I
C. C. DELWICHE Kearney Foundation of Soil Science, University o f California, Berkeley, Calif.
Biological Transformations of Nitrogen Compounds The processing of sewage has two primary aims-elimination of health hazards due to the pollution of water and avoidance of nuisances resulting from ineffective disposal of waste waters. The mechanism of breakdown of nitrogenous compounds is of particular interest to sanitary engineers because the degradation products of nitrogenous materials can influence the nuisance level (especially odors) of processing methods. There are other indirect means by which the nitrogen compounds resulting from this degradation may influence the over-all process.
T H E nitrogen of sewage is largely in the form of ammonium ion or urea from which ammonia is readily derived whereas about 10% (in some cases more) of the total nitrogen is in the form of more complex organic materials such as proteins and amino acids. There are deviations from this generality usually due to the effect of large volumes of industrial waste on sewage composition and, indeed. in some cases the nitrogen content may be of such a low level as to adversely affect the processing of the sewage. Most modern methods of sewage processing use a combination of anaerobic and aerobic degradation of organic materials. and the decomposition products will vary depending on the degree of aerobiosis. In the various forms of activated sludge treatment, conditions are usually aerobic and unlikely to result in the production of noxious nitrogenous compounds. The sludge from this process or other sedimented material is frequently subjected to anaerobic digestion. Here, because of the necessity of maintaining anaerobic conditions, hoivever, materials are normally in a closed system, and the liberation of noxious compounds to the atmosphere is not a problem. The effluent Ivater from the sedimentation phase of activated sludge processes usually is sufficiently loll in organic material that aerobic conditions can be maintained. One of the primary aims of processing is to lower the concentration
of readily oxidized materials to such a point that the maintenance of aerobic conditions is ensured. I n some sedimentarion units the extent of prior excessive aeration may be important, since the complete oxidation of dissolved materials, including the autotrophic oxidation of ammonium ion to nitrate, can sometimes result in mechanical difficulties later in the process \\here denitrification in sedimented materials with the production of nitrogen gas causes the buoying up of particles, thus hampering sedimentation. This report does not deal with the ramifications of these problems nor the complicated reactions that tvould be anticipated under various processing conditions but rather with the specific degradative pathways which \could be expected in the destruction of organic nitrogenous materials under aerobic and anaerobic conditions. A comparative study of rates of these various processes as related to other oxidative and fermentative degradations taking place in the system would be desirable, but sufficient data are not available to make possible a comparative interpretation of this nature. T h e conversion of nitrogenous compounds to simpler forms and eventually ammonia and nitrate is discussed only in a general way, and the interpretation of these conversions as they apply to the processing of sewage are considered in the ‘‘Di~cu~sion” following this paper.
Nitrogenous Constituents of S e w a g e
The organic nitrogen of sewage, ehclusive of urea, constitutes perhaps 10%; of the total combined nitrogen. Of this 10cc all species of nitrogenous compounds from amino acids through proteins and including the protoplasmic nitrogen of a large and varied microflora can be expected. The degradation of these nitrogenous compounds will invariably be by way of hydrolysis of proteins and other complex nitrogenous compounds and the eventual decomposition of the loner molecular weight residue resulting therefrom These can be classified as follows for the purposes of our consideration: ( u ) amino acids, ( b ) purines and pyrimidines, (c) other nitrogenous compounds of low molecular weight. The degradation of these compounds will vary depending on whether conditions are aerobic or anaerobic and varying \\ith the p H of the medium and the different intermediates resulting from such degradation pose quite different problems to the engineer. Microbial Degradation of A m i n o Acids
Much of the combined nitrogen of sewage is in the form of protein and its degradation \vi11 be by way of amino acids. The degradation products of amino acids are also among those most likely to create a nuisance in the way of VOL. 48, NO. 9
SEPTEMBER 1956
142 1
odor, and it is for this reason that control of their degradation is necessary. As indicated above, the main objective is to eliminate from sewage readily oxidizable materials in order that the effluent water will not contain a sufficient quantity of them as to ever create anaerobic conditions which would result in the production of these noxious intermediates or products. It is not possible at this time to treat the pathlvays or mechanisms of degradation of amino acids in detail, but the subject is considered in its broader aspects in order that the general processes of amino acid degradation can be related to the practical problems of the engineer. For a more detailed coverage of the decomposition of nitrogenous compounds the reader is referred to reviews by Gale (34) and the recent Symposium on A4minoAcid Metabolism edited by McElroy and Glass (56). Some amino acids serve directly as units in the synthesis of bacterial cell material, and others, through transamination, provide amino nitrogen for newly synthesized amino acids. The remaining amino acids are degraded oxidatively or fermentatively to provide energy for the microflora involved. End products of the fermentative reactions and intermediates of the fermentative and oxidative reactions are potential nuisance compounds. I n the oxidative processes few intermediates will accumulate in quantit>-, so the principal source of objectionable products \vi11 be fermentation reactions. Fermentation may involve the oxidation of one amino acid molecule concomitant with the reduction of another, the oxidation of one part of a molecule with the reduction of another part, or the oxidation of a molecule with some other inorganic substance acting as hydrogen acceptors-for example, carbon dioxide, sulfate, or nitrate. At a neutral to alkaline pH most amino acids will be deaminated early in the reaction resulting in the abundant production of ammonia, whereas in more acid media decarboxylation will proceed rapidly with the production of amines. The reactions considered in the following pages are typical of those which could be expected to occur in the microbial population of sewage. I t would be pointless a t this time to consider all known reactions or to speculate on the nature of many as yet undiscovered reactions. Moreover, it should be borne in mind that although each reaction is demonstrated with only one or two organisms this is a n expression of the limited extent to which it has been possible to investigate these degradative processes. A reaction demonstrated with one organism in most cases is probably present in a number of species and only for some of the more specialized reactions would it be
1422
expected that the number of responsible organisms is limited to several species. Deamination, Depending on conditions, deamination of an amino acid can take several courses, either oxidative or reductive, a combination of oxidation and reduction, or by desaturation or hydrolysis, with no net oxidation or reduction. Oxidative Deamination. The oxidative deamination of amino acids is effected by a number of organisms-for example, the reaction carried on by Hemophilus ParainJuentae ( 4 7 ) . The general reaction as formulated below results in the production of ammonia and the corresponding oc-keto acid.
+
R . CHNHz. COOH '/20:! + R . CO . COOH
+ NH3
Related to this but differing from it in the sense that oxygen is not utilized is a type of deamination described by Woods and Clifton (702) in lvhich molecular hydrogen is produced. Desaturation Deamination. Deamination of this type has becn descrihed by Quastel and iVoolf (63) \.iith E. coli and results in the formation of the a-8 unsaturated acid, thus the deamination of aspartic acid yields fumaric acid and ammonia according to HOOC , CH?.CHNH:!. COOH HOOC. CH CH . COOH
+ NH3
Similarly, the formation of urocanic acid from histidine has been reported as has the reductive deamination of tyrosine (42, 64) to form p-hydroxyphenylpropionic acid. Hydrolytic Deamination. E. coii and other organisms are able to produce the a-hydroxy acid from amino acids-for example, malic acid from aspartic acid according to
+
HOOC , CH?.CHNH:!.COOH HrO + HOOC, CH2. CHOH . COOH 4-KHs This has been reported by IYoolf (703) and Gale (35) and with Pseudomonas Juorescens by Virtanen and Erkama (90). iVhether such hydrolytic deamination occurs directly or is actually a desaturation reaction followed by hydration is not clear and for this report is unimportant. The net result is the same in that ammonia is the form of nitrogen liberated to the culture medium and malic acid will eventually appear through the action of fumarase. Reductive Deamination. The reductive deamination of a n amino acid resulting in the production of the corresponding fatty acid or dicarboxylic acid also has been reported; thus succinic acid can be produced from aspartic acid (22). Here, again, it is not clear whether the primary deamination results in the production of fumaric acid which is then hydrogenated by succinic dehydrogenase, or \\hat the precise pathLvay is.
INDUSTRIAL AND ENGINEERING CHEMISTRY
HOOC . CHe. CHXH:! . COOH Aspartic Acid
+
HOOC CHz. CH2, COOH Succinic Acid
+ NHa
Similarly the reductive deamination of glycine, tryptophane, and ornithine has been reported (43). Mutual Oxidation Reduction Reactions. A n interesting reductive reaction has been studied in some detail by Stickland (76, 77) w.ith Clostridium @roSeiies in Xvhich one amino acid is osidatively deaminated a t the expense of another \vhich is reduced. Thus alanine. valine, leucine: and phenylalanine are oxidized, Lvhereas glycine, proline, hydroxyproline, ornithine, and arginine are reduced. H?C-CHz 2
I
CH.COOH
H ~ C
+
\, /,
N H
Proline
C:H? CHNH:! COOH Alanine +
+ 3HlO
2 HaXCH? (CH2)a.COOH &.Amino Valeric Acid
CH3. COOH Xcetic Acid
+
+
+ KHs $- CO:!
The precise mechanism of this reaction is not known and the mechanism of deamination of both amino acids could be the same with one of the deaminated products being oxidized and the other reduced. A variation of this t)pe of reaction is one in Xvhich alanine is simultaneously oxidized and reduced in a type of mutase reaction-2 moles of alanine being reduced to propionic acid whereas 1 mole is oxidized to acetic acid and carbon dioxide according to 3CH3.CHNH2.COOH Alanine
2CH3CH2COOH Propionic .4cid
+ 2H20 -,
+ CH3.COOH Acetic Acid 3NH3
T
+ CO?
Similar mutase decomposition of serine, threonine, and glycine have been reported (79, 20). -411 of these deamination reactions and undoubtedly others may take place depending on conditions. The oxidative reactions will occur predominantly under aerobic conditions, whereas the reductive ones will more likely be found under anaerobic conditions. The keto acids produced aerobically will be further oxidized. Those amino acids which result in deamination products closely related to the tricarboxylic acid cycle are probably converted to carbon dioxide and water via the classical citric acid cycle. Examples of these are aspartic and glutamic acids, and alanine. Other amino acids of the categories cited earlier. those having branch chains or aromatic or heterocyclic nuclei: \vi11 result in the production of derivatives the oxidation of
WATER PURIFICATION which is less clearly understood. Reductive deamination may result in the production of fatty acids of low molecular weight which, if present in sufficiently large quantities, may have some nuisance properties. Examples are propionic, butyric, and valeric acids. These, ho\vever, are not normally found in large quantities and are probably not such objectionable products as those ivhich \\rould result from the decarboxylation of amino acids. The nitrogen derived from these various deaminations is invariably in the form of ammonia, and its further disposition is discussed later. Of the various types of deamination cited, certainly oxidative deamination has been studied most thoroughly. Different type enzymes exist in different systems. I t is beyond the scope of this report to treat them in any detail, and only the most general reference to them is made here. Distribution of enzymes affecting this reaction, the so-called amino acid oxidases. is general among animals? plants, and microorganisms. The enzyme exhibits distinct optical specificity, and some sources rich in Damino acid oxidase may contain little or no L-amino acid oxidase, whereas other sources \vi11 contain predominantly enzymes acting on the L-amino acid. For description of the D-amino acid oxidase, the reader is referred to studies of Krebs (57,52),Bernheim and coworkers (77), and it'ebster and Bernheim (94). ~-.4minoacid oxidase has also been studied, including early reports by Stumpf and Green (78) and Blanchard and coworkers (73). T h e latter \\-orkers obtained evidence implicating riboflavin phosphate in the reaction \vith the mammalian enzyme and Singer and Kearney (68) have demonstrated the participation of flavine-adenine dinucleotide. Decarboxylation reactions have not been studied in as much detail, but it appears tliat amino acid decarboxylations are idely distributed. particularly among the microorganisms (3:I?36, 37). Decarboxylation of
Amino
Acids.
T h e deamination of amino acids by bacteria is favored at neztral to alkaline hydrogen ion concentrations, \vith an optimum in the neighborhood of pH 7 to 8 (75). In contrast, a t loij-er h>-drogenion concentrations, according to the work of Gale ( 3 6 ) . decarboxylation is more likely to occur, the optimum for this reaction being in the neighborhood of pH 4 to
5. The occurrence of amines as products of bacterial action has long been recognized, having been reported by Ellinger (30), Ackermann (3:3 ) !and others. Gale (36. 37) has demonstrated the formation of a variety of amines by the decarboxJ.1ation of amino acids by E. [di:
CH?.CHNHz. COOH +
Tyrosine
for deamination and decarboxylation. it is possible that with these organisms deamination or decarboxylation could be favored, depending on the p H of the medium. Decomposition of Sulfur-Containing Amino Acids, These discussions of de-
Tyramine HOOC. C H ? .CHNH?.COOH + Aspartic Acid H O O C . CH?. C H ~ N H I CO, p Alanine
+
Specificity as to amino acids acted upon varies with different organisms (33, 38), and a number of cell-free preparations have been made. I n those cases where a dissociablecoenzyme can be demonstrated by dialysis pyridoxal phosphate appears to be involved (39). These decarboxylations are favored by acid pH: and the amines produced are particularly noxious, both with regard to their unpleasant odors and their toxicity to animals. The latter is generally nullified by effective detoxication mechanisms in the animal system, usually by acetylation or oxidation. .4lthough the amines resulting from these and other decarboxylation reactions are particularly objectionable when liberated to the atmosphere, their extensive production requires anaerobic conditions. I n mixed flora under aerobic conditions they will be further oxidized, the nitrogen being liberated as ammonia by other deamination reactions. Deamination Combined with Decarboxylation. The deamination combined
\vith decarboxylation of amino acids has been reported by Brasch (75, 76) with Clostridium putrijicum. Propionic acid is formed from aspartic acid according to the empirical formulation:
+
H O O C C H ? CHNHz COOH [2H]+ HOOC.CH2.CH3 NH3 COn
+
+
Similarly, butyric acid is formed from glutamic acid by this organism. The deamination observed here is reductive. Examples of hydrolytic deamination combined with decarboxylation have been reported. Bre\vers yeast was observed by Thorne (84) to decompose glutamic acid and valine, in this maii yielding 6 hydroxybutyric acid and isobutyl alcohol, respectively. The type reaction can be formulated : R , CHNH? COOH NH3
+ HZ0 COI
+
+ RCHzOH
Other examples involving aromatic and heterocyclic amino acids have also been reported (26-29) and are discussed later. These, again, are the over-all reactions observed and simultaneous deamination and decarboxylation probably do not take place. I n view of the different optimal pHs which most organisms exhibit
amination and decarboxylation are applicable to aliphatic amino acids containing only carbon, oxygen, hydrogen, and nitrogen. Although the same reactions apply to sulfur-containing amino acids. the presence of the sulfur atom in cystine and cysteine warrants additional consideration of these amino acids because of reactions producing hydrogen sulfide. Little detailed information is available with regard to the mechanism of breakdo\vn of these amino acids. The products of their decomposition are well knoivn, hoivever. Desnuelle and Fromageot (25) studied their anaerobic decomposition. Cystine is first converted to cysteine which is then fermentativelJbroken down-for example, by Proleu3 uulgaris-lvith the liberation of hydrogen sulfide and ammonia and the production of acetate and formate according to H S . C H ? .CHNHI.COOH Cysteine CH3COOH HCOOH
+
+ 2H2O + NH3 + HIS --t
This reaction may \vel1 take place uith 0-mercapto pyruvate as an intermediate which would result either from the action of a transaminase system as has been reported for animal systems (78) or by oxidative deamination. K u n (.W) has demonstrated the dependence of hydrogen sulfide production upon carbohydrate oxidation with Endamoeba hi.itolytica. There are certain indications that the hydrogen sulfide production i n this case is by way of thiol radicals. Hydrogen sulfide formed in any of these reactions, besides being one of the more objectionable products of amino acid decomposition, is x?-c~~-".y of further consideration because 01 ihe variety of reactions in which it may participate. hloreover, sulfur-containing amino acids are not the only source of hydrogen sulfide. This gas can be produced in large quantities in \vastes containing appreciable quantities of sulfate, sulfite, hyposulfite. and thiosulfate, such as those of paper and other industries in which sulfur compounds are used as bleaches or for other purposes. Organisms carrying on this type of reaction were studied in some detail by van Delden (85) as early as 1904. Under conditions where a n adequate organic substrate is available and oxygen tensions are low, they will utilize oxidized sulfur compounds as hydrogen acceptors for the oxidation of organic compounds resulting in the production of hydrogen sulfide. sometimes in considerable quantities. A typical reaction is that VOL. 48, NO. 9
SEPTEMBER 1956
1423
whereby sulfate is reduced to hydrogen sulfide according to the equation
+ l'/zHzSO4 + 3C02 + 3H20
C H I .CHOH. COOH Lactic Acid l'/pHzS
+
The hydrogen indicated in this equation can be presumed to be that derived from some organic substrate. The numerous subsequent reactions which may then involve hydrogen sulfide are discussed in detail by Starkey, p. 1429. Suffice it to say that it may serve
trophic organisms whereby it is again converted to sulfur or more oxidized sulfur compounds [(86-88), Cohn (27), Beijerinck (9, 70), Winogradsky (96), and Starkey (73, 741. Because of its intense odor and common occurrence in sewages containing moderate quantities of sulfur, this gas is of considerable concern to the engineer. The avoidance of its production from amino acid or other sources requires the maintenance of aerobic conditions. Degradation of Basic Amino Acids.
The basic amino acids are likewise deserving of special consideration because of additional reactions not already considered. These may be involved in their degradation and also because some particularly offensive amines may be produced by their decarboxylation. One of the more common basic amino acids from both plant and animal sources is arginine. The observation by early workers ( 2 ) that ornithine frequently was formed in the microbial degradation of arginine led to the supposition that a system similar to the classical KrebsHenseleit (53) urea cycle was operative in these organisms and responsible for the formation of ornithine. Hills (47) has made a quantitative study of the conversion of arginine to ornithine by a number of cocci, and it does follow the stoichiometry of the reaction which would be consistent .ith this hypothesis: HN " C . N H . ( C H Z ) ~ . C H N HCOOH ~ H zN
/
lactis are able to convert arginine to citrulline plus ammonia, presumably without ATP, magnesium ion. or aspartate ( 4 9 ) . The degradation of arginine, therefore. probably is not via the classical Krebs-Henseleit system but rather involves arginine desimidase and citrullinase or citrulline phosphorylase. Preparations of various streptococci, including S.lactis and other organisms, are also capable of converting citrulline to ornithine and ammonia according to the reaction (5. 50,70)
+
H 1:?j.(CHz).:.CHNH?COOH CO2 Ornithine
This reaction also requires magnesium ion, adenosine diphosphate (ADP), and some exogenous source of orthophosphate, and the conversion results in the production of high energy phosphate in the form of ATP. The reaction can be coupled to the phosphorylation of glucose by hexokinase yielding glucose-6phosphate. This suggests that it provides a limited energy source for the organism (77). The system is a reversible one and citrulline can be formed from ornithine and an appropriate source of ATP. The further degradation of ornithine resulting from these reactions can follow a number of courses. Thus. the interrelationship between glutamate and ornithine via their .\'-acetyl derivatives through the ,\-=-acetyl ?-glutamic semialdehyde is presumably reversible, and the degradation of ornithine by such a mechanism would thence follow the same course as the metabolism of glutamate. Systems effecting this degradation have been described ( 7 , 92). O n the basis of specific activity data gleaned using CI4labeled ornithine, Yogel: .4belson, and Bolton conclude that an alternative pathkvay exists in E. coli for the metabolism of exogenous ornithine resulting in the conversion of this compound to
+ 2H20
H Z N . ( C H ~ ) ~ . C H N H ? . C Of OH 2NH3 Ornithine
1424
+
acids derived from proteins and other organic nitrogen sources are histidine, tryptophan, proline, and hydroxyproline. Considerable work has been done recently on the degradation of histidine by various microorganisms (82). Work with Ps. Jflorescens by Tabor and Hayaishi (83) and Suda and coworkers (80) indicates that it is converted to glutamic acid via formyl-L-glutamic acid as a n intermediate. Lt'achsman (93) has also studied the fermentation with Clostridium tetanomorphum. The products of the fermentation by this organism are ammonia, carbon dioxide, hydrogen, acetate, butyrate, formamide, and possibly formate. Wachsman further suggests urocanic acid, .V-formamidinoglutaric, and glutamic acid as intermediates in the reaction. The decarboxylation of histidine yields the corresponding amine histamine which? although it has potent physiological effects, probably is not of too much direct concern to the sanitary engineer. The formation of this compound from histidine has long been known (3) and the specific decarboxylase carrying on this reaction has been prepared from C'lostridium welchii by Epps (37). The degradation of tryptophan also may take a number of courses?depending on the organism and culture conditions. The generalizations made earlier with reference to decarboxylation and deamination apply as well to this compound. The oxidative degradation of tryptophan may follow a number of alternate pathiiays. One characteristic reaction is its cleavage to indole, pyruvate, and ammonia according to the reaction (40, 700).
' 2 0 2
proline (97). Glutamic semialdehyde. holvever, appears to be intermediate in this reaction also. so the point of divergence between this pathway and the ornithine glutamate pathway is not clear. The deamination of ornithine to yield &amino valeric acid by ClosO L ~ C H ~CHNHz. , COOH
+ H?O--.
0,
.---
\
N
Tryptophan
INDUSTRIAL AND ENGINEERING CHEMISTRY
+ NH3
Metabolism of Heterocyclic Amino Acids. The principal heterocyclic amino
-
Arginine
However, Hills found that the streptococci did not possess any urease activity and the staphylococci he studied, although having urease activity, decomposed urea less rapidly than they did arginine. From this he concluded that a combination of arginase and urease activity was not responsible for the formation of ornithine in these organisms. Moreover, whereas the synthesis of arginine from citrulline in mammalian systems requires adenosine triphosphate (ATP), magnesium ion, and aspartate (65, 6 6 ) , dialyzed preparations from S.
tridium sporogenes has been reported by LYoods (707). ,4 number of organisms are also known to be capable of decarboxylation of ornithine to the corresponding diamine putrescine (30). This is analogous \vith the formation of cadaverine from lysine, a reaction also affected by a variety of organisms. Both of these diamines are likely products of anaerobic decomposition, particularly in mildly acid media. Their further degradation, as well as that of other amines. is discussed later.
+ NHI + CH2 CO.COOH
\
N
Indole
Pyruvic Acid
WATER P U R I F I C A T I O N This reaction has been demonstrated in
E. coli and other organisms and is probably the most common pathway whereby tryptophan is oxidized. Little is known about the further degradation of indole. I t is also known that E. coli can produce other oxidative products of tryptophan such as indole propionic acid which Lvould result from its reductive deamination and indole acetic acid by oxidative deamination and decarboxylation. Both these compounds were demonstrated to be formed by this organism as early as 1903 by Hopkins and Cole (44). Still another pathway for the oxidation of this amino acid results in a cleavage of the indole ring with the formation of kynurenine (72, 79). The kynurenine upon further oxidation will yield nicotinic acid. The decarboxylation of tryptophan to yield a corresponding amine, although reported by Berthelot and Bertrand (72), has not been studied. Degradation of Purines and Pyrimidines. The degradation of purines and
pyrimidines is of interest from a quantitative point of view since these compounds are intermediate in the breakdown of nucleic acids, nucleo proteins, and other purine and pyrimidine derivatives common to all organic life forms. Barker and Beck (7>8) have isolated Clostridia fermenting some of these compounds. Clostridium midi-urici and Clostridium cjlindrosporum are able to oxidize uric acid, xanthine, and guanine completely and rapidly. Xanthine is decomposed more slo~vlyby these organisms, and the degradation is not so complete. In general, ammonia, carbon dioxide, and acetic acid are end products of the fermentation with Cl. acidi-urici, whereas cjlindrosporum yields these compounds as well as some glycine. Streptococcus allantoicus, reported by Barker (6). is able to utilize allantoin as a sole carbon and energy source. Products of the fermentation include ammonia, urea, carbon dioxide, formate, acetate, lactate, and glycolate. Oxidation of Amines. Although systems capable of oxidizing both monoamines and diamines of the type resulting from the various decarboxylations mentioned above occur in various animal tissues (23. 87) and microorganisms are known to be able to decompose them, little work has been done in studying their reactions. Early in the degradative process is the removal of ammonia from the molecule, probably oxidatively, to yield in addition to ammonia, the corresponding alcohol. .Although the reactions discussed in the preceding paragraphs by no means include all the types of reactions involved in the degradation of nitrogenous compounds in as heterogeneous a system as sewage: they summarize in a general way some of the more common and better un-
derstood reactions from which it is possible to draw certain broad conclusions. These considerations indicate that an adequate supply of oxygen can prevent the formation of nuisance products in the degradation of amino and other organic nitrogenous compounds in selvage. Under sufficient aeration these materials \\ill be converted rapidlv either to slovly decomposed compounds or to carbon dioxide. water, and ammonia. Aforeover, as discussed later, the ammonia is further oxidized to nitrate. the ultimate product of aerobic digestion. The principal difficulties to be expected are those due to the inadequate provision of oxygen to the system under conditions where evolved degradation products are not confined but escape to the atmosphere. This can take place, for example, I\ here sewage must be conducted for considerable distances before processing, thus enabling an active microflora to develop. Under such conditions it may become necessary either to provide completely closed svstems for its conduction to the processing point or to provide aeration a t stations en route, thus maintaining a n adequate oxygen level. This may also take place in settling basins which contain excessive quantities of readily metabolized materials, or in the terminology of the sanitary engineer, have a high B.O.D. The entire industry of sewage processing is built around this concept of producing a n effluent water sufficiently low in dissolved metabolites as to prevent the development of anaerobic conditions with the consequent formation of undesirable products.
The Fate of Ammonium Ion As indicated earlier, most of the nitrogen of sewage will be in the form of ammonium ion or other compounds (e.g.. urea) from which ammonium ion is readily derived by hydrol>-sis. The remaining organic nitrogen likewise is soon converted by microbial action to ammonium ion and reactions involving this ion eventually must include essentially all the nitrogen of sewage excepting that removed as insoluble material in the form of aggregated bacterial cells in the sludge itself. Provided oxygen is available: a vigorous nitrifying flora can be expected to develop in effluent waters containing ammonium ion. This nitrifying flora consists in the main part of organisms belonging to the genera ,I'itrosomonas and ,Vitrosococcus (74, 77, 97, 98) \vhich are responsible for the oxidation of ammonia to nitrite and the genus A-itrobacter of which there appear to be a t least two species (62, 98) oxidizing nitrite to nitrate. These organisms exist autotrophically, obtaining their energy
through the oxidation of their inorganic substrate, ammonium ion or nitrite, as the case may be. L'nder suitable conditions large quantities of ammonia can thus be converted to nitrate by mixed flora and, indeed, the availability of oxygen permitting, it can be expected that ammonia will rapidly undergo such a conversion by mixed flora in typical outflow \vaters from sewage processing systems. Certain features of the physiology of these organisms are of interest to the sanitary engineer. In so far as is known, they are obligatory autotrophs and will not grow in the absence of their characteristic substrate, ammonium or nitrite ions. However, claims of inhibition due to organic materials have perhaps been overemphasized. Although certain compounds, particularly amino acids and amines in higher concentrations, do inhibit their growth (67, 99), the organisms are quite tolerant to concentrations of organic material of the order of magnitude normally found in sewage material (32, 45'). I t can be expected that because of their ubiquitous distribution, they will appear wherever ammonium and/or nitrite ions are present and aerobic conditions prevail. Meyerhof (60) early made studies on the effect of oxygen on these organisms and found that a t oxygen tensions below 50% of normal (11'10 atmosphere 0 2 ) the development oflVitrosomonaswas measurably suppressed and iVitrobacter was even more sensitive to oxygen concentrations. For this reason the development of these organisms probably will not be abundant until the oxygen demand of sewage waters has been brought to a reasonably low level. .4 number of heterotrophic organisms have been cited as capable of nitrification (24, 46). However, the amount of nitrite or nitrate formed in this manner has not been established and probably is not significant compared with the contributions of autotrophic organisms. For coverage of the literature with regard to nitrification, the reader is referred to recent reviews by hleiklejohn (59): Lees (55):and Delwiche in (57). Provided conditions remain aerobic thereafter, nitrogen will remain in the form of nitrate. If, hoxvever, there is a n abundance of organic substrate and some zones, either of sedimented material or of solution, become deficient in oxygen. nitrate may further participate in the biological chain of events as hydrogen acceptor for denitrifying organisms subsisting on oxidizable organic materials present. This process is one carried on by heterotrophic organisms Lvhich derive their energy from the oxidation of the organic substrate and ivhich are capable of mediating electrons from the organic substrate to nitrate as hydrogen acceptor. This is directly analogous to the situation VOL. 48, NO. 9
SEPTEMBER 1956
1425
alluded to a n d discussed in more detail in the article by Starkey, wherein sulfates and other oxidized forms of sulfur are reduced to the level of hydrogen sulfide by various heterotrophic organisms. \Vith nitrate, however, the more common reduction product is nitrogen gas rather than ammonia. With denitrifiers, nitrate serves as a n alternative hydrogen acceptor making it possible for the organisms to grow in the absence of oxygen. Xormally the activation of nitrate is a n adaptive phenomenon and cells Jvhich have been grown under aerobic conditions exhibit some lag in the development of denitrifying abilities. Recent work by Wijler and Delwiche (95) and Skerman and coworkers (69) indicates that denitrification will not take place until the medium is essentially oxygen free. Marshall and coworkers (58)working with aerated cultures of Pseudomonas have observed the disappearance of nitrate of low concentrations. Nitrous oxide, a common if not universal product of denitrification, has been reported in denitrifying systems frequently in high concentrations (48, 67> 95). This gas is also reabsorbed and further reduced to nitrogen gas as the concentration of other hydrogen acceptorse.g., nitrate and nitrite-diminishes. Nitric oxide and its oxidation products have also been reported as products of denitrification, particularly under more acid conditions (89, 95). Nitrogen and other gases produced by denitrification are quite innocuous. T h e principal difficulty which may be expected from their presence is that of a mechanical hindrance to settling by denitrification in sludges or sedimented materials resulting in a mixing or buoying up of particles by the gas. Conceivably, nitrogen so formed could also be objectionable in systems ivhere the other gaseous products of sewage processinge.g., hydrogen and methane-are collected for use as fuel. Here, if large quantities of nitrate \rere present during the early anaerobic stages of processing, nitrogen formed by denitrification might dilute other gases, making them less effective as fuels. For a further discussion of the literature on denitrification see (57). Barring excessive losses of nitrogen due to denitrification, nitrate, the final oxidation product of the nitrogenous constituents of sewage, can play a n important role in subsequent complications in the processing of water. Because other mineral elements probably are in adequate supply in the effluent water to support a n abundant algal flora, nitrogen may in some cases be the limiting factor in determining the extent to which algae will grow in such waters. Ho\vever: since even a small quantity of nitrogen will support a rather large growth of algae and since, in addition, numerous
1426
photosynthetic organisms probably possess the capacity for nitrogen fixation. mechanisms necessary for the control of algal blooms probably are necessary regardless of the nitrate or ammonia nitrogen content of processed waters.
literature Cited (1 ) hbelson, P. H.: Vogel, H. J.. J . Biol. Chem. 213. 355 119551. ( 2 ) .-lckermann,’D., 2.Physiol. Chrrn. 5 6 , 305 11908‘1 ( 3 ) Ibid:,- 65: 504 .( 19 10 ). ( 4 ) Ibid., 69, 273 (1910). ( 5 ) Akamatsu, S.,Sekine, T., J . Biochem. ( J a p a n ) 38, 349 (1951) (6) Barker, H. .4., J . Bacteriol. 46, 251 11943’1. ( 7 ) Birker, H. .A,, Beck, J. V.. Ibid., 43, 291 ( 1 9 4 2 ~ . (8) Barker, H . A , , Beck, J. V., J . Biol. Chem. 141. 3 (1941). ( 9 ) Beijerinck. i,l. Zentr. Bakteriol., Abt. 11; 11, 593 (1904). (10) Beijerinck: hl. W.,Proc. Soc. Sci.. Kon. Akad. Il.‘etenschap, rlmstrrdani, 22, 899 (1920). (11) Bernheim, F., Bernheim, hl. L. C.: Webster, M. D., J . B i d . Chem., 110. 165 11935). (12) Berthilot, A:, Bertrand, D. hf., Compt. Rend. &ad. Sci. 154, 1826 (1912). (13) Blanchard. M., Green, D. E., Nocito. V., Ratner, S.,J . Biol. Chem. 155. 421 (1944). (14) Bomeke, H.. Arch. ‘tfikrobioi. 15, 414 11951). ( 1 5 ) Brasch, ‘Ll‘.: Blochem. Z. 18, 380 (1909 1. (16) Ibtd.,22; 403 (1909). (17) Breed, R. S., Slurray, E. G. D.. Hitchens, .A. P., “Bergey’s Manual of Determinative Bacteriology,”
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6th ed., IVilliarns and Wilkins. Baltimore. 1948. (18) Cammarata; P. S.,Cohen, P. P., J . B i d . Chem. 187, 439 (1950). (19) Cardon, B. P., Barker, H. .%,> Arch. Biochem. 12, 165 (1947). (20) Cardon, B. P., Barker, H. .A,, J . Bacteriol. 52. 629 (1946). Cohn, F., Behr. Biol. PJan:. 1, 141 (1875).
Cook, R. P., Woolf, B., Biochem. J . 22, 474 (1928).
Cotzias, G. C.! J . Biol. Chern. 190, 665 (1951).
Cutler, D. Ll’., Crump, L. M., Ann. z4pplied B i d . 20, 291 (1933). Desnuelle, P., Frornageot, C., E n q moloeia 6. 80 (1939). (26) Ehrlic;, F.; Be;. deut: chem. Ges. 40, 2538 (1907). (27) Ibid., 44, 139 (1911). (28) Ibid., 45, 833 (1912). ( 2 9 ) Ehrlich, F., Breslauer chem. Ges. 11 (February 1910). ( 3 0 ) Ellinger; -1.. Ber. drut. chem. Ges. 31, 3183 (1898). (31) Epps, H . hl. R., Biochem. J . 39, 42 (1945). (32) Fred, E.’ B.. DaLVenport, A., Soil .S’cz. 11, 389 (1921). (33) Gale, E. F., .4dzances in Enqmol. 6, 1 (1946). (34) Gale. E. F., Bacteriol. Rei. 4 , 135 (iio 4-n )
(35) Gale. E. F., Biochem. J . 32, 1583 (1 938 ). ( 3 6 ) Ibid., 34, 392, 846, 853 (1940). (37) Zbid., p. 846. (38) Zbid., p. 400. (39) Gale, E. F.. Epps, H. h i . R.: Ibid., 32, 232 (1944).
INDUSTRIAL AND ENGINEERING CHEMISTRY
( 4 0 ) Happold. F. C., Hoyle, L., Zbid., 29, 1918 (1935). (41) Hills, G . ~ M . , i b i d . 34, , 1057 (1940). (42) Hirai, K., Biochem. Z . 114, 71 (1921). (43) Hoogerheide, J. C., Kocholaty, LV., Biochem. J . 32, 949 (1938). (44) Hopkins, F. G., Cole, S. \V., J . Physiol. 29, 451 (1903). (45) Jensen, H. L., ,Vature 165,974 (1950). (46) Jensen, H. L., Gundersen, K., Ibid., 175, 341 (1955). (47) Klein, J. R., J . Biol. Chem. 134, 43 (1 940). (48) Kluyver, A . J., Verhoeven, W., Antonie isan Leeuwenhoek 20, 241 (1954). (49) Korzenovsky, hl., Werkman, C. H., Arch. Biochem. Biophys. 41, 283 (1952). (50) Ibid., 46, 174 (1953). 151) Krebs. H. A , . Biochem. Sac. Svm~, posia., Cambridge, Eng., 1 , 1948. (52) Krebs; H. A , , Z. Physiol. Chem. 217, 191 (1933). (53) Krebs, H. A , , Henseleit, K., Ibid., 210, 33 (1932). (54) , . Kun. E.. Biochim. Biobhvs. iicta. 11. 312 (1953). (55) Lees, H., Autotrophic Microorganisms, 4th Symposium, SOC. Gen. Microbiol., p. 85, Cambridgc Univ. Press, London, 1954. ( 5 6 ) McElroy, W. D., Glass, B., A Symposium on Amino Acid Metab(57) (58) (59)
(60) (61) (62) (63)
olism. Johns Hopkins Press, Baltimore, 1954. McElroy, 1%’. D.> Glass, B., “Inorganic Xitrogen Metabolism.” Johns Hopkins Press, Baltimore, 1955. Marshall, R. O., Dishburger, H . J., MacVicar, R., Hallmark, G. D., J . Bact. 66, 254 (1953). Meiklejohn, J., Autotrophic Microorganisms, 4th Symposium, Soc. Gen. hlicrobiol., p. 68, Cambridge Univ. Press, London, 1954. Meverhof, O., Pfriigers Arch. srs. Physiol. 165, 229 (1916). Ibid., 166, 240 (1917). Nelson, D. H., Zentr. Bakteriol., Abt. 11, 83,280 (1931). Quastel, J. H., Woolf, B., Biochem. J .
20, 545 (1926). (64) Raistrick, H., Ibid., 11, 71 (1917). (65) Ratner. S., Federation Proc. 8 , 603 (1949). (66) Ratner, S., Petrack, B., J . Bioi. Chem. 191, 693 (1951). (67) Sacks, L. E., Barker, H. A , , ,I, Bacteriol. 64, 247 (1952). ( 6 8 ) Singer, T. P., Kearney, E. B., .Irch. Biochem. 27, 348 (1950). (69) Skerman, V. B. D., Lack, J., Millis, N., Australian J . Sci. Research 4B, 511 (1951). (70) Slade, H. D., Arch. Biochern. Bi,$hys. 42, 204 (1953). (71) Slade, H. D., Doughty: C. C.. Slarnp. W.C., Ibid.! 48, 338 ( 1 9 5 4 ~ . ( 7 2 ) Stanier, R . Y . , Hayaishi, O., ’Tsuchida, Xf.: J . Bacttriol. 62, 355 (1951). ( 7 3 ) Starkey, R. L., Ibid., 28, 365 (1934). ( 7 4 ) Starkey, R . L., J . Gen. Physioi. 18, 325 (1935). (75) Stephenson, M., Gale, E. F., Biochem. J . 31, 1316 (1937). (76) Stickland, L. H., Ibid., 28, 1746 119341. (77) Ibihl, 29; 288, 889 (1935). ( 7 8 ) Stumpf, P. K., Green. D. E., J . B i d . Chem. 153, 387 (1944). (79) Suda, M.,Hayaishi, O., Oda, Y., J . Biochem. ( J a p a n ) 37, 355 (1950 i . ( 8 0 ) Suda. hl.. Nakiya, X . , Hara. SI.. Kato, .A,, Idenaka, T., .lied. J . Osaka 17nn2c.4, 107 (1953). (81) Tabor, H., J . B i d . Chern. 188, 125 (19501.
WATER P U R I F I C A T I O N 182) Tabor. H.. Pharniacoi. Rets. 6, 299 (1954). ( 8 3 ) Tabor, H., Hayaishi. O., J . Biol. Chem. 194. 171 11952). ( 8 4 ) Thorne. R. ’S. \V.~. J . Znst. Brewing 43, 288 (1937). 1851 Van Delden. A. Zentr. Bakteriol.. Xht. 11: 11, 811 113 (1904). ( 8 6 ) Van Niel. C. B., .lrch. .Ifikrobiol. 3, 1 (1 931 ).. ( 8 7 ) Van Siel. C. B., .4rrh. .\.fikrobioi. 7 , 323 (1 936 j. ( 8 8 ) Van Niel. C. B., Cold Spring Harbor Symposia Quant. Biol. 3, 138 (1935.) ( 8 9 ) Verhoeven, LV., “Aerobic Spore\
,
DISCUSSION
forming Nitrate Reducing Bacteria,” Thesis, Delft, Holland, 1952. (90) Virtanen, .A. I.? Erkama, J., Nature 142, 954 (1938). (91) Vogel, H. J., Ahelson, P. H., Bolton, E. T.. Biochim. Biophvs. Acto 11, 584 (1953). (92) Vogel, H. J., Bonner, D. M., Prod. N o t l . d c a d . Sci. 40, 688 (1954). ( 9 3 ) Wachsrnan, J. T., Ph.D. thesis, Univ. of Calif., Berkeley, 1955. ( 9 4 ) Webster, M. D.. Bernheim, F., J . Biol. Chem. 114, 265 (1936). (95) Wijler, J., Delwiche, C. C., Plant and Soil 5 , 2 ( 1 9 5 4 ) .
( 9 6 ) Winogradsky, S., d n n . inst. Pastmr 1, 548 (1887). (97) Zbid., 4, 213, 231. 760 (1890). ( 9 8 ) Zbid., 5 , 92, 577 (1891). ( 9 9 ) Winogradsky, S., Omelianski, V., Zentr. Bakteiiol., Aht. 11, 5 , 329, 349 (1899). (100) \$roods, D. D.. Biochein. J . 29, 640 (1935). (101) Zbid., 30, 1934 (1936). (102) Woods. D. D., Clifton, C . E , Zbid., 31. 1774 (1937). (103) Ltoolf, B., Zbzd., 23, 472 (1929).
RECEIVED for review January 12, 1956 ACCEPTED April 18, 1956
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Biological Transformations of Nitrogen Compounds
1
n the design and operation of Lvorks for the treatment of selvage and industrial \castes and in evaluating: understanding, and solving problems in the natural and artificial purification of polluted Lvaters, the sanitar)- engineer relies heavily on the basic principles of biochemistry. Most certainly. De1Tviche.s excellent review \vi11 prove a valuable reference article for the engineer in making progress in the intelligent application of basic principles toivards the solurion of difficult problems. In a general \vay the importance of the different forms of nitrogen as indexes of the quality of a Lvater supply has been recognized for some time. In fact, in the chemical examination for water quality, many workers have regarded the forms of nitrogen as the most satisfactory indicators. The presence of organic nitrogen, ammonia, nitrites, and nitrates has indicated contamination from sewage. Changes in the state or content of the compounds from that considered normal for the water in question is interpreted as a warning signal. Delwiche has pointed out several ways in which the general processes of amino acid degradation can he related to the practical problems of the engineer. I n particular he has clearly shown that the end products of the fermentative reactions, and the intermediates of the fermentative and oxidative reactions are potential nuisance compounds. The control of nuisance odors is a n
ever-present problem to the sanitary engineer, and it is a well-knoivn fact that the olfactory acuity of the layman is keenly sharpened on the mere sight of a sewage treatment plant or on mere mention of the word servage. It must be admitted, however, that perhaps there are times when those of us who are frequent visitors to sewage treatment plants are suffering from chronic olfactory fatigue. None the less, selvage is a real as \vel1 as a potential source of odors. The principal method for control of nuisance odors: however, has been the use of chemicals? and in particular chlorine has been used extensively (2). Its application under proper control provides additional oxidation potential for the oxidation of hydrogen sulfide and retards degradation that results from bacterial metabolism. Chlorination both in the seiverage system (called upstream chlorination) and a t seLvage treatment plants is in widespread use. The fate of the chlorinated amino acids in sewage treatment processes is not knolvn, but it might be of interest to extend this part of the discussion to the related problem of taste and odors in \Yarer supplies with particular reference to the role of nitrogenous compounds of biological origin. The source of the nitrogen would include not only sewage or sewage effluent but also algae and other microorganisms normally present in surface waters. The odors produced by algae may be odors of growth (72), but they may be in-
tensified by decomposition or by destruction of the algae by the use of algacides or by other means. The presence in algae of large percentages of protein has been noted by many authors. Domogalla: Fred, and Peterson ( / , 7) have noted the forms in which both organic and inorganic nitrogen are present in lake waters and have reported the presence of the amino acids-tryptophan, tyrosine. histidine, and cystine. The natural microbial population of the Ivater utilizes, as nutrient material, the dead algae; and the degradation of the nitrogenous compounds, through the reactions revieived by DelIviche, produces a variety ofcompounds,many of which are capable of producing odor (8). Ingols and coworkers (5) have reported on a study of tastes and odors produced by chlorination of simple nitrogenous compounds. Four of twentyfive amino acids studied produced taste on reaction with hypochlorous acid and monochloramine. These were alanine, phenylalanine, arginine, and proline. Proline and phenylalanine also produced taste with chlorine dioxide. .4dditional investigations on the presence of amino acids in surface \vaters and their relationship to the taste and odor problem will undoubtedly be a fruitful area of research. An understanding of the cycle of nitrogen in aerobic and anaerobic decomposition has been of value to the sanitary engineer in the design and operation of VOL. 48, NO. 9
SEPTEMBER 1956
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unit processes for the treatment of secvage and industrial wastes. Among others Snell (71) has reported on nitrogen changes and losses during anaerobic digestion, a subject of concern where nitrogen content of sludges to be used as fertilizer is under consideration. Heukelekian’s studies (3, 4) on nitrification show that sewage alone nitrifies slo\vly because the number of nitrifying organisms is limited. With the addition of activated sludge the deficiency is corrected. and nitrification proceeds rapidly. The studies also shoxv that the oxidation of carbonaceous material does not retard nitrification which can take place simultaneously, provided that there is no deficiency in oxygen, that the demand for nitrogen, created by the rapid utilization of carboh>-drate, does not exceed the supply, and that a n active nitrifying flora is present. This work also pointed out the influence of a n active nitrifying flora on the B.O.D. test in which a n additional oxygen demand is exerted from the beginning of the test as a result of nitrification taking place simultaneously \vith carbon oxidation. The importance of nitrogen in the nutritional requirements in the biological oxidation of wastes has been reported by Saivyer (9) and Kilgore and Sawyer (6). The ability of an activated sludge to nitrify is dependent on the relationship bet1veen the B.O.D. and nitrogen in the sewage (9). Sludges fed on
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diets containing 8.2 p.p.m. B.O.D. for 1 p.p.m. of ammonia nitrogen developed the greatest ability to oxidize nitrogen. while sludges fed on diets containing 16 p.p.m. B.O.D. or more per unit of ammonia nitrogen lost most of their ability to oxidize nitrogen. Among other factors: Sawyer and Bradney (70) relate the rising sludge problem, mentioned by Delwiche, with low B.O.D. to nitrogen ratios. The Sioux Falls studies (70) shoxv that biological denitrification of nitrates \vith resultant production of nitrogen gas results in the buoying of the sludge; the Lvork indicates the importance of controlled nitrification and the design of final clarifier mechanisms in reducing the floating sludge problem. A further relationship bet\reen engineering design and a n understanding of the biochemistry of the process is apparent from these studies which indicate that short circuiting in the aeration tanks carries a plentiful food suppl>- to the sludge in the final tanks which accelerates denitrification and shortens the rising time of the sludge. Increasing attention is being devoted to the problem of fertilization of receiving \vaters v i t h the subsequent groivth of algae and other aquatic plants \vhich results from the discharge of sewage and sewage effluents containing nitrogen and other nutrients. The reviews presented by Delwiche and others in this symposium \vi11 be appreciated by sanitary
INDUSTRIAL AND ENGINEERING CHEMISTRY
engineers. \vho must make use of such knowledge in the development of new processes designed to remove nutrients from se\vage effluents and in the control of se\vage treatment Xvorks.
literature Cited ( 1 ) Domogalla, B. P.: Fred, E. B.. Peterson, i V . H., J . Am. Il.hte7 [ t b r k s -4ssoc. 15, 369 (1926). ( 2 ) Fed. Sew. and Ind. Wastes .lssoc. Comm. Rept. “Chlorination of
Sewage and Industrial IVastes,“ Manual of Practice S o . 4, 1951. ( 3 ) Heukelekian, H., Sewage T1.o~X-s J . 14, 964 (1 942). ( 4 ) Heukelekian, H., Littman, 51. L., Zbid., 11, 226 (1939). ( 5 ) Ingols, R. S.: Hodgden, H. I V . , Hildebrand, J. C., J . 4 7 . Food Chem. 2, 1068 (1954). ( 6 ) Kilgore, H. D., Sawyer, C . X., Sewage and Znd. Ft’astes 25, 596 (1953). (7) Peterson, LV. H., Fred, E. B., Domogalla, B. P., J . Bid. Chern. 63, 28(1925). ( 8 ) Rohlich, G. A,, Sarles, W.B., Proc. Inservice Training Course for rVater
LVorks Personnel, Univ. of hfichi?an, Ann Arbor, May 1947. ( 9 ) SLwyer, C. N., Sewage ll’orks -J. 12, 3 (1940). Bradney, L.: Zbid., 17, (10) Sawyer, C. h-., 1191 (1945). (11) Snell, J. R., Ibid., 15, 56, (1943). (12) \tJhipple, G. C., “Microscopy of Drinking Water,” 4th ed. Lt’iley, New York, 1927.
GERARD A. ROHLICH University of Wisconsin, Madison, Wis.
