WALTER J. NICKERSON Institute of Microbiology, Rutgers University, The State University of New Jersey, New Brunswick, N. J.
Transformations of Carbon Compounds
by Microorganisms
As an aid in presenting the complex type reactions considered here, liberal use is made of biochemical flow sheets or metabolic maps as guides for the “type pathways” exhibited by certain microorganisms in their transformation of carbon compounds
F O R practical reasons it is necessary limit our consideration of transformations of carbon compounds to those containing carbon. hydrogen. and oxygen. Among the main classes of substances containing carbon, hydrogen, and oxygen are carbohydrates. including sugars, p o l y c c h a r i d e s , and sugar alcohols and acids; lipids. including fats and \\axes; and aromatic ring substances: including simple aromatic compounds and complex substances such as steroids. resins. and lignin. AS more is learned about the decomposition of natural materials by microorganisms, which are the chief agents for the decomposition of carbon compounds in nature, it becomes apparent that the generalization can be made that for w e r y carbon compound formed in nature there exists some microbial agency Tor its decomposition. ,And. as more is learned about the biochemical mechanisms involved in the decomposition of a \vide variety of substrates, there is emerging the generalization that the initial stage of attack on a carbon compound. no matter how complex, converts it into a substance that feeds into one of the relatively few metabolic throughways common to all cells. Let us examine some of the bases for these t\vo generalizations. \Ve m a y see to 10
phenanthrene
what extent the factual information on the decomposition of aromatic ring substances and of carbohydrates support them. I t is true, of course, that vast accumulations of aromatic compounds-for example, lignin in peat and coal and hydrocarbons in petroleum-have survived decomposition. Apparentl). these substances have been removed from an aerobic environment (where microbial agencies certainly exist for the decomposition of lignin, hydrocarbons, and aromatic compounds) into a n anaerobic state Jvherein such agencies apparently d o not exist. Cellulose, on the other hand, is decomposed readily under both aerobic and anaerobic conditions and does not accumulate in nature (the cellulose theory of the origin of coal has fe\v supporters today). T h e apparent lack of agencies for the anaerobic decomposition of lignin and hydrocarbons, considered with the existence of such agencies for the decomposition of cellulose, raises some intriguing questions of biochemical evolution. I t \vould undoubtedly be interesting to pursue the biogeochemical and cosmological implications of these facts, as chemists and microbiologists seem prone to d o these days, but they \\-odd no doubt lead us into even deeper and darker mysteries
saligenin Figure 1
salicylic acid
catechol
than we are considering in this symposium. Decomposition of Aromatic Ring Compounds
O n e of the first indications rhat microorganisms are capable of decomposing aromatic rings was the report of Emmerling and Abderhalden (75), in 1903. 01’ their isolation from soil of ~ M i c i o c o c c u ~ rhinicus that oxidized quinic acid b!. aromatization to protocatechuic acid :
quinjc acid
protocatechuic acid
Subsequently, several investigators including Gray and Thornton (23) (1 328) isolated soil bacteria capable of oxidizing phenol. tyrosine. and other aromatic rings. Happold (27) has presented a valuable survey of early work on this subject. In a series of papers, Tausson (67) described the isolation of soil bacteria by a n enrichment technique that attacked phenanthrene, naphthalene, and toluene. T h e phenanthrene organism grew well only on saligenin, salicylic acid, catechol, and phenanthrene as carbon sources and the chain of reactions shoTvn in Figure 1 \vas postulated. I n 1932, Happold and Key (28) began a study of the decomposition of phenols. thiosulfates, and thiocyanates in spent gas liquors by sewagr eiRuent bacteria. They isolated a n oxidase-positive strain of Vibrio which grew rvith phenol but not with cresol as sole source of carbon. Evidence \vas obtained in this work for the occurrence of catechol as a n intermediate in the oxidation of phenol and benzoic acid. Traces of a keto acid were also detected; this acid was subsequentl>VOL. 48, NO. 9
SEPTEMBER 1956
14 1 1
6
COOH
*
bH
k+J OH
OH
p-cresol
p-hydroxy benzyl alcohol
CHOHCOOH
p-hydroxy benzaldehyde
COCOOH
benzoyl
benzaldehyde
.
OH
~
mandelic acid
CGGH
tonization of the enol corresponding to the p, y-unsaturated lactone.
F\\,
Herbicides
b
p-hydroxy
protocatechuic
benzoic acid
acid
6-
o?/c\CO
OH
I
H2C\C,cO~ Hz
/>&-keto
QJ/
benzoic acid
Herbicides are mainly synthetic aromatic compounds and are daily finding more widespread use. Decomposition of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in soil was reported by Nutman, Thornton, and Quastel (46),Audus ( Z ) , Jensen and Petersen (33); it is attributed to activities of Bacterium glo biforme, Flavobacterium aquatile, and Corynebacterium sp. T o investigate the disappearance of 2,4-D in soil, Audus (1950) employed the valuable soil perfusion technique of Lees and Quastel (36) and succeeded in isolating a strain of Bacterium globgorme that grew well on a n agar medium with 2,4-D as the sole source of carbon. '4 garden soil required 14 to 28 days of continuous perfusion with 0.01% sodium2,4-D solution before a n enrichment population developed the maximum rate of 2,4-D decomposition (0.01 mg./gram of dry soil per hour). After seeding the soil with a suspension of the 2,4-Dadapted strain of B. globiforme this maximum rate of decomposition was obtained during the first day of perfusion. Evans and Smith (78) isolated from soil a small Gram-negative bacterium that grew well under aerobic conditions in a mineral salts medium containing p-chlorophenoxyacetic acid ( u p to 0.1%) as the sole source of carbon. From the culture filtrates they isolated 2-hydroxy-4-chlorophenoxyacetic acid (I) and 4-chlorocatechol (11).
Hi
adipic acid
OH
catechol
formic acid COOH
Figure 2
identified as P-ketoadipic acid by Kilby (34). By grinding bacterial cells with powdered glass, Parr and co-workers (48) in 1949, also obtained a cell-free enzyme system from Pseudomonas Juorescens which accomplishes ring cleavage with catechol as substrate. I n 1947 Stanier (60) described his technique of simultaneous adaptation; its use clearly brought out that essentially distinct pathways are involved in the initial attack on benzoic acid and p-hydroxybenzoic acid. Microbial attack on any simple aromatic ring compound is apparently the result of adaptive enzyme formation. This adaptation exhibits a high specificity and complete simultaneous adaptation to two or more aromatic substances after exposure to one of them occurs, only if the compounds are in the same reaction chain. By this method Stanier (1948) showed that the initial attack on benzoic acid was different from that on p-hydroxybenzoic acid; this been confirmed by several other workers. One reaction chain demonstrated by Stanier in pseudomonads com-
OH /
OH
phenol
salicylic acid
prised : mandelate+benzoyl formate+ benzaldehyde-tbenzoate. I n nature the decomposition of aromatic rings appears to be primarily a n oxidative process. Phenol and o-cresol are decomposed more slowly under anaerobic conditions, as found in some streams and lagoons, than under aerobic conditions (76). Two important reaction sequences in the enzymatic oxidation of aromatic rings by microorganisms are shown in Figure 2 . Evans, Smith, Linstead, and Elvidge (77) have examined the mechanism of oxidative decomposition of aromatic compounds by microorganisms and present evidence for the occurrence of cis-cis muconic acid and unsaturated lactonic acids as intermediates between catechol 8-ketoadipate (Figure 3). Both cis-cis muconic acid and ycarboxymethylene butenolide gave rise to P-ketoadipic acid when incubated with the ring-splitting enzyme obtained from a catechol oxidizing strain of Pseudomonas j'7uowscens. The course of this oxidation, therefore, involves addition of water, migration of the double bond, and ke-
cis-cis muconic acid
O
o
H
I
p-chlorophenoxy acetic acid
O H
J(OTH c1 I
CI
II 4-cMorocatechoJ
The latter substance, when added to adapted cells, underwent ring fission by
H2
/c\ C-OH
-
I1
H~+,COOH
H"
H2O
JT 52
migration
% double bond
TLQOOH
catechol
G
HC\ C'
C,O
CVooH H2
1
2,
COOH
Y - carboxy methylene butenolide
H2
H2
%/c\OOH
Jr
0 ' 8\>COOH
H
Figure 3
--
COOH
Hac\
AOOH
C H2
& -keto adipic acid
WATER PURIFICATION which chloride ions and organic acids were produced. Chlorine is not liberated until the aromatic nature of the ring is destroyed.
Guayule Resins It was shown over 40 years ago by Sohngen and Fol (58) that microorganisms can decompose rubber as well as the olefinic rubber hydrocarbon. T h e process has been studied by many investigators and the early literature is briefly reviewed by Busnell and Haas (7). Allen, Naghski, and Hoover (7) (1 944) isolated several aerobic microorganisms, including three different fungi, Streptomyes fradiae, and several bacteria that proved capable of clearing resin emulsions prepared from the guayule shrub (Parthenium argentatum Gray). .4 resin comprising the crude rubber extract was most readily attacked, whereas the unsaponifiable portion of this extract \vas cleared by only three of the isolates. An interesting aspect of this work: reminiscent of the difficulties encountered in studies on lignin decomposition, was the inability of any of the organisms to act on important constituents of the native resin when these were prepared in pure form. T h e cinnamic acid ester of a sesquiterpene alcohol, parthenylcinnamate (I), constitutes about 2070 of the exuded resin (67). Although resin emulsions containing I or partheniol (11) were cleared, I or I1 alone were not decomposed even on prolonged incuhation.
I t ivould appear to be of interest to take u p this question again, employing the techniques of soil perfusion, and enzyme adaptation. Oxidation of the cinnamate derivatives would most likely proceed by the benzoic acid route.
numerous species of basidiomycetese.g., so-called white rot of wood-few aerobic organisms seem to be capable of attacking lignin (66). Although some evidence has been advanced that mesophilic and thermophilic anaerobes can decompose lignin slowly, in general, under anaerobic conditions there are very few agencies capable of attacking lignin. The net result is that the lignin content of plant residues increases. particularly in peat bogs. Fischer and Schrader (79) consider that the accumulation of lignin under such conditions constitutes the origin of peat and of coal. There is some evidence that the presence of lignin interferes with the anaerobic decomposition of cellulose by bacteria, but this is still a matter of debate. The presence of cellulose and even of lower carbohydrates in preglacial woods has been reported; indeed, Olson, Peterson, and Sherrard (47) found that thermophilic anaerobes which carried out a n almost quantitative fermentation of cellulose could not ferment ground wood. A pulpwood of more than 1% lignin content \vas not fermented satisfactorily in their experience. Although lignin is the third most abundant constituent of the cell walls of plants, the principal reason so little has been definitely established concerning its decomposition has been, until recently, the difficulty of isolating lignin from plant material in a chemically unmodified form, .Although in its native form lignin is certainly attacked by aerobic microorganisms, especially mixed populations (62) and by basidiomycetes, it is clear that the drastic procedures which have generally been employed for the isolation of lignin yield a variety of modified materials.
CHO
By avoiding drastic extractants, high temperatures, and exposure to air, conditions which cause self-condensation or other modifications, Brauns (5) succeeded in the isolation of native spruce lignin. This was accomplished by taking wood from a freshly cut tree and pulverizing it to a meal: avoiding high temperatures in the process. Only inert organic solvents were employrd as extractants. T h e final product was native spruce lignin of constant methoxyl content (14.8%) and C = 63.87,> H = 6.1cT,. The methoxyl content is idrntical with that of spruce protolignin. Lignin has high carbon content and a relatively low hydrogen content; oxygen atoms are present mainly as -OH or OCH3, thus indicating that a considerable part a t least of lignin is unsaturated. There is considerable evidence indicating that as much as half of lignin may consist of aromatic rings of a benzene nature with a 3-carbon side chain. Harris and coworkers (29) isolated propylcyclohexane derivatives on hydrogenolysis of methanol maple lignin. This phenylpropane structure may be a basic unit in a lignin “polymer” in the opinion of many students of lignin chemistry. Relatively large amounts of vanillin (I) are obtained on alkaline nitrobenzene oxidation of gymnosperm lignin, while yields of vanillin plus syringaldehyde ( I I) amounting to 45% of angiosperm lignin have been obtained by this process. By treating maple wood meal with acid alcohol, Hibbert and coworkers (30) isolated the propyl vanillyl derivatives (I11 and I\’) as well as the corresponding syringaldehyde derivatives. The mounting evidence points more and more clearly to the accuracy of the view of Klason, a pioneer student of lignin chem-
CHO
COCHOHCH,
COCOCH,
lignin
To conclude this brief consideration on the decomposition of aromatic ring compounds, let us examine what is probably the largest natural reservoir of such compounds-lignin. Among the substances most resistant to decomposition in nature are the lignins, complex materials the composition of which is not yet fully understood. From the extensive investigations of Klason, Freudenberg, Erdtman, Hibbert, and Brauns, however, they are assumed to be high polymers composed of substituted aromatic rings. (Brauns has reviewed the chemistry of lignin in great detail.) il.’ith the exception of the action of
I vanillin
m
IT syringaldehyde
7p
$H=CH C H, CH=CHCH,OH
6H
AT
Y coniferyl alcohol
dehydroisoeugenol
V O C H ,
bH Figure
4
VOL. 48, NO. 9
SEPTEMBER 1956
14 13
istry, that the basic structure of gymnosperm lignin resembles that of coniferyl alcohol (V) which is widely distributed as its glucoside, coniferin (Figure 4). In angiosperm lignin the basic structure may comprise the monomethoxy- and dimethoxyphenylpropane structure found in dehydroisoeugenol (VI). Gottlicb and Pelczar (22) have revieived the extensive literature on microbiological decomposition of lignin and have pointed to numerous technical difficulties inherent in this work, such as failure to recognize that some material other than lignin in the "lignin substrate" might actually be responsible for supporting microbial growth and employment of nonspecific methods to assay lignin utilization. Pelczar and his coworkers a t Maryland appear to be the first to have employed native lignin, prepared after Brauns, in microbiological studies. This material was incorporated into a medium-containing glucose and used for the cultivation of white rot fungi. O n gradually \vithdrawing glucose from the medium, Poria subncida and Polyporus rihietinus could gro\v ivith native lignin as the sole source of carbon. Several strains of Polyporus imersicolor greiv \vel1 on the lignin on initial isolation, without benefit of the glucose-withdrawal acclimatization. Pelczar and coworkers also report the growth of aerobic bacteria in a mineral salts medium containing native lignin. pure sodium lignosulfonate, or coniderdrin as sole source of carbon. In continuously agitated cultures utilization of 90% of the conidendrin in 10 days was observed. From this short survey on the microbiological decomposition of lignin it is apparent that much of the confusion which has marked this field has been the result of technical deficiencies. Investigations may now employ a native lignin of reasonable purity that closely approximates the material found in nature. Application of the knowledge gained in the study of the decomposition of simple aromatic compounds to the lignin problem may prove of value. Employment of the technique of simultaneous adaptation \sill doubtless aid in tracing the metabolic pathways involved in the degradation of this aromatic polymer.
ance of nylon to microbial attack is \vel1 known. Microorganisms-bacteria, actinomycetes, 5-easts, molds, higher fungi, algae, and protozoa-are the chief agencies for the decomposition of carbon compounds in nature. I n the ultimate analysis. the mechanism of microbial decomposition of a carbon compound is that of enzymatic action. T h e question at issue is then, do microorganisms possess the enzymatic equipment to oxidize, hydrolyze. reduce, or in somr manner decompose materials that have never been seen in nature? In view of ivhat is known of the specificity of enzyme action, it would appear most unlikely that a synthetic polymer could be attacked by presently existing cnzymes unless the synthetic had great structural similarity to some natural material. Hon. great the structural similarity need be is considered in the discussion on decomposition of modified cellulose. The phrase "attack by presently existing enzymes" as used presupposes that enzymes not now in existence mighr somehow come onto the scene to o f f m the prosprct of the \i.orld being covered a t some future date Lvith a saran ivrapping. \V>-ss has ably discussed the subject of adaptive enzyme formation and its bearing on the subject of this symposium. Hotvever, attention is called to recent Xvork of Lederberg (,?5) and of Cohn and hlonod (9) on the induction of 8galactosidase formation in E. coli by the synthetic p-nitrophenyl-$-galactoside> Fvhich is hydrolyzed by the enzyme, and to the even more striking result obtained bvith a-galactosides (such as melibiose) and substituted @-galactosides which induce enzyme formation and yet are not substrates for the enzyme. Likewise, the herbicides 2,4-D and p-chlorophenoxyacetic acid are synthetics that appear LO induce enzyme formation for their oxidative metabolism along pathirays for the oxidation of aromatic rings, These observations bear only indirectly on the problem of microbial decomposition of synthetic polymers but do show that unnatural substances can induce adaptive enzyme formation in microorganisms.
T h e mesophilic cellulolytic anaerolrs. most likely to participate in stream purification, are the subject of a revieiv by. Hungate (32), Lvhile the thermophilic cellulolytic anarrobes have been treated by hlcBee (40). Somr years ago Fulmer (20) pointcd t o the potential industrial importance 0 1 treatment of cellulosic rrastes through a high temperature Clostridium fermentation. The combination of obtaining valuable by-products from waste matcrials that pose a difficult disposal problem is inherently attractive. Hajny. Gardner: and Retter (25'), have recentl!. revived interest in this field. The mechanism of cellulose decoinposition has been studied intensively it1 many laboratories in recent years. Siu (Ti?) has presented a detailed treatmcnt of the results obtained in the Philadelphiq laboratory of the Quartermaster C : O I , ~ S ivhere special attention has been paid i o the decomposition of cotton fibers b! filamentous fungi. Siu pointed to the stringent relationship betlveen molecular structure of ii cellulose and its susceptibility to rnicrobial attack. If there is a t least one substituent per anhydroglucose unit, rhc. modified cellulose is completely resistant. This is brought out in Table I for cyanoethylcellulos~ Tvhich was made b y the action of acr)-lonitrile on cellulose :
HCX
-+
ROCH?CHz(:N
It is \vorth noting that cyanoethylation of starch makes that normally easily dccomposed material relatively immunc. to bacterial ferrnen ta tion. .idditional rxamples of the prowcti\-c.
Table I. Decomposition of Modified Cellulose (Cyanoethylcellulose) b y Myrofhecrum verrucaria (56) so. (
yarioethyl
90
11 eayht
Loss aft?,. -liihi/rlroylucose 13 D a y s T nit r?t.cubatL O I I 0 80 1.02 29 1.43 6
( c .\- a i
Sfibstztucnts'
lfodificti eiiulosc
(
0 6.64
8.43 9.98 11.44
1.85
2
2.35
0
Cellulose Decomposition Decomposition of Synthetic Polymers
At this point it appears to be appropriate to insert a note on the resistancr of synthetics to decomposition by natural agencies. The total output of high polymer synthetics by the chemical industry of the world may be small in comparison with total s>-nthesis of pol! mers by plants on land and in the sed, nevertheless synthetic waste materials may pose a problem to the natural agencies of decomposition. T h e resist-
14 14
'The microorganisms responsible for decomposition of cellulose in ri\ws and other flowing water systems are chiefly anaerobic bacteria, including such species of Clostridium as C. cellulosne-dissolL'ens, C. ce!lulosoluens, and c'. celluloljticuni (65). T h e degradation of cellulose is apparently brought about by extracellular enzymes released by these bacteria. Two stages in the cellulolytic decomposition are recognized: cellulase action results in the appearance of cellobiose, followed by cellobiase Xvhich c,onverts the disaccharide to glucose.
INDUSTRIAL AND ENGINEERING CHEMISTRY
Table II. Decomposition of Cellulose Derivatives by Myrothecrum verrucaria
(57) 4t '\O \ubstztuents Ircrliaiillli
If f
f7?11t
r,oa
f
Filter paper Oxldized cellulose Methylcellulose Ethylcellulose Cellulose acetate Tosylcellulose Carboxymethylcellulose
c(
d7ihgdroglitcoic 0
..
2.0 2.25-2 - 5 8 1 . 0 t22.3C', AcO)
1.18 2.01 0.067
69 11 8 0 0 0
56
WATER P U R I F I C A T I O N effect exerted by a variety of substituents on the anhydroglucose ring are shown in Table 11. Cell-free preparations lvith cellulolytic activity are readily obtained. .A cellfree filtrate of a culture of ,$I. z'prrucaria gro\rn on cellulose contains cellulase activity. T h e enzyme is apparently of a n adaptive nature since filtrates from cultures gro\vn on other substrates d o not possess cellulase activity. Reese and Gilligan (57) ha\,e separated components o f fungal cellulolytic systems by paper chromatograph!.. Cellulolytic activity [defined as the production of reducing sugar from carboxymethylcellulose) in culture filtrates showed three components o n paper chromatograms, depending on species employed and the conditions of gro\vth. In contrast to the findings reported for fungal cellulase, formation of bacterial cellulase by species of Cellulomonas, Cellz'ibrio: and Clostridium appears not to be adaptive. Harnmerstrom! McBee, Claus, a n d Coghlan ( 2 6 ) found that cellulase is formed even when these cellulosedecomposing bacteria were groivn on glucose or cellobiose as the source of carbon. T h e appearance of phosphorylated cellobiose as a n intermediate in the microbial decomposition of cellulose has also been reported by these authors. This observation opens the possibilit). that the initial attack on cellulose may! in some instances, be phosphorolytic rather than hydrolytic. I t must be borne in mind that the tivo modes of attack on a polysaccharide are not mutually exclusive. Starch and glycogen are depolymerized by extracellular diastatic hydrolysis lvhile glycogen (a microbial assimilation product) is also depolymerized by intracellular phosphorolysis as a result of the action of phosphorylase.
A
\E\
Decomposition of Chitin and Keratin
Other examples of microbial decomposition of relatively resistant high polymers are seen in the breakdown of chitin of keratin as a result of the action of hydrolytic enzymes secreted by a few organisms. Reynolds (52) sho\ved that a species of Streptomyces produces a n exocellular chitinase. Formation of this enq-me was induced specifically by the presence of chitin in the culture medium. As products of the hydrolysis, Rc!-nolds demonstrated chromatographically the appearance of AY-acetylglucosamineand the corresponding disaccharide, . diacetylchitobiose. This latter amino sugar was subsequently proved by isolation. Several bacterial types have been obtained from soil-enrichment cultures ivhich are capable of utilizing keratin. A species of Streptomyces has been found particularly active in this respect: 1- to 3-gram quantities of hoof meal per 100 ml. of culture medium are completely solubilized in 24 hours by this organism during growth (J5). Undenatured \roo1 (extracted with water and ether and sterilized with ethylene oxide) is somewhat more resistant to attack, but in cultures containing such ~voolas the sole source of carbon and nitrogen, SO%, or more of the ~ ~ o was o l solubilized in 8 days. Heat-labile keratinase activity is present in cell-free filtrates from cultures groivn on wool or hoof meal and the activity of the enzyme is not decreased on dialysis. Elaboration of this exocellular enzyme is induced by some constituent grouping of keratin. Filtrates from cultures grown on glutamic acid possess typsinlike proteolytic activity hut sho\v no keratinolytic activity:
\.
Fermentation products CO2 Assimilation products and cell s y n t h e s i s
G E N E R A L I Z E D , OMNIVOROUS M I C R O B I A L CELL
Figure
5
cultures grown on peptone show almost as much keratinolytic activity as those grown \vith keratin as the solc source of nitrogen. Although soluble disulfide increases during the digestion of \voo1 by the filtrate, no free -SH has been detected. even Lvhen the digestion is carried out under nitrogen. Optimum digestion of both ~vooland casein occurs a t p H 8.5 io 9.0. T h e chelating agents, disodium ethylenediaminetetraacetic acid (Sa,EDTA) (1 O-4a\.I)and 8-hydroxyquinoline (10-4,M)% inhibit both proteolytic activities. T h e activities are differentiated by c)-anide (lO-RAld) which stimulates and p-chloromercuribenzoate (1 0 -4.Mj which inhibits ~vooldigestion; neither of these substances has a n effect on casein digestion. Intracellular Transformations of Carbon Compounds
\Ve have seen how the routes of oxidative decomposition of a wide variety of aromatic ring substances converge through catechol and protocatechuic acid into P-ketoadipate. I n like manner the initial hydrolytic attacks on a variety of polysaccharides lead in a few steps to one of the fe\v naturally occurring hexoses or to one of the few pentoses. Similarly, enzymatic hydrolysis of any fat yields glycerol and one or two of the few fatty acids occurring in nature. This amounts to stating the converse of that fundamental proposition of comparative biochemistry that from a few building stones nature has. by suitable permutation of a few seemingly simple procrsses of condensation, constructed a vast array of polymers. T h e action of hydrolytic enzymes secreted by the microbial cell causes the depolymerization of a variety of large molecules, including many too large to permeate the cell boundary. Cellulose is hydrolyzed by cellulase, chitin by chitinase, starch by amylases, and so on. These exoenzymatic decompositions d o not yield any energy to the cell hut d o serve to produce substratcs of smallel, size that can permeate the ccll boundary. I n some instances a second battery of hydrolytic enzymes, localized in the cell surface (and not diffusing or sccreted out of the cell) may come into plal- to decompose oligopolymers into monomers. Examples that come to mind are invertasc. acting on sucrose and $-galactosidase on lactose. T h e fascinating area in cellular physiology concerned \vith study of the mechanism of uptake of substrates by thy cell has made notable advances in recent years, due in large measure to the employment olisotopically labeled materials, but Lve must forego discussion of these details and assume that the small molecules produced by enzymatic hydrolysis enter the cell and become sub,ject to thr VOL. 48, NO. 9
0
SEPTEMBER 1956
1415
oxido-reductive action of enzymes localized within the cell. T h e patterns of reactions discussed thus far are illustrated schematically in Figure 5. These patterns, generalized symbolically, are shown in Figure 6.
C - c p d P\ I
Dissimilation of Carbon Substrates
1
Om
C - c p d PN)
=
=
=
Figure 6. P polymer, 0 oligopolymer, and M monomer, N i s indeterminately large, rn i s a much smaller number perhaps of the order l o 3 or lo4, and k i s some potentially determinable number of the order lo'?; a and u indicate distinct pathways of which an order of 10 may exist in a cell
PATHWAY O F ALCOHOLIC F E R M E N T A T I O N GLYCOGEN
D-GLUCOSE
11
phosphorylase
'
[G-l.b-diP]
GLUCOSE- I - P (Cori ester)
'
phosphoglucomutase
G L U C O S E - 6 - P (Robison e s t e r ) phosphohexoisomerase
::>
F R U C T P S E - 6 - P (Neuberg e s t e r ) phosphohexokmase
FRUCTOSE 1 , 6 - d 1 P (Harden-Young e s t e r ) tal
",zHJ 1
D-NYDROXY AC E T O N E - P (Kiessling e s t e r )
L-
( F i s ch e r - B a e r e s t e r )
isomerase
b e h ydrogenase
a -GLYCEROPHOSPHATE
1. 3 DI-PHOSPHOGLYCERATE (NegelemlBromel ester) ~
phosphatase GL*EROL
t H ~ P O ~
glycerikinase
ATP
I1
3-PHOSPHOGLYCERATE (Embden-Nilsson e s t e r )
1,3-:;,:-glyceric
1
11
phosphoglyc e r o r n u t a s e
IVe cannot go into detail concerning the intermediate metabolism of the various kinds of substrates that are brought into the cell. An intensive symposium in the Division of Biological Chemistry is being devoted a t these meetings to a few topics in this fast developing field. and, in a recent excellent review Gunsalus, Horecker, and Wood (2.1) devote 50 pages to the consideration of pathwa!.s of carbohydrate metabolism in microorganisms. We can, however, examine some of the main features of intracellular transformation of carbon compounds. I n the 60 years that have elapsed since Buchner accidentally discovered cell-free alcoholic fermentation of yeast, the numerous intermediate reactions involved in this transformation of sugar into ethanol and carbon dioxide have been elucidated. I n similar rnanner the metabolism of glucose by coliform organisms to yield a variety of products has been intensively studied. These pioneering investigations, with which the names of Harden, Neuberg, hfeyerhof, and Kluyver are intimately associated, contributed not only important factual information on microbial metabolism but established the generalizations that substrates are transformed enzymatically by single-step reactions, involving phosphor)-lation and coenzyme transfer of hydrogen and electrons (Figure 7). For man)- of the major groups of microorganisms that have been grown in pure culture, information is now available on the principal metabolic pathways employed in their aerobic or anaerobic degradation of substrate. The phosphorylative glycolytic route characteristic of yeast is found to be operative in certain other organisms. each employing variations in pattern following the "pyruvate interchange" to provide the products characteristic of the organism in question. Recently, important nonglycolytic pathways have been found to operate in some organisms.
2 - P HOSPHOGLY C E R X T E
2 HzO
eEi::e
P-ENOLPYRUVATE
carboxylase
DpN2 J1
ATP PYRUVATE
a l c o h o l dehydrogenase DPN ETHANOL
Figure 7. Embden, Meyerhof, Parnas scheme of glucose metabolism passing through hexose diphosphate
14 1 6
INDUSTRIAL AND ENGINEERING CHEMISTRY
Metabolism of Glucose
\Ye may focus attention on a few of the path\vays for decomposition of glucose that have been established as operative in certain microorganisms. There are certain features in common among many of these, as well as fascinating instances of species individuality. Phosphorylation of glucose by hexokinase to yield glucose6-phosphate (G-6-P) is a reaction carried on in many microbial types. From G-6P there runs the well known glycolysis
WATER P U R l F l C A t l O N route (Embden, Myerhof! Parnas scheme) which has been demonstrated to be operative in a wide variety of microorganisms including yeast (Saccharomyces cerevisiae), Escherichia coli, Streptococcus fecalis: Clostridium thermoaceticum, and Lactobacillus casei. A detailed "metabolic map" of the system shown in Figure 7. Another point of departure from G-6-P is the ribulose phosphate pathway, the existence of which in yeast and in E. coli was indicated some time ago by studies of Warburg ( 6 9 ) ,Lipmann (37, 38), and Dickens (73) and, more recently. intensively studied, especially in Azotobacter r'ineiandii, by Horecker (37),Racker (50), and Cohen (55). I n Pseudomonassaccharophila the operation of yet another road leading from G-6-P has been uncovered by Doudoroff and designated the 2-keto, 3-deoxy-6-phosphogluconate pathway (47). The occurrence of the closely related substance, 2-keto-~-gluconate-6phosphate (2-K-6-P). as a n intermediate in the oxidation of 2-ketogluconate by adapted cells of Aerobacter cloacae has been demonstrated by DeLey ( 7 7 , 72), and in Pseudomonasfluorescens by Wood (77). An adaptive enzyme, 2-ketoglucokinase, catalyzes transphosphorylation from adenosine triphosphate (,4TP) to 2-ketogluconate yielding 2-K-6-P. Comparison of the guide maps for these routes may be made as in Figure 8. To facilitate the comparisons, some known intermediate steps are omitted.
actual mechanism of this beta oxidation has become clear (42). A union of fatty acid with Coenzyme A occurs between the carboxyl group of the acid and the sulfhydryl group of Coenzyme A to yield a thioester termed acyl CoA :
only a fraction of the carbon substrate that disappeared could be accounted for as carbon dioxide while the remainder was incorporated into a cellular material with the empirical composition of a carbohydrate (CHZO),. For the colorless alga Prototheca Zopfii Barker showed CHa(CHn),CH2CHnCOOH that the over-all reaction could be exHS-COA + CHa( CH~),CH~CHICOSCOA pressed as (1) for glycerol oxidation and (2) for acetate: This is in turn dehydrogenated by a 2C3HsOa 202 + metalloflavoprotein (termed acyl CoA COS 3 H 2 0 j(CIH.0) (1) dehydrogenase) of which two varieties are CHzCOOH On + involved: acyl CoA with chain lengths CO.2 HyO (CHzO) ( 2 ) from CISdown to C? is oxidized by what is apparently an iron flavoprotein, while The term oxidative assimilation was proposed for the process. Many Ivorkers from Ca to Cs a copper flavoprotein is involved : have since studied the process in a wide variety of microorganisms; both oxidaCH,(CH~),CH,CH,COSCO.~ CH $(C H ~ ) , , C H = ~ C H C O S C O Ative and fermentarive assimilations have been found, and assimilation reactions The unsaturated product is hydrated are observed wherever sought for and with most metabolizable substrates. and, by reaction with another molecule Thiinann (63) in his delightful volume, of Coenzvme A, releascs a two-carbon fragment as acetyl (20.4: "The Life of Bacteria" has a most interesting account of this chapter in microbial CH3( CHr),,CHOHCHlCOSCo.I + physiology. HSCnX + In most short term experiments not all the substrate carbon supplied to a "resting-cell" suspension can be accounted for In this manner the fatry acid undergoes as metabolically produced carbon dibeta oxidation with the generation of the oxide-that is, total COz liberated by energy-rich substance acetyl CoA a t each the time the substrate disappears from two-carbon step down the ladder. the medium, which coincides with the decline in substrate-induced rate of oxygen consumption, or fermentation. Carbon Assimilation However. if the period of observation is and Cell Synthesis prolonged, say to 24 hours, one can freFatty Acid Oxidation The work of Marjorie Stephenson quently recover as C o t all the carbon about 25 years ago provided indications The classical scheme for the biological supplied. This is actually the basis of that nonproliferating suspensions of oxidation of a fatty acid was termed beta the quantitative analysis for mixtures of microorganisms do not metabolize suboxidation by its proponent, Knoop. I n sugars that Kluyver introduced in 1914. strates to completion in short term experithe past few years as a result of the neoA succession of yeasts is employed to ments. Both Barker (3) and Giesberger classical investigations of Lipmann (37, effect the removal first of glucose, fruc(27) in 1936 demonstrated with non38),Lynen (39), Stadtman and Barker tose, and mannose, then of galactose, proliferating cells of microorganisms that (59) and Green and coworkers ( 4 ) the and subsequently of sucrose, or maltose, or lactose, or raffinose. The amount of carbon dioxide produced in 24 hours by glucose each of the succeeding yeasts gives a n ATP Hexokinase accurate measure of the amount of each sugar present in the mixture. An interglucose - 6 - P esting and valuable application of this I 3 dual principle of assimilation-that is (l), rapid temporary storage of intracellular polysaccharide reserve, glycogen 2 - keto, 3- deoxy, 6 P - gluconate fructose I>6 - di P in some instances, and (2) subsequent 6 P - gluconate slow oxidation of the store after external metabolites have been utilized-has been made by Porges, Jasewicz, and Hoover ribulose 5 - P (49). By calculated adjustment of the triose - P + pyruvate tr lose - P alternating phases of feeding and starvation, a microbia1 population was enabled triose - P + "diose" to dispose of large amounts of dairy waste without proliferating so as to necessitate removal of the cell debris as py ruvot e py r uvot e pyruvate sludge. It has been known for some time that the relative rates of oxidation EMP route Ribulose phosphate route 2 k - 3d - 6 P route and assimilation in microorganisms are Figure 8. Comparison of three routes of carbohydrate oxidation originating affected by variation in the culture from glucose-6-phosphate history (44).
+
+
+
+ +
+ +
+
I
\I/
I
1
-
1
-
1
1
-
I
I
VOL. 48, NO. 9
SEPTEMBER 1956
1417
mannan
glycogen
ma nn o sc'- I- p ho s pha t e
II
trehalose
glucan
-+ g 1u c o s e
NnF
\
J/
fructose-6-phospha t e Figure 9. Diagram of pathways leading to polysaccharide synthesis in growing yeasts, illustrating loci of fluoride-sensitive reactions
Although assimilation of substrate to polysaccharide stores may occur as a n oxidative or fermentative process, the subsequent utilization of the intracellular store can be accomplished only aerobically by most facultative organisms. For example, anaerobic fermentation in the absence of added substratethat is, carbon dioxide production a t the expense of reserve stores-is unknown in all but a very few species of yeasts (43). It is therefore significant that in the process described by Hoover and coworkers a high degree of aeration is maintained during the starvation period in which assimilated products are being oxidized. Both polysaccharide and nitrogenous assimilation products are involved in the mixed population handling dairy wastes and the follo\ving relationship was obtained experimentally for utilization of skim milk: Ci2Ha?Oii
+
+
CiHi~Ii;a03 6 0 2 + 2C5HjNO2 6'202 7Hr0
+
+
(3)
\vhere C1?Hz?OI1 = lactose, and C&IIrN?Os = empirical formula for casein. The assimilated product: expressed as CbH7TY02: is undoubtedly a composite of assimilated polysaccharide and nitrogenous product. It is interesting to compare these results with those obtained some years earlier by Schade and Thimann 1.53) for the water mold Lrptomitus lactrus, \vhich is an important organism in the ultimate decomposition of dilute se\vage in brackish v'aLer. Cultures supplied with the amino acid 1-leucine show a n increase in oxygen consumption, but the substrate is not completely oxidized, nor is it deaminated. T h e net equation indicates assimilation of a nitrogenous material with the state of oxidation of alanine :
+
C4HSCHNH2COOH 4 1 / 2 0 2+ (CsH,NO?)
14 1 8
+ 3H:O + 3CO2
(4)
Biosynthesis of Carbon Compounds-Polysaccharides
it-hereas reactions involved in the degradation of substrates are becoming more and more thoroughly understood, our knowledge of the synthesis of cellular components is, by comparison. extremely meager. Significant information is available on the biosynthesis of a few polysaccharides. but for the other poly-mers the route of synthesis is still retained as a secret by the cell. O n e direction of synthesis essential to the gro~vthof yeasts (and. quite likely, \vith some variation to many other organisms) may hc depicted as in Figure 9. T h e in vitro synthesis of glycogen from glucose-1-phosphate has been intensively studied by the Cori's (70) and by other workcrs. The synthesis results through glycosyl transfer from glucosr- 1 -phosphate to a pre-existing polymer chain, the coordinated operation of t\\o enz)-mes being necessary to ubrain the characteristic linking and branching of glycogen. The in vitro synthesis of mannan, glucan. or chitin has not yet been realized. but studies on the hexoseI-phosphate reversal of fluoride inhibition of growth support the vie\v that these pol>-saccharides also originate from hexose-1-phosphate esters (8). The coordinated operation of sevrral enzymes might hc necessar!' to achievc the s!mthesis ol' polysaccharides lvith thr branching characteristic of yeast mannan or of' yeast glucan (70). Carbon Skeleton of Amino Acids
Conversion of carbohydrate substrate into the carbon skeletons of ct'rtain key amino acids has been sho1i.n for bakers' yeast: 7'orulopsis utilis. Eschrtichici coli, and other microorganisms to result from the operation of the tricarboxylic acid cycle
INDUSTRIAL AND ENGINEERING CHEMISTRY
(Krebs cycle). Studies employing labeled glucose, pyruvate, or acetate have shoivn that aspartic and glutamic acids are spthesized by condensation of ammonia \vith the respective keto acids, oxaloacetic and a-ketoglutaric, which are derived from oxidation of substrate via the Kreb's cycle (74, 53, 68). These key dicarboxylic amino acids are themselves precursors of families of amino acids. Both proline and arginine arise from glutamic acid. while from aspartic acid there are derived lysine, homoserine. methionine. threonine, and isoleucine (53). By a n ingenious use of prolineless mutants of E. coli. Davis has demonstrated that glutamic y-semialdehyde, and pyrroline-5-carboxylic acid are intermediate stages in rhe conversion of glutamic acid into proline (6J). Detailed knoxvledge of the routes of biosynthesis of amino acids, purines, and other important building stones for cellular polymers is becoming increasingly available. A s this information becomes assimilated. the formidable intricacy of the cellular regulation of this multiplicity of reactions is increasingly apparent. T h e biochemical patterns involved in the processes, Xvhich in their totality comprise cellular growth and differentiation, are gradually being revealed. Bibliography
( 1 ) Allen, P. .I..Piaqhski, J., Hoover, S . R., J. Bacterial. 47, 559 (1944). ( 2 ) Audus, L. J., ,l'a!ure 166, 356 (1950). ( 3 ) Barker, H. A . , J . Cdl. Comp. - Physiol. .
8, 231 (1936). ( 4 ) Beinert, H., Bock, R., Goldman, D., Green. I).E.. Lfahler. H. R.. X i . S.;Stansley,'P. G., iVaki1, 8. J.: J . .4m. Chern. SOL.75, 4111 (19533. ( 5 ) Brauns, F. E., Zbid., 61, 2120 (1939). ( 6 ) Brauns, F. E., T h e Chemistry of Lignin," .Academic Press, N. Y . , 1952. ( 7 ) Busnell, L. D., Haas, H. F., J. Barteriol. 41, 653 (1941). ( 8 ) Chung, C. \V., Nickerson. W. .I.: J . Bioi. Chem. 208, 395 (1954). ( 9j Cohn, bf.,Monod, ,J., in "Adaptation
in Slicro-Organisms," University Press. CamhridTe, Eng., 1953. I, 10) Cori, G. T., Colowick, S. P., Cori, C . F., J . B i d Chem. 123,375 ( 1 9 3 8 ) ; 124, 543 (1938). ( 1 1 ) DeLey, J.: Enqmologia 15, 55 (1954;l. ( 12) DeLey, J., .\htiir~issensch~j~en42, 96 (193). ( 1 3 ) Dickens, F., Hiochrm. J . 32, 1626 (1938). I 1 4 ) Ehrensvgrd, I,.. Reio, L.. Saluste. F'., Stjernholm, R., J . B i d . Chern. 189, 9'-3 (1951 ). \ - - - - ,
L.;rnmmlin~, O., .\bderhalden,
f;.?
Zent. f,bok!.II! 10, 339 (1903). Ettinger, I f . B., ?\.loore, W. :L, Leshka, R. J., IXD.C s C . CIIEM. 43, 1132 (1952). Evans, \$'. C . , Smith, 13. S. \,V., Linstead, K. P., Elvidse, J. A., .Ya/ure
Evans: LV. chem. J. 5 7 , xxx (1954). 1 9 ) Fischer, F., Schrader, H., "Entsrehuna und chemische Struktur der Kohle." Girardet, Essen. Ger., 1922.
WATER P U R I F I C A T I O N Fulmer, E. I.: ISD. ENC.CHEM.28, 778 (1936).
Giesberger, G., Dissertation, Utrecht, Ger.. 1936. Gottlieb, S., and Pelczar, M. J., Jr., Bacterid. Rezs. 15, 55 (1951). Gray. P. H. H., Thornton, H. G., Zentr. Bakterioi. 11, 73, 7 4 (1928). Gunsalus, I. C., Horecker, B. L., \Yood, \V. .4.,Bacterial. Reos. 19, 79 (1955).
Hajny, G.! Gardner, C. H., Retter. G. J., IND. ENG. CIIEM.43, 1384 (1951).
Hammerstrom. R. .4., McBee, R. H.. Claus, K. D.? Coghlan. J. W.. Bacterzol. Procs. 1954, p. 106. Happold, F. C., in “Biological Oxidation of Aromatic Rings,” University Press, Cambridge, Eng., 1950. Happold, F. C., Key, X., J . Hygiene, Comb. 32, 573 (1932). Harris, E. E., D’Ianni, J., and Adkins, H., J. =ini. Ciiem. SOC. 60, 146’ (1 938 ).
Hibbert. H., I n n . Rer,. Biochem. 11, 183 119421. -, \
Horecker, B. L., Smyrniotis. P. Z . , Seegmiller, J. E., J . Biol. Chetn. 193, 383 (1951).
Hungate, R. E., Bacterid. Rew. 14, 1 (1950).
Jensen, H. L., Petersen, H. I.: ;Icto .4gri. Scand. 2, 215 11952). Kilbj., B. X . , i s t Znt.‘ Conp. Biochem. Abstracts, p. 315: 1949. Lederberg, J.? J . Bnctrriol. 60, 381 (1950).
Lees. H.. and Quastel. J. H., Bioc h i . J . 40, 803 (1946). Lipmann. F., Bocteriol. Rers. 17, 1 (1953 ).
DISCUSSION
(38’1 Lipmann: F.? .\-atrue 138, 588 (1936). ( 3 9 ) Lynen. F., Federation Proc. 12, 683
(56) Siu, R. G . H., “Microbial Decomposition of Cellulose,” Reinhold, York, 1951. ( 5 7 ) Siu, R. G. H., Darby, K. T.? Burkholder: P. K.. Barghoorn, E. S., 7extile Research J . 19, 484 (1949). (58) S8hnqen. S . L.. Fol, J. G., ZeJlt. j. Bmit. 11, 40, 87 (1914). (59) Stadtman. E. R.: Barker, H. ‘4..J . Bud. Chem. 180, 1095 (1949): 184, 769 (1950). (60) Stanier. R. \-., J . Bactrr~ol. 54, 339 ( 1 9 4 7 ) : 55, 4-’7 (1948). (61) Tausson: W.C . , P ~ U J 4, Z ~214 R (192:); 5, 239 ( 1 9 2 8 ) ; 7, 735 (1929). (62) Tenney, F. G.. IVaksman, S. A . , Soil Sci. 28, 55 ( 1 9 2 9 ) ; 30, 143 (1930). (63) Thimann. K. V., “The Life of Bacteria,“ 12\lacrnillan, New York, 1955. (641 Vogel, H. J.. Davis. B. D., J . .4m. Chem. Sgc. 74, 109 (1952). (65) Waksman. S. -4,. Botan. Reo. 6 , 637 (1940). (66) IVaksman. S. .\,, in “Wood Chemistry,“ Reinhold, S e l v York, 1944. Chem. Soc. 66, ( 6 7 ) IValter, E. D.. J . :h. 419 (1944). (68) IVanq, C. H.. Christensen, B. E., Cheldrlin. V , H.. J . Bioi. ChPm. 201, 683 (1953): 213, 365 (1955). (69) tt‘arburg-, O., Christian. tV., K O diem. %. 242, 206 (1931 I ; 292, 287 (1937). (70) IVhistler, R. L.. Smart, C:. I>.,“Polysaccharide Chemistry,” Academic Press: Scw York, 19.53. (71) \l‘ood, \V. .A,, Rncteriol. h’et~s. 19, 222 (1 955 11.
(1953). (40) LlcBee, R. H., Bacterioi. Reis. 14, 51 (19SO). --, \ -
h4acGee, J., Doudoroff. XI.. J . Bioi. Chem. 210, 617 (1954). Mahler, H. R., Green. D. E., Science 120, 7 (1954). Nickerson, I$‘. J., in “Biology of Pathogenic Fungi,“ Chronica Botanica (26.%M’althi%n. Mass.. 194’. ( 4 4 ) Sickerson, \V. J., Carroll, I V . R.? J . Cell. Comp. Physiol. 22, 21 ( I 943). ( 4 5 ) Noval, J., Nickerson, LV. J., R ~ c f . Procs. 1956, p. 125. ( 4 6 ) Nutman, P. S.. Thornton, H . G., Quastel; .J. H., .Vatitre 155, 498 (1945 ). ( 4 7 ) Olson. F. R.. Peterson, It‘. H.. Sherrard, E. C;.? IND.ENG. CHEM.29, 1026 (1937). ( 4 8 ) Parr, \V. H., Evans, R. .I., Evans, \\-. C., Biochem. J. 45, vsis (1949). ( 4 9 ) Porges, N.: Jasewicz, L.: Hoover, S. R.. Abbl. .26icrobiol. 1. 262 11953). ( 5 0 ) Racker, E., .idronces i n knzwid. 15, 141 119541. Reese, E. T., Gillig.an, \V.. .4rch. Biochem. Biophys. 45, 74 ( 1 9 5 3 ) . Reynolds. DI 121.. Bacterid. Procs. 1954, p. lo7.
\
‘ 1
Roberts, R. B.. Abelson. P. H., CoLvie. D. B.. Bcdton, E. T.,Britten. R. J., ‘.Studies of biosvnthesis in Escherichia coli,” Publ.’ 607, Carnepir Institution of LVashington. 1955. ( 5 4 ) Schade, A. L., ‘Thimann: K . \’.. Am. J . BO/RIIV 27. 659 11940). (55) Scott, I). B. xi.,Cohen, S: s.,J . Hioi. Chrm. 188, 509 (1951 ).
I
RECEIVED for rrview January 12: 1 9 5 6 . ; ~ C E P T E DApril 25, 1956.
...
Transformations of Carbon D r . Nickerson’s excellent review can be related to waste disposal in a direct manner, for he discusses the fundamental biochemical reactions that are carried out by pure cultures of microorganisms in the oxidation of individual chemical compounds. I n waste disposal practice complex mixtures of chemical compounds are oxidized by mixed cultures. Let us attempt to see where the theory and practice coincide. Oxidation of Carbon Compounds
I n considering the oxidation of compounds containing carbon, hydrogen. and oxygen, \ve shall divide them into three classes--carbocyclic compounds. detergents and related compounds. and resistant small molecules. Carbocyclic Compounds. T h e fact that phenol, cresol, and related carbocyclic compounds have long been known to undergo rapid biochemical oxidation in trickling filters and activated sludge
by Microorganisms
systems comes as a shock to the bacteriologist !Tho thinks of such compounds only in relation to the “phenol coefficient.!’ Nevertheless. pioneer studies demonstrated the oxidation of phenolic compounds in municipal treatment plants in England before 1900. Similar and quite detailed studies Icere carried out a t hlilivaukee? a t Chicago, and recently a t Gary: I n d . ? all demonstrating that such Isastes are readily oxidized in conjunction lvith sewage in activated sludge plants. T h e first industrial waste disposal plant designed to oxidize phenolic \castes ivas that of the Dow Chemical Co. a t Llidland, Slich. (6). Recent installations. patterned after the Slidland studies, are in operation a t Sarnia. Ont.. and there are doubtless a number of Ivhich I a m not aivare. -4noteivorthy feature is that chemical-grade phenol is added to such units \\-hen the plant is shuc down. thus keeping the culture happy a n d well fed (3! 7 7 ) .
An important ieature of the biologica oxidation of phenols is that these compounds arc cvidcnt to taste in remarkably low concentrations, and the taste threshold of their derivatives produced by chlorination of tvater is yet lower. Therefore. they must be essentially removed by biochernical oxidation from ivater that might later be used for a city supply. T h e terpenes and rosin waste from tvood naval stores production is a related lvaste that has been oxidized successfully in pilot plant studies ( 7 ) . I t is almost certain that these can be treated when the need arises. Paper mill \vastes: consisting of lignin derivatives. pentose. and hexose sugars Lvere long considered to be resistant to biochemical oxidation. The solution to this problem rested on the demonstration by various Lvorkers. notably Sa\vyer and his students (7, 8). that nitrogen and phosphorus must be supplied to meet [he nutritional requirements of the 1VOL.
48, NO. 9
SEPTEMBER 1956
14 19
organisms. High rates of oxidation have been obtained in pilot plant studies and a t least one commercial installation is in operation a t the \%’est1-irginia Pulp and Paper Co., Covington, Va. (72). Detergents and Related Compounds. These important compounds that have the common property of being surface active or of containing a polar and a nonpolar portion Xvithin the molecule are mentioned as a class, but it is essential to emphasize that they do not constitute a group of compounds that have a comparable ease of oxidation. They differ widely in susceptibility of oxidation because they differ in chemical nature. This point has been made before, especially by Bogan and Sawyer (2). Their work and that of Mills and Stack (73, 74) has shown that synthetic organic chemicals can be roughly classified as follows:
important in industrial waste practice, to which I wish to call attention. Formaldehyde is a potent bactericide, yet Dickerson and associates have successfully treated it by aerobic oxidation (4). Pentaerythritol, C(CHZOH)I, is also oxidized in the same treatment plant. This compound shows no B.O.D. by the usual technique and also passes through animals without toxicity or chemical change. The presence of readily available carbon compounds \vas shoivn to assist the oxidation of pentaerythritol. The development of a culture which oxidizes these compounds \vas a relatively slow and difficult job; acclimatization of the culture to the unusual compound was shown to be important both here and in the laboratory studies of Mills and Stack (73. 74). Common Pathways of Oxidation
Alkyl sulfates Fatty acid derivatives Alkylaryl sulfonates Alkylphenoxy sulfonates Polyethoxy fatty acid esters Ethers and seconddry amines Acrylonitrile Heterocyclic ring structures Polyglycol ethers
‘\ Readily Oxidized
J
1 I
1
Oxidized ;ikulty
J Resistant / to
J
Oxidation
Oxidation of Synthetic Organic Compounds
Branching of the alkyl group, not indicated in the above listing, has been proved to reduce markedly the susceptibility of alkyl groups to aerobic assimilation (2). hlost of these compounds are detergents; they occur in the wastes from the textile and chemical industries and from the use of these compounds in industry and in the home. They and related synthetic compounds yet to come into large scale use will furnish a continuing challenge to the sanitary engineer, one in which he can draw upon the results of basic investigators of bacterial metabolism to great advantage. A noteworthy example i s the high molecular weight polyethylene glycols, lvhich contain the repeating unit, (-CH2--CH~-O-),, and are thereby polymeric ethers. As yet no one has demonstrated the necessary extracellular enzyme that must be secreted before these compounds can be broken down sufficiently to enter the cell and be metabolized aerobically. Resistant Small Molecules. Almost all of the compounds containing 2-5 carbon atoms are metabolites in the cell or contain groups readily oxidized to cell intermediates. Nickerson has indicated these relationships very clearly. There are also several noteworthy exceptions,
1 420
T h e common path\vays of oxidation were clearly shown by Nickerson. T h e compounds in which carbon atoms are combined with oxygen (carbohydrates) or nitrogen (proteins and amino acids) as well as the simple aliphatic residues (from fats and amino acids) readily enter the metabolic pool of the organism and are used for its o\vn vital processes. Many of the enzyme systems depicted d o not occur in every microorganism. I t is for this reason we find a mixed culture in a n activated sludge tank----one that changes in composition \vith the changing constituents of the \vaste. Several years ago it was shown thar the rate of oxidation of lactose and of casein by an aerated culture developed on milk waste is strictly additive ( g ) , This result can best be interpreted as indicating that one group of organisms oxidizes lactose and another group oxidizes casein. Isolation of organisms Lvhich gro\v on one constituent but do not attack the other seemed to confirm this conclusion. Therefore it must be remembered that, although these probably are common path\vays in metabolism! they are not all possessed by one organism. This idea is implicit in Nickerson‘s paper, wherein he speaks of many special transformations carried out by particular organisms.
Cell Synthesis Finally let u s consider cell synthesis-the primary carbon transformation of all microorganisms. In recent years there have been a number of studies of the yield of cell material in aerobic treatment systems (5, 70, 76). During the rapid growth phase slightly over 50% of the carbon utilized is transformed to cell tissue : the remaining half is oxidized to obtain energy. Wesron and Eckenfelder have recently concluded that a far higher efficiency of cell synthesis can be calculated from
INDUSTRIAL AND ENGINEERING CHEMISTRY
thermodynamic data than this observed value (76). It would appear that the cells do not exhibit the thermodynamic efficiency predicted; further study will be required to explain this discrepancy between theory and observation. I n such studies care must be taken to prove that the composition of the cells produced does not change-that there is a thermodynamically steady state. Recent evidence that activated sludge can storr carbohydrates in a n amount equal to about half its original weight indicates that the composition of the cells may vary rapidly (7.5). I n such cases the theoretical calculations must be different for short time experiments from those used in developing the equations for a balanced-groivth process producing cells of constant composition. These ideas of the efficiency of cell synthesis and the subsequent endogenous oxidation are of more than theoretical value. They account for the constancy of operation of the mixed culture in aerobic oxidation systems. The concept can be stated as a n illustration of the biological principle of selection of spccies: The organisms that produce the greatest yield of their oLvn cell tissue will thereby prevail in a mixed culture. Literature Cited
(1) Black, H. H., Minch, V. A , Seaoqr and Ind. Wastes 25, 462 (1953). (2) Bogan, R. H., Sawyer, C. N., Ibid., 26, 1069 (1954); 27, 917 (1955). (3) De Laporte, A. V.,Proc. 9th Incl Waste Conf., Purdue Univ., Lafdiette, Ind., p. 352, 1954. (4) Dickerson, B. W.,Campbell, C. J , > Stankard, M., Ibid.,p. 331. (5) Gellman, I., Heukelekian, H., Sereu,or and Ind. Ll’astes 25, 1196 (1953). (6) Harlow, I. F., Powers, T. J., Ehler?, R. B., Sewage W o r k s J. 10, 1043 (1938). (7) Helmers, E. N., Anderson, E. J., Kilgore, H. D., Jr., Weinberger, L. W., Sawyer, C. N., Scwage and Ind. Tl’askes 22. 1200 11950). Helmers, E.”., Frkne, J. D., Greenberg, A. E., Sawyer, C. N., Ibid., 23, 884 (1951). Hoover, S. R., Jasewicz, L., Pepinsky, J. B., Porges, N.,Ibid., 23, 16’ (1951). Hoover, S. R., Porges, N., Ibid., 24, 306 (1952). McRae. A . D., Proc. 9th Ind. Waste Conf.’, Purdue Lniv., Lafayettc, Ind., p. 440, 1954. (12) Miller, H. E., Kniskern, ,J. M., I&/.! p. 531. (13) Mills, E. J., Jr., Stack, V. T., Jr.! Ibid., 8th Conf., p. 492, 1953. (14) Mills? E. J.: Jr., Stack, V. T., J r . , Zbid., 9th Conf., p. 449, 1954. (15) Porges, N., Jasewicz, L., Hoover, S. R., 10th Ind. Waste Conf.. Purdue Univ., Lafayette, Ind., 195%
(16) Weston, R. F., Eckenfelder, W. LV., Sewage and Ind. Wastes 27, 802
(1955).
SAM R. HOOVER Eastern Regional Research Laboratory, Philadelphia, 18, Pa.
