associated with ammonia nitrogen include promotion of eutrophication,toxicity to aquatic organisms and depletion of dissolved oxygen in receiving streams due to bacterial oxidation of ammonia to nitrite and nitrate (Klees &Silverstein 1992).The biological elimination of nitrogen in waste-water treatment plants results from the process of nitrification and denitrification (Helmer &Kunst 1998).The two process have been thought to be two parate reactions by different groups of microorgan-isms in activated sludge.Aerobic,autotrophic nitrifiers oxi
dize ammonia to nitrite and nitrate,with molecular oxygen as electron acceptor.Nitrite and nitrate are reduced to dinitrogen gas by heterotrophic denitrifying bacteria that u NO Àx instead of oxygen as electron acceptor(Helmer&Kunst 1998).A typical bacter ium,Thiosphaera pantotropha isolated by Robertson &Kue-nen (1983),has been widely studied forits peculiar enzyme system to explain the properties of heterotrophic nitrification (Robertson &Kuenen 1988;Robertson et al .1989)and aerobic denitrification (Bell et al .1990;Bell &Ferguson 1991;Berks et al .1993;Moir et al .1993).Moreover,it was capable of conducting simulta-neous heterotrophic nitrification and aerobic denitrifica-tion (Robertson et al .1988;Gupta 1997).Simultaneous nitrification and denitrification,which has advantages
over the parated nitrification and denitrification pro-cess,means that nitrification and denitrification occur concurrently in the same reaction vesl under identical operating condition (Munch et al .1996).
In this study,a mixed culture of isolated nitrifying bacteria and denitrifying bacteria was investigated for simultaneous nitrification and denitrification.Bad on the investigation of the appropriate concentration of carbon and nitrogen sources,the degradation ability of the mixed culture towards ammonia,nitrite and nitrate was examined in aerobiosis as well as in anaerobiosis.
Materials and methods Media and culture conditions
The growth medium for the screening of nitrifying bacteria contained (g/l):(NH 4)2SO 41.0,NaHCO 31.0,Na 2HPO 4 1.35,KH 2PO 40.70,MgSO 4Á7H 2O 0.10,CaCl 2Æ2H 2O 0.0184,FeCl 3Æ6H 2O 0.40,agar20.0.The pH was 7.2.The medium forthe scr eening of denitr i-fying bacteria contained (g/l):CH 3COONa 1.0,KNO 31.0,NaNO 31.0,Na 2HPO 41.35,KH 2PO 40.70,MgSO 4Á7H 2O 0.10,agar20.0.The pH was 7.4.The medium for the ed contained (g/l):NaHCO 31.75(0.25carbon),CH 3COONa 1.71(0.25carbon),(NH 4)2SO 41.0(0.212nitrogen),NaCl 1.0,FeSO 40.016,K 2HPO 4Æ3H 2O 1.3,
World Journal of Microbiology &Biotechnology 19:433–437,2003.433
Ó2003Kluwer Academic Publishers.Printed in the
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MgSO40.25.The pH was7.4.For the preparation of slants,2.0%(w/v)of agarwas added.The ed was prepared in500mlflasks containing100ml of ed medium,and incubated in a rotary shaker at200rev/ min and30°C for24h.
Screening of nitrifying and denitrifying bacteria
Five millilitres of aerobic activated sludge from a nitrifying tanker,taken from the wastewater plant of an enzyme production company in Wuxi,was added into100ml of distilled water.After complete mixing, the solution was diluted to10)5–10)7,and distributed onto petri dishes containing nitrifying medium and denitrifying medium,respectively.After incubation at 30°C for around8days,colonies from both media were identified and stored at4–8°C forfur therstudy.
Shakingflask culture
职业经理人培训The fermentation medium contained(g/l):NaCl 1.0, FeSO40.016,K2HPO4Æ3H2O1.3,MgSO40.25,pH7.4, and carbon source and nitrogen source were added as needed.In the experiments of aerobic degradation of nitrogen compounds,a mixture of Na
HCO3and CH3COONa was ud as the carbon source and their concentrations were1.75and1.71g/l,respectively.5ml of the ed culture was inoculated into the500mlflasks containing50ml fermentation medium and incubated in a rotary shaker at200rev/min with temperature con-trolled at30°C for24h.Aerobic degradation of nitrogen compounds was conducted in shakingflask cultures with1.75g NaHCO3/l and1.71g CH3COONa/ l as carbon sources.
Anaerobic degradation of nitrogen compounds
The fermentation medium contained(g/l):NaCl 1.0, FeSO40.016,K2HPO4Æ3H2O 1.3,MgSO40.25,Na-HCO31.75,CH3COONa1.71,pH7.4,and nitrogen sources were added as needed.5ml of ed culture was inoculated into500mlflask containing50ml fermen-tation medium and theflasks were covered with a rubber plug.Air was replaced by nitrogen by repeated vacuum-nitrogenflushing cycles(five times)to ensure anaerobic conditions in theflasks.Theflasks were incubated at 30°C.After42h,the rubber plug was replaced with a sponge one and theflasks were incubated in a rotary shakerat200r ev/min and30°C.
Analytical methods
Dry cell weight(DCW)was determined as follows:5ml culture broth was centrifuged at10,000·g for10
min. 5ml of0.1M HCl solution was added to the precipitate when CaCO3was ud in the medium.Aftershaking for 10min,the broth was centrifuged at10,000·g for 10min again,and the precipitate was dried at80°C to constant weight.Ammonia,nitrite and nitrate were determined as described by Graaf et al.(1996). Results
Screening of nitrifying bacteria and denitrifying bacteria and their subculture
Three nitrifying bacteria were identified from nitrifying medium and two denitrifying bacteria from denitrifying medium.When the three nitrifying bacteria were transferred parately for subculture,the cells grew very slowly and colonies were very small as well.This indicated that,with NHþ
4
as the nitrogen source,the formation of intermediates such as nitrite or nitrate would result in the decrea of pH of the medium,and the accumulation of intermediate as well as the drop of pH were unfavourable for cell growth.Nitrite or nitrate, however,was an appropriate substrate for denitrifying bacteria and could be reduced to dinitrogen gas.So it was decided to mix nitrifying bacteria with denitrifying bacteria during the culture for the removal of ammonia nitrogen in order tofind out if the
mixed bacteria could grow very well on the medium with ammonium sulphate as the sole nitrogen source during subculture.The mixed culture was transferred more than10times and reiso-lation was done on nitrifying medium and denitrifying medium.Again three and two bacteria were isolated from nitrifying medium and denitrifying medium as nitrifying bacteria and denitrifying bacteria,respective-ly.It indicated that afixed symbiotic relationship existed between nitrifying bacteria and denitrifying bacteria. Further identification of the bacteria is proceeding in our laboratory.
Investigation of appropriate carbon sources for the mixed culture
Most nitrifying bacteria are autotrophic bacteria,and
can grow using CO2À
3
orCO2as carbon sources,while denitrifying bacteria are heterotrophic bacteria,and can grow only if organic substrates are supplied.The determination of a suitable carbon source for the mixed culture should take into account the requirements of both nitrifying and denitrifying bacteria simultaneously. So it is very important to investigate the effect of different carbon sources on cell growth and the rem
oval rate of ammonia nitrogen.Several inorganic and organic carbon sources were examined and the results are shown in Table1.When NaHCO3orCaCO3was ud as the sole carbon source,only nitrifying bacteria could grow, and denitrification could not proceed,resulting in the low DCW and low removal rate of ammonia nitrogen. On the otherhand,when CH3COONa was ud as the sole carbon source,nitrifying bacteria could only grow byfixing the limited carbon source CO2from the air, which also resulted in the low DCW and low ammonia nitrogen removal rate.However,with the supply of a
434G.Du et al.
mixture of organic and inorganic carbon sources, enhanced cell growth and ammonia nitrogen removal rate could be achieved.The highest DCW of2.8g/l and ammonia nitrogen removal rate(R)of97.6%were obtained with NaHCO3and CH3COONa as carbon sources.
Effects of the concentration of carbon and nitrogen source on cell growth and nitrogen removal
Cell growth and ammonia nitrogen removal would not only be affected by the types of carbon sources,but by their concentrations as well.A mixture of NaHCO3and CH3COONa in different concentrations was ud as carbon source and the results are shown in Table2. With the increa of
carbon concentration,the ammonia nitrogen removal rate incread and the highest DCW and ammonia nitrogen removal rate were achieved at the concentration of0.5g carbon/l.The residual am-monia nitrogen decread with the increa of cell growth.The cell growth,however,was inhibited by the higher concentration of carbon source.
When ammonium sulphate was ud as the nitrogen source by nitrifying and denitrifying bacteria,it could be utilized as energy for cell growth and oxidized to nitrite and then further reduced to dinitrogen gas.The effect of different nitrogen concentrations on cell growth and ammonia nitrogen removal was investigated and the results are shown in Table3.Cell growth incread on increasing the nitrogen concentration,but was inhibited at high nitrogen concentration.Ammonia nitrogen removal rate also decread at high nitrogen concentra-tion,resulting in the increa of residual ammonia nitrogen concentration in the culture broth.
Aerobic degradation of nitrogen compounds
Nitrite and nitrate would be the intermediates of ammonia oxidation in the conventional nitrogen remov-al process.They are the products of nitrification and also the substrates for denitrification.To investigate the utilization of ammonia,nitrite and nitrate would be helpful forthe under standing of the
mechanism of the ammonia nitrogen removal process under aerobic con-ditions.Different combinations of ammonia,nitrite and nitrate were ud as nitrogen source and the results are shown in Figure1.The degradation of ammonia was not affected by the addition of nitrite or nitrate,the concentrations of ammonia nitrogen in all three cas dropped to zero at30h and their degradation rates were almost the same(Figure1A).When nitrite was ud as the sole nitrogen source,it could be utilized rapidly and its concentration decread to zero at24h.However, the degradation of nitrite was inhibited by the prence of ammonia,and almost no nitrite degradation was obrved in thefirst12h,though it dropped to zero at 30h(Figure1B).When nitrate and ammonia were ud as nitrogen sources,though the degradation of ammonia was not affected by the prence of nitrate,utilization of nitrate by the mixed culture was not obrved(Fig-ure1C).
The same situation happened when nitrate was ud as the sole nitrogen source,no cell growth and no nitrate degradation were obrved(data not shown).It indi-cated that nitrate could not be ud by the mixed culture as the substrate.When ammonia was ud as the sole nitrogen source,no nitrite or nitrate was detected in the culture broth as shown in Figure1B and C.It is suggested that nitrite was the intermediate of nitrifica-tion and could be further utilized by denitrifying bacteria.Muller(1995)reported that it was nitrite,not nitrate that was the direct substrate of denitrifying bacteria during the aerobic denitrification process.
Table1.Effect of carbon sources on cell growth and ammonia nitrogen removal.一先令
Carbon source DCW
(g/l)Ammonia
nitrogen
removal rate(%)
Residual
NHþwhite collar
4
-N
(g/l)
CaCO3 1.748.60.11 NaHCO3 1.351.90.102
CH3COONa 2.276.90.049 NaHCO3+CH3COONa 2.897.60.005 CaCO3+CH3COONa 2.589.20.023
*When only one carbon source was ud,its concentration was 0.5g carbon/l.When two carbon sources were ud,the concentra-
tion of each was0.25g carbon/l.The initial NHþ
4-N concentration
was0.212g/l.
水的痕Table2.Effect of carbon concentration on cell growth and ammonia nitrogen removal.
Carbon concentration (g/l)DCW
(g/l)
Ammonia nitrogen
removal rate(%)edg是啥意思
Residual
6410NHþ
4
-N
(g/l)
0.1 1.876.40.050
0.2 2.386.30.029
0.3 2.496.20.008
0.5 2.998.60.003
0.8 2.698.60.003
*At each total carbon concentration,the same carbon concentra-tions of NaHCO3and CH3COONa were ud.The initial
concentration of NHþ
4-N was0.212g/l.
Table3.Effect of nitrogen concentration on cell growth and ammonia
nitrogen removal.
NHþ
4
-N
concentration
(g/l)
DCW
(g/l)
Ammonia
nitrogen removal
rate(%)
Residual
NHþ
4
katherine什么意思-N
(g/l)
0.05 1.495.30.001
0.1 2.096.20.003
0.2 2.997.60.004
0.3 2.989.20.024
0.4 1.886.90.040
0.5 1.776.70.092
*Both carbon concentration of NaHCO3and CH3COONa were
0.25g/l.
Mixed culture for nitrification/denitrification435
The addition of nitrite enhanced cell growth and DCW reached the highest value of 3.4g/l at 24h when ammonia and nitrite were ud as nitrogen source.Although nitrate could not be utilized by the mixed culture,the prence of nitrate in the culture broth did not affect cell growth compared to the ca with ammonia as the sole nitrogen source.Less cell growth was obtained with nitrite as the sole nitrogen source (Figure 1D).
Anaerobic degradation of nitrogen compounds
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Anaerobic degradation of nitrogen compounds such as ammonia and nitrite was investigated.The results are shown in Figure 2.Under anaerobic conditions,the concentration of ammonia nitrogen remained almost unchanged,and little degradation of ammonia was obrved within 42h (Figure 2A).When oxygen was supplied at 42h,however,the degradation of ammonia was started and the concentration of ammonia nitrogen decread to zero after 26h in both cas (Figure 2A).It indicated that the growth of nitrifying bacteria was inhibited by the abnce of oxygen and the supply of oxygen recovered the nitrification ability of nitrifying bacteria,which led to the degradation of ammonia.It emed that anaerobic condition resulted in the inhibi-tion of nitrifying bacteria instead of their death.
Nitrite could be reduced by denitrifying bacteria and the concentration of nitrite decread to zero at 42h in both cas (Figure 2B).The prence of ammonia in the culture broth emed to have little influence on the degradation of nitrite.This phenomenon was quite different from that obrved under aerobic conditions in
which the existence of ammonia inhibited the degrada-tion of nitrite to some extent (Figure 1B).Nitrate could not be reduced anaerobically (data not shown).It is suggested that nitrite was the unique electron acceptor of denitrifying bacteria in the mixed culture.
Less cell growth was obrved under anaerobic conditions with ammonia as the sole nitrogen source.Nitrite was ud as the electron acceptor for denitrifying bacteria under anaerobic conditions.With nitrite as the sole nitrogen source,DCW reached the highest value of 1.8g/l at 42h and the complete consumption of nitrite resulted in the cea of cell growth.With nitrite and ammonia as the co-nitrogen sources,however,DCW incread continuously becau the growth of nitrifying bacteria when oxygen was supplied at 42h,which could utilize ammonia to support the cell growth (Figure 2C).
Discussion
When ammonia was ud as the substrate by nitrifying bacteria,no nitrite and nitrate were detected as shown in Figures 1B,C and 2B.Nitrite and nitrate are the intermediates of ammonia oxidation in the conventional process and can be further ud by denitrifying bacteria.However,in the mixed culture,only nitrite could be utilized by denitrifying bacteria aerobically and anaero-bically.So it emed that,in ourstudy,only nitr ite was the intermediate of ammonia oxidation,and it was produced by nitrifying bacteria and consumed by denitrifying bacteria.The consumption rate of nitrite was higher than its formation rate,resulting in its abnce in the culture broth.In this way,
ammonia
Figure 1.Aerobic degradation of nitrogen compounds.}:0.212g/l NH þ4-N;s :0.212g/l NH þ4-N +0.055g/l NO À2-N;n :0.055g/l NO À
2-N;
u :0.212g/l NH þ4-N +0.023g/l NO À
3-N.A:utilization of ammonia;B:utilization of nitrite;C:utilization of nitrate;D:increa of biomass.
436
G.Du et al.
could be converted to dinitrogen gas without the accumulation of nitrite by the mixed culture.It is suggested that aerobic nitrification and denitrification could be ascribed to two reactions,in which ammonia is first oxidized to nitrite and then reduced to nitrogen as shown in Figure 3.Geraats et al .(1990)demonstrated this process in Thiosphaera pantotropha .
Ammonia could only be oxidized aerobically by the mixed culture.Nitrite,however,could be reduced both aerobically and anaerobically.The consumption rate of nitrite in aerobic conditions was much higher that in anaerobic conditions when nitrite was ud as the sole substrate (Figures 1B and 2B).The prence of nitrite or nitrate in culture broth did not affect the degradation of
ammonia.Becau of the good degradation ability to ammonia and nitrite,the mixed culture is assumed to have a good potential forthe pr oduction of a denitr i-fying preparation in the treatment of wastewater con-taining ammonia and nitrite.Besides the removal rate of nitrogen compounds,DCW was also an important consideration during the cultivation of the mixed culture.Further investigation on high cell density cultures of the mixed culture by fed-batch fermentation is being conducted in ourgr oup.
References
Bell,L.C.&Ferguson,S.J.1991Nitric and nitrous oxide reductas
are active under aerobic conditions in cells of Thiosphaera pantotropha .European Journal of Biochemistry 273,423–427.
Bell,L.C.,Richardson,D.J.&Ferguson,S.J.1990Periplasmic and
membrane bound nitrate reductas in Thiosphaera pantotropha .FEBS Letters 265,85–87.
Berks,B.C.,Baratta,D.,Richardson,D.J.&Ferguson,S.J.1993
Purification and characterization of a nitrous oxide reducta from Thiosphaera pantotropha .European Journal of Biochemistry 272,467–476.
Geraats,S.G.M.,Hooijmans, C.M.,van Niel, E.W.J.,Robertson,
L.A.,Heijnen,J.J.,Luyben,K.CH.A.M.&Kuenen,J.G.1990The u of a metabolically structured model n the study of growth,nitrification,and denitrification by Thiosphaera pantotropha .Bio-technology and Bioengineering 36,921–930.
Graaf,A.A.,van de Bruijn,P.,Robertson,L.A.,Jetten,M.S.M.&
Kuenen,J.G.1996Autotrophic growth of anaerobic,ammonium-oxidising microorganisms in a fluidized bed reactor.Microbiology (UK)142,2187–2196.
Gupta, A.B.1997Thiosphaera pantotropha :a sulphurbacter ium
henrietta
capable of simultaneous heterotrophic nitrification and aerobic denitrification.Enzyme and Microbial Technology 21,89–595.Helmer,C.&Kunst,S.1998Simultaneous nitrification/denitrification
in an aerobic biofilm system.Water Science and Technology 37,183–187.
Klees,R.&Silverstein,J.1992Improved biological nitrification using
recirculation in rotating biological contactors.Water Science and Technology 26,545–553.
Moir,J.W.B.,Baratta,D.,Bichardson,D.J.&Ferguson,S.J.1993The
purification of a cd 1-type nitrite reducta from and the abnce of a copper-type nitrite reducta from,the aerobic denitrifier Thiosphaera pantotropha :the role of pudoazurin as an electron donor.European Journal of Biochemistry 212,377–385.
Muller, E.B.,Stouthamer, A.H.&Van Verveld,H.W.1995
Simultaneous NH 3oxidation and N 2production at reduced O 2tensions by wage sludge subcultured with chemolithotrophic medium.Biodegradation 6,339–349.
Munch,E.V.,Lant,P.&Keller,J.1996Simultaneous nitrification and
denitrification in bench-scale quencing batch reactors.Water Rearch 30,277–284.
Robertson,L.A.&Kuenen,J.G.1983Thiosphaera pantotropha gen nov
sp nov,a facultatively anaerobic,facultatively autotrophic sulphur bacterium.Journal of General Microbiology 129,2847–2855.
Robertson,L.A.&Kuenen,J.G.1988Heterotrophic nitrification in
Thiosphaera pantotropha :oxygen uptake and enzyme studies.Journal of General Microbiology 134,857–863.
Robertson,L.A.,Cornelis,R.,De Vos,P.,Hadioetomo,R.&
Kuenen,J.G.1989Aerobic denitrification in various heterotrophic nitrifiers.Antonie van Leeuwenhoek 56,289–299.
Robertson,L.A.,van Niel,E.W.J.,Torremans,R.A.M.&Kuenen,
J.G.1988Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaera pantotropha .Applied and Environmental Microbiology 54,
2812–2818.
Figure 2.Anaerobic degradation of nitrogen compounds.d :0.212g/l
NH þ4-N;j :0.212g/l NH þ4-N +0.055g/l NO À2-N;m :0.055g/l NO À
2-N.A,B and C have the same significance as A,B and D in Figure
1.
Figure 3.Simultaneous aerobic nitrification and denitrification by the mixed culture.
Mixed culture for nitrification/denitrification 437
