RICHARD HATFIELD' Department o f Chemistry and Chemical Engineering, Southwest Research Institute, Son Antonio, Tex.
Biological Oxidation of Same Organic Compounds
Oxidation patterns of individual components in organic wastes can point out materials unfavorable to oxidation b y acclimated microorganisms. O f 23 compounds frequently found in such wastes, primary and secondary alcohols and aldehydes are readily oxidized. But tertiary alcohols, methylal, and glycols are resistant.
THE
PURPOSE of this study was to evaluate the ability of acclimated microorganisms to stabilize various organic compounds present in many organic and typical petrochemical wastes. It was believed that establishing their individual biological oxidation patterns would point out individual compounds that give trouble in biological purification of industrial wastes. The 23 compounds used were :
Methanol n-Propyl alcohol Isopropyl alcohol n-Butyl alcohol Isobutyl alcohol sec-Butyl alcohol tert-Butyl alcohol n-Amyl alcohol Isoamyl alcohol sec-Amyl alcohol tert-Amyl alcohol Formaldehyde
gen and phosphorus nutrients. The amount of organic feed added daily was slowly increased until, a t the end of 1 month, most of the units were receiving a concentration in the aeration media of 333 p.p.m. of organic feed daily, Some of the units were acclimated to about 500 p.p.m. concentration. These feed concentrations were not necessarily the maximum that could be added daily. Most of the units had a well formed sludge with protozoa present. Slant cultures were made of the microorganism populations in several units. Each unit seemed to have several different species present on the culture media. These species will be isolated, identified, and further studied to determine their significance in the different biological oxidation steps. Then a comparison is to be made of the microorganism population present in the units acclimated to the various organic compounds. I t is also planned to make an oxidation
run on formaldehyde using the microorganisms acclimated to methanol as well as a run on acetic acid using the activated sludge acclimated to acetaldehyde. This may help determine if formaldehyde is a step in the oxidation of methanol, and if acetic acid is involved in the oxidation of acetaldehyde. The analytical methods used in following the oxidation rates of the various compounds studied were the biochemical (B.O.D.) (7) and chemical (C.O.D.) oxygen demand ( 4 ) tests. In only one unit (formaldehyde) was the actual disappearance of the organic feed material followed. This was done by modified Schiff's reagent (2) for determination of formaldehyde. The ultimate destruction of organic wastes in the biological treatment method is brought about by life activities of the microorganisms which break down complex organic substances into materials they can metabolize. This oc-
Paraformaldehyde Acetaldehyde Propionaldehyde Methylal Ethylene glycol Diethylene glycol Triethylene glycol Propylene oxide Acetone Acetic acid Calcium formate
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2 Apparatus and Procedure
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The method used for this study was the batchwise fill-and-draw method commonly used for laboratory studies of this nature. Each gallon jug unit was started by adding 3 liters of settled domestic sewage and a small amount of the organic compound to be studied. Each unit was then aerated continuously for several days until a floc began to form, and then fed once daily by removing the air source and letting the sludge settle. Then 2 liters of the supernatant was discarded and replaced by 2 liters of the diluted organic feed along with nitro-
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Present address, Texas Butadiene and Chemical Corp., Channelview, Tex.
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Figure 1.
Biological oxidation of some alcohols Compound
0 Methanol E n-Propyl alcohol A Isopropyl alcohol Q, Isopropyl alcohol
Suspended Solids, P.P.M. 1520
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Figure 2.
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Biological oxidation of butyl alcohol isomers
Figure 3.
Suspended Solids,
A
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curs by diffusion of the simpler molecules through their cell walls. However, for colloidal, complex, or larger molecules, the bacteria synthesize extracellular enzvmes which break these molecules into simpler structures by hydrolytic cleavage or oxidation. These materials
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Isomer 0 nW isoA sec0 ferf-
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Biological oxidation of amyl alcohol isomers
Suspended Solids,
Isomer 0 nW iso-
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can then be absorbed through the cell membrane for intracellular reactions.
Discussion of I n Figure 1, showing oxidation patterns for methanol and two propyl
P.P.M.
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alcohols, the plots show a general type oxidation curve. In some instances, more than one B.O.D. and C.O.D. removal curves are shown because they rearesent different acclimated units. One oxidation rate may be faster than another because of different degrees of
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Biological oxidation of formaldehyde
Figure 5.
A E R A T I O N TI M E , H O U R S BiologicaI oxidation of formaldehyde VOL. 49, NO. 2
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Figure 6.
Oxidation-reduction of formaldehyde
acclimation of the microorganisms. For n-propyl alcohol, the oxidation is virtually complete within 4 hours of aeration. This was true of most oxidation runs where the microorganisms seemed to have little trouble utilizing the organic material. Isopropyl alcohol, however, required more time for oxidation than its primary isomer; also, there appears to be a toxic effect on the B.O.D. seed for zero-hour B.O.D. incubation (Figure 1). These two sets of data for isopropyl alcohol are from different acclimated sludges. The next four chemicals studied were the four isomers of butyl alcohol (Figure 2). The n-, iso-, and sec- isomers give the general type oxidation curves, with most of the B.O.D. reduction taking place within 4 hours of aeration. However, the C.O.D. curve in Figure 2 indicates little biological oxidation in 8 hours of aeration of the tert-butyl alcohol unit. No appreciable B.O.D. was exerted by any of the samples incubated during the 8-hour aeration period. Thus, microorganisms in the B.O.D. incubation were unable to utilize the tert-butyl alcohol. Similar isomers of amyl alcohol were also run, and their removal patterns are similar to those obtained for isomers of butyl alcohol (Figure 3). However, for the primary and secondary isomers, there was a more pronounced
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difference in B.O.D. removal rates; this was true also for the primary and secondary propyl isomers. Another result of the amyl alcohol units was that from the start no solids were wasted; on the day the oxidation runs were made, a total suspended solids analysis of each unit was made. The results were : Total Suspended Solids, P.P.M.
Amyl Alcohol Unit
nIso-
2550 3000 1750 900
sec-
tert-
The reduced amounts of solids grown in the secondary and tertiary alcohols appear to substantiate reduced micro-
Table I.
organism activity which becomes more marked in the tertiary alcohols. Thus, biological oxidation of alcohols appears to follow the same pattern as chemical oxidation-primary alcohols are easily oxidized, secondary alcohols with a little more difficulty. and tertiary alcohols with great difficulty. Table I also shows differences in the ability of microorganisms to utilize primary. secondary, and tertiary alB.O.D. determinations on cohols. 100-p.p.m. solutions of the various isomers were made at two different times with sewage seed and four different times at various stages of acclimation with the acclimated organisms as seed. No B.O.D. was obtained for tert-amyl alcohol during any of the incubations. Probably the most interesting reaction followed was that of formaldehyde; the only compound studied in which the disappearance of the feed substance was measured. The unit represented in Figure 4 was acclimated to 500 p.p.m. of formaldehyde, and that shown in Figure 5 to 333 p.p.m, of formaldehyde a t each feeding. Figure 4 shows that formaldehyde concentration had become zero within 3 hours of aeration, although approximately 400 p.p.m. of B.O.D. still remained. This suggests that formaldehyde readily goes to some intermediate state-possibly formic acid, or a Cannizzaro dismutation to formic acid and methanol, which in turn are oxidized further or assimilated by the bacteria. I t took approximately 8 to 10 hours of aeration for the B.O.D. to approach zero. Figure 6 shows possible chemical and biological enzymatic oxidation-reduction reactions for formaldehyde ( 3 ) . Figure 7 gives data obtained for acetaldehyde and propionaldehyde. Each of these compounds was oxidized at a rapid rate by the acclimated organisms. The polymer of formaldehyde (paraformaldehyde) was also studied, and its oxidation pattern resembled that for formaldehyde. Methylal, the acetal formed between formaldehyde and methanol also gave interesting results. The author has never obtained a B.O.D. value for pure methylal solutions when seeding the
B.O.D. of Some Amyl Alcohols (100 p.p.m. Solution)"
5-Day B.O.D., P.P.M. Acclimated Seed Run 2 Run 3 Run 4
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Domestic Sewage Seed Run 1 Run 2
Compound
Run 1
+Amyl alcohol Isoamyl alcohol sec-Amyl alcohol tert-Amyl alcohol
125 137 0
142 147 106
132 174 115
218 140 155
129 146 32
124 134 0
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Theoretical oxygen demand of a 100-p.p.m. amyl alcohol solution is 284. B.O.D. ran a t various intervals during acclimation of batchwise fill-and-draw activated sludge unit.
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Figure 8.
Figure 7. Biological oxidation of acetaldehyde and propionaldehyde
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Biological oxidation methylal
Propionaldehyde, suspended solids, 3460 p.p.m.
incubations with sewage organisms, probably because of their toxicity. Run 1 in Figure 8 shows a C.O.D. removal curve; however, there were no dissolved oxygen depletions in the B.O.D. incubations because of toxicity of the material incubated. These B.O.D. incubations were seeded with sewage seed as the effluents for analysis were filtered to remove suspended solids which would add to the C.O.D.
However, for run 2, the portion of effluent for the B.O.D. analysis was not filtered, but values were obtained because the incubations contained some acclimated organisms in the B.O.D. bottle. But these values were small compared to the theoretical oxygen demand. Again, toxicity causes the initial zero-hour B.O.D. to be less than the 2-hour B.O.D. By the time the unit had aerated for 2 hourg, the C.O.D.
was reduced by about 200 p.p.m. After 2 hours of aeration, larger amounts of sample were incubated for B.O.D. determinations; this gave greater populations of acclimated organisms. Ethylene glycol, diethylene glycol, and triethylene glycol were also run. Of the two runs made with units acclimated with ethylene glycol (Figure 9) run 1 indicates virtually no B.O.D. reduction within 8 hours of aeration
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Figure 9.
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Biological oxidation of ethylene glycol
Diethylene glycol, suspended solids, 1 1 50 p.p.m.
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Figure 10. glycols
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Biological oxidation of di- and triethylene Triethylene glycol, suspended solids, 1 2 5 0 p.p.m.
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the C.O.D. of acetic acid is only a fraction of the theoretical amount by the dichromate reflux method ( 4 ) . As the acetic acid was converted to other molecules, the C.O.D. of these materials was higher than for acetic acid. Calcium formate was the last compound studied and two oxidation runs were made on the same unit at about a 3-week interval. Its theoretical oxygen demand is small compared to the other compounds studied. After 24 hours of aeration, the oxidation process for the second run was more complete than for the first. Conclusions
In organic wastes containing many toxic-type organic compounds, oxidation
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Figure 13. Biological oxidation of acetic acid, suspended solids, 2570 p.p.m,
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Figure 12. Biological oxidation of acetone, suspended solids, 2370 p.p.m.
Figure 1 1. Biological oxidation of propylene oxide, suspended solids, 2000 p.p.m.
while run 2, acclimated a t a slower rate, shows some removal. The zero-hour B.O.D. for ethylene glycol was small compared to the theoretical oxygen demand. The author has run B.O.D. determinations many times on the various pure glycols using sewage seed. Some incubations have given small B.O.D. values as compared to the theoretical oxygen demand, while others were completely toxic to the sewage organisms ( 5 ) . When diethylene glycol was used to acclimate sewage organisms (Figure 10) no B.O.D. or C.O.D. reduction occurred in 8 hours of aeration. Possibly, however, slower acclimation may have resulted in microorganisms capable of removal. With triethylene glycol (Figure lo), there was a slight B.O.D. and C.O.D. removal rate. I t is believed that a properly acclimated microogainism culture can oxidize the three glycols a t a low rate. Propylene oxide (Figure 11) was another compound studied, and its B.O.D. was small compared to the theoretical oxygen demand. Acetone was the only ketone studied and it was easily oxidized by the acclimated organisms (Figure 12). No B.O.D. values were obtained because the incubations contained too much of the acetone mixture which proved toxic to the B.O. D. seed organisms. B.O.D. incubations of pure acetone solutions using sewage seed gave 64y0 of theoretical yield (5); however, more than 2 p.p.m. of acetone in the B.O.D. incubation was toxic to the nonacclimated organisms. ASshown by the B.O.D. removal curve in Figure 13, acetic acid was easily stabilized by the acclimated organisms; however, the C.O.D. curve shows a slight increase during the 8 hours of aeration. This can probably be explained by the fact that
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patterns of the individual components should be studied because materials unfavorable to biological oxidation by the acclimated organisms may be eliminated from total waste. Of the chemicals studied, primary and secondary alcohols and aldehydes were readily oxidized biologically, with a major portion of the B.O.D. and C.O.D. removed within a 4-hour aeration time. Tertiary alcohols, methylal, and glycols, however, were more resistant. The degree of acclimation of the sludge seems to have an important effect on the oxidation rate. Different rates were obtained for a given compound by using differently acclimated sludges. Because of toxicity of the organic feed to seed microorganisms in B.O.D. incubations, in many instances C.O.D. is an important method of following oxidation rates. Literature Cited (1) American Public Health Association, “Standard Methods for the Examination of Water and Sewage,” 9th ed., 1946. ( 2 ) Hatfield, R . , Strong, E. R., “Determinations of Formaldehyde in the Presence of Dichromate Ion,” 6th Southwest Regional Meeting, ACS, Antonio, Tex., 1950. (3) McGavock, W. C., “Organic Oxidation-Reduction Reactions,” 2nd ed., Clegg Go., San Antonio, Tex., 1945. (4) Moore, W. A., Kroner, R. C., Ruchhoft, C. C., Anal. Chem. 21, 953-6 (1 949). ( 5 ) Strong, E. R., Hatfield, R., “Biochemical Oxygen Demand of Some Common Organic Compounds Present in Chemical Wastes,” 5th Southwest Regional Meeting, ACS, Oklahoma City, Okla., 1949.
RECEIVED for review March 14, 1956 ACCEPTED July 13, 1956 10th Annual Southwest Regional Meeting, ACS, Fort Worth, ‘rex., 1954.
