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Denitrification with methanol: microbiology and biochemistry
Water Research Vo[. 14, pp. 531 to 537 Pergamon Press Ltd 1980. Printed in Great Britain
DENITRIFICATION WITH METHANOL: MICROBIOLOGY AND BIOCHEMISTRY GEORGE R. NURSE Department of Biochemistry, University of Natal, Pietermaritzburg, Republic of South Africa
(Received March 1978) Abstract--Although methanol is frequently chosen as carbon and energy source for denitrifieation of nitrate polluted effluents, all kinetic parameters thus far reported have been for an unidentified biomass in the belief that a more specialized knowledge of the bacterial species present in the reactors has not been necessary. However, it has now been adequately demonstrated that denitrification with methanol results in a selective enrichment for bacteria belonging to the genus Hyphomierobium.Based on current available biochemical knowledge of nitrate reduction and assimilation, methanol oxidation and assimilation and the energy yield and requirements for these reactions, it is theoretically possible to develop an equation which describes the stoichiometry of denitrification with methanol.
INTRODUCTION
During the last 25 years considerable research has been conducted into denitrification of nitrate polluted waters using methanol as sole carbon and energy source [1-12]. Since denitrification can be effected by a wide variety of facultative bacteria [13-16], it has always been concluded by workers in this field that little can be achieved from identification of species actively performing in their reactors. Although the concept of working with an unidentified biomass may be justified in those investigations using heterogenous carbon and energy sources, it has unfortunately persisted in those situations where methanol has been used. The first evidence that the biomass in a denitrifying reactor had an unusual population when methanol was used, was in fact reported by the first investigators to use methanol [1]. They commented: "So far as the organisms are concerned, they consist of an almost pure culture of as yet unidentified very small ciliated protozoa with large numbers of bacteria". In an investigation into the denitrification of agricultural wastewaters using various carbon sources, it was noted that a lag resulted before denitrification occurred with methanol and it was mentioned that this was "presumably due to a need for biological acclimation to this substrate" [3]. In 1971 it was reported that denitrification with methanol resulted in a selective enrichment for bacteria belonging to the genus Hyphomicrobium [17]. The same result was obtained a year later [18] and has since been confirmed by the present author [19]. A photograph showing the characteristic morphology of the species is shown in Fig. 1. Unfortunately these observations have been overlooked by workers in the field although they have retained the terminology in referring to the biomass as a "modified activated sludge". The present author, however, has carried out a kinetic investigation into the denitrification of a high level nitrate industrial effluent bearing the above w.R. 14/5--1
results in mind [19]. The kinetic parameters obtained have not only been used for plant design purposes but also to characterize quantitatively Hyphomicrobium spp. growing anaerobically on methanol, as data concerning this aspect of their growth are very limited. In order to increase confidence in the experimental growth yield, Y, (0.195 g biomass per g methanol consumed), this investigation also included a theoretical study into the efficiency with which methanol is converted into biomass. This involved developing equations which describe anaerobic growth on methanol by Hyphomicrobium spp. using our existing knowledge of the biochemical pathways involved. The principles underlying the calculations involved and their validity are discussed in greater detail by van Dijken and Harder [20] who developed similar equations for microorganisms growing aerobically on methane and methanol in order to determine theoretical growth yields. It is the purpose of this paper to describe in simple biochemical terms the development of a theoretical equation that adequately describes the stoichiometry of denitrification with methanol. INORGANIC NITROGEN METABOLISM
Hyphomicrobium spp. can utilize nitrate for the synthesis of all the nitrogen containing compounds of the cell and as the terminal electron acceptor in place of oxygen under anaerobic conditions [17-19, 21]. The two processes are called nitrate assimilation and nitrate respiration respectively. Unfortunately, the enzymes involved in both processes have not been studied in Hyphomierobium spp. and in the discussion below it is assumed that the concept of unifying metabolism exists between nitrate utilizing bacteria. Nitrate assimilation In a growth medium in which nitrate is the sole source of nitrogen, a preliminary reduction of the nitrate to ammonia must occur before assimilation can take place [15, 16]. The steps involved in the
531
532
GEORGER. NURSE
Fig. 1. Hyphomicrobium spp. liom an enrichment culture growing anaerobically on methanol (pH 7.4, 28°C) in continuous culture (phase-contrast, ca 3000 x)E19 ].
conversion are depicted in Fig. 2, where (Z), the intermediate between nitrite and hydroxylamine remains unknown although some researchers have proposed nitroxyl (NOH) or its dimer nitramide (N202H2) or hyponitrite [15]. NO3 -~ N O r --, (Z)---, NH2OH --~ NH3 Fig. 2. Pathway of nitrate assimilation assumed to occur in Hyphomicrobium spp. where (Z) represents an unknown intermediate. Since some of the enzymes responsible for the conversion have been shown to use NADH as electron donor in a wide variety of microorganisms [16], it is assumed that 4 NADH2 are required for the conversion as represented by Eq. (1) below: HNO3 + 4 NADH2 ---,NH3 + 3 H20
(1)
Nitrate respiration The reduction of nitrate to nitrogen gas proceeds through a number of steps as shown in Fig. 3. Many researchers have been devoted to studies of this reaction sequence [15, 16]. NO3 --~ NO2 --* NO
--~
N20
--+ N 2
Fig. 3. Pathway of nitrate reduction assumed to occur in Hyphoraicrobium spp. Although nitrous oxide is sometimes formed during denitrification, it is assumed in the calculations below
that all nitrate consumed for respirometric purposes is quantitatively recovered as nitrogen gas. In the calculations performed by van Dijken and Harder [20], it was assumed that the electron transport chain in methanol utilizing bacteria shows 3 phosphorylation sites, so that theoretically 3 moles of ATP can be formed during the oxidation of 1 mole of NADH2. However, this assumption is only valid under aerobic conditions and the amount of ATP assumed to be generated under anaerobic conditions requires careful consideration. One-step reduction of nitrate [15, 16] and nitrous oxide [22, 23] are known to support the growth of various bacteria. It is therefore intuitively obvious that reduction of these compounds is coupled to oxidative phosphorylation. However, those bacteria that grow when supplied with nitrite may conceivably benefit from coupling reduction with phosphorylation only during the reduction of nitrous oxide and not during the reduction of nitrite or nitric oxide. Based on the assumption that ATP generating systems remain membrane-bound when cells are ruptured, several workers have claimed that nitrate and nitrous oxide reduction is coupled to oxidative phosphorylation whereas nitrite and nitric oxide reduction is not [23, 24]. These findings, however, are inconsistent with results obtained when esterification of radioactively labelled phosphate with concomittant reduction of nitrite was demonstrated in cell-free extracts [25, 26"1and no esterification was observed in reaction mixtures supplied with nitric or nitrous oxide
Denitrification with methanol: Microbiology and biochemistry [27, 28]. The observed lack of esterification with nitrous oxide was accounted for by mentioning that the sensitive coupling of phosphorylation to the electron transport chain may have been uncoupled during the physical disruption of the cells in order to prepare the cell-free extracts. In order to overcome the disadvantages associated with the interpretation of results from in vitro studies, an in vivo method for determining the efficiency of oxidative phosphorylation has been developed. The method is based on the conclusion of Bauchop & Elsden [29] that the molar growth yield of cells per mole of ATP generated from the oxidation of the energy source, designated YATP,is a constant for different microorganisms, the value being 10.5 g dry weight of cells per mole of ATP produced. More recently, however, it has been shown that YATPvalues can be very different for different microorganisms and that the value for a particular organism growing on a particular substrate is also determined by the cell composition, the specific growth rate and the maintenance coefficient [30]. However, if YArP for a particular organism is known and the factors which affect its value are taken into consideration, then it is possible to calculate the ATP formed per electron pair transferred to oxygen (also known as the P/O ratio) or nitrate (P/2 e ratio) by dividing the growth yield per g atom O or per amount of nitrate reduced by YATPrespectively. Results with Aerobacter aero#enes grown aerobically and anaerobically gave a P/O of 3 and a P/2e of 1.8 respectively using glucose as energy source [30, 31]. In a well conceived and conducted experiment, Koike & Hattori [32] have determined the difference in molar yield of Pseudomonas denitrificans when cultured aerobically and anaerobically on glutamate. Results showed that nitrate respiration is about 40% less efficient than aerobic respiration from which it was inferred that there are fewer phosphorylation sites in the electron transport chain associated with the reduction of nitrate to nitrogen gas than in that associated with aerobic respiration. In order to resolve the controversy as to the extent of phosphorylation associated with the reduction of the intermediates involved in the conversion of nitrate to nitrogen gas, the same workers cultured Pseudomonas denitrificans under nitrate-, nitrite- and nitrous oxidelimited conditions [33]. The results obtained showed that the energy yield, expressed on an electron basis, was proportional to the oxidation state of the nitrogen: nitrate (+ 5) nitrite (+ 3) and nitrous oxide (+ 1). They concluded that phosphorylation occurs to a similar extent in each of the electron transport chains associated with the reduction of nitrate, nitrite and nitrous oxide. Unfortunately, owing to the technical difficulties involved, the growth yield under nitric oxide-limited conditions was not determined and therefore it remains uncertain whether or not phosphorylation is associated with the reduction of nitric oxide. However, it is claimed that circumstantial evi-
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dence suggests that phosphorylation is associated with the reduction of nitric oxide [33]. Additional support in favour of the reduction in the number of phosphorylation sites associated with nitrogenous oxide reduction has been obtained from studies comparing the components of the electron transport chains with those involved in aerobic oxidations. Several studies have revealed that cells grown anaerobically contain a lower concentration of cytochrome a + a3 and higher concentrations of cytochrome b and c than aerobically grown cells [34-37]. Based on the above discussion, it is assumed in the theoretical development of the equation which describes anaerobic growth on methanol below that the electron transport chain in Hyphomicrobium spp. shows, maximally, 2 phosphorylation sites when the cells are cultured anaerobically so that theoretically only 2 moles of ATP can be formed from the oxidation of 1 mole of NADH2. Furthermore it is assumed that phosphorylation occurs to a similar extent with the reduction of each nitrogenous oxide involved in the conversion of nitrate to nitrogen gas. METHANOL METABOLISM In contrast to the situation regarding inorganic nitrogen metabolism, the pathways of methanol oxidation and assimilation in Hyphomierobium spp. have been studied. However, only those aspects which are relevant to the development of the theoretical equation that describes denitrification with methanol are discussed below. Methanol oxidation The pathway of methanol oxidation is shown in Fig. 4 and has been investigated in cells growing aerobically and anaerobically ['17, 38]. CH3OH --* HCHO --* HCOOH --* CO2 Fig. 4. Pathway of methanol oxidation in Hyphomierobium spp. grown aerobically or anaerobically. The enzyme responsible for the oxidation of methanol to formaldehyde and formaldehyde to formate in Hyphomierobium spp. is an NAD-independent enzyme that requires ammonium ions for activity and has a relatively high pH optimum [38, 39]. A similar enzyme has been found in serveral other species of methanol utilizing bacteria 1-39--48]. In cell-free extracts the enzyme can only be assayed in the presence of phenazine methosulphate (PMS), an auxilliary electron carrier required for the non-enzymic reduction of 2,6-dichlorophenolindophenol (DCPIP) or molecular oxygen. Unfortunately, very little is known about the exact nature and role of the pteridine-like cofactor found in methanol dehydrogenase and two reaction mechanisms have been postulated: a dehydrogenation in which methanol is bound to the pteroate ring and is oxidized completely to the level of formate [43] and a hydroxylation [39].
534
GEORGER. NURSE [ ~p~sp~glycerate ]
2 =eth~aol~ 2 Xlt2 2 fomaldehyde
I
I
|
tetra~/hydra-
] metMnol ]
2 ADP 2 q 2 AT~I 2 -phpsphoglycerate--~ 2 glycerate
/ 2 tavu2 2 hydroxypyruvat~e ,~ [
x Xlt2~ ~ormaldehyde phosphoenolX~[ pyruvate XH~ ~.--.~rbon~---~---formate Sdioxide ~N ' AD
o~loacetate
NADH2
~ folate~ i n e
2 N5'10 methylene-'-'~ tetrahydra2 glycine folate ~ ' - 81yoxylate l ~ . glyoxylate ~.
ma1yl - C~ ~
--~ a e e t y l - C ~
L o~loacetate
citrate
,~lete £umarate
FAD 2
8ucct~te
t~cItrate
~ J Fig. 5. The icl-serine pathway of methanol assimilation in Hyphoraicrobium slap. (based upon [55]), showing all reducing power and energy producing and requiring reactions when X represents the unknown physiological electron acceptor for methanol dehydrogenase. In an attempt to obtain information concerning the energy yield from the oxidation of methanol and formaldehyde, s~verai investigators have studied the nature and components of the respiratory pathways found in methanol utilizing bacteria [49-52]. Initial studies indicated that cytochrome c may be involved in methanol and formaldehyde oxidation; however, results have not been able to demonstrate unequivocally a reaction between methanol dehydrogenase and the cytochrome [49]. The possibility that cytochrome c may have an oxidase or oxygenase function in methanol oxidation has also been investigated but results indicated that this is probably not so [50]. Since the redox couples for CHaOH/HCHO and HCHO, HCOOH have E~ (pH 7) values of - t 8 2 and - 4 5 0 m V respectively [53] and the redox potential for the purified cytochrome c is + 260 mV [50], it is thermodynamically possible that some ATP could be synthesized from the oxidation of methanol and formaldehyde, if cytochrome c is an obligatory component of the electron transport chain involved in these particular oxidations. Furthermore, if the last assumption is correcL then it is possible that some ATP could also be produced during the reoxidation of the reduced cytochrome c. It is evident from the discussion above that it is difficult to assess the amount of ATP that could be formed from the oxidation of methanol and formaldehyde. However, the present author [!9], using the in vivo method of estimating the amount of ATP produced by measuring the molar growth yield and comparing the value obtained with a theoretical yield, found the
two values in good agreement only when O moles of ATP are produced from the oxidation of methanol and formaldehyde. Based on this result, it is concluded that O moles of ATP are produced during the anaerobic oxidation of methanol and formaldehyde in Hyphomicrobium spp. Furthermore, since the end-products of denitrification with methanol are carbon dioxide, nitrogen gas and cells, it is assumed that the unknown physiological electron acceptor involved in the oxidation of methanol and formaldehyde is reoxidized by the nitrogenous oxides and that the reactions involved may be represented by the following equation: 5 XH 2 t 2 N O r --' N2 + 2 O H - -r 4 H20
(2)
In contrast to the oxidation of methanol and formaldehyde, the evaluation of the ATP yield from the oxidation of formate is relatively easy. Formate has been shown to be oxidized by a NAD-dependent dehydrogenase [38]; thus, it may be assumed that maximally 2 moles of ATP can be formed from the anaerobic oxidation of 1 mole of formate in Hyphomicrobium spp.
Methanol assimilation The pathway of methanol assimilation in Hyphomicrobium spp. has been elucidated by the elegant studies carried out by Quayle and his colleagues [54, 55]. Assimilation can he divided into two parts: the synthesis of 3-phosphoglycerate (3-PGA) from a net input of 2 molecules of methanol and 1 molecule of carbon dioxide (derived from the oxidation of I mol-
Denitrification with methanol: Microbiology and biochemistry ecule of methanol) via the icl-serine pathway as depicted in Fig. 5, and the synthesis of cell constituents from 3-PGA via established pathways of intermediary metabolism [56].
DEVELOPMENT OF AN EQUATION WHICH DESCRIBES THE STOICHIOMETRY OF DENITRIFICATION WITH METHANOL
In order to develop an equation that describes the stoiehiometry of denitrification with methanol, it is necessary to determine the amount of methanol and nitrate that is needed to supply the total energy and reducing power requirements for the biosynthesis of cell material.
Eneroy requirementfor synthesis of 3-PGA Using the icl-serine pathway as depicted in Fig. 5, the overall reaction leading to the synthesis of 3-PGA from methanol may be described by the following equation: 2 CH3OH + CO2 ---, 3-PGA.
(3)
However, since the source of carbon dioxide is from methanol, Eq. (3) becomes: 3 CH3OH ---, 3-PGA.
(4)
The total energy requirement, where it is assumed that maximally 2 moles of ATP can be produced from the oxidation of 1 mole of NADH2, 1 mole of ATP from the oxidation of FADH2 and 0 mole from the oxidation of the unknown physiological electron acceptor for the enzyme methanol dehydrogenase, is obtained by summing the ATP required for the overall reaction and subtracting the amount of ATP produced from the overall reaction as shown below: Total ATP required for synthesis of 3-PGA is: hydroxypyruvate reductase giycerate kinase malate dehydrogenase malate thiokinase
2 NADH2 = 4ATP 2ATP 1 NADH2 = 2ATP 1A T P
9ATP Total ATP produced during synthesis of 3-PGA is: formate dehydrogenase malate dehydrogenase succinate dehydrogenase methanol dehydrogenase
1 NADH2 = 1 NADH2 = 1 FADH2 = 4 XH2 = 4 XH 2 +
2ATP 2ATP 1ATP 0ATP 5ATP
The synthesis of 3-PGA from methanol via the serine pathway, accounting for the total energy requirement, may therefore be described by the following balanced equation: 3 CH3OH + 4X + 4 ATP + 4 H 2 0 --~ CaH7OTP (3-PGA) + 4 XH2 + 4 ADP + HPO~ + 2 H2PO4.
(5)
535
Energy and reducing power requirementfor synthesis of cellsfrom 3-PGA with nitrate as nitrogen source Based on the cell composition C4HvO2N [19], it is observed from the following balanced equation that 16 NADH2 are required to synthesize cells from 3-PGA when nitrate is the sole nitrogen source (12 NADH2 are required for the conversion of 2 N O r to 3 NH 3 before assimilation of nitrogen can occur and an extra 4 NADH2 are required to reduce the 3-PGA to the oxidation level of cell material): 4 C3H707P + 3 HNO~ + 16 NADH2 ---, 3 C4H702N + 4 H3PO4 + 16 NAD + 15 H20.
(6)
The energy requirement for the synthesis of cells from 3-PGA, however, can only be approximated by dividing the g molecular weight of the cells by an assumed value for YArvon 3-PGA, taken as 10.5 g dry weight cells per mole ATP [20]. Using this method, it is estimated that for the synthesis of 3 x 101 g dry weight cells, the energy requirement is 29 moles of ATP (from 3 x 101/10.5 = 29 moles ATP). Hence the synthesis of all material from 3-PGA and nitrate, accounting for the reducing power and energy requirements, can be represented by the following equation: 4 C3H7OTP + 3 HNO3 + 16 NADH2 + 29 ATP + 29 H20--* 3 C4H702N + 4 H3PO4 + 16 NAD + 15 H 2 0 + 29 ADP + 29 H2PO2.
(7)
Eneroy and reduciny power requirementfor synthesis of cells from methanol and nitrate Taking Eqs (5) and (7), the total energy and reducing power requirement for cell synthesis can be deduced and is given by the following equation: 12 CHaOH + 16 X + 3 HNO3 + 16 NADH2 + 45 ATP + 45 H 2 0 --, 3 C4H702 N + 16 XH 2 + 16 NAD + 15 H20 + 45 ADP + 45 H2PO~.
(8)
From Eq. (8) it is possible to derive an equation which describes the stoichiometry of denitrification with methanol by calculating the amount of methanol and nitrate that has to be oxidized and reduced respectively in order to generate the NADH2 and ATP required for cell biosynthesis. In order to meet the reducing power requirement alone, 16 moles of methanol have to be completely oxidized since NADH2 is generated only in the oxidation of formate to carbon dioxide. An additional 22.5 moles of methanol have to be oxidized to meet the ATP requirements (from 45/2 = 22.5) since it is assumed that the electron transport chain shows only
536
GEORGE R. NURSE
two phosphorylation sites and that 0 mole of ATP are obtained from the oxidation of methanol and formaldehyde. Using Eq. (2) in order to determine the amount of nitrate required to regenerate the reduced acceptor of methanol dehydrogenase and NADH2, the following equation is derived: 50.5 C H a O H + 3 H N O 3 + 46.2 NO3 --, 3 C4H702N + 38.5 CO2 + 23.1 N 2 + 46.2 O H - + 68.9 H20.
(9)
DISCUSSION Although methanol dominates as electron donor for denitrifying purposes and the fact that Hyphomicrobium spp. are now known to be the sole type of bacteria responsible for the reactions involved, there is little data in the literature which describes the biochemistry of the process. By assuming a YATPof 10.5 on 3-PGA ['20-] and that no ATP is produced from the oxidation of methanol and formaldehyde under anaerobic conditions [19], one of the main goals of this paper was to develop a theoretical equation, based on currently available knowledge of the biochemical reactions involved, which would describe the stoichiometry of denitification with methanol. When used to calculate a theoretical growth yield (0.188 g cells/g methanol on an ash-free basis) or a consumptive ratio (1.31; defined as the ratio of the total quantity of methanol consumed during denitrification to the stoichiometric requirement for denitrification [energy purposes] alone), the values thus obtained are found to be in close agreement with experimental values (Y = 0.195 g cell/g methanol with an ash-content of 3.7% [19] and 1.30 + 0.06 [3] respectively). This suggests that the assumptions made in the theoretical development of Eq. (9) are not far from reality. Another equation which adequately describes the stoichiometry of denitirification with methanol has, however, been available to workers in the field for several years and was derived from half reactions for the oxidation of methanol and the reduction of the nitrogenous oxides and a half reaction for the formation of bacterial cells [3]. The development of this equation, however, offered no information regarding the biochemistry of the process. Owing to the high cost of methanol required to denitrify high level nitrate containing wastes, the present author has investigated the potential of the waste biomass as a source of single-ceU protein and obtained favourable results [19]. Consequently t h e development of an equation which describes the upper limit to the yield parameter becomes of greater importance in the optimization of the process. REFERENCES 1. C. W. Christenson, E. H. Rex, W. M, Webster & F. A. Vigil, U.S. Atom. Energy Comm TID 75t7 (Pt 1A) (1957).
2. E. F. Barth, R, C. Brenner & R. F. Lewis, J W P C F 40, 2040-2054 (t968). 3. P. L. McCarty, L. Beck & P. St. Amant, 24th Ind. Waste Conf Purdue Univ 24, 1271-1285 (1969). 4. D. F. Seidel-& R. C. Crites, Proc .4SCE, J. San. Engn~r Div., 96, 267 277 (1970). 5. Mulbarger, J WPCF, 43, 2059-2070 (1971). 6. C. E. Adams, P. A. Krenkel & E. L. Bingham, Proc. 5th Int. Wat. Poll. Res. Conj'. (Ed. Jenkins) 1-13 (1970). 7. S. F. Moore & E. D. Schroeder, Water Res. 4, 685-694 (1970). 8. S. F. Moore & E. D. Schroeder, Water Res. 5, 445--452 (1971). 9. W. K. Johnson Proc ASCE, J. San. Engng. Div., 98, 623 (1972). 10. H. D. Stensel, R. C. Loehr & A. W. Lawrence J W P C F 45 (2), 249-261 (1973). 11. W. J. Jewell & R. C. Cummings, J W P C F 47 (9), 2281-2291 (1975). 12. C. W. Francis & J, B, Mankin. Water Res. 11,289-294 (1977). 13. M. H. Christensen & P. Harremo~s, Biological Denitrification in Water Treatment-- a literature stud},. Rep 2-72 Dept. of San. Engng., Tech Univ of Denmark (1972). 14. M. H. Christensen & P. Harremo~s, A literature Review of Biological Denitrification of Sewage (Conf. on nitrogen as a water pollutant) Tech Univ of Denmark, August (1975). 15. H. A, Painter, Water Res. 4, 393-450 (1970). 16, W. J. Payne, Bact. Reviews 37 (4), 409-452 (t973). 17. G. T. Sperl & D. S. Hoare, J. Bact. 108 (2), 733-736 (1971). 18. M. M. Attwood & W. Harder, Ant. van Leeuw 38, 369-378 (1972). 19. G. R. Nurse. Unpublished results. 20. J. P. van Dijken & W. Harder, Biotech. Bioeng. 17, 15-30 (1975). 21. P. Hirsch, Ann. Rev Microbiol 28, 393 (1974). 22. T. Matsubara, J. Biochem 69, 991-1001 (1971). 23. W. J. Payne, P. S. Riley & C. D. Cox Jr, J. Bact. 106, 356-361 (1971). 24. C. D. Cox & W. J. Payne, Can. J. Microbiol 19, 861-872 (1973). 25. P. John & F. R. Whatley, Biochim. biophys. Acta 216, 34~352 (1970). 26. T. Oshnishi, J. Biochem. (Tokyo)48, 406-411 (1963). 27. K. lmai, A. Asano & R. Sato, J. Biochem. 63, 207-218 (1968). 28. M. S. Naik & D. J. D. Nicholas, J. Biochem. 4, 129-130 (1967). 29. T. Bauchop & S. R. Elsden, J. gen Microbiol. 23, 457-469 (1960). 30. A. H. Stouthamer & C. Bettenhausen, Biochim. bit)phys. Acta 301, 53-70 (1973). 31. L. P. Hadjipetrou & A. H. Stouthamer, J. gen. Microbiol. 38, 29-34 (1965). 32. I. Koike & A. Hattori, J. gen. Microbiol. 88, 1-10 (1975). 33. I. Koike & A. Hattori, J. gen. Microbiol. 88, 11-t9 (1975). 34. R. J. Porra & J. Lascelles, J. Biochem. 94, 120 (1965). 35. P. B. Scholes & L. Smith, Biochim biophys. Acta 153, 363-375 (1968). 36. R. J. Downey, D. F. Kiszkiss & J. H. Nuner, J. Bact. 98, 1056-1062 (1969). 37. L. M. Sapshead & J. W. T. Wimpenny, Biochim. biophys. Acta 267, 388-397 (1972). 38. W. Harder & M. M. Attwood, Ant. van Leeuw 41, 421-429 (1975). 39. G. T. Sped, H. S. Forrest & D. Gibson, J Bact 110, (2) 541-550 (1974).
Denitrification with methanol: Microbiology and biochemistry 40. C. Anthony & L. J. Zatman, Biochem. J. 92, 614-621 (1964). 41. C. Anthony & L. J. Zatman, Biochem. d. 96, 808-812 (1965). 42. C. Anthony & L. J. Zatman, Biochem. J. 104, 953-959 (1967). 43. C. Anthony & L. J. Zatman, Biochem. J. 104, 960-969 (1967). 44. J. Heptinstall & J. R. Quayle, Biochem. J, 117, 563-572 (1970). 45. R. N. Patel, H. R. Bose, W. J. Mandy & D. S. Hoare, J. Bact. 110, 570-577 (1972). 46. I. Goldberg, Eur. J. Biochem. 63, 233-240 (1976). 47. R. J. Mehta, Ant. van Leeuw 39, 303-312 (1973). 48. A. M. Wadzinski & D. W. Ribbons, J. Bact, 122, 1364-1373 (1975).
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49. C. Anthony, Biochem. J. 146, 289-298 (1975). 50. D. Widdowson & C. Anthony, Biochem. J. 152, 349-356 (1975). 51. G. M. Tonge, C. J. Knowles, D. E. F. Harrison & I. J. Higgins, FEBS Letts 44, 106-110 (1974). 52. G. M. Tonge, D. E. F. Harrison, C. J. Knowles & I. J. Higgins, FEBS Letts 58, 293-299 (1975). 53. D. W. Ribbons, J. E. Harrison & A. M, Wadzinski, Ann. Rev. Microbiol. 24, 135-158 (1970). 54. P. J. Large, D. Peel & J. R. Quayle, Biochem. J. 81, 470-480 (1961). 55. W. Harder, M. M. Attwood & J. R. Quayle, J. gen. Microbiol. 78, 155-163 (1973). 56. J. R. Quayle, Process Biochem. Febr. (1969).
DENITRIFICATION WITH METHANOL: MICROBIOLOGY AND BIOCHEMISTRY GEORGE R. NURSE Department of Biochemistry, University of Natal, Pietermaritzburg, Republic of South Africa
(Received March 1978) Abstract--Although methanol is frequently chosen as carbon and energy source for denitrifieation of nitrate polluted effluents, all kinetic parameters thus far reported have been for an unidentified biomass in the belief that a more specialized knowledge of the bacterial species present in the reactors has not been necessary. However, it has now been adequately demonstrated that denitrification with methanol results in a selective enrichment for bacteria belonging to the genus Hyphomierobium.Based on current available biochemical knowledge of nitrate reduction and assimilation, methanol oxidation and assimilation and the energy yield and requirements for these reactions, it is theoretically possible to develop an equation which describes the stoichiometry of denitrification with methanol.
INTRODUCTION
During the last 25 years considerable research has been conducted into denitrification of nitrate polluted waters using methanol as sole carbon and energy source [1-12]. Since denitrification can be effected by a wide variety of facultative bacteria [13-16], it has always been concluded by workers in this field that little can be achieved from identification of species actively performing in their reactors. Although the concept of working with an unidentified biomass may be justified in those investigations using heterogenous carbon and energy sources, it has unfortunately persisted in those situations where methanol has been used. The first evidence that the biomass in a denitrifying reactor had an unusual population when methanol was used, was in fact reported by the first investigators to use methanol [1]. They commented: "So far as the organisms are concerned, they consist of an almost pure culture of as yet unidentified very small ciliated protozoa with large numbers of bacteria". In an investigation into the denitrification of agricultural wastewaters using various carbon sources, it was noted that a lag resulted before denitrification occurred with methanol and it was mentioned that this was "presumably due to a need for biological acclimation to this substrate" [3]. In 1971 it was reported that denitrification with methanol resulted in a selective enrichment for bacteria belonging to the genus Hyphomicrobium [17]. The same result was obtained a year later [18] and has since been confirmed by the present author [19]. A photograph showing the characteristic morphology of the species is shown in Fig. 1. Unfortunately these observations have been overlooked by workers in the field although they have retained the terminology in referring to the biomass as a "modified activated sludge". The present author, however, has carried out a kinetic investigation into the denitrification of a high level nitrate industrial effluent bearing the above w.R. 14/5--1
results in mind [19]. The kinetic parameters obtained have not only been used for plant design purposes but also to characterize quantitatively Hyphomicrobium spp. growing anaerobically on methanol, as data concerning this aspect of their growth are very limited. In order to increase confidence in the experimental growth yield, Y, (0.195 g biomass per g methanol consumed), this investigation also included a theoretical study into the efficiency with which methanol is converted into biomass. This involved developing equations which describe anaerobic growth on methanol by Hyphomicrobium spp. using our existing knowledge of the biochemical pathways involved. The principles underlying the calculations involved and their validity are discussed in greater detail by van Dijken and Harder [20] who developed similar equations for microorganisms growing aerobically on methane and methanol in order to determine theoretical growth yields. It is the purpose of this paper to describe in simple biochemical terms the development of a theoretical equation that adequately describes the stoichiometry of denitrification with methanol. INORGANIC NITROGEN METABOLISM
Hyphomicrobium spp. can utilize nitrate for the synthesis of all the nitrogen containing compounds of the cell and as the terminal electron acceptor in place of oxygen under anaerobic conditions [17-19, 21]. The two processes are called nitrate assimilation and nitrate respiration respectively. Unfortunately, the enzymes involved in both processes have not been studied in Hyphomierobium spp. and in the discussion below it is assumed that the concept of unifying metabolism exists between nitrate utilizing bacteria. Nitrate assimilation In a growth medium in which nitrate is the sole source of nitrogen, a preliminary reduction of the nitrate to ammonia must occur before assimilation can take place [15, 16]. The steps involved in the
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GEORGER. NURSE
Fig. 1. Hyphomicrobium spp. liom an enrichment culture growing anaerobically on methanol (pH 7.4, 28°C) in continuous culture (phase-contrast, ca 3000 x)E19 ].
conversion are depicted in Fig. 2, where (Z), the intermediate between nitrite and hydroxylamine remains unknown although some researchers have proposed nitroxyl (NOH) or its dimer nitramide (N202H2) or hyponitrite [15]. NO3 -~ N O r --, (Z)---, NH2OH --~ NH3 Fig. 2. Pathway of nitrate assimilation assumed to occur in Hyphomicrobium spp. where (Z) represents an unknown intermediate. Since some of the enzymes responsible for the conversion have been shown to use NADH as electron donor in a wide variety of microorganisms [16], it is assumed that 4 NADH2 are required for the conversion as represented by Eq. (1) below: HNO3 + 4 NADH2 ---,NH3 + 3 H20
(1)
Nitrate respiration The reduction of nitrate to nitrogen gas proceeds through a number of steps as shown in Fig. 3. Many researchers have been devoted to studies of this reaction sequence [15, 16]. NO3 --~ NO2 --* NO
--~
N20
--+ N 2
Fig. 3. Pathway of nitrate reduction assumed to occur in Hyphoraicrobium spp. Although nitrous oxide is sometimes formed during denitrification, it is assumed in the calculations below
that all nitrate consumed for respirometric purposes is quantitatively recovered as nitrogen gas. In the calculations performed by van Dijken and Harder [20], it was assumed that the electron transport chain in methanol utilizing bacteria shows 3 phosphorylation sites, so that theoretically 3 moles of ATP can be formed during the oxidation of 1 mole of NADH2. However, this assumption is only valid under aerobic conditions and the amount of ATP assumed to be generated under anaerobic conditions requires careful consideration. One-step reduction of nitrate [15, 16] and nitrous oxide [22, 23] are known to support the growth of various bacteria. It is therefore intuitively obvious that reduction of these compounds is coupled to oxidative phosphorylation. However, those bacteria that grow when supplied with nitrite may conceivably benefit from coupling reduction with phosphorylation only during the reduction of nitrous oxide and not during the reduction of nitrite or nitric oxide. Based on the assumption that ATP generating systems remain membrane-bound when cells are ruptured, several workers have claimed that nitrate and nitrous oxide reduction is coupled to oxidative phosphorylation whereas nitrite and nitric oxide reduction is not [23, 24]. These findings, however, are inconsistent with results obtained when esterification of radioactively labelled phosphate with concomittant reduction of nitrite was demonstrated in cell-free extracts [25, 26"1and no esterification was observed in reaction mixtures supplied with nitric or nitrous oxide
Denitrification with methanol: Microbiology and biochemistry [27, 28]. The observed lack of esterification with nitrous oxide was accounted for by mentioning that the sensitive coupling of phosphorylation to the electron transport chain may have been uncoupled during the physical disruption of the cells in order to prepare the cell-free extracts. In order to overcome the disadvantages associated with the interpretation of results from in vitro studies, an in vivo method for determining the efficiency of oxidative phosphorylation has been developed. The method is based on the conclusion of Bauchop & Elsden [29] that the molar growth yield of cells per mole of ATP generated from the oxidation of the energy source, designated YATP,is a constant for different microorganisms, the value being 10.5 g dry weight of cells per mole of ATP produced. More recently, however, it has been shown that YATPvalues can be very different for different microorganisms and that the value for a particular organism growing on a particular substrate is also determined by the cell composition, the specific growth rate and the maintenance coefficient [30]. However, if YArP for a particular organism is known and the factors which affect its value are taken into consideration, then it is possible to calculate the ATP formed per electron pair transferred to oxygen (also known as the P/O ratio) or nitrate (P/2 e ratio) by dividing the growth yield per g atom O or per amount of nitrate reduced by YATPrespectively. Results with Aerobacter aero#enes grown aerobically and anaerobically gave a P/O of 3 and a P/2e of 1.8 respectively using glucose as energy source [30, 31]. In a well conceived and conducted experiment, Koike & Hattori [32] have determined the difference in molar yield of Pseudomonas denitrificans when cultured aerobically and anaerobically on glutamate. Results showed that nitrate respiration is about 40% less efficient than aerobic respiration from which it was inferred that there are fewer phosphorylation sites in the electron transport chain associated with the reduction of nitrate to nitrogen gas than in that associated with aerobic respiration. In order to resolve the controversy as to the extent of phosphorylation associated with the reduction of the intermediates involved in the conversion of nitrate to nitrogen gas, the same workers cultured Pseudomonas denitrificans under nitrate-, nitrite- and nitrous oxidelimited conditions [33]. The results obtained showed that the energy yield, expressed on an electron basis, was proportional to the oxidation state of the nitrogen: nitrate (+ 5) nitrite (+ 3) and nitrous oxide (+ 1). They concluded that phosphorylation occurs to a similar extent in each of the electron transport chains associated with the reduction of nitrate, nitrite and nitrous oxide. Unfortunately, owing to the technical difficulties involved, the growth yield under nitric oxide-limited conditions was not determined and therefore it remains uncertain whether or not phosphorylation is associated with the reduction of nitric oxide. However, it is claimed that circumstantial evi-
533
dence suggests that phosphorylation is associated with the reduction of nitric oxide [33]. Additional support in favour of the reduction in the number of phosphorylation sites associated with nitrogenous oxide reduction has been obtained from studies comparing the components of the electron transport chains with those involved in aerobic oxidations. Several studies have revealed that cells grown anaerobically contain a lower concentration of cytochrome a + a3 and higher concentrations of cytochrome b and c than aerobically grown cells [34-37]. Based on the above discussion, it is assumed in the theoretical development of the equation which describes anaerobic growth on methanol below that the electron transport chain in Hyphomicrobium spp. shows, maximally, 2 phosphorylation sites when the cells are cultured anaerobically so that theoretically only 2 moles of ATP can be formed from the oxidation of 1 mole of NADH2. Furthermore it is assumed that phosphorylation occurs to a similar extent with the reduction of each nitrogenous oxide involved in the conversion of nitrate to nitrogen gas. METHANOL METABOLISM In contrast to the situation regarding inorganic nitrogen metabolism, the pathways of methanol oxidation and assimilation in Hyphomierobium spp. have been studied. However, only those aspects which are relevant to the development of the theoretical equation that describes denitrification with methanol are discussed below. Methanol oxidation The pathway of methanol oxidation is shown in Fig. 4 and has been investigated in cells growing aerobically and anaerobically ['17, 38]. CH3OH --* HCHO --* HCOOH --* CO2 Fig. 4. Pathway of methanol oxidation in Hyphomierobium spp. grown aerobically or anaerobically. The enzyme responsible for the oxidation of methanol to formaldehyde and formaldehyde to formate in Hyphomierobium spp. is an NAD-independent enzyme that requires ammonium ions for activity and has a relatively high pH optimum [38, 39]. A similar enzyme has been found in serveral other species of methanol utilizing bacteria 1-39--48]. In cell-free extracts the enzyme can only be assayed in the presence of phenazine methosulphate (PMS), an auxilliary electron carrier required for the non-enzymic reduction of 2,6-dichlorophenolindophenol (DCPIP) or molecular oxygen. Unfortunately, very little is known about the exact nature and role of the pteridine-like cofactor found in methanol dehydrogenase and two reaction mechanisms have been postulated: a dehydrogenation in which methanol is bound to the pteroate ring and is oxidized completely to the level of formate [43] and a hydroxylation [39].
534
GEORGER. NURSE [ ~p~sp~glycerate ]
2 =eth~aol~ 2 Xlt2 2 fomaldehyde
I
I
|
tetra~/hydra-
] metMnol ]
2 ADP 2 q 2 AT~I 2 -phpsphoglycerate--~ 2 glycerate
/ 2 tavu2 2 hydroxypyruvat~e ,~ [
x Xlt2~ ~ormaldehyde phosphoenolX~[ pyruvate XH~ ~.--.~rbon~---~---formate Sdioxide ~N ' AD
o~loacetate
NADH2
~ folate~ i n e
2 N5'10 methylene-'-'~ tetrahydra2 glycine folate ~ ' - 81yoxylate l ~ . glyoxylate ~.
ma1yl - C~ ~
--~ a e e t y l - C ~
L o~loacetate
citrate
,~lete £umarate
FAD 2
8ucct~te
t~cItrate
~ J Fig. 5. The icl-serine pathway of methanol assimilation in Hyphoraicrobium slap. (based upon [55]), showing all reducing power and energy producing and requiring reactions when X represents the unknown physiological electron acceptor for methanol dehydrogenase. In an attempt to obtain information concerning the energy yield from the oxidation of methanol and formaldehyde, s~verai investigators have studied the nature and components of the respiratory pathways found in methanol utilizing bacteria [49-52]. Initial studies indicated that cytochrome c may be involved in methanol and formaldehyde oxidation; however, results have not been able to demonstrate unequivocally a reaction between methanol dehydrogenase and the cytochrome [49]. The possibility that cytochrome c may have an oxidase or oxygenase function in methanol oxidation has also been investigated but results indicated that this is probably not so [50]. Since the redox couples for CHaOH/HCHO and HCHO, HCOOH have E~ (pH 7) values of - t 8 2 and - 4 5 0 m V respectively [53] and the redox potential for the purified cytochrome c is + 260 mV [50], it is thermodynamically possible that some ATP could be synthesized from the oxidation of methanol and formaldehyde, if cytochrome c is an obligatory component of the electron transport chain involved in these particular oxidations. Furthermore, if the last assumption is correcL then it is possible that some ATP could also be produced during the reoxidation of the reduced cytochrome c. It is evident from the discussion above that it is difficult to assess the amount of ATP that could be formed from the oxidation of methanol and formaldehyde. However, the present author [!9], using the in vivo method of estimating the amount of ATP produced by measuring the molar growth yield and comparing the value obtained with a theoretical yield, found the
two values in good agreement only when O moles of ATP are produced from the oxidation of methanol and formaldehyde. Based on this result, it is concluded that O moles of ATP are produced during the anaerobic oxidation of methanol and formaldehyde in Hyphomicrobium spp. Furthermore, since the end-products of denitrification with methanol are carbon dioxide, nitrogen gas and cells, it is assumed that the unknown physiological electron acceptor involved in the oxidation of methanol and formaldehyde is reoxidized by the nitrogenous oxides and that the reactions involved may be represented by the following equation: 5 XH 2 t 2 N O r --' N2 + 2 O H - -r 4 H20
(2)
In contrast to the oxidation of methanol and formaldehyde, the evaluation of the ATP yield from the oxidation of formate is relatively easy. Formate has been shown to be oxidized by a NAD-dependent dehydrogenase [38]; thus, it may be assumed that maximally 2 moles of ATP can be formed from the anaerobic oxidation of 1 mole of formate in Hyphomicrobium spp.
Methanol assimilation The pathway of methanol assimilation in Hyphomicrobium spp. has been elucidated by the elegant studies carried out by Quayle and his colleagues [54, 55]. Assimilation can he divided into two parts: the synthesis of 3-phosphoglycerate (3-PGA) from a net input of 2 molecules of methanol and 1 molecule of carbon dioxide (derived from the oxidation of I mol-
Denitrification with methanol: Microbiology and biochemistry ecule of methanol) via the icl-serine pathway as depicted in Fig. 5, and the synthesis of cell constituents from 3-PGA via established pathways of intermediary metabolism [56].
DEVELOPMENT OF AN EQUATION WHICH DESCRIBES THE STOICHIOMETRY OF DENITRIFICATION WITH METHANOL
In order to develop an equation that describes the stoiehiometry of denitrification with methanol, it is necessary to determine the amount of methanol and nitrate that is needed to supply the total energy and reducing power requirements for the biosynthesis of cell material.
Eneroy requirementfor synthesis of 3-PGA Using the icl-serine pathway as depicted in Fig. 5, the overall reaction leading to the synthesis of 3-PGA from methanol may be described by the following equation: 2 CH3OH + CO2 ---, 3-PGA.
(3)
However, since the source of carbon dioxide is from methanol, Eq. (3) becomes: 3 CH3OH ---, 3-PGA.
(4)
The total energy requirement, where it is assumed that maximally 2 moles of ATP can be produced from the oxidation of 1 mole of NADH2, 1 mole of ATP from the oxidation of FADH2 and 0 mole from the oxidation of the unknown physiological electron acceptor for the enzyme methanol dehydrogenase, is obtained by summing the ATP required for the overall reaction and subtracting the amount of ATP produced from the overall reaction as shown below: Total ATP required for synthesis of 3-PGA is: hydroxypyruvate reductase giycerate kinase malate dehydrogenase malate thiokinase
2 NADH2 = 4ATP 2ATP 1 NADH2 = 2ATP 1A T P
9ATP Total ATP produced during synthesis of 3-PGA is: formate dehydrogenase malate dehydrogenase succinate dehydrogenase methanol dehydrogenase
1 NADH2 = 1 NADH2 = 1 FADH2 = 4 XH2 = 4 XH 2 +
2ATP 2ATP 1ATP 0ATP 5ATP
The synthesis of 3-PGA from methanol via the serine pathway, accounting for the total energy requirement, may therefore be described by the following balanced equation: 3 CH3OH + 4X + 4 ATP + 4 H 2 0 --~ CaH7OTP (3-PGA) + 4 XH2 + 4 ADP + HPO~ + 2 H2PO4.
(5)
535
Energy and reducing power requirementfor synthesis of cellsfrom 3-PGA with nitrate as nitrogen source Based on the cell composition C4HvO2N [19], it is observed from the following balanced equation that 16 NADH2 are required to synthesize cells from 3-PGA when nitrate is the sole nitrogen source (12 NADH2 are required for the conversion of 2 N O r to 3 NH 3 before assimilation of nitrogen can occur and an extra 4 NADH2 are required to reduce the 3-PGA to the oxidation level of cell material): 4 C3H707P + 3 HNO~ + 16 NADH2 ---, 3 C4H702N + 4 H3PO4 + 16 NAD + 15 H20.
(6)
The energy requirement for the synthesis of cells from 3-PGA, however, can only be approximated by dividing the g molecular weight of the cells by an assumed value for YArvon 3-PGA, taken as 10.5 g dry weight cells per mole ATP [20]. Using this method, it is estimated that for the synthesis of 3 x 101 g dry weight cells, the energy requirement is 29 moles of ATP (from 3 x 101/10.5 = 29 moles ATP). Hence the synthesis of all material from 3-PGA and nitrate, accounting for the reducing power and energy requirements, can be represented by the following equation: 4 C3H7OTP + 3 HNO3 + 16 NADH2 + 29 ATP + 29 H20--* 3 C4H702N + 4 H3PO4 + 16 NAD + 15 H 2 0 + 29 ADP + 29 H2PO2.
(7)
Eneroy and reduciny power requirementfor synthesis of cells from methanol and nitrate Taking Eqs (5) and (7), the total energy and reducing power requirement for cell synthesis can be deduced and is given by the following equation: 12 CHaOH + 16 X + 3 HNO3 + 16 NADH2 + 45 ATP + 45 H 2 0 --, 3 C4H702 N + 16 XH 2 + 16 NAD + 15 H20 + 45 ADP + 45 H2PO~.
(8)
From Eq. (8) it is possible to derive an equation which describes the stoichiometry of denitrification with methanol by calculating the amount of methanol and nitrate that has to be oxidized and reduced respectively in order to generate the NADH2 and ATP required for cell biosynthesis. In order to meet the reducing power requirement alone, 16 moles of methanol have to be completely oxidized since NADH2 is generated only in the oxidation of formate to carbon dioxide. An additional 22.5 moles of methanol have to be oxidized to meet the ATP requirements (from 45/2 = 22.5) since it is assumed that the electron transport chain shows only
536
GEORGE R. NURSE
two phosphorylation sites and that 0 mole of ATP are obtained from the oxidation of methanol and formaldehyde. Using Eq. (2) in order to determine the amount of nitrate required to regenerate the reduced acceptor of methanol dehydrogenase and NADH2, the following equation is derived: 50.5 C H a O H + 3 H N O 3 + 46.2 NO3 --, 3 C4H702N + 38.5 CO2 + 23.1 N 2 + 46.2 O H - + 68.9 H20.
(9)
DISCUSSION Although methanol dominates as electron donor for denitrifying purposes and the fact that Hyphomicrobium spp. are now known to be the sole type of bacteria responsible for the reactions involved, there is little data in the literature which describes the biochemistry of the process. By assuming a YATPof 10.5 on 3-PGA ['20-] and that no ATP is produced from the oxidation of methanol and formaldehyde under anaerobic conditions [19], one of the main goals of this paper was to develop a theoretical equation, based on currently available knowledge of the biochemical reactions involved, which would describe the stoichiometry of denitification with methanol. When used to calculate a theoretical growth yield (0.188 g cells/g methanol on an ash-free basis) or a consumptive ratio (1.31; defined as the ratio of the total quantity of methanol consumed during denitrification to the stoichiometric requirement for denitrification [energy purposes] alone), the values thus obtained are found to be in close agreement with experimental values (Y = 0.195 g cell/g methanol with an ash-content of 3.7% [19] and 1.30 + 0.06 [3] respectively). This suggests that the assumptions made in the theoretical development of Eq. (9) are not far from reality. Another equation which adequately describes the stoichiometry of denitirification with methanol has, however, been available to workers in the field for several years and was derived from half reactions for the oxidation of methanol and the reduction of the nitrogenous oxides and a half reaction for the formation of bacterial cells [3]. The development of this equation, however, offered no information regarding the biochemistry of the process. Owing to the high cost of methanol required to denitrify high level nitrate containing wastes, the present author has investigated the potential of the waste biomass as a source of single-ceU protein and obtained favourable results [19]. Consequently t h e development of an equation which describes the upper limit to the yield parameter becomes of greater importance in the optimization of the process. REFERENCES 1. C. W. Christenson, E. H. Rex, W. M, Webster & F. A. Vigil, U.S. Atom. Energy Comm TID 75t7 (Pt 1A) (1957).
2. E. F. Barth, R, C. Brenner & R. F. Lewis, J W P C F 40, 2040-2054 (t968). 3. P. L. McCarty, L. Beck & P. St. Amant, 24th Ind. Waste Conf Purdue Univ 24, 1271-1285 (1969). 4. D. F. Seidel-& R. C. Crites, Proc .4SCE, J. San. Engn~r Div., 96, 267 277 (1970). 5. Mulbarger, J WPCF, 43, 2059-2070 (1971). 6. C. E. Adams, P. A. Krenkel & E. L. Bingham, Proc. 5th Int. Wat. Poll. Res. Conj'. (Ed. Jenkins) 1-13 (1970). 7. S. F. Moore & E. D. Schroeder, Water Res. 4, 685-694 (1970). 8. S. F. Moore & E. D. Schroeder, Water Res. 5, 445--452 (1971). 9. W. K. Johnson Proc ASCE, J. San. Engng. Div., 98, 623 (1972). 10. H. D. Stensel, R. C. Loehr & A. W. Lawrence J W P C F 45 (2), 249-261 (1973). 11. W. J. Jewell & R. C. Cummings, J W P C F 47 (9), 2281-2291 (1975). 12. C. W. Francis & J, B, Mankin. Water Res. 11,289-294 (1977). 13. M. H. Christensen & P. Harremo~s, Biological Denitrification in Water Treatment-- a literature stud},. Rep 2-72 Dept. of San. Engng., Tech Univ of Denmark (1972). 14. M. H. Christensen & P. Harremo~s, A literature Review of Biological Denitrification of Sewage (Conf. on nitrogen as a water pollutant) Tech Univ of Denmark, August (1975). 15. H. A, Painter, Water Res. 4, 393-450 (1970). 16, W. J. Payne, Bact. Reviews 37 (4), 409-452 (t973). 17. G. T. Sperl & D. S. Hoare, J. Bact. 108 (2), 733-736 (1971). 18. M. M. Attwood & W. Harder, Ant. van Leeuw 38, 369-378 (1972). 19. G. R. Nurse. Unpublished results. 20. J. P. van Dijken & W. Harder, Biotech. Bioeng. 17, 15-30 (1975). 21. P. Hirsch, Ann. Rev Microbiol 28, 393 (1974). 22. T. Matsubara, J. Biochem 69, 991-1001 (1971). 23. W. J. Payne, P. S. Riley & C. D. Cox Jr, J. Bact. 106, 356-361 (1971). 24. C. D. Cox & W. J. Payne, Can. J. Microbiol 19, 861-872 (1973). 25. P. John & F. R. Whatley, Biochim. biophys. Acta 216, 34~352 (1970). 26. T. Oshnishi, J. Biochem. (Tokyo)48, 406-411 (1963). 27. K. lmai, A. Asano & R. Sato, J. Biochem. 63, 207-218 (1968). 28. M. S. Naik & D. J. D. Nicholas, J. Biochem. 4, 129-130 (1967). 29. T. Bauchop & S. R. Elsden, J. gen Microbiol. 23, 457-469 (1960). 30. A. H. Stouthamer & C. Bettenhausen, Biochim. bit)phys. Acta 301, 53-70 (1973). 31. L. P. Hadjipetrou & A. H. Stouthamer, J. gen. Microbiol. 38, 29-34 (1965). 32. I. Koike & A. Hattori, J. gen. Microbiol. 88, 1-10 (1975). 33. I. Koike & A. Hattori, J. gen. Microbiol. 88, 11-t9 (1975). 34. R. J. Porra & J. Lascelles, J. Biochem. 94, 120 (1965). 35. P. B. Scholes & L. Smith, Biochim biophys. Acta 153, 363-375 (1968). 36. R. J. Downey, D. F. Kiszkiss & J. H. Nuner, J. Bact. 98, 1056-1062 (1969). 37. L. M. Sapshead & J. W. T. Wimpenny, Biochim. biophys. Acta 267, 388-397 (1972). 38. W. Harder & M. M. Attwood, Ant. van Leeuw 41, 421-429 (1975). 39. G. T. Sped, H. S. Forrest & D. Gibson, J Bact 110, (2) 541-550 (1974).
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