Biochemical Oxidation Cha racteristi PolILJta nt Or M. B. ETTINGER Roberf A. Taft Sanitary Engineering Center, Cincinnati, Ohio
U. S. Public Healfh Service,
Organic chemicals resistant to biochemical oxidation processes occurring in watercourses may travel long distances or persist indefinitely as harmful pollutants once they enter a stream. In contrast, a stream has a useful capacity to assimilate easily oxidized organic chemical wastes. A limited laboratory investigation may fail to reveal the susceptibility of CI compound to biological destruction, as the compound’s resistance in a stream i s affected by variables. An index to the typical behavior o f organic chemicals as stream pollutants under favorable and unfavorable conditions for biochemical oxidation i s suggested, and profile charts are drawn showing the change in oxygen demand factors with time, with an explanation offered for the persistence o f a biochemical oxygen demand caused b y the chemical pollutant even after the chemical itself has disappeared.
STRESRI-POLLUTISG
organic chemicals fall into b o gioups, those rapidly destroyed in streams, and those that travel long distances or persist indefinitely once they enter a stream. Since the tolerance of a stream to chemicals of the first group is usually subject t o estimation in terms of biochemical oxygen demand (B.O.D.) loading and stream f l o ~ their , effect 011 the health of a stream can usually be determined by its dissolved oxygen content. l’otential damage to a stream by chemicals of the second type, however, is not subject to such analysis. Damage may occur in the stream as undesirable color, toxicity to aquatic life, tainting of fish, and impairment of esthetic or recreational value. Persistent pollutants not oxidized in vatercourses have caused taste and odor difficulties, coagulation problems, and higher chlorine demand in municipal m-ater plants Impairment of water for industrial use varies from industry to industry. It may lead to higher water costs or t o less attractive products. I n many important inland waters progressive deterioi stion in the quality of water has occurred without serious oxygen depletion. Clearly, any chemical pollutant that persists in a stream and travels along with it is a t least as important as one that undergoes early biochemical oxidation. With the resistant pollutants the stream has little or no self-purification capacity, and one must assay pollutional damage in terms of permanent degradation of stream quality. With an easily oxidized pollutant, use can be made of the assimilative capacity of streams. A series of investigations of the persistence of specific organic compounds as part of the total pollutional complex in aerobic surface waters have been carried on by the author and his associates. These data, coupled with the findings of others regarding the B.O.D. of specific chemicals, provide some index t o the types of behavior of organic stream pollutants. Persistence of Specific Chemicals
Ettinger and Ruchhoft ( 6 ) described the destruction of phenol and cresols added t o surface waters and studied factors governing behavior of these materials therein. Biological oxidation was found to be the principal cause of the destruction. Under favor-
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able conditions, phenol destruction was complete in a relatively brief period. At least six factors affected the metabolic rates of the organisms destroying the phenolic materials: temperature, metabolic lag, the nature of the initial microbiological population of the water, the concentration of the phenolic compound, the specific compound involved, and the presence of nutrients such as nitrogen and phosphorous to permit utilization of the pheiiolir material in microbiological metabolism. These influences are reflected in Figure 1, a conservative estimate of the persistence of phenol residues in variously polluted and clean waters a t low temperatures. The behavior of pyridine and several pyridine bases as pollutants was found to be governed by the same factors that affected the phenols (3). The chief difference noted was the relative scarcity of seed organisms attacking the piccolines and even pyridine. In the presence of efficient seeds, pyridine and the piccolines were destroyed almost as quickly as the phenols. In sewage and surface maters, seed organisms attacking the pyridine bases were scarcer than seed organisms attacking phenols. Furfural as a pollutant proved t o be subject t o chemical oxidation, but a t a relatively slo~vrate (less than 10% in 30 days a t 20” C.) ( 4 ) . Furfural Ras also lost from substrates by vaporization, especially under aeration. When added to stored samples of surface waters, furfural was dissipated largely by biochemical oxidation, Rith chemical oxidation and vaporization playing minor roles. Seed attacking furfural appeared plentiful in surface waters investigated. Disposition of Material Destroyed
Although chemicals of the type studied may be destroyed in a few hours or days, the organisms that have metabolized the chemical may produce some new cell substance or soluble metabolic products that cause a residual B.O.D. after the original chemical is gone. An experiment partially reported by Lackey and others (0) illustrates several factors involved in biochemical take-up of a specific nutrient. A substrate was used containing 500 p.p.m. of
INDUSTRIAL AND ENGINEERING CHEMISTRY
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STREAM POLLUTION PROBLEMS glucose, 100 p.p.m. of glycine, a 50-p.p.m. total of nine other amino acids, and adequate mineral nutrients. The initial inoculum consisted of 3 ml. per liter of stale sewage. The substrate was maintained a t 20" C. and kept aerobic with minimal aeration. Figure 2 gives the glucose content, total solids, and 5day B.O.D. of the substrate a t intervals. The glucose suffered substantially complete destruction in 3 days. Meanwhile 158 p.p.m. of cell substance was formed as biologically created solids which represented much of the &day B.O.D. of the corresponding substrate, Figure 2 shows also that, once formed, the cell material was destroyed more slowly than the glucose which had caused it t o grow. A second substrate, identical to the first except for the absence of phosphate, received a like inoculum. Phosphate in the system (0.04 p.p.m.) was limited to that introduced by the inoaulum or by impurities of substrate components. Figure 2 shows the glucose residue, solids content, and 5-day B.O.D. of this substrate a t intervals also. Comparing the results in two media, it is evident t h a t the lack of the essential nutrient caused extreme reduction in the rate of attack on glucose, retarded the satisfaction of B.O.D., and drastically limited the development of cell solids. It is well known that metabolism of a substrate by microorganisms causes growth of cellular material that is more resistant t o biological destruction than dissolved foods, and that phosphate and other minerals are essential for cell growth. The experiments cited demonstrate these effects in a case of acute phosphate deficiency in a n aerobic substrate undergoing biological oxidation. They further demonstrate that B.O.D. originally caused by a chemical does not disappear altogether when the chemical itself is completely assimilated.
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Estimate of persistence of phenol residues in river waters
1000 p.p.m. phenol at 4' C. under aerobic conditions
The organic compounds discussed so far have been materials subject to rapid aerobic attack in the presence of suitable seed organisms in substrate containing mineral materials necessary for metabolism. Ranging from glucose used aerobically by a multitude of microorganisms to certain of the pyridine bases for which suitable seed organisms appear t o be relatively uncommon, these compounds undoubtedly resemble numerous other compounds in their watercourse behavior.
Persistence of Resistant Chemicals Some compounds or groups of compounds show high resistance to biological destruction. Ortho and parachlorophenol showed stability in several 20-liter samples of 1% dilutions of sewage given extended incubation (6). Apparently the chemicals were destroyed biologically a t slow rates when added t o several different polluted river waters. Efforts t o develop seeds that would
February 1956
destroy the chemicals at a n accelerated rate had limited success. It was concluded that the dissipation of the two monochlorophenols required a highly specialized seed, and that such removal proceeded at a much slower rate than removal of phenols or cresols (6). A chemical resistant t o aerobic destruction in surface waters need not be a new product of man. Lignin and lignin derivatives (Kraft wastes) were found to be extremely resistant t o biological destruction in 20-liter samples of polluted surface waters, including waters with a long history of pollution by Kraft wastes (8).
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Algal growths in samples stored in sunlight apparently accelerated the dissipation of the lignin. The data presented (8) did not establish biological activity as the mechanism of the observed attack on lignin compounds. A number of the changes observed might have been caused by simple chemical oxidation. Under the conditions investigated, which included favorable temperature (20' C. or above), the lignin compounds had a half life of the order of 3 t o 6 months. I n terms of surface streams in this country, lignin might be considered a relatively permanent pollutant once it enters a watercourse.
Indirect Evidence of Persistence of Specific Chemicals Studies of the biological oxidation of specific chemicals in terms
of measurement of oxygen utilization offer some basis for prediction of their persistence as pollutants. T o interpret such data, it must be assumed that metabolic derivatives of a chemical, such as cell substance, will exert B.O.D. after the chemical itself has disappeared. Placak and Ruchhoft (IS) reported many carbohydrates, alcohols, amino acids, and organic acids to be biologically oxidized or converted to cell substance a t rapid rates by activated sludges. The proportions of material oxidized and material converted t o sludge solids varied widely. Among the compounds found by Placak and Ruchhoft to be resistant to activated sludges were methanol, tyrosine, formaldehyde, oxalic acid, and compounds containing SH and CN groups. Placak and Ruchhoft used feeds containing about 1000 p.p.m. of the chemical investigated, and in most cases no attempt was made t o acclimate the sludge to the food added. I n later investigations, a number of the chemicals found resistant by Placak and Ruchhoft proved susceptible t o biological attack. Dickerson ( 2 ) reported efficient destruction of formaldehyde in a plant waste by a system consisting of a trickling filter with a 40 to 1 recirculation followed by a lagoon. Dickerson's filter feeds contained as much as 200 p.p.m. of formaldehyde. Gellman and Heukelekian (7) developed a seed t h a t oxidized formaldehyde in concentrations up t o 1750 p.p.m. in a Warburg apparatus. Nelson and others (18)reported laboratory development of activated sludges t h a t destroyed oxalic acid in batch feeds con-
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Figure
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Observed B.O.D. for synthetic organic chemicals 1. /I. 111. IV.
Curve Curve Curve Curve
Chemical persistence patterns corresponding to B.O.D. patterns of Figure 3
demonstrating ready susceptibility to biochemical oxidation showing slow oxidation, with or without lag indicating rapid biochemical oxidation after recovery from lag indicating no biochemical attack on organic material by seed used
taining up to 2000 p.p.m. of the neutralized chemical, Lamb and Jenkins (IO)found that methanol in 2.5 p.p.m. concentration readily exerted an oxygen demand. These observations on oxalic acid, formaldehyde, and methanol demonstrate that a limited laboratory investigation may fail to reveal the susceptibility of a chemical to biological destruction. Bogan and Sawyer ( I ) studied the biochemical oxidation of synthetic detergents with B.O.D. technique using normal and acclimated seeds and with Warburg technique. It was concluded that all principal anionic and nonionic detergents are susceptible in some degree to biochemical degradation with considerable variation in the rate of attack, the rate being influenced by small changes in the molecular arrangement. Collectively, the “alkyl benzene and petroleum aromatic derivatives” show susceptibility to biological attack, but such attack was slow. Lamb and Jenkins ( I O ) studied the B.O.D. of a number of organic chemicals produced by Carbide and Carbon Chemicals Corp. a t South Charleston. Their data (Table I) showed few chemicals not subject t o biochemical destruction with long term incubation, using sewage as seed. Table I shows five substances, diethanolamine, triethanolamine, morpholine, diethylene glycol, and triethylene glycol for which it would not be possible to predict eventual complete destruction. Lamb and Jenkins included oxidation of all nitrogen to nitrogen trioxide in estimation of theoretical B.O.D. values. Acclimated seeds %’ere reported to be superior to settled sewage and river mater. I t was not stated IT hether the river water was taken a t the plant or a t a downstream point where the biological population of the river would reflect the biological reaction of the river to the plant wastes. The work of Lamb and Jenkins \vas done in an effort to explain the difference between B.O.D. loads on the Kanawha River estimated on the basis of analysis of waste streams and the larger loads actually observed in the river. Laboratory examination applied to a small sample of x-aste n ith an experimentally limited inoculum may not reveal all the susceptibility of the material to biological destruction bv the array of organisms and environments a receiving stream can provide. T17hile such work has shown that a wide range of resistance to biochemical attack exists, it is impossible to prove conclusively that any chemical has complete biological stability. However, there are materials such as T K T that have defied extensive searches for organisms capable of destroying them. Mills and Stack ( I I ) showed four tvpee of B.O.D. curves for individual organic chemicals. Figuie 3, taken from the n orlr of these authors, illustrates each type of curve. Figure 4 qhons the Triter’s estimates of specific chemical persistence based on Figure 3 and taking into account the time lag between chemical assimila-
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Figure 4.
tion and B.O.D. destruction discussed earlier. For materials indicating no biochemical attack on the organic material by the seed used, isopropyl ether, diethanolamine, triethanolamine, polyethylene glycols, and morpholine were cited. The four typical curves, however, can result from differences in stream water as well as from different chemicals. Three of the four general types of residue curves are presented in Figure 1 for a single chemical (phenol), and the fourth type is covered by the observation (6) that the chemical was persistent in steriie solution. Biochemical attack on any given material present a s a pollutant represents the interaction of a complex biological population and the chemical. It is affected by the seed, the chemical, other materials in the substrate, and by the physical conditions of the environment. A single chemical can ehon a wide range in persistence patterns and susceptibility to biochemical destruction. Compounds show further differences in susceptibility to biochemical attack which cannot be explained solely by environmental differences or the specific effectiveness of seed? present in the environment,
Biochemical Oxidation Course in Complex Substrates The mechanisms that produce the B.O.D. patterns observed have been the subject of much discussion. No completely satisfactory explanation has yet been developed, but summation of the information presented thus far leads directly t o a t least it helpful description of mechanisms involved in the biochemical oxidation reaction in streams under favorable and unfavorable conditions. In general, in organic material in a given sewage or n aste theie is a fraction readily available to microorganisms for food. 4 second portion of the B.O.D. present is accounted for by living microorganisms, although in some cases this fraction may be no greater than that introduced in the seed. A third portion of the B.O.D. consists of chemical materials resistant t o attack in the substrate This fraction includes materials not readily dissolved by the substrate or enzymes produced therein, and materials (exotic foods) not utilized as food by many of the microorganismi present. Figures 5 and 6 present schematically a qualitative picture of the interaction of the seed and the substrate. There usually nil1 be a number of overlapping phases, but stepwise the process is a. follons: Before metabolism starts, there is an initial lag period which may range from a few hour. a t high temperatures to a week or more a t Ion. temperatures. The readily available food materials in the substrate undergo a
I N D U S T R I A L AND E N G I N E E R I N G CHEMISTRY
Vol. 48,No. 2
STREAM POLLUTION PROBLEMS
[ I ) OXYGEN USED (Moleriol destroyed by biochemical oxidation) ( I ) OXYGEN U S E D
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Summary and Conclusions Table I.
B.O.D. of Synthetic Organic Chemicals
2.5 p,p,m. of chemical in B.O.D. bottle mineralized dilution water and settled sewage seed Days Incubation a t 20' C. 5 10 15 20 30 40 50 Chemical % of Theoretical B.O.D. Satisfied Monethanolamine Diethanolamine Triethanolamine Monoisopropanolamine Butylamine Morpholine Methanol E t h y l alcohol Butanol-2 Allyl alcohol Ethylene glycol Diethylene glycol Triethylene glycol Propylene glycol Butyraldehyde Methyl isobutyl ketone Diethyl ketone Acetone Pentanedione-2,4 E t h y l acetate Butyl acetate Isopropyl acetate Carbitol acetate Butyl carbitol acetate Ethylene chlorhydrin
0 0.9 0 5.1 26.5 0.9 53.4 44.2 0 9.1 12.5 1.5 1.4 2.2 43.4 4.4 0 55.4 5.6 36.0 23.5 12.7 23.1 13.3 0
58.4 1.4 0.8 34.0 48.8 0.9 62.7 65.4 44.2 55.0 51.8 5.6 3.7 56.7 59.8 49 3 12.3 71.8 40.0 50.4 50.7 40.0 44.0 18.4 16.1
61.2 3.5 2.6 43.4 50.0 4.0 69.4 71.2 69.2 78.2 71.0 9.0 11.5 72.1 61.5 55,Q 50.8 78.2 62.8 51.6 46.6 40.0 82.4 24.6 74.4
64.0 6.8 6.2 46.0 48.8 5.1 67.0 71.2 72.3 81.8 78.0 18.8 17.0 77.8 66.4 56.6 56.9 78.2 69.6 53.8 57.4 40.0 90.1 67.6 88.6
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fairly rapid biological attack and hence disappear quickly. Part oxidizes, but some synthesizes into cell structure, or adsorbs b y cell structure, causing an increase in the total amount of carbonaceous material present as living protoplasm or as material adsorbed thereon. Various fractions of the less readily available food materials (exotic food) undergo attack not necessarily smooth or continuous. When a group of microorganisms starts to use these fractions of the substrate, this material may be used up rapidly like the readily available materials referred to in the first step. Concurrently, there is a progressive reduction in the B.O.D. represented by living matter, through death and enzymatic solution, the usage of bacteria as food by their predators, encystment of organisms, and other processes of this sort. Figures 5 and 6 are essentially similar except in rates of attack represented. This might simply reflect temperature difference. Seed differences combined with food characteristics of substrates could also cause the evident divergence in courses. Unfavorable p H or the presence of toxicants can also cause such a difference. For instance, Ruchhoft and others (14) observed this in comparing the B.O.D. of a sewage dilution at normal p H with that obtained at p H 2.7. Their data indicate also that the low p H caused substantial increase in the material surviving biological attack as resistant material and/or cell substance.
February 1956
There can be a tremendous variation in the rate a t which a specific chemical compound undergoes biological attack. Variables such as seed, temperature, concentration, mineral nutrients, and pH have been discussed. Different chemical compounds show a wide range of resistance t o biochemical attack. Closely related chemicals such as groups of detergents with a high degree of structural similarity can differ widely in resistance t o biological destruction. A limited laboratory investigation may fail to reveal the susceptibility of a compound t o biological destruction, The process of biological purification of a polluted substrate has been characterized as the sum of a number of sequential biochemical changes and biological transformations. Stream pollutants cannot be evaluated satisfactorily in terms of B.O.D. alone, A chemical resistant t o biological destruction may be capable of doing much more damage than mere depletion of the oxygen in a stream.
literature Cited Bogan, R. H., and Sawyer, C. N., Sewage and I n d . Wastes 26, 1069 (1954). Dickerson, B. W., Ibid., 22, 537 (1950). Ettinger, M. B., Lishka, R. J., and Kroner, R. C., IND.ENG. CHEM.46, 791 (1954). Ettinger, M. B., Lishka, R. J., and Moore, W. A., Extension Series 83,Eng. Ext. Dept. Purdue Univ., p. 206 (1954). Ettinger, M. B., and Ruchhoft, C. C., IND.ENG.CHEM.41, 1422 (1949). Ettinger, M.B.,and Ruchhoft, C. C., Sewage and I n d . Wastea 22, 1214 (1950). Gellman, J., and Heukelekian, H., Ibid., 22, 1321 (1950). Kroner, R. C., and Moore, W. A.,Extension Series 83,Eng. Ext. Dept., Purdue Univ., p. 122 (1954). Lackey, J. B., Wattie, Elsie, Kachmer, J. F., and Placak, 0. R., A m . Midland Naturalist 30,403 (1943). Lamb, C. B., and Jenkins, G. F., Extension Series 79, Eng. Ext. Dept., Purdue Univ., p. 326 (1952). Mills, E. J., Jr., and Stack, V. T.,Extension Series 83, Eng. Ext. Dept., Purdue Univ., p. 492 (1954). Nelson, D. J., Reading, L. M., and Christenson, C. W., Sewage m d I n d . Wastes 26, 1126 (1954). Placak, 0. R., and Ruchhoft, C. C., Sewage Works J . 19, 423 (1947). Ruchhoft, C. C., Ettinger, M. B., and Walker, W. W., IND. ENQ.CHEM.32, 1394 (1940). RECEIVEDfor review April 25, 1955.
INDUSTRIAL AND ENGINEERING CHEMISTRY
ACCEPTED October 17, 1955.
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