The effect of electron acceptor variations on the behaviour of Thiosphaera pantotropha and Paracoccus denitrificans in pure and mixed cultures

FEMg; Microb,ologyEcology86 (1992) 221-228 © 1992 Federation of European MicrobiologicalSocieties0168-6496/92/$05 0(1 Pubhshed by Elsevier

FEMSEC 00367

of

The effect of electron acceptor variations on the behaviour Thiosphaera pantotropha and Paracoccus denitrificans in pure and mixed cultures Lesley A. R o b e r t s o n a n d J G o s K , t e n e n

Kluyter Laboratoryfor Btote~hnology,Delft Unnerslty o[ Technology, Delft, The Netherlands Received 20 July 1991 Accepted27 September 1991 Key words Aerobic denttrtficatton, Oxygen, Nttrate, Nitrite, Selection

1 SUMMARY

totropha did, indeed, dominate the population when expected However, when Pa denttnficans

The competitive advantages provided by a capactty for aerobic denttrtftcat,on have been tested by compar,ng Thlosphaera pantotropha (which denttrtfies aerobically and anaerobically), with a stram of Paracoccus demtnffcans (which only demtrtfles under anaerobic conditions) m acetate-hmited chemostats A comparison of /.t-C s curves based on K s and ~mdx measurements mdtcated that Pa demtrificans could be expected to dominate mLxtures of the two species at high growth rates when the dissolved oxygen was above 80% of air saturation and NH 3 was the sole source of nitrogen The compar,son also suggested that at lower growth rates, lower dissolved oxygen tensions, a n d / o r in the presence of nitrate, Tsa pantotropha should have the compet,tlve advantage Chemostat experiments with mixtures of the two s p e o e s showed that Tsa pan-

was expected to dominate, only a small increase tn the Pa demtnficans numbers was possible before Tsa pantotropha formed a btofilm on the walls of the chemostat instead of washing out, and was agam able to out-compete Pa demtnficans for acetate Experiments v.lth axentc chemostat cultures subjected to aerobic/anaerobic sw,tches showed that Tsa pantotropha, wtth its constitutive denttrffymg system, was able to adjust smoothly to the changing envtronmental condttlons and thus continued to grow. Pa denttnficans does not have constitutive denttrtfylng enzymes, and could consequently not adjust its metabohsm to the lack of oxygen rapidly enough It therefore washed out at a rate equivalent to the d,lutton rate

2 INTRODUCTION

Correspondence to

L A Robertson, Kluyver Laboratory for Biotechnology, Delft Un,verstty of Technology, Juhanalaan 67, 2628 BC Delft, The Netherlands

The occurrence of aerobic denttrtficatlon is now well documented (see ref 1 for a review) Thiosphaera pantotropha Is among the co,~stttu-

222 rive demtrifiers able to demtrdy at dissolved oxygen concetratmns approachmg air saturation Tsa pantotropha was isolated as one of the dominant organisms in a den.tnfymg waste-water treatment reactor which was generally anaerobic but received occasional pulses of oxygen [2] Against this background, and given some of the (eco)phys~ological properties of Tsa pantotropha and other similar strains, the postulated ecological niche for these organisms appears to lie m situations where oxygen ~s hmltmg, or when the environment goes through relatively short cycles of aeroblosls and anaerobiosis [1,3,4]. Tsa pantotropha is, of course, also a heterotrophlc nltnfier [5,6,7] Both nitrification and demtriftcat~on are of ~mportance m soil fertdlty (where they are undesirable) and waste water treatment (where both phenomena play an important part m nitrogen removal). An understanding ¢f the factors which will select for bacteria that can simultaneously nitrify and denltrlfy ts thus desirable Because of the complexity of experiments to test the combined phenomena and the number of varmbles involved, it was deczded that their ecological ~mportance should be .nvestlgated separately Experiments were therefore set up to discover whether, and under what condmons, its abilities as an aerobic demtntier would give Tsa l~antotropha a selective advantage over Paracoccas denttnficans, a species which only demmfies under anaerob.c conditions The problem was approached in two ways" firstly, the outcome of compeuuon between the two species in different acetate-hmited chemostat cultures was checked, secondly, the effect of aerobic/anaerobic switches o r axemc cultures of Tsa pantotropha and Pa de~anfica~s was measured in order to test their ac[aptablhty. This 16aper reports the results of these experiments.

3 MATERIALS AND METHODS

Organisms Thtosphaera pantotropha LMD 82 5 was originally isolated from a demtnfying, sulphide oxidlzmg wastewater treatment system [2] Paracoccus den,trtficans LMD 22.21 was obtained from the

culture collection of this laboratory, and is the strata isolated by Beijermck [8].

Me&a The medium for the chemostats contamed (g IOl). KzHPO4, 08, KH2PO 4, 03, NH4CI, 04, MgSO 4 7HzO, 0 4 and 2 ml of trace element solution Sodium acetate (20 raM) was supplied as the limiting substrate When appropriate, 32 mM KNO~ was used The medmm described for the growth of Thtobactllus versutus (formerly Thtobacdlus A2) by Taylor and Hoare [9] was used for batch cultures It contamed (in g ! - I ) Na2HPO4 7HzO, 7.9, KH2PO 4, 1 5, NH4CI, 0 3, MgSO 4 7H20, 0 1, 2ml of trace element solution The MgSO4, trace element soluuon, substrates and KNO3 (20 raM) were added after stenllzauon The initial concentration of the sodium acetate in the batch cultures was 10 mM The trace element solution [10] used with all media contained (as g I - I ) EDTA, 50; ZnSO 4, 22, CaCI2, 5.5; M n C I 2 - 4 H 2 0 , 5.06, FeSO4" 7H20, 5 0; (NH 4) 6Mo7024 4H20, I 1; CuSO 4 5HaO, 1 57, CoCl 2 6H20, 1.61 For colony counting, 2% (w/v) Difco bacto agar was added to the batch culture medmm The substrate In these plates was a m~xture of 0 3 g I - ' yeast extract, 0 1 g I-~ fructose and 0.1 g - ' sodium acetate As Tsa pantotropha .s more sensitive to amp]cdhn than Pa demmficans, plates with and without 0 15 mg I - ' amplcdlln were used to obtam Pa denltnficans and total counts, respectively

Contmuous cultures Continuous cultures were made m chemostats fitted with dissolved oxygen and pH control. The temperature was mamtalned at 30°C and the pH at 8.0. For the compeutlon experiments, Tsa pantotropha and Pa. denltnficans were grown axenically to steady state at the appropriate dtluUon late and dissolved oxygen concentraUon (80% of mr unless otherwise specified). Once the steady state had been confirmed by analysis of the medium and bmmass, the two cultures were mixed To a v i d potential contamination prob-

223 lems durmg the mixing, the chemostats had been linked before sterdlzatlon by tubes through wh,ch the cultures could be pumped In a control experiment, It was confirmed that each of the test species grew normally m filter-stermhzed medmm in which the other had previously been grown, indicating that neither produced excretion products which would be inhibitory to the other For the a e r o b i c / a n a e r o b i c and anaerob i c / a e r o b i c experiments, the aerobic (80% of a~r saturation) and anaerobic cycles m the cultures were achieved by sparglng with air or ultra-pure mtrogen Cultures were grown to steady state before a switch was achieved by suddenly changmg the sparglng gas The behaviour of the cultures was monitored with continuous, o n - l i e optical density measurements and periodic samphng for off-line analysis

Miscellaneous The mammum specific growth rates for the two species under each set of growth condltmns were determined during the batch culture start-up phase of each chemostat experiment Oxygen uptake was measured using a Clarktype electrode mounted in a thermostatically controlled cell which is closed except for a small hole through which addmons may be made. Apparent K s values for acetate were determined using direct linear plots [15] of the oxygen uptake rate as a function of various acetate concentrations Washed cells were resuspended m 0 05 M phosphate buffer, pH 8 0, and the measurements were done m the presence or absence of ammonia and nitrate lmmuno-fluorescent staining was carried out using antibodies raised against Tsa pantotropha by the method described by Muyzer et al [16]

Btomass analysis Protein was measured spectrophotometrically, by means of the Micro-Biuret method [11] Total orgamc carbon was measured using a Beckman Tocamaster Model 150B Because Tsa pantotropha tends to make poly-B hydroxybutyrate (PHB) under some growth conditions, both dry weight and total organic carbon measurements could give an artificially high y~eld The yield estimates for the nitrogen balances were therefore based on the protein determinatmns, as previously described [7]

Analysts o f culture medium Acetate was determined with acetyl-coenzyme A synthetase using a test kR (Boehrlnger) NRrlte was measured colounmetncally, w~th the Griess-Romun reagent [12] or by means of an H P L C fitted with a ionosphor-TMA column (Chrompak) and a Waiters RI detector Nitrate was also measured with the HPLC Hydroxylamme and ammonia were determ,ned colounmetr,cally by means of the methods described by Frear and Burrell [13] and Fawcett and Scott [14], respectively. As, at the pH values used m these experiments, ammoma and ammonium would both be present, the term 'ammonia' will be used throughout to indicate both the protonated and unprotonated forms

4 RESULTS A N D DISCUSSION

4 1 Competition studies 4 1 1 Theoretical models The effect of various environmental factors on the outcome of competltmn between two species can frequently be predicted from the comparison of theoretical plots of the growth rates versus the residual substrate concentration ( / ~ / C s curves) constructed according to the Monod formula [17]. Examples of such curves, in which the chemostat dilution rate ( D ) is plotted against C s (as D = p. in a chemostat), for Tsa pantotropha and Pa demtnficans, when grown aerobically on acetate in the presence and absence of nitrate, are shown in Fig 1 From these curves, it appeared that Pa demmficans should dominate aerobic mixed cultures of the two species at high growth rates when ammonia was the sole source of nitrogen, and Tsa pantotropha should do better at lower dilution rates (Fig la) If mtrate and oxygen were both present m the medium, the increase in the /~m~ of Tsa pantotropha [3] should confer a selective advantage over Pa. demmficans at all growth rates (Fig lb). A t lower dissolved oxygen concentrations ( < 25% of air saturation), the/Zm~

224

D (h-l) 05 t

I "s

vantage, even at high dilution rates The posttton was reversed In anaerobic cultures, where Pa demtrt~cans had a much htgher /~m~x than Tsa pantotropha, and should therefore dominate at all growth rates (not shown)

A -

~ -.~--

:

"-

-" - -" - ' - - ' " a

0 251/ ,

ot

4 1 2 Experimental verification

o4s

Cs

0 5 O~h-ll

B

. ..

02 t ~

O'

........

O-:~s

D(h-ll

0[ Fig

"" "

a

'

Cs

C .........

o:~s

1 Curves of ddutlon

o~5

a

Cs rate (D)

against residual

substrate

concentration (Cs) showingthe theoretical outcome of comI~tmon for acetate between Tsa pantotropha (a) and Pa demmJicans(b) in the chemostat Ks aceta,e values of 28 and 63 ~M. respectively, were used IA ffi80% air saturation, NH s as sole mtrogen source, ~raa.~=042 and 047 h -I, respectively, 1Bffi95% air sa,ura,lon. NH3 and NO~" both supphed, ttma, ffi0 32 and 0 25h- I, respec,,vely.IC = 25% air saturation, Nil 3 as sole mtrogen source,/zm.~ffi0 47 and 0 38 h- I, respectively of Tsa. pantotropha increased to such an extent that, even when oxygen was the sole electron aceeptor provided, ,t should have a selective ad-

Experimental testing of these models was carn e d out by compet,t,on exper,ments m the chemostat The changes tn the numbers of cells from the two species were followed after m~xmg until a steady state was reached Cell ratios were determined by selectwe plating and by immunofluorescent labelling (see MATERIALS AND r~E'rHODS for details). The exper,ments gave the predicted results whenever Tsa pantotropha was expected to dominate (F~g. l a - c ) , and the cultures remained well suspended and homogenous. In these mixtures, Pa. denunfwans amounted to less than 1% of the population after more than 10 volume changes, ,rrespecttve of the ratios of the two species m the ong,nal culture. Confirmation that the majority populaaon wvs Tsa. pantotropha c a ~ e from the measurements of the yields and mtrogen balances For example, as the percentage of Pa denttn.ficans ,n the commumty reeewmg oxygen as the sole electron acceptor fell from Its ortgmal 90% value, the b,omass concentration also fell It eventually reached a value s~mtlar to that obtained with axen,c Tsa pantotropha cultures, that ts changing from 8.1 to 8 7 g protein tool a c e t a t e - t at the start to 6 2 to 6 6 g protein tool acetate-I at the end Moreover, the ammonia loss from the axemc Pa denttr,ficans cultures, which had been very low (equivalent to a nitrification rate of 6 nmol r a m - I m g p r o t e l n - I at a ddutton rate of 0 07 h - l), rose after mutmg to values equivalent to those in the Tsa pantotropha cultures (e g 18 n m o l - ' r a m - J mg protein - t at a dilution rate of 007 h - l ) The results obtamed with the cultures m which Pa demtnficans was expected to dominate were less clear cut. For example, when the dilut,on rate was increased to a value above the cross point of the curves shown m Fig la (and thus at which Tsa pantotropha might be expected to wash out), the population of Pa demtnficans began to recover, increasing from 1% to 13% of

225 the community after 5 volume changes. Unfortunately, at this point, a rapidly thickening blofilm became apparent on the wall of the chcmostats, and the suspended Pa demtnpcans populauon fell back to below 1% lmmunofluorcscent staining showed that this btofllm consisted almost entirely of Tsa pantotropha Of course, Tsa pantotropha was originally isolated from a flutdlzed bed column where the biomass was attached to sand. One of the main selectwe pressures in such a system is the abihty to attach to surfaces when the dilution rate exceeds the /~m~ of the organIsm [18] As Tsa pantotropha has a much lower K s for acetate than Pa demtnficans (28 and 63 p.M, respectively), it is clear that the attached Tsa pantotropha was able to build a populanon sufficiently large to successfully compete for substrate even though the Pa denttnficans remained In s u s p e n s i o n

4 2 Aerobzc/anaerobzc cycles To test the prediction that Tsa pantotropha (as an aerobic demtrlfier) would have an advantage over Pa demtnficans (as a specialist which only denitrifies anaerobically) In situations where the oxygen supply fluctuated, cultures were grown m chemostats undergoing alternate cycles of aeroblosls and anaeroblosls To allow the study of the behawour of Pa denltnflcans under this

regime, without the masking effect caused by potential Tsa pantotropha blofilm formation, the experiments were run with pure cultures.

4 21 Aerobic to anaerobic switch When aerobic cultures were grown to steady state and then suddenly made anaerobic, the mtrate concentrations in both cultures began to fall immediately, but this fall was much more dramatic in the Pa denttnficans cultures (Fig 2a) While mtrlte did not accumulate in the Tsa pantotropha medium (Fig. 2b), its concentration In the Pa denttnficans cultures rose in an almost 1 1 ratio with the decrease in th,: mtrate, reaching 15 mM after 4 h A similar, transient (3 h) accumulation of nlmte, accompanied by a fall-off m cytochrorne oxJdase activity, was observed with batch cultures of Pa denttnficans which had been switched from aerobic to oxygen-limited growth in the presence of mtrate [19] Regardless of the ddution rate, the protein content of the Pa denttnficans cultures fell substantlally during the experiment (by 25% at D = 015 h -~, by 50% at D = 0 3 0 h - I ) , confirming the wash out indicated by previous optical density measurements (Fig. 3) In contrast, the protein content of the Tsa pantotropha cultures changed very little untd the dilution rate was increased to a value above the anaerobic/Zma ,, when the culture washed out The total organic carbon of all Tsa

,oa ~ l t r l f l c a n s (A)

Aerobic

to

anaerobic

--(~- nitrate

(B)

transition

Aerobic - - e - - r, trale

---0-- rHtrlle

to

,oantotro~Ja anaerobic --i-

trans,tlon hilt,re

30

ilt ....... 4........ o

.......

i' i ,j

-60

2

=

i

t

6 ¢1

126

t i~11~

250

Fig 2 The response of steady state, acetate-limited, aerobic cllemostaI LulIures ( D = 0 1 5 h - i) of Pa deflttrtflcafls (2a) and Tsa pantotropha (2b) to a sudden switch to anaerobzosts, as demonstrated by the changes in the nitrate and mtrlle concentrations The bar indicates the change in the gas stream from air to nitrogen at t = 0

226 o f t h e s u p e r n a t e n t s rose after t h e c u l t u r e s w e r e m a d e anaerobic, indicating t h a t a c e t a t e h a d c e a s e d to be limiting A l t h o u g h this w a s a t e m p o rary s t a t e o f affairs In t h e Tsa pantotropha cultures, w h i c h s o o n b e c a m e a c e t a t e - l i m i t e d again, this w a s n o t t h e case in t h e Pa denimficans cultures. E l e c t r o n m i c r o s c o p y revealed t h e prese n c e of poly-fl-hydroxybutyrate In b o t h Tsa pantotropha a n d Pa denzmficans cells at t h e e n d , b u t no*. t h e start o f t h e e x p e r i m e n t s

Table 1 The effect of nitrite on acetate-dependent oxygen uptake by washed cells of Tsa pantotropha and Pa denitrzficans m the presence and absence of ammonia (A cells grown aerobically in an acetate.llmded chemosat in the presence of ammonia and mtrate B cells grown aerobically in an acetate-limited chemosat in the presence of ammonia and nitrite ) Additive NH~(mM)

NO~" (raM)

4 2 2 Anaerobw to aerobic swztch A n a e r o b i c c u l t u r e s o f Tsa pantotropha a n d Pa denzmficans w h i c h h a d b e e n g r o w n to s t e a d y

0 0 0 0 0 0 75 75 75 75 75 75

0 25 50 to 0 t5 0 200 0 25 50 10 0 15 0 202

a d j u s t e d s m o o t h l y w h e n s u d d e n l y switched to a dissolved oxygen c o n c e n t r a t i o n 8 0 % t h a t o f air s a t u r a t i o n , c o n f i r m i n g t h e constitutive n a t u r e o f b o t h aerob;c respiratory c h a i n s l m m e d l a t e d l y

state

t h e air supply w a s c o n n e c t e d , t h e n i t r a t e c o n c e n tratlons In both c u l t u r e s b e g a n to rise N e i t h e r s p e c i e s a c c u m u l a t e d nitrite T h e p r o t e i n c o n t e n t o f b o t h c u l t u r e s increased, c o n f i r m i n g t h e switch to t h e energetically m o r e efficient u s e o f oxygen as a t e r m i n a l e l e c t r o n a c c e p t o r O f c o u r s e , Tsa pantotropha c o n t i n u e d to partially respire by m e a n s o f t h e denltrlfiCatlon pathway, a n d its inc r e a s e in yield was t h e r e f o r e relatively s m a l l e r ( f r o m 84 m g / I , anaerobically, to 110, aerobically) t h a n t h a t o f Pa demtrtficans (98 to 138, r e s p e c tively)

OU (%) - - ~ . ~

100"

-~

.........

-'-'---._J/ l~r

75

Tp

~.

llir ..* "2

5'0

100

Pd

150

time (ram)

Fig 3 The outcome of switching steady-stale, acelale-hmlled, chemoslat cultures (D = 0 25 h- i) of Tsa pantotropha ([ p ) and Pa denzinficans (P d ) from aerob,osm (80% air saturation) to anaerobloSlS

Oxygen uptake rate (nmol 02 m m - ~(mg protein)- ')

Tsa pantotropha Pa denztnficans A B A 311 301 301 284 283 254 284 315 313 316 nd 293

336 349 343 334 336 nd 344 nd 343 349 334 334

208 172 146 105 87 nd 182 155 126 95 68 48

4 3 The influence of nitrite D u r i n g t h e e a r h e r e x p e r i m e n t s s h o w n in Fig 3, t h e Pa demtnficans c u l t u r e s c o n t i n u e d to w a s h o u t a f t e r aeroblOSlS h a d b e e n restored. In t h e h g h t o f t h e results r e p o r t e d above, a n d b e c a u s e t h e rate o f a c e t a t e - d e p e n d e n t oxygen u p t a k e by w a s h e d , m t r l t e - f r e e cells o f Pa demtrtficans s a m pled a f t e r d i f f e r e n t p e r i o d s o f anaeroblOSlS did n o t c h a n g e significantly, p e r m a n e n t inactivation of the cytochrome oxldases during anaerobic g r o w t h did n o t s e e m very likely It t h e r e f o r e s e e m e d possible t h a t t h e failure o f t h e c u l t u r e to recover was d u e to t h e level o f nitrite in t h e culture Nitrite h a s previously b e e n s h o w n to inhibit oxygen u p t a k e by Pa denttrificans [5,20], a n d this also p r o v e d to be t h e case with r e s t i n g cells f r o m t h e s e c u l t u r e s . T h e rate o f a c e t a t e - d e p e n d e n t oxygen u p t a k e by w a s h e d Pa denttraficans cells fell as t h e nitrite c o n c e n t r a t i o n i n c r e a s e d , while Tsa pantotropha r e m a i n e d relatively u n a f f e c t e d ( T a b l e 1) Tsa pantotropha w h i c h h a d b e e n g r o w n in c u l t u r e s w h e r e nitrite r e p l a c e d t h e n i t r a t e was c o m p l e t e l y u n a f f e c t e d by t h e nitrite c o n c e n t r a tions u s e d (Table 1).

227

Nitrate reductase actwlty m Pa denttnficans Is clearly much less sensitwe to oxygen than m t n t e reductase (Fig 2; [21,22]) If oxygen uptake was inh~b~ted to some extent by the high m t n t e concentration, the cytochrome chain would become more reduced Electrons would then be able to flow to mtrate, generating more mtrtte and creating a 'v~cious circle' Presumably, tf permitted a longer period of anaeroblosts (for example, tn batch cultures where the culture density could not fall to too low a level for a vmble culture) Pa denttrtficans would be able to generate its complete demtrffymg pathway and reduce the mtrtte concentration

5 CONCLUSION As judged by theoretical predictions of the type shown in Fig 1, the fate of Tsa pantotropha in competition with Pa demtnficans for acetate depends very much on the growth rate, and thus on the avadable electron acceptors Pa demmficans appears to have an advantage ,f oxygen or mtrate are avadable as single electron acceptors, especmlly at higher growth rates However, Jn the presence of both electron acceptors, the tim.~ of Tsa pantotropha becomes greater than that of Pa demtnficans and it wdl gain a competitive advantage A n o t h e r ~mportant factor was the concentration of the d~ssolved oxygen m the cultures. Even when oxygen was the sole electron acceptor, Tsa. pantotropha grew faster than Pa denanficans at low ( < 30% air saturation) oxygen tensions, with the result that it should also dom~hate mixed cultures under these condRions The results of the experimental venficatlon of these models by measuring population changes m communities competing for acetate emphasize the need for caution m the design of experiments of this type Although the inmal experiments appeared to confirm the models, with Tsa pantotropha dominating the commumties when expected, it would appear that the abihty to adhere to a surface, forming a btofilm, gwes a major competitive advantage which outweighs the influence of the growth rate The models will therefore be tested using another heterotroph~c mtrl-

fler/aerohlc denlmfier which does not form btofilms so readdy The response of the two types of denitrifier to a sudden onset of anaeroblOSJS revealed, as expected, that the possession of a constRut~ve denitrificatio~: pathway can be a seleetwe advantage when sudden changes m the available electron acceptor occur The advantage gamed was twofold tt prevented the braid-up of inhibitory amounts of nitrite and it allowed the smooth transmon to anoxlc growth it therefore appears that the possession of a constitutive denitrtficatton system provides Tsa. pantotropha with a competltwe advantage for hfe under (relatively short-term) oxygen fluctuation, whereas its mabdtty to use more than one electron acceptor at a time makes this Pa denanficans strata better suited to semi-steady-state conditions

ACKNOWLEDGEMENTS The authors are grateful to Anke de Bruyn for help with the ~mmunofluorescent staining, and to Prashant Gupta, Jan den Ouden and Adele van Houwel,ngen for experimental assistance

REFERENCES [1] Robertson. L A and Kuenen,J G 0990) Combinedheterotrophlc flttrlflCatlon and aerobic denltni~catlon in Thwsphaerapanlotropha and other bacteria Antoine van Leeuwenhoek 57,139-152 [2] Robertson, LA and Kuenen, J G (1983) Thtosphaera pantotropha gen nov sp nov, a facultallvelyanaerobic, facultatJvely auIolrophic sulphur bacterium J Gen Microblol 129,2847-2855 [3] Robertson, L A and Kuenen, J G (1984) Aerobic denlIrnficattur, a controversy revived Arch Mlcroblol 139,351-354 [4] Robertson, L A and Kuenen, J G (1984) Aerobic demlrlfiCatlon - old wine in new bon]es"~Ant van I..ceuwenhock 50,525-544 [5] Robertscn, L A and Kuenen, J G (1988) Heterotrophlc nitrification in Ttuosphaerapantotropha oxygen uptake and enzymestudies J Gen Microblol 134,857-863 [6] Kuenen, JG and Robertson, LA (1987) Ecolo~ of nitrification and denltrllicatlon In The Nitrogen and Sulphur Cycles(Cole, J A and Ferguson,S, Eds ) Cambridge UmversityPress pp 162-218

228 [7] Robertson, L A , van N,el, E W J , Torremans, R A M and Kuenen, J G (1988) Simultaneous mtrtficatlon and demtnficatton in aerobic chemostat cultures of Thlosphaerapantotropha Appl Env Mtcroblol 54,2812-2818 [8] Beuertnck, W M (1910) BiIdung uod verbrauch yon st,ckoxydul dutch Bakterien Centrallblat fur Bakterlol (Abt II) 25,30-63 [9] Taylor, B F and Hoare, D S (1969) New facultatlve Thtobacdlus and a reevaluatmn of the heterotrophlc POtcnt,al of Thtobacdlus novellus J Bact 100, 487-479 [10] Vishnlac, W and Santer, M (1957) The Thlobacdh Bact Rev 21,195-213 [11] Goa, J (1953) A microbiuret method for protein determination, determination of total protein in cerebrospmal fluid J Chn Lab Invest 5,218-222 [12] Gness-Romtln van Eck (1966) Physiological and chemical tests for dnnkmg water NEN 1056, IV-2, Nederlands Normahsatte lnstltuut Rt.lSwljk [13] Frear, D S and Burrell, R C (1955) Spectrophotometnc method for determlmng h~,droxylamzne reductase activity m higher plants Anal Chem 27,1664-1665 [14] Fawcett, J K and Scott, J E (1960) A rapid and precise method for the determznauon of urea J Chn Path 13,156-159 [15] Williams, P (1984) Enzpack A demonstratmn and calculation program for enzyme kinetics (BBC version) Elsevier Biosoft, Elsevier, Amsterdam [16] Muyzer, G , de Bruyn, J C , Schmeddlng, D J M, Bos, P,

Westbroek, P and Kuenen, J G (1987) A combined immunofluorescence-DNA fluorescence staining technique for enumeration of Thlobacdlus ferrooxtdans in a population of acsdophshc bacteria Appl Env Microbiol 53,660-664 [17] Monod, J (1942) Recherches sur la Crolssance des Cultures Bacterlennes Hermann, Paris [18] Heijnen, J J (1984) Biological industrial waste-water treatment minimizing blomass production and maximizing blomass concentration PhD Thesis Delft University of Technology, Delft, The Netherlands [19] Kucera, I , Boubhkova, P al~':lDadak, V (1984) Function of terminal acceptors in the biosynthesis of demtrlflcatlon pathway components in Paracoccus dentlnficans Foha Microbsol 29,108-114 [20] McCarthy, J E and Ferguson, S J (1981) Estimation with an son-selective electrode of the membrane potential in cells of Paracoccus demtnficans from the uptake of the butyltrlphenylphosphomum cation during aerobic and anaerobic respiration BIochem J 196,311-321 [21] Lain, Y and Nicholas, D J D (1969) Aerobic and anaerobic respiration in Micrococcus demtnficans Biochtm Biophys Acta 172,450-461 [22] Alefounder, P R , McCarthy, J E O and Ferguson, S J (1981) The basis of the control of mtrate reduction by oxygen In Paracoccus demtrlficans FEMS Mlcroblol Letts 12,321-326