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Effect of oxygen concentration on the rates of denitratification and denitrification in the sediments of an eutrophic lake
K~h:er Re~ Vol 18, No 3, pp 335-338. i984 Pnnted in Great Britain. All rights rese,.".ed
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{~a'~- 15a 8-4' 53 :-~)+00+~ 19S4 P:_'r~amon Pr_~ Lid
EFFECT OF OXYGEN C O N C E N T R A T I O N ON THE RATES OF D E N I T R A T I F I C A T I O N AND D E N I T R I T I F I C A T I O N IN THE SEDIMENTS OF AN E U T R O P H I C LAKE MITSUTOSHI NAKAJIMA ~. TERUYOStll HAYAMIZU: a n d HAJ[MI: NISHIMURA-" Department of Chemical Engineering, Faculty of Engineering. Kyushu L niversity, Higashi-ku. Fukuoka S 12 and :Department of Chemical Engineering, Faculty of Engineering, University of Tok.',o, Bunk)o-ku. To~,yo 1t3, Japan
(Recei,'ed June 1983)
Abstract--The effect of oxygen concentration on the rates of denitratification and dcnitritification was investigated by the acetylene inhibition method during initial t-h incubation period after prccuiture under complete anaerobic conditions for wide ranges of nitrate and nitrite concentrations using sediment sample collected from a highly eutrophic lake. A maximum denitratification rate of 4 #mol (g-dry mud)-~hwas obtained under anaerobic conditions. The denitratification rate was found to be a decreasing function of the oxygen concentration below 60 t~ M. Approximately the same rate ~as observed t\-~rdenitritification in the range below 30 # M O,. Beyond 30 l~ M O.,, this rate dropped to the half of the maximum, and remained almost constant until a critical oxygen concentration was attained. The critical concentration. above which denitritification was suppressed thoroughly, depended on nitrite concemration, Key word,--denitrification, denitritification, dcnitratification, ox,vgen, critic-d ox,,gen concentration. sediment, eutrophic lake, acetylene inhibition method
INTRODUCTION
T h e m a j o r nitrogen t r a n s f o r m a t i o n processes by bacteria such as nitrification, denitrification, nitrogen fixation, a m m o n i f i c a t i o n and i m m o b i l i z a t i o n may occur in a w a t e r - s e d i m e n t system. T h e bacterial reduction of nitrate a n d nitrite is responsible to removal of nitrogen in n a t u r a l waters. M a n y types o f aerobic bacteria use nitrate a n d nitrite as the terminal electron acceptor under a n a e r o b i c c o n d i t i o n s (Payne, 1973). Payne (1973) reported that denitrification simply proceeded in the sequence: NOf-NO.;--NO-N20-N:. Denitratification and denitritification are defined as the respective reduction of nitrate a n d nitrite to gaseous p r o d u c t s (Prak a s a m and Loehr, 1972). T h e rates o f denitratification a n d denitritification are mainly governed by the p o p u l a t i o n o f denitrifying bacteria, the a m o u n t of available organic matter, oxygen c o n c e n t r a t i o n a n d the levels o f nitrate a n d nitrite concentrations. F o c h t a n d C h a n g (1975) rep o r t e d in their reviews that the critical oxygen concentration for denitratification, above which denitratification is suppressed completely, ranged from 6 to 6 3 p M . However, Voets et al. (1975) reported that denitritification occurred u n d e r b o t h aerobic and a n a e r o b i c c o n d i t i o n s in activated sludge. N a k a j i m a a n d T e z u k a (1974) reported t h a t cells of P s e u d o m o n a s previously g r o w n anaerobically reduced nitrite u n d e r aerobic conditions. A l t h o u g h these two reports showed t h a t the critical oxygen R
Ix?,
F
c o n c e n t r a t i o n for denitritification can be sufficiently high, it is not well k n o w n whether significant denitrification occurs under aerobic conditions as c o m p a r e d with denitratification a n d denitritification u n d e r a n a e r o b i c conditions. F u r t h e r investigation on the effect of oxygen c o n c e n t r a t i o n on denitratification or denitritification is required. The purpose of the present study is to investigate the effect of dissolved oxygen c o n c e n t r a t i o n s on the rates of denitratification and denitritification under various c o n c e n t r a t i o n s o f nitrate or nitrite, respectively.
MATERIAI~% AND METHODS Sediments
Core sediments were collected from the bottom of Lake Teganuma on 8 J a n u a ~ 1981. using a rigid acrylic plastic tube 36 mm in diameter and 50 cm long. The top 5 cm of the core sediments was mixed, refrigerated at 0-1 C and then divided into subsamples tbr further use in the experiment. The contents of volatile matter, particulate carbon and particulate nitrogen were 19.0. 8.5 and 1.0°~, respectively (Nakajima et al., 1984). Lake Teganuma (mean depth: 1.5 m, surface area: 5 kmZ). located in Chiba prefecture, is a highly eutrophic lake owing to the large amount of domestic waste water discharged into it. E.werimental proce&+re
The rates of denitratification and denitritification were determined by the acetylene inhibition method (Yoshinari et al., 1977: Batdestron et al., 1976: M{iller et al., 19801. An amount of 2.5 or 5 cm ~ of each sediment sample (0.34 or 0.68 g dry weight) was placed into a 60-ml glass bottle and 335
10cm' ot either KNO. or N a N O , soiuuon was added The c o n c e n t r a t ~ n of KNO, m the soluuon was either 2fJ~) (,r i 0 t ~ ) , M , and that o( N a N O . ~ a s rather I00. 21~9 or I(XR) , a M . The aclua~ concentrauon of m~ratc or nitrite '*a~ reduced b3 approx. 20",, because of mtxing with interstitial water m the ~edimem The ~alues of pH of some samples ~ere ~...e~-6..,. The atmosphere v,as replaced vath mtrogen gas lot se,,era! minutes to assure complete gas exchange, and each bottle was plugged with a rubber _,topper and aluminum seal. The content of oxygen in gas phase was reduced to less than 0.01 atm. which corresponded to anaerobic conditions "n water. I n order to get aerobic conditions in water, the replacement of atmosphere wxth nitrogen gas was unneccessa.w. An amount of I CTn+ of acetylene gas was added to each bottle with a s~nnge. After vtgorous shaking, the bottles were incubated m the dark at 13 C, the temperature of the sediment when the samples were collected. The sediment was agitated with a magneuc surrer d u n n g I-h incubation period. Gas sampies of I c m ' were withdrawn with a syringe at intervals of 5-30 rain. utter vigorous shaking of the bottles, These samples were analyzed for nitrous oxide by gas chromatography tShimadzu GC-3BT. equipped with a T C D and a stainless steel column 2 m in length and 2 m m i.d packed with Porapak Q 80/100 mesh). The temperature in the oven was 5 0 C , The bridge current was 114mA. The determinatmn of the a m o u n t o f N , O formed in the bottlez was based on the calibration curve of N , O corrected for solubility of N , O m water by the Henry constant (Kagaku Benran. 1975) -About 20'!,, o f the total N , O was estimated to be dissohed in the water-sediment suspension. The a m o u n t of ratratc or nitrite added to the sedtments was recovered almost stoichiometricaltv as N , O under anaerobic conditions (Nakajima et al., 1984). Oxygen concentration was also nwasured by gas chromatography (Shimadzu GC-3BT; Molecular Sieve 13X 60, 80 mesh: carrier gas (He) flow rate. 62 cm ~ min + ~: and the other conditions were same as in N+O). Both at the beginning and the end of incubation period. 0.1 cm J o f the gas phase was sampled with a syringe. The concentration of dissolved oxygen in the water was calculated from the oxygen content m the gas phase, assuming vapor-liquid equilibrium. Prior to the expenment dealing w~th denitrititication dependence on the oxygen concentration, the effects of mtrate and nitrite concentrations were investigated under anaerobic conditions. The concentrations of KNO~ and N a N O , solutions added ranged from 30 to 2 0 0 0 # M . Temperature dependence was studied under anaerobic condiuons. setting temperature at Ibur levels: 5. 13.20 and 3 0 C . The concentration of added nitrate or nitrite was 200 u M.
', ] •
" r"+O~ : 6 0 - 3 5 ~ M
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Fig. I. Producnon of N , O m sednment collected f r o m a highly eutrophic lake, under anaerobic conditions. Initial concentranons were 24 and 140 .u M nitrite, and 80. 120 and 1601tM nitrite IltM = ~mol l-t). Incubated temperature was 13:C. N u m b e r s in parenthesis denote initial concentrations and remaining concentrations were estimated from the atnount of recovered N-O r a t h e r o n t h e initial c o n c e n t r a t i o n o f n i t r a t e o r m t r i t e All t h e r a t e s were o b t a i n e d f r o m the o b s e r v e d d a t a d u r i n g initial l - h i n c u b a t i o n period. B o t h t h e d e n i t r a t i f i c a t i o n a n d d e n i t r i t i f i c a t i o n rates u n d e r a n a e r o b i c c o n d i t i o n s at 1 3 C a r e p l o t t e d as a f u n c t i o n o f t h e r e s p e c t i v e initial c o n c e n t r a t i o n o f n i t r a t e or nitrite in Fig. 2. A t l o w e r c o n c e n t r a t i o n s . t h e r a t e s i n c r e a s e d a p p r o x i m a t e l y p r o p o r t i o n a l l y to t h e initial c o n c e n t r a t t o n o f n i t r a t e o r nitrite. T h e y a t t a i n e d m a x i m a a t a b o u t 1 4 0 / ~ M ( = ~ u m o l l 1) for d e n i t r a t i f i c a t i o n a n d 1 6 0 , a M for d e n i t r i t i f i c a t t o n . T h e d e n i t r i t i f i c a t i o n rate w a s h i g h e r t h a n the denitratification for c o n c e n t r a t i o n s lower t h a n 2 0 0 - - 3 0 0 # M . In t h e r a n g e h i g h e r t h a n 2 0 0 - 3 0 0 , a M .
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RESULTS
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N i t r a t e o r nitrite a d d e d to t h e s e d i m e n t w a s detected in t h e r e c o v e r y o f N,.O a f t e r a v e r y s h o r t lag t i m e as s h o w n in Fig. I, w h i c h w a s o b s e r v e d u n d e r anaerobic conditions. The amount of produced N.,O i n c r e a s e d w i t h time, a n d l e v e l l i n g - o f f w a s n o t o b s e r v e d in m a n y cases. N u m b e r s in p a r e n t h e s i s d e n o t e initial c o n c e n t r a t i o n s a n d r e m a i n i n g c o n c e n t r a t i o n s e s t i m a t e d f r o m the a m o u n t o f p r o d u c e d NzO+ bec a u s e t h e a d d e d n i t r a t e o r nitrite w a s r e c o v e r e d a l m o s t s t o i c h i o m e t r i c a l l y as N : O ( N a k a j i m a e t al.. 1984). T h e rates o f d e n i t r a t i f i c a t i o n a n d d e n i t r i t i f i c a t i o n were c a l c u l a t e d f r o m t h e s l o p e s o f t h e s t r a i g h t lines in this figure. T h e s l o p e s o b v i o u s l y d o n o t d e p e n d o n the i n s t a n t a n e o u s c o n c e n t r a t i o n , b u t
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~500
NO3 - N , NO2 - N (u.M] Fig. 2. Effects o f initial concentrauons of nitrate and nitrite on the rates o f denitratification and denitritificadon under anaerobic conditions during l-h incubation period, at temperature 1 3 C
Effect of ox)gen concentration both rates gradually decreased with concentration. At concentrations of 1600 # M of nitrate and nitrite, the rate of denitritification decreased to 40'~;, of the maximum value, and that of denitratification to 65~o. Nitrate was found to be reduced faster than nitrite at higher concentrations. The effect of dissolved oxygen concentration on the denitratification rate at temperature of 13'C is shown in Fig. 3 and that on the denitrititication rate m Fig. 4. The rates were shown against initial and final oxygen concentrations observed during incubation. Both rates were high (4-6 u m o l g -~ h -~) for concentrations lower than 20-30
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Fig. 5. Efl'cct of temperature on the rates of denitratification and denitritification under anaerobic conditions. Initial concentrations of nitrate and nitrite were 160 # M.
i z O Z
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Fig. 3. Effect of dissolved oxygen concentration on the denitratifieation rate during l-h incubation period, at temperature 13C. Initial nitrate concentrations were 160 and 800 # M. Open and solid symbols correspond to initial and final concentrations of dissolved oxygen during incubation.
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300
0 z (~.M) Fig. 4. Effect of dissolved oxygen concentration on the denitritification rate during I-h incubation period, at temperature 13:C. Initial nitrite concentrations were 80, 160 and 800 u M. Open and solid symbols correspond to initial and final concentrations of dissolved oxygen during incubation.
completely under aerobic conditions. The critical oxygen concentration for denitratification was about 60 # M Oz, independent of nitrate concentration. In contrast, denitritification occurred even under aerobic conditions during incubation period. The critical oxygen concentration for denitritification depended on nitrite concentration. Under 80/~ M nitrite concentration, denitritification ceased at about 7 0 # M O,. The critical oxygen concentration for denitritification increased to 2 4 0 # M O, under 160 p M nitrite concentration. Under 800 # M nitrite concentration, denitritification occurred even at 300 # M 02, which corresponds to saturation of oxygen at this temperature. Thus, the critical oxygen concentration for denitritification was found to increase with nitrite concentration. The rate of denitritification under aerobic conditions was approximately halved l¥om 4 to 2/~ mol g-~ h z, under both 160 and 800/~M nitrite concentration. The effect of temperature on the rates of denitratification and denitritification under anaerobic conditions is shown in Fig. 5. Both rates increased approximately proportionally to the temperature between 5-30°C. The rate of denitritification was always higher than that of denitratification at each temperature under 160 # M nitrate or nitrite concentration. DISCUSSION
The critical oxygen concentration for denitratification has been reported to be in the range of 6--63 # M (Focht and Chang, 1975). They suggested that the difference in the critical oxygen concentration may reflect difference in bacterial species, having different affinity to oxygen. Nakajima { 1981)
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,~,I|TStl"OSHi NAKAJIMA ('t ~al.
observed denitratification by sesstte microbes under almost complete saturation of oxygen, a n d ~t was con.lectured to be due to denttratification in anaerooic microsites in attached substrate. T h e denitratification rate m the present study ~ a s I\:und to be halved at a b o u t 20--30.uM O , . and the crmcat c o n c e n t r a t t o n for denitratification was 60 u 3,1 O , Few reports of the critical oxygen c o n c e n t r a t i o n for denitritification are availab!e. N a k a j i m a and Tezuka (1974) reported that cells ol" Pseudomon~s No. - previously grown anaerobically reduced nitrite even under 90"., sutural,on of oxygen at 1.7 m M o f initial n i m t e c o n c e n t r a t t o n Voets et al. (1975) reported that the occurrence of denitritification was estimated from the mass balance under 11-32 m M of nitritc c o n c e n t r a t i o n a n d 160-230~,M of average oxygen c o n c e n t r a t i o n in activated sludge containing 1-6 g 1-' suspended solids. These conclusions agree with our results. It is very interesting to note that under aerobic c o n d i t o n s sngnificant denitritification was observed while denitratification was totally suppressed. Pacaud et al, (1982) investtgated the stability of denitritification activity using pure bacterium Paracocus denitrificuns previously grown under anaerobic conditions, and reported that under aerobic conditions denitritification activity was high during lirst 2 h a n d decreased gradually. The rates o f denitratification a n d denitritification were reported for activated sludge were 0.1-10 nmol (g-volatile suspended solid J - ~ h - ~( Voets et al.. 1975). According to J o h n s o n a n d Schrepfer (1964), activated sludge c o n t a i n i n g 4 g 1-~ suspended solids was capable o f denitrification ( b o t h denitratification a n d denitritification) at a b o u t 1 4 0 p m o l g ' h -~ u n d e r anaerobic conditions. In our investigation, denitratification a n d denitritification rates were found to be 4--6 # mol (g-dry m u d ) - ' h - ' under anaerobic c o n d i t i o n s at 13~C. As the c o n t e n t of volatile matter in the sediment was 19.0% ( N a k a j i m a et al.. 1984), the rates were equal to 2 0 - 3 0 / a m o l (g-vo "latile m a t t e r ) ' h - ' in terms o f volaltile matter. Even u n d e r high oxygen c o n c e n t r a t i o n , denitritification occurred at a considerably high rate of 2 u m o l (g-dry m u d ) - h -~ (10 p m o l (g-volatile matter) -~ h-~). Such denitritification, therefore, would find i m p o r t a n t application in the nitrogen removal from sewage a n d natural e u t r o p h i c waters. As the t e m p e r a t u r e effect was investigated u n d e r almost the same c o n c e n t r a t i o n o f nitrate or nitrite, we could simply calculate the activation energies from an A r r h e n i u s plot o f reaction rates without assuming zero-order kinetics. T h e a c t i v a t m n energies of denitratification a n d denitritification in this study were calculated to be 39 a n d 3 4 k J m o l - ' . respectively. T h e activation energaes were calculated to be 67 ___20 kJ m o l - ~ (from the d a t a o f F o c h t a n d Chang, 1975). and 41 kJ m o l - ' for denitratification (from the d a t a o f N a k a j i m a . 1981). As the activation
energy of an enzyme r e a c n o n ~s m the range of 21-63kJmol( C o n n a n d Stumpi\ 1976). all the a b o v e data show that physical processes such ~t~, diffusion, which have much lower activation enermes, are not controlling processes m denitrification. [ h e possnble effect on denitritificatlon ot" the add> tion of glucose was investigated using the same sediment. The a m o u n t o f available c a r b o n was found not to be a d e t e r m i n i n g factor on the denitrification rates ( N a k a j i m a et al.. 1984J. Acknowledgements We express sincere apprecmuon to Dr Nicholas C. Kraus. Nearshore Environmental Research Center. for a helpful review of the manuscript, t.)ae of u~ (M.N.) w a s supported bv a scholarship from the Sakkokai Foundation.
REFERENCES
Balderston W. L., Sherr B. and Payne W, J. t 1976~ Blockage by acetylene of nitrous oxide reduction m Pseudomona~ p(zfectomarimts. Appt. em'ir. Mierohiol. 31, 504-508. Corm E. E. and Stumpf P. K. (1976) Outlines of Biochemtstry. 4th Edition. p, 157. Wiley, New York. Focht D. D. and Chang A. C (1975) Nitrification and denitrification processes related to waste water treatment Adv. appL Microhiol. 19, 153 t86 Johnson W. K. and Schroeprzr G. J ¢1964J N~trogcn removal by nitrification and denitrification. J. ~f27t Poltut Control Fed. 36. 101_'~-1036. Kagaku Benran (1975) Solubilities ol Inorganic Compounds, 2rid Edition. p. 770. Maruzen (in Japanese). MfiUer M M.. Sundman V. and Skujins L 11980~ Denitrification in low pH spodosols and peats determined with the acetylene inhibition method. Appt. envtr, microbiol. 40, 235--239 Nakajima T. {1981 } Denitrification in a polluted river ~,rn Int. Verein Limnol. 21. Nakajima T. and Tezuka Y, (1974) The possibility of gaseous nitrogen release from water bodies under aerobic conditions. Jap. J. LimnoL 35, 117-123. Nakajima M., Hayamizu T. and Nishimura H. 11984) Relation between the denitrification activities and the organic matters m the sediments of Lake Teganuma. In preparation. Pacaud B., Kawakami Y. and Nishmura H I1982) Environmental factors affecting biological denitrification. Autumnal Meeting 05 Chemical Engineering at Kanazawa. p. 268. Painter H. A. t1970) Review of literature on morgamc nitrogen metabolism m microorgamsms. Water Res. 4. 393--450 Payne W. J (1973) Reduction of nitrogeneous oxides by microorgamsms. Bact. Rev 37, 409--452. Prakasam T. B. S. and Loehr R C. (1972) Microbiai nitrification and denitrification in concentrated wastes. Water Res. 6, 859--869. Voets J P.. Vanstaen H. and Verstraeta W. (1975) Removal of nitrogen from highly nitrogenous waste waters. J. War Pollut. Control Fed. 47, 394-398. Yoshida T., Aizaki M., Asami T. and Makishima N. ~1979~ Biological nitrogen fixation and denitrification in Lake Kasumigaura. Jap. J. LimnoL 40. I-9. Yoshinari T., Hynes R. and Knowles R. (1977) Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil, Soil Biol. Biochem. 9, 177-183.
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{~a'~- 15a 8-4' 53 :-~)+00+~ 19S4 P:_'r~amon Pr_~ Lid
EFFECT OF OXYGEN C O N C E N T R A T I O N ON THE RATES OF D E N I T R A T I F I C A T I O N AND D E N I T R I T I F I C A T I O N IN THE SEDIMENTS OF AN E U T R O P H I C LAKE MITSUTOSHI NAKAJIMA ~. TERUYOStll HAYAMIZU: a n d HAJ[MI: NISHIMURA-" Department of Chemical Engineering, Faculty of Engineering. Kyushu L niversity, Higashi-ku. Fukuoka S 12 and :Department of Chemical Engineering, Faculty of Engineering, University of Tok.',o, Bunk)o-ku. To~,yo 1t3, Japan
(Recei,'ed June 1983)
Abstract--The effect of oxygen concentration on the rates of denitratification and dcnitritification was investigated by the acetylene inhibition method during initial t-h incubation period after prccuiture under complete anaerobic conditions for wide ranges of nitrate and nitrite concentrations using sediment sample collected from a highly eutrophic lake. A maximum denitratification rate of 4 #mol (g-dry mud)-~hwas obtained under anaerobic conditions. The denitratification rate was found to be a decreasing function of the oxygen concentration below 60 t~ M. Approximately the same rate ~as observed t\-~rdenitritification in the range below 30 # M O,. Beyond 30 l~ M O.,, this rate dropped to the half of the maximum, and remained almost constant until a critical oxygen concentration was attained. The critical concentration. above which denitritification was suppressed thoroughly, depended on nitrite concemration, Key word,--denitrification, denitritification, dcnitratification, ox,vgen, critic-d ox,,gen concentration. sediment, eutrophic lake, acetylene inhibition method
INTRODUCTION
T h e m a j o r nitrogen t r a n s f o r m a t i o n processes by bacteria such as nitrification, denitrification, nitrogen fixation, a m m o n i f i c a t i o n and i m m o b i l i z a t i o n may occur in a w a t e r - s e d i m e n t system. T h e bacterial reduction of nitrate a n d nitrite is responsible to removal of nitrogen in n a t u r a l waters. M a n y types o f aerobic bacteria use nitrate a n d nitrite as the terminal electron acceptor under a n a e r o b i c c o n d i t i o n s (Payne, 1973). Payne (1973) reported that denitrification simply proceeded in the sequence: NOf-NO.;--NO-N20-N:. Denitratification and denitritification are defined as the respective reduction of nitrate a n d nitrite to gaseous p r o d u c t s (Prak a s a m and Loehr, 1972). T h e rates o f denitratification a n d denitritification are mainly governed by the p o p u l a t i o n o f denitrifying bacteria, the a m o u n t of available organic matter, oxygen c o n c e n t r a t i o n a n d the levels o f nitrate a n d nitrite concentrations. F o c h t a n d C h a n g (1975) rep o r t e d in their reviews that the critical oxygen concentration for denitratification, above which denitratification is suppressed completely, ranged from 6 to 6 3 p M . However, Voets et al. (1975) reported that denitritification occurred u n d e r b o t h aerobic and a n a e r o b i c c o n d i t i o n s in activated sludge. N a k a j i m a a n d T e z u k a (1974) reported t h a t cells of P s e u d o m o n a s previously g r o w n anaerobically reduced nitrite u n d e r aerobic conditions. A l t h o u g h these two reports showed t h a t the critical oxygen R
Ix?,
F
c o n c e n t r a t i o n for denitritification can be sufficiently high, it is not well k n o w n whether significant denitrification occurs under aerobic conditions as c o m p a r e d with denitratification a n d denitritification u n d e r a n a e r o b i c conditions. F u r t h e r investigation on the effect of oxygen c o n c e n t r a t i o n on denitratification or denitritification is required. The purpose of the present study is to investigate the effect of dissolved oxygen c o n c e n t r a t i o n s on the rates of denitratification and denitritification under various c o n c e n t r a t i o n s o f nitrate or nitrite, respectively.
MATERIAI~% AND METHODS Sediments
Core sediments were collected from the bottom of Lake Teganuma on 8 J a n u a ~ 1981. using a rigid acrylic plastic tube 36 mm in diameter and 50 cm long. The top 5 cm of the core sediments was mixed, refrigerated at 0-1 C and then divided into subsamples tbr further use in the experiment. The contents of volatile matter, particulate carbon and particulate nitrogen were 19.0. 8.5 and 1.0°~, respectively (Nakajima et al., 1984). Lake Teganuma (mean depth: 1.5 m, surface area: 5 kmZ). located in Chiba prefecture, is a highly eutrophic lake owing to the large amount of domestic waste water discharged into it. E.werimental proce&+re
The rates of denitratification and denitritification were determined by the acetylene inhibition method (Yoshinari et al., 1977: Batdestron et al., 1976: M{iller et al., 19801. An amount of 2.5 or 5 cm ~ of each sediment sample (0.34 or 0.68 g dry weight) was placed into a 60-ml glass bottle and 335
10cm' ot either KNO. or N a N O , soiuuon was added The c o n c e n t r a t ~ n of KNO, m the soluuon was either 2fJ~) (,r i 0 t ~ ) , M , and that o( N a N O . ~ a s rather I00. 21~9 or I(XR) , a M . The aclua~ concentrauon of m~ratc or nitrite '*a~ reduced b3 approx. 20",, because of mtxing with interstitial water m the ~edimem The ~alues of pH of some samples ~ere ~...e~-6..,. The atmosphere v,as replaced vath mtrogen gas lot se,,era! minutes to assure complete gas exchange, and each bottle was plugged with a rubber _,topper and aluminum seal. The content of oxygen in gas phase was reduced to less than 0.01 atm. which corresponded to anaerobic conditions "n water. I n order to get aerobic conditions in water, the replacement of atmosphere wxth nitrogen gas was unneccessa.w. An amount of I CTn+ of acetylene gas was added to each bottle with a s~nnge. After vtgorous shaking, the bottles were incubated m the dark at 13 C, the temperature of the sediment when the samples were collected. The sediment was agitated with a magneuc surrer d u n n g I-h incubation period. Gas sampies of I c m ' were withdrawn with a syringe at intervals of 5-30 rain. utter vigorous shaking of the bottles, These samples were analyzed for nitrous oxide by gas chromatography tShimadzu GC-3BT. equipped with a T C D and a stainless steel column 2 m in length and 2 m m i.d packed with Porapak Q 80/100 mesh). The temperature in the oven was 5 0 C , The bridge current was 114mA. The determinatmn of the a m o u n t o f N , O formed in the bottlez was based on the calibration curve of N , O corrected for solubility of N , O m water by the Henry constant (Kagaku Benran. 1975) -About 20'!,, o f the total N , O was estimated to be dissohed in the water-sediment suspension. The a m o u n t of ratratc or nitrite added to the sedtments was recovered almost stoichiometricaltv as N , O under anaerobic conditions (Nakajima et al., 1984). Oxygen concentration was also nwasured by gas chromatography (Shimadzu GC-3BT; Molecular Sieve 13X 60, 80 mesh: carrier gas (He) flow rate. 62 cm ~ min + ~: and the other conditions were same as in N+O). Both at the beginning and the end of incubation period. 0.1 cm J o f the gas phase was sampled with a syringe. The concentration of dissolved oxygen in the water was calculated from the oxygen content m the gas phase, assuming vapor-liquid equilibrium. Prior to the expenment dealing w~th denitrititication dependence on the oxygen concentration, the effects of mtrate and nitrite concentrations were investigated under anaerobic conditions. The concentrations of KNO~ and N a N O , solutions added ranged from 30 to 2 0 0 0 # M . Temperature dependence was studied under anaerobic condiuons. setting temperature at Ibur levels: 5. 13.20 and 3 0 C . The concentration of added nitrate or nitrite was 200 u M.
', ] •
" r"+O~ : 6 0 - 3 5 ~ M
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Fig. I. Producnon of N , O m sednment collected f r o m a highly eutrophic lake, under anaerobic conditions. Initial concentranons were 24 and 140 .u M nitrite, and 80. 120 and 1601tM nitrite IltM = ~mol l-t). Incubated temperature was 13:C. N u m b e r s in parenthesis denote initial concentrations and remaining concentrations were estimated from the atnount of recovered N-O r a t h e r o n t h e initial c o n c e n t r a t i o n o f n i t r a t e o r m t r i t e All t h e r a t e s were o b t a i n e d f r o m the o b s e r v e d d a t a d u r i n g initial l - h i n c u b a t i o n period. B o t h t h e d e n i t r a t i f i c a t i o n a n d d e n i t r i t i f i c a t i o n rates u n d e r a n a e r o b i c c o n d i t i o n s at 1 3 C a r e p l o t t e d as a f u n c t i o n o f t h e r e s p e c t i v e initial c o n c e n t r a t i o n o f n i t r a t e or nitrite in Fig. 2. A t l o w e r c o n c e n t r a t i o n s . t h e r a t e s i n c r e a s e d a p p r o x i m a t e l y p r o p o r t i o n a l l y to t h e initial c o n c e n t r a t t o n o f n i t r a t e o r nitrite. T h e y a t t a i n e d m a x i m a a t a b o u t 1 4 0 / ~ M ( = ~ u m o l l 1) for d e n i t r a t i f i c a t i o n a n d 1 6 0 , a M for d e n i t r i t i f i c a t t o n . T h e d e n i t r i t i f i c a t i o n rate w a s h i g h e r t h a n the denitratification for c o n c e n t r a t i o n s lower t h a n 2 0 0 - - 3 0 0 # M . In t h e r a n g e h i g h e r t h a n 2 0 0 - 3 0 0 , a M .
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N i t r a t e o r nitrite a d d e d to t h e s e d i m e n t w a s detected in t h e r e c o v e r y o f N,.O a f t e r a v e r y s h o r t lag t i m e as s h o w n in Fig. I, w h i c h w a s o b s e r v e d u n d e r anaerobic conditions. The amount of produced N.,O i n c r e a s e d w i t h time, a n d l e v e l l i n g - o f f w a s n o t o b s e r v e d in m a n y cases. N u m b e r s in p a r e n t h e s i s d e n o t e initial c o n c e n t r a t i o n s a n d r e m a i n i n g c o n c e n t r a t i o n s e s t i m a t e d f r o m the a m o u n t o f p r o d u c e d NzO+ bec a u s e t h e a d d e d n i t r a t e o r nitrite w a s r e c o v e r e d a l m o s t s t o i c h i o m e t r i c a l l y as N : O ( N a k a j i m a e t al.. 1984). T h e rates o f d e n i t r a t i f i c a t i o n a n d d e n i t r i t i f i c a t i o n were c a l c u l a t e d f r o m t h e s l o p e s o f t h e s t r a i g h t lines in this figure. T h e s l o p e s o b v i o u s l y d o n o t d e p e n d o n the i n s t a n t a n e o u s c o n c e n t r a t i o n , b u t
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~500
NO3 - N , NO2 - N (u.M] Fig. 2. Effects o f initial concentrauons of nitrate and nitrite on the rates o f denitratification and denitritificadon under anaerobic conditions during l-h incubation period, at temperature 1 3 C
Effect of ox)gen concentration both rates gradually decreased with concentration. At concentrations of 1600 # M of nitrate and nitrite, the rate of denitritification decreased to 40'~;, of the maximum value, and that of denitratification to 65~o. Nitrate was found to be reduced faster than nitrite at higher concentrations. The effect of dissolved oxygen concentration on the denitratification rate at temperature of 13'C is shown in Fig. 3 and that on the denitrititication rate m Fig. 4. The rates were shown against initial and final oxygen concentrations observed during incubation. Both rates were high (4-6 u m o l g -~ h -~) for concentrations lower than 20-30
tA tl i
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Fig. 5. Efl'cct of temperature on the rates of denitratification and denitritification under anaerobic conditions. Initial concentrations of nitrate and nitrite were 160 # M.
i z O Z
2
:k _
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o
Fig. 3. Effect of dissolved oxygen concentration on the denitratifieation rate during l-h incubation period, at temperature 13C. Initial nitrate concentrations were 160 and 800 # M. Open and solid symbols correspond to initial and final concentrations of dissolved oxygen during incubation.
T
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80016080N0 z (,~M)c_eo._.
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200
300
0 z (~.M) Fig. 4. Effect of dissolved oxygen concentration on the denitritification rate during I-h incubation period, at temperature 13:C. Initial nitrite concentrations were 80, 160 and 800 u M. Open and solid symbols correspond to initial and final concentrations of dissolved oxygen during incubation.
completely under aerobic conditions. The critical oxygen concentration for denitratification was about 60 # M Oz, independent of nitrate concentration. In contrast, denitritification occurred even under aerobic conditions during incubation period. The critical oxygen concentration for denitritification depended on nitrite concentration. Under 80/~ M nitrite concentration, denitritification ceased at about 7 0 # M O,. The critical oxygen concentration for denitritification increased to 2 4 0 # M O, under 160 p M nitrite concentration. Under 800 # M nitrite concentration, denitritification occurred even at 300 # M 02, which corresponds to saturation of oxygen at this temperature. Thus, the critical oxygen concentration for denitritification was found to increase with nitrite concentration. The rate of denitritification under aerobic conditions was approximately halved l¥om 4 to 2/~ mol g-~ h z, under both 160 and 800/~M nitrite concentration. The effect of temperature on the rates of denitratification and denitritification under anaerobic conditions is shown in Fig. 5. Both rates increased approximately proportionally to the temperature between 5-30°C. The rate of denitritification was always higher than that of denitratification at each temperature under 160 # M nitrate or nitrite concentration. DISCUSSION
The critical oxygen concentration for denitratification has been reported to be in the range of 6--63 # M (Focht and Chang, 1975). They suggested that the difference in the critical oxygen concentration may reflect difference in bacterial species, having different affinity to oxygen. Nakajima { 1981)
'~"
,~,I|TStl"OSHi NAKAJIMA ('t ~al.
observed denitratification by sesstte microbes under almost complete saturation of oxygen, a n d ~t was con.lectured to be due to denttratification in anaerooic microsites in attached substrate. T h e denitratification rate m the present study ~ a s I\:und to be halved at a b o u t 20--30.uM O , . and the crmcat c o n c e n t r a t t o n for denitratification was 60 u 3,1 O , Few reports of the critical oxygen c o n c e n t r a t i o n for denitritification are availab!e. N a k a j i m a and Tezuka (1974) reported that cells ol" Pseudomon~s No. - previously grown anaerobically reduced nitrite even under 90"., sutural,on of oxygen at 1.7 m M o f initial n i m t e c o n c e n t r a t t o n Voets et al. (1975) reported that the occurrence of denitritification was estimated from the mass balance under 11-32 m M of nitritc c o n c e n t r a t i o n a n d 160-230~,M of average oxygen c o n c e n t r a t i o n in activated sludge containing 1-6 g 1-' suspended solids. These conclusions agree with our results. It is very interesting to note that under aerobic c o n d i t o n s sngnificant denitritification was observed while denitratification was totally suppressed. Pacaud et al, (1982) investtgated the stability of denitritification activity using pure bacterium Paracocus denitrificuns previously grown under anaerobic conditions, and reported that under aerobic conditions denitritification activity was high during lirst 2 h a n d decreased gradually. The rates o f denitratification a n d denitritification were reported for activated sludge were 0.1-10 nmol (g-volatile suspended solid J - ~ h - ~( Voets et al.. 1975). According to J o h n s o n a n d Schrepfer (1964), activated sludge c o n t a i n i n g 4 g 1-~ suspended solids was capable o f denitrification ( b o t h denitratification a n d denitritification) at a b o u t 1 4 0 p m o l g ' h -~ u n d e r anaerobic conditions. In our investigation, denitratification a n d denitritification rates were found to be 4--6 # mol (g-dry m u d ) - ' h - ' under anaerobic c o n d i t i o n s at 13~C. As the c o n t e n t of volatile matter in the sediment was 19.0% ( N a k a j i m a et al.. 1984), the rates were equal to 2 0 - 3 0 / a m o l (g-vo "latile m a t t e r ) ' h - ' in terms o f volaltile matter. Even u n d e r high oxygen c o n c e n t r a t i o n , denitritification occurred at a considerably high rate of 2 u m o l (g-dry m u d ) - h -~ (10 p m o l (g-volatile matter) -~ h-~). Such denitritification, therefore, would find i m p o r t a n t application in the nitrogen removal from sewage a n d natural e u t r o p h i c waters. As the t e m p e r a t u r e effect was investigated u n d e r almost the same c o n c e n t r a t i o n o f nitrate or nitrite, we could simply calculate the activation energies from an A r r h e n i u s plot o f reaction rates without assuming zero-order kinetics. T h e a c t i v a t m n energies of denitratification a n d denitritification in this study were calculated to be 39 a n d 3 4 k J m o l - ' . respectively. T h e activation energaes were calculated to be 67 ___20 kJ m o l - ~ (from the d a t a o f F o c h t a n d Chang, 1975). and 41 kJ m o l - ' for denitratification (from the d a t a o f N a k a j i m a . 1981). As the activation
energy of an enzyme r e a c n o n ~s m the range of 21-63kJmol( C o n n a n d Stumpi\ 1976). all the a b o v e data show that physical processes such ~t~, diffusion, which have much lower activation enermes, are not controlling processes m denitrification. [ h e possnble effect on denitritificatlon ot" the add> tion of glucose was investigated using the same sediment. The a m o u n t o f available c a r b o n was found not to be a d e t e r m i n i n g factor on the denitrification rates ( N a k a j i m a et al.. 1984J. Acknowledgements We express sincere apprecmuon to Dr Nicholas C. Kraus. Nearshore Environmental Research Center. for a helpful review of the manuscript, t.)ae of u~ (M.N.) w a s supported bv a scholarship from the Sakkokai Foundation.
REFERENCES
Balderston W. L., Sherr B. and Payne W, J. t 1976~ Blockage by acetylene of nitrous oxide reduction m Pseudomona~ p(zfectomarimts. Appt. em'ir. Mierohiol. 31, 504-508. Corm E. E. and Stumpf P. K. (1976) Outlines of Biochemtstry. 4th Edition. p, 157. Wiley, New York. Focht D. D. and Chang A. C (1975) Nitrification and denitrification processes related to waste water treatment Adv. appL Microhiol. 19, 153 t86 Johnson W. K. and Schroeprzr G. J ¢1964J N~trogcn removal by nitrification and denitrification. J. ~f27t Poltut Control Fed. 36. 101_'~-1036. Kagaku Benran (1975) Solubilities ol Inorganic Compounds, 2rid Edition. p. 770. Maruzen (in Japanese). MfiUer M M.. Sundman V. and Skujins L 11980~ Denitrification in low pH spodosols and peats determined with the acetylene inhibition method. Appt. envtr, microbiol. 40, 235--239 Nakajima T. {1981 } Denitrification in a polluted river ~,rn Int. Verein Limnol. 21. Nakajima T. and Tezuka Y, (1974) The possibility of gaseous nitrogen release from water bodies under aerobic conditions. Jap. J. LimnoL 35, 117-123. Nakajima M., Hayamizu T. and Nishimura H. 11984) Relation between the denitrification activities and the organic matters m the sediments of Lake Teganuma. In preparation. Pacaud B., Kawakami Y. and Nishmura H I1982) Environmental factors affecting biological denitrification. Autumnal Meeting 05 Chemical Engineering at Kanazawa. p. 268. Painter H. A. t1970) Review of literature on morgamc nitrogen metabolism m microorgamsms. Water Res. 4. 393--450 Payne W. J (1973) Reduction of nitrogeneous oxides by microorgamsms. Bact. Rev 37, 409--452. Prakasam T. B. S. and Loehr R C. (1972) Microbiai nitrification and denitrification in concentrated wastes. Water Res. 6, 859--869. Voets J P.. Vanstaen H. and Verstraeta W. (1975) Removal of nitrogen from highly nitrogenous waste waters. J. War Pollut. Control Fed. 47, 394-398. Yoshida T., Aizaki M., Asami T. and Makishima N. ~1979~ Biological nitrogen fixation and denitrification in Lake Kasumigaura. Jap. J. LimnoL 40. I-9. Yoshinari T., Hynes R. and Knowles R. (1977) Acetylene inhibition of nitrous oxide reduction and measurement of denitrification and nitrogen fixation in soil, Soil Biol. Biochem. 9, 177-183.