This dissertation has been 63—2038 microfilmed exactly as received

G E H H O L D , Robert Murray, 1931- STRUCTIJRAL DETERMINANTS IN OXIDATIVE BREAKDOWN OF ORGANIC ALIPHATIC COMPOUNDS BY DOMESTIC ACTIVATED SLUDGES.

The Ohio State University, Ph.D., 1962 Bacteriology

University Microhlrnr, Inc Ann Arbor, Mchkpn Copyright by

Hobert Murray Gerhold 1963

% STRUCTURAL DETERMINANTS IN OXIDATIVE BREAKDOWN

OF ORGANIC ALIPHATIC COMPOUNDS BY

DOMESTIC ACTIVATED SLUDGES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of Jhe Ohio State University

By

Robert Murray Gerhold, B.A., M.S.

The Ohio State University 1962

Approved by

Adviser ■ Department of Microbiology ACKNOWLEDGMENT

The author is deeply grateful to Dr. George W. Malaney for his aid in selection of this problem, for hLs understanding and guidance throughout the work, and for making a research assiatantship possible during 1960 to 1962.

Appreciation is also extended to Mr. George P. Hanna, Jr., of the Department of Civil Engineering, for the opportunity of utilizing the excellent laboratory facilities of the Water Resources Center and for his aid in preparation of the figures for this dissertation.

Special thanks are expressed to Mr. V. D. Sheets, of the Engi­ neering Experiment Station, for his patient technical assistance.

The author sincerely appreciates the encouragement and patience of his wife.

ii /

CONTENTS

Page

INTRODUCTION...... 1

MATERIALS AND METHODS ...... 4

General Experimental procedure

REVIEW OF THE LITERATURE...... S

Development of Methods Oxygen Demand as a Measure of Diodegradation Biodegradation of Specific Aliphatic Compounds

RESULTS ...... 21

Presentation of Data Groups of Compounds Constant Weight Versus Constant Molarity and Substrate Oxidation I DISCUSSION...... 155

S W * i A R Y ...... 161

APPENDIX...... 164

Flow Diagrams of Treatment Plants

BIBLIOGRAPHY...... 168

AUTOBIOGRAPHY...... 174

i ii Tabic bage

1. Alkane Oxidation by Activated diulge...... 24

2. Olefin Oxidation by Activated sludge...... 29

3. Olefin Oxidation by Activated kludge...... 32

4. Chloro- and Nitro- Substituted Alhane Oxidation by Activated Sludge ...... 3 7

6. Alcohol Oxidation bv Activated H u d c c . 39

6. Alcohol Oxidation by /ctivated Sludge ...... 41

7. "econdary, Tertiary, and tranched-Ghain 'lcohol Oxidation by Activate 1 ’lulgc...... 44

Secondary Alcohol Oxidation by Activated ; lu d g e ...... 46

o . I'olyhvdric Alcohol Oxidation by Activated 'ludge...... 49

10. Aldehyde Oxidation by Activated sludge...... 5 2

11. Aldehyde Oxidation by ' ctivated Sludge...... r‘5

12. betopc Oxidation by Activated Sludge...... 3,°

I7 . hot one Oxidation by Activated Sludge...... 61

14. Ketone Oxidation by Activated Sludge...... 67

15. Fatty Acid Oxidation by Activated -’ludge ...... 66

16. Fatty Acid Oxidation by Activated 'lulgc...... 68

17. bnsnturated Fatty A-icil Oxidation by Activated ."ludge. . . . 72

1n. bicarbnxylic Acid Oxidation by Activated Sludge ...... 74

19. b’nsaturatcd Dibasic and Hydroxy Acid Oxidation by Activated Sludge ...... 60

29, Glyceride Oxidation by Activated 'ludge ...... 83

21. Methyl Aster Oxidation by Activated Slul e. . .'...... <7

iv TABLES (Continued) Table Page

22. Acetate Ester Oxidation by Activated Sludge...... 90

23. Alkyl and Allyl Ester Oxidation by Activated Sludge. . . . 93

24. Nitrile Oxidation by Activated Sludge...... 96

25. Amide Oxidation by Activated Sludge...... 100

26. Amine Oxidation by Activated Sludge...... 101

27. Ainino Acid Oxidation by Activated Sludge ...... 106

28. Amino Acid Oxidation by Activated Sludge ...... 10P

29. Amino Acid Oxidation by Activated Sludge ...... 110

30. Ainino Acid Oxidation by Activated Sludge ...... 112

31. Amino Acid Oxidation by Activated Sludge ...... 114

32. Protein Oxidation by Activated Sludge...... 119

33. Protein Oxidation by Activated Sludge...... 121 \

34. Protein Oxidation by Activated Sludge...... 123

35. Enzyme Oxidation by Activated Sludge ...... 125

36. Enzyme Oxidation by Activated Sludge ...... 127

37. Hexose Oxidation by Activated Sludge ...... 130

39. Pentose Oxidation by Activated Sludge...... 133 a 39. Hexose Disaccharide Oxidation by Activated Sludge. .... 135

40. Di- and Trisaccharide Oxidation by Activated Sludge. . . . 138

41. Maltose and Polysaccharide Oxidation by Activated Sludge . 140

42. Polysaccharide Oxidation by Activated Sludge...... 142

43. Sugar Alcohol Oxidation by Activated Sludge...... 145

44. Thiol Oxidation by Activated Sludge...... 148

45. Veight Versus Molecular Basis of Comparing Substrates. . . 150

46. Compound of Each Carbon Chain Length Showing the Highest Percentage of Theoretical Oxidation in 24 hr ... 157 v ILLUSTRATIONS

Figure Page

1. Alkane Oxidation by Activated.Sludge...... 23

2. Olefin Oxidation by Activated.Sludge...... 23

3. Olefin Oxidation by Activated.Sludge...... 31

4. Chloro- and Nitro- Substituted Alkane Oxidation by Activated Sludge ...... 34

5. Alcohol Oxidation by Activated Sludge ...... 3<'

6. Alcohol Oxidation by Activated Sludge ...... 40

7. Secondary, Tertiary and Branched-Chain Alcohol Oxidation by Activated Sludge...... 43

H. Secondary Alcohol Oxidation by Activated Sludge ...... 4 5

9. l’olyhydric Alcohol Oxidation by Activated Sludge...... 48

10. Aldehyde Oxidation by Activated Sludge...... 51

11. Aldehyde Oxidation by Activated Sludge...... 54

12. Ketone Oxidation by Activated Sludge...... 57

13. Ketone Oxidation by Activated Sludge...... 60

14. Ketone Oxidation by Activated.Sludge...... 62

15. Fatty Acid Oxidation by Activated Sludge...... 6 5

16. Fatty Acid Oxidation by Activated Sludge...... 67

17. Unsaturated Fatty Acid Oxidation by Activated Sludge. .. . 71

18. Dicarboxylic Acid Oxidation by Activated Sludge ...... 73

19. Unsaturated Dibasic and Hydroxy Acid Oxidation by Acti­ vated Sludge ...... 7u

20. Glyceride Oxidation by Activated Sludge ...... 82

21. Methyl Ester Oxidation by Activated Sludge...... i6

22. Acetate Ester Oxidation by Activated Sludge ...... 89 ILLUSTRATIONS (Continued)

Figure Page

23. Alkyl and Ally! Ester Oxidation by Activated Sludge. .. . 92

24. Nitrile Oxidation by Activated Sludge...... 95

25. Amide Oxidation by Activated Sludge...... 99

26. Amino Acid Oxidation by Activated Sludge ...... 105

27. Amino Acid Oxidation by Activated Sludge ...... 107

28. Amino Acid Oxidation by Activated Sludge ...... 109

29. Amino Acid Oxidation by Activated Sludge ...... Ill

30. Amino Acid Oxidation by Activated Sludge ...... 113

31. Protein Oxidation by Activated Sludge...... 118

32. Protein Oxidation by Activated Sludge...... 120

33. Protein Oxidation by Activated Sludge...... 122

34. Enzyme Oxidation by Activated Sludge ...... 124

35. Enzyme Oxidation by Activated Sludge ...... 126

36. Hexose Oxidation by Activated Sludge ...... 129

37. Pentose Oxidation by Activated Sludge...... 132

38. Hexose Disaccharide Oxidation by Activated Sludge.... 134

39. Di- and Trisaccharide Oxidation by Activated Sludge. .. . 137

40. Maltose and Polysaccharide Oxidation by Activated Sludge . 139

41. Polysaccharide Oxidation by Activated Sludge ...... 141

42. Sugar Alcohol Oxidation by Activated Sludge...... 144

43. Thiol Oxidation by Activated Sludge...... 147

44. Weight Versus Molecular Basis of Comparing Fatty Acid Oxidation by Brookside Activated Sludge ...... 151

vii ILLUSTRATIONS (Continued)

Figure Tage

45. Weight Versus Molecular Basis of Comparing Fatty Acid Oxidation by Columbus Activated Sludge...... 152

46. Weight Versus Molecular Basis of Comparing Fatty Acid Oxidation by Hilliard Activated Sludge...... 155

47. Flow Diagram of the Brookside Estates Sewage Treatment P l a n t ...... 164

48. Generalized Flow Diagram of the Columbus, Ohio Sewage Treatment Plant ...... 165

49. Block Flow Diagram of the Columbus, Ohio Sewage Treatment Plant ...... 166

50. Flow Diagram of the Hilliard, Ohio Sewage Treatment P l a n t ...... 167

viii INTRODUCTION

Formerly the goal of waste treatment was to eliminate physical nuisances. Successively other goals were added: to reduce the numbers of disease organisms entering streams, to avoid the contamination of water needed for municipal and industrial purposes, and to avoid the debasement of streams which provide natural beauty and opportunities for recreation such as boating, swimming, and fishing. Population statisticians increasingly caution us against depletion of natural re­ sources resulting from our enormous increase in population with its concurrent demand for more space, more food, more recreational facili­ ties, and for better water supplies.

The practice of discharging untreated or partially treated wastes into streams and rivers is still widely followed in the United

States. It is not unusual for the water for household use to come from waterways the contents of which have been used many times (I). Adding to the seriousness of the water supply problem is the fact that these streams are often grossly polluted. For this reason, strong emphasis must be placed on waste treatment in order to eliminate pollution.

Although streams have a remarkable capacity for self purification, this capacity depends upon whether or not the stream can acquire enough dissolved oxygen to satisfy the demands for self-purification. The ability to acquire dissolved oxygen is a function of stream temperature, and flow. The pollutional load of many streams often is in excess of the stream's natural ability to purify itself. "The sanitary cere and economic efficiency with which a people handles its own wastes are two 2

of the more valuable criteria for measuring the level of its civili­

zation" (2).

Modern waste treatment demands a process which is highly effi­

cient, uniform in operational characteristics, economical, and

aesthetically non-objectionable, and one that does not require extensive

land areas. A process meeting these requirements was developed in 1914

by Ardern and Lockett (3) who termed the new process "activated sludge

treatment." This method of treatment came into widespread use in the

United States in the late 1930*s (4).

Activated sludge possesses the power to adsorb and metabolize a wide variety of organic compounds. In many cases, the organisms in

domestic sludges can either oxidize a compound immediately or after

short periods of acclimatization. Elimination of the biological demand

for oxygen by activated sludge is due partly to adsorption and pertly to

oxidation.

The objectives of this investigation were (a) to determine the

amount of oxygen uptake that could be attributed to the biological oxida­

tion of aliphatic compounds in the aeration chamber during conventional

detention periods, (b) to elucidate possible relationships between

oxidizability and the structural configuration of the aliphatic com­

pounds, and (c) to relate the rates of oxygen uptake by different

domestic sludges to plant design and operational peculiarities.

Although much data are available that are based on the dilution method for the determination of biological oxygen demand of individual

chemical compounds, few studies have been made on the oxidative 3 abilities of activated sludges taken directly from the aeration tanks of plants in operation. It is hoped that this investigation may provide operators of waste treatment plants with fundamental knowledge regarding the biological oxygen demand of individual aliphatic compounds. The re­ sults may also suggest when acclimatization of activated sludge to these compounds may be expected within short periods of aeration.

Heukelekian (5) defined the problem:

A knowledge of the BOD of pure organic chemicals is of importance from the standpoint of treatment of in­ dustrial wastes. Chemicals used in the production of a certain product are known. The amount of these chemicals lost to the waste water can be estimated. If the BOD of the particular chemical is known, it is possible to estimate the BOD load contributed by the chemical. MATERIALS AND METHODS

General

The experimental apparatus used was the l.arburg Constant Temper­ ature Respirometer.

Selection of Compounds

The following types of compounds were chosen for study: (a) com­ pounds commonly found in living tissue; such as amino acids, fatty acids, glycerides, carbohydrates and proteins; (b) compounds having a common functional group, but varying in numbers of carbon atoms; and (c) com­ pounds with the same number of carbon atoms, but having a functional group in a different position.

Selection of Treatment PI ants

The activated sludge from any one treatment plant is only par­ tially representative of activated sludges from all waste treatment plants. It was decided to study sludges from three treatment plants of different sizes and designs and fed by differ ent sewerage systems:

1. The Columbus, Ohio, municipal sewage treatment plant was the largest. Sewage treated by this plant carried domestic as well as in­ dustrial wastes. Activated sludge from the Columbus treatment plant was the most uniform in quality and never carried large particles. Bull;inf was never encountered and nixed liquor from this plant filtered easily, settled rapidly, and gave a clear supernatant.

2. The Hilliard, Ohio, municipal plant was smaller than the

Columbus plant and received only domestic sewage. This plant was hindered by operational difficulties, one of vrhich was the diluting effect on the sludge by moderate to heavy rainfall. 4 5

3. The Brookside Estates treatment plant was a small "total oxidation** type plant receiving domestic sewage. This plant had no pri­ mary settling or grit-removal units, consequently the activated sludge carried heavy particles. Poor filtration was often encountered and bulking of this sludge was an occasional problem. Plow diagranta of the three plants are shown in the Appendix.

Sampling

Samples of activated sludge were collected from the aeration tanks with a #303 tin can attached to a mop handle. The time from collection to the beginning of the analysis was never less than 3 hr nor more than 6 hr. When the sludge failed to settle rapidly or when deter­ mination of suspended solids concentration was prolonged by slow filtration, the sample was held in a refrigerator at 6 C.

Experimental Procedure

Warburg Flasks

The Warburg flasks were made at the Ohio State University glass- blowing shop. They were modified 125 ml Erlenmeyer flasks fitted with

1.5 ml center-wells and female ground glass joints.

Cleaning of G1aaaware

Glassware for mixed liquor samples was cleaned with detergent, then rinsed three times each with tap and then distilled water. Pipettes were cleaned by rinsing in tap water, followed by distilled water. They were dried and submerged in chromic acid cleaning solution for 24 hr.

Since a film of chromic acid tends to bind to glassware surfaces, pipettes received twenty rinses in tap-water followed by eight rinses in distilled-water. Warburg flasks were cleaned by the following procedure: (a) flasks were rinsed once with tap water, and dried In the

103 C oven; (b) flsaks were washed with two rinses of chloroform to re­ move fats and greases, then dried; (c) the flasks were submerged in potasaiun dlehrornate cleaning solution for 24 hr, rinsed In the same manner as the pipettes, and dried in an inverted position.

Calibration of Flasks

Flasks and manometers were calibrated so that manometer readings multiplied by the flask constant (6) gave the mg O2 utilized by a liter of the mixture in the flask.

Adjustment of Sol ids Concen tr at ion

The number and types of organisms added to each Warburg flask could not be standardized, but by adjusting the mixed liquor suspended solids to the same concentration in each flask, reasonable uniformity in numbers of organisms was achieved. The term "suspended solids" includes all particles retained by a type HA 047 nun membrane filter. The method used for determination of the concentration of suspended solids was the technique of Engelbrecht and McKinney (7). The sludge for each analysis was adjusted to 5000 mg/liter. While the solids determination was being carried out, a 1-liter portion of the mixed liquor was allowed to settle

in a graduated cylinder. After determination of the suspended solids, sufficient supernatant liquid was drawn off to give the required 5000 mg/liter concentration of solids. The sludge was not washed. Mineral salts were not added. Following the adjustment of sludge solids, a

250-700 ml portion of the well-mixed material was homogenized for 10 seconds in a blendor. 7

Preparation of Substrates

Nearly three hundred aliphatic compounda were used as experi­ mental substrates. Those substrates easily soluble in water were made up in 0.1 per cent concentration with distilled water and stored at 6 C until needed. Poorly soluble substrates were made up and stored in the same manner, but prior to addition to Warburg flasks the substrate sus­ pensions were shaken so as to achieve an even distribution. If the melting point of the insoluble substrate was above room temperature, the suspension was heated to 80-90 C, shaken vigorously, and pipetted into the Warburg flask.

Each flask received 10 ml of substrate solution or suspension delivered with a volumetric pipette. Next, 10 ml of blended sludge were added to each flask. The final concentration of substrate was 500 mg/liter. The final concentration of sludge solids was 2500 mg/liter.

The control for endogenous respiration contained 10 ml of distilled water and 10 ml of adjusted sludge. Endogenous respiration was defined as the amount of accumulative O 2 uptake observed in the control flask containing sludge and distilled water.

After 10-20 min of shaking for temperature equilibration the

flasks were closed off to the atmosphere and shaken for 24 hr at 7R oscillations per rain. From 9 to 16 readings were made during each ex- per iment.

Although care was taken in all manipulations, no attempt was made to maintain sterility of glassware, diluent or substrates. REVIEW OF THE LITERATURE

Development of Methods

The dilution method for the determination of biological oxygen

‘demand (BOD) is a videly employed standard procedure for the determina­

tion of the organic load of waste waters, but it has several disadvantages

that limit its usefulness. Research directed toward refinement of this

test has been extensive (8,9,10,11,12,13).

In an attempt to devise a better method, Wooldridge and Standfast

applied Barcroft manometry to activated sludge studies (14,15,16,17).

Another method for recording the continuous utilization of oxygen was

introduced in 1928 by Sierp (18). This method was studied in detail by

Gellman and Heukelekian (19). Caldwell and Langelier (20) reviewed the

prior use of manometry in sewage investigations, and reported their ovn

studies on sewage and activated sludge using the Warburg apparatus.

Dawson and Jenkins (21,22) confirmed previous work which had shown that

constant agitation caused increased oxidation of organic material. They

also reported that (a) the rate of oxygen uptake by activated sludge is

directly proportional to the amount of sludge solids; (b) constant shak­

ing is necessary for maximum oxidation even in a pure oxygen atmosphere;

and (c) oxygen uptake occurs entirely in the biological portion of the

sludge.

In a comparison of the BOD, Sierp, and Warburg techniques,

Gellman (23) found that 15-20 per cent more oxygen was consumed in the

direct methods than in the indirect BOD method. Other workers showed

that the rate of 0j uptake was the same for all methods (19,24,25).

8 9

Although the Warburg apparatus affords a "direct** approach to the determination of BOD, it is still primarily a research tool, and initial cost prohibits use of the instrument by many laboratories.

Oxygen Demand as a Measure of Blodegradation

Compounds similar in structure may show variations in resistance to biological attack <26). Since 02 uptake rates vary directly vith the organic load of activated sludge (22), it follows that O 2 uptake is an accurate measure of the ox'ldation of single organic compounds by acti­ vated sludge. Few chemicals utilized by bacteria are degraded completely to CO2 and HjO; part of the substrate is assimilated, "... the amount being dependent on the nature of the substrate and independent of its concentration" (27). Kountz and Forney (28) showed that 63-82 per cent of the oxygen taken up by activated sludge was used for oxidation of organic substrates, and that the higher percentages of oxidation were obtained when a low ratio of substrate/cell solids was used.

Biodegradation of Specific Aliphatic Compounds

In this report, the terms "percentage oxidized," or "percentage of oxidation," or "X per cent oxidized" mean the ratio of the amount of oxygen taken up by the sludge in the presence of that concentration of the substrate to the amount of oxygen required for complete oxidation of that concentration of substrate, i.e., oxidation to carbon dioxide, water, nitrate, and sulfate. This ratio is also referred to as the "percentage of total theoretical oxygen demand."

Alkanes

Zobell (29) concluded that, within limits, long chain alkanes 10 vere more readily degraded than ahort chain molecules. Marlon (30), working with a pure culture of Alcallgenea faecalIs and alkanes from

5 to 12 carbons In length, reported that only the 12-carbon dodecane was oxidized and only after extended periods of aeration.

Olefins

Zobell (29) and Marion (30) both reported that the olefins were more susceptible to biodegradation than the saturated hydrocarbons.

Chloroalkanea

The chloroalkanea studied by Marion (30) were oxidized.

Nitroalkanes

The nitro-substituted alkanes tested by Marion were apparently toxic to Alcaljgenes faecal is (30).

Alcohols

Cook (31) found that ethanol was not oxidized by a culture of

Bacterium coli, while Heukelekian (32) reported that ". . . ethyl alcohol has . . . no retarding effect on oxygen utilization within the narrow range of concentrations [0.001 to 0.010 cc/liter] in which dissolved oxygen could be maintained."

Butterfield, Ruchhoft and McNamee (33) used pure cultures of bacteria isolated from activated sludge. They developed niltuv.T that formed a floe and apparently oxidized wastes as natural sludge. For example, using 1000 mg/liter of substrate, oxidation of ethanol exceeded that of methanol. Placak and Ruchhoft (34) concluded that, in general, the alcohols were 24 to 38 per cent oxidized in one day. However, methyl alcohol and ethylene glycol were not readily attacked by their activated sludge. 11

In a study using substrate concentrations of 1000 mg/liter, the

Southwest Research Institute (35) found that the 5-day BOD of several alcohols when calculated as the percentage of total theoretical oxida­ tion (PTO) was as follows:

Substrate PTO Substrate PTO

methyl alcohol 60 butyl alcohol 56 ethyl alcohol 69 amyl alcohol 59 propyl alcohol 63 iso-amyl alcohol 55 iso-propyl alcohol 0

Lamb and Jenkins (36) used 2.5 mg/liter of alcohol in the dilution method BOD and obtained the following percentages of oxidation:

PTO PTO PTO Substrate 5 days 20 days 50 days

methanol 53.4 # 67.0 97.7 ethanol 44.2 71.2 77.0 butanol-2 0 72.3 77.0

They also showed that isopropanol in concentrations of 1-15 mg/liter exerted a negligible BOD in 5 days. Waldmeyer found that "60 per cent purification" of methanol was obtained in 4 hr using activated sludge as seed (37).

Studying direct methods of BOD determination on alcohols at

1000 mg/liter concentration, Gellman (38) obtained the following values for percentage of oxidation (PTO) by normal activated sludge:

PTO PTO PTO Substrate 6 hr 12 hr 24 hr

methanol 1.3 4.7 12.7 ethanol 7.9 26.6 45.2 isopropanol 2.7 6.3 10.0 butanol 5.0 14.5 39.8

These values were calculated from Gellman*s data. Gellman also showed 12 ethanol waa probably the best alcohol for increasing suspended solids content in the aeration tank.

Hatfield (39) confirmed Ge11man's conclusion that acclimatiza­ tion of activated sludge greatly increased the rate of oxidation of the corresponding substrate. He adapted activated sludges to 333-500 mg/liter of alcohol per day by the fill and draw method. His results showed that "... biological oxidation of alcohols appears to follow the same pattern as chemical oxidation— primary alcoholb are easily oxidized, secondary alcohols with a little more difficulty, and tertiary alcohols with great difficulty."

Polyhydric Alcohols

Several workers have determined the BOD of the glycols by differ­ ent methods (1,5,9,30,34,36,39).

In BOD tests, Masselli and Burford (40) found that polyethylene glycols and diethoxy derivatives were poorly oxidized. Monoethoxy derivatives were oxidized and greater oxidation was observed with shorter chain compounds than with the long chain compounds. Acetate derivatives of the lower glycols were oxidized to a greater extent than were the long chain derivatives.

Aldehydes

Formaldehyde, the first member in the aliphatic aldehyde series, was toxic to unacclimatized systems. However, sewage and sludge organ­ isms readily adapted to formaldehyde (5,23,32,34,41) and oxidized it almost stoichiometrically (35,42), thus disking possible the treatment of formaldehyde wastes. 13

McKinney and Jeria (43) showed that activated sludges acclima­ tised to 2- and 3-carbon normal alcohols oxidised the corresponding 2- or 3-carbon aldehydes. Isopropanol-adapted sludge, however, attacked propionaldehyde very slowly. On the other hand, sludges acclimatised to straight-chain and branched 4-carbon alcohols oxidized the corresponding aldehydes, but sludges acclimatized to tert. butanol did not oxidize butyraldehyde significantly.

Ketones

The ketones were susceptible to biodegraaation and many investi­ gations contributed information regarding the oxidation of these compounds

(1,9,23,30,32,35,36,39,43).

Saturated Fatty Acids

Oxidation of the saturated fatty acids was investigated by numerous workers (5,23,31,33,34,35,43).

Gaffney and Heukelekian (44) shoved that the lower fatty acids, including caprate, exerted Oj demands averaging 67+3 per cent of the theoretical demand in 5 days, and 91+4 per cent of the theoretical demand in 20 days. Furthermore, they showed that the Warburg method of

BOD determination gave results comparable to the standard dilution technique. In a later paper, the same authors reported on a new graph­ ical method for evaluating BOD data on the lower fatty acids (45). Long lag periods were observed, followed by rapid rates of oxidation, after which an abrupt cessation of measurable oxygen uptake occurred. These authors could not devise a method of eliminating the lag periods in BOD tests on fatty acids. Carbon balance studies seemed to indicate that 14

72-78 per cent of the lower fatty acids, including caprate, were oxidized to carbon dioxide and water.

Pngaturated Fatty Acids

Dawson and Jenkins (41), and Heukelekian and Rand (4) presented evidence that the unsaturated fatty acids less than 18-carbons in length were oxidized.

Recent work by Viswanathan, Bai, and Pillai (46) showed that, in the aerobic phase of sewage treatment, linolenic acid was synthesized by the protozoan Carchesium sp. Oleic acid was found in large Quantities

in influent sewage, but the concentration was reduced approximately 98.6 per cent during activated sludge treatment.

Dicarboxylic Acids

Cook and Stephenson (31), Placak and Ruchhoft (34), Gellman (23), and Dawson and Jenkins (41) presented conflicting reports on the oxida­ tion of oxalic acid. These studies showed conclusively, however, that most of the other dicarboxylic acids were susceptible to oxidation by sewage and sludge organisms.

Unsaturated Dibasic Acids and Hydroxy Acids

Placak and Ruchhoft (34) showed that tartaric acid was oxidized

to a greater extent than citric or lactic acid. Dawson and Jenkins (41)

found that citric acid was oxidized to a slight extent by a washed acti­ vated sludge. From Heukelekian*s survey of Warburg-technique data (5)

the following averages were obtained:

Substrate PTO

fumaric acid 00.0 malic acid 63. 0 maleic acid 46.0 15

Glycerides

There wee e much greater oxidation of glycerides in the aerobic

system then in the anaerobic units. No fatty matter appeared in the effluent from the aerobic system after the third day of treatment, according to Viswanathan et.al. (46).

Methyl Esters

One study (35) In which the 5 day BOD dilution method was used

showed that methyl formate in a concentration of 1000 mg/liter exerted no oxygen demand.

Acetate Eaters

Dawson end Jenkins (41) reported that ethyl acetate was toxic to activated sludge, but the other acetate esters were readily oxidized

(23,36,30).

Allyl Compounds

Heukelekian and Rand (5) reported that allyl alcohol in the dilu­ tion BOD test was 9.1 per cent oxidized in 5 days, 55.0 per cent oxidized

in 10 days and 81.8 per cent oxidized in 20 days, but they used low con­ centrations of substrate (2.5 mg/liter).

Nitrites

Schaffer and Bloomhuff (47) used the measurement of CO^ produc­

tion as an indication of the extent of oxidation of several nitrites.

All the compounds tested showed at least 2-day lag periods, but acclima­

tization was possible. Nitrogen in the molecule apparently provided

adequate nitrogen for growth. Marion found that Alcaligenes faecalis

oxidized all nitriles, except malononitrile which appeared to be toxic 16

to the culture (30'. Marlon's results indicated extended lag periods

for the nitriles as reported for the nitriles in Ludzack's system.

In a later study by Ludzack et al. (48) on "cross acclimatiza­

tion" it was shown that aerobic treatment was most efficient in

oxidizing nitriles and that lactonitrile-acclimated sludges adapted readily to oxidation of other nitriles.

Amides

Placak and Ruchhoft (34) found that thioacetaraide, CH^CSNT^.

exerted no O2 demand in 5 or 24 hr. Likewise, formamide did not exert

an oxygen demand in the dilution BOD test (5).

Amines

Methylamine and aniline caused a slight O2 uptake in short-term

Warburg experiments using washed activated sludges (41). Glucosamine was also slightly oxidized. The most extensive study of amine oxida­

tion using the standard BOD with substrates at 2.5 mg/liter was that

of Lyon (1). His results indicated the following PTO values:

PTO PTO Substrate 5 days 10 days

butylamine 26.5 50.0 ethylenediamine 0.0 monoethanolamine 0.0 58.0 diethanolamine 0.3 triethanolamine 0.7

Marion (30) showed that at 500 mg/liter the 1-, 2-, and 3-

carbon aliphatic amines and sec.-butylamine were apparently toxic in

extended Warburg experiments using Alcaligenes faecalia. Tert.-butyl-

amine was neither oxidized by nor toxic to this organism. After lag

periods of less than 24 hr, the 4- and 5-carbon compounds were readily

oxidized. 17

Amino Acids

Placak and Ruchhoft (34) reported that cyatlne was only slightly oxidized in 24 hr, due probably to the toxicity of its sulfur groups.

Dawson and Jenkins (41) found that the 0 2 uptake was greatest with those amino acids containing the most amino nitrogen. Busch (49), working with glutamic acid, showed that the plateau of oxygen consumption which corresponds to the first stage of bacterial decomposition could be used as a reference point for BOD studies. Myrick and Busch (50) showed that glutamic acid was forced in the direction of complete oxidation to CO 2 and H 20 by the addition of 5 mg/liter sodium azide. The azide acted as a selective stimulant of respiration.

Other workers, such as Sawyer et al. (11), Lee and Oswald (25), and Heukelekian and Rand (5), showed that the amino acids are signifi­ cantly oxidized by activated sludge.

Proteins

In studies on the clarification stage of the activated sludge process, Heukelekian and Ingols (51) showed that protein hydrolysis occurred readily. Albumin was more resistant than gelatin, and gelatin was more resistant than peptone. The authors concluded that "... the rate of ammonia production varies inversely with the degree of complex­ ity and directly with the degree of hydrolysis of the materials. . ."

Other workers showed that proteins were more resistant than the products of protein hydrolysis (34,41).

Thirteen purified proteins including keratin were found to be readily oxidized by pure cultures of Alcaligenea faecal is (30). 18

Monosaccharides

Glucose is an excellent source of carbon and energy, and has been used extensively for studying BOD activity. Enough work has been done with glucose to reveal a definite pattern of metabolism. Besed on oxygen uptake alone, glucose appears to be poorly utilized, but based on removal from solution by activated sludge, it is rapidly utilized. Many investigators have shown that glucose is absorbed by the sludge floe and is assimilated within the first 20 min of contact. It is not a simple absorption, but an oxidative assimilation. Based on rates of 02 uptake in 24 hr, glucose never exerts more than 26 per cent of the theoretical demand necessary for complete degradation to carbon dioxide and water.

Over half of the 0 2 demand of glucose may be exerted in the first 50 min of aeration. The glucose taken from solution is stored and is utilize! by the cell for energy and carbon over a much longer period (52,53,54,

35,54,55).

Most studies followed the removal of glucose from solution by activated sludge with tests for the presence of reducing sugars. How­ ever, Porges, Wasserman, Hopkins, and Jasewicz (54) used glucose labeled with carbon-14 and demonstrated that a considerable amount of carbon­ aceous material remained in the supernatant fluid after short periods of aeration. They concluded that C - 6 was removed most rapidly but that

C-l was more readily oxidized. In the early stages of aeration 37 per cent of the labeled C-l was found in the evolved C0 2 but only 4.5 per cent of the labeled C - 6 was found in the evolved CO 2.

Using the Warburg techniaue and glucose-acclimatized activated sludge, Gaudy and Engelbrecht (56) showed that glucose was oxidized 19

28.1 per cent in 6 hr, 32.8 per cent in 12 hr, and 37.7 per cent in 24 hr.

In general, carbohydrates as well as many other organic chemicals were adsorbed to the activated sludge £loc immediately after mixing with

the sludge. Prom 4-7 per cent of the xylose, glucose, and lactose added

to sludge was removed from solution; 13-15 per cent of the sucrose and maltose added to sludge was removed from solution; and 30-80 per cent of

the starch and dextrin added to sludge was removed from solution. Thus

. . it would seem that size, chemical structure and solubility of the

carbohydrate molecules are all important factors in their removal from the supernatant liquor by activated sludge. . .** (34)

Disaccharides

Using the Warburg technique, Hoover e t al. demonstrated that

500 mg/liter of lactose exerted an 02 demand that was 37 per cent of the

theoretical value for complete oxidation (57,58). The 5 day BOD data of

Heukelekian and Rand (5) showed oxidation of sucrose and lactose to the

extent of 59.6 and 53.6 per cent of the theoretical demand, respectively.

Although diastase activity was present in both sewage and acti­ vated sludge, starch was not normally found in sewage or sludge in a

significant concentration (59). Oxidation of polysaccharides was not

rapid in activated sludge since other carbon and energy sources were

usually more readily available (41).

Hurwitz et al. (60) showed that at 12-13 C, 7 per cent of cellu­

lose added to activated sludge was degraded in 72 hr and 20 per cent in

96 hr. At 23 C, 87 per cent of the added cellulose was degraded in

72 hr, but only 8 8 per cent was degraded in 96 hr. No effort was made 20 to show how much of the degraded cellulose was oxidized.

Sugar Alcohols

The work reported by Placak and Ruchhoft (34), Gellman (23), and Heukelekian and Rand (5) showed that, in general, the sugar alco­ hols were readily oxidized by activated sludge. RESULTS

Presentation of Data

Explanation of Reporting Method

Figures. All figures were drawn to the same scale. Accumula­ tive oxygen uptake corrected for endogenous respiration was plotted. In a few instances, the ordinate scale chosen was not adequate to include the 24-hr value. When this occurred, the final reading was shown on the figure.

Symbols. The curves were identified by line-symbols, not data- points. The large number of curves plotted on each grtiph precluded the use of each data-point.

Toxicity. Negative symbols appear in the "Percentage of Theore­ tical C>2 Uptake" columns of the tables. These symbols represent in­ stances in which the (>2 uptake of the endogenous control flask exceeded the O2 uptake of the substrate flask. Curves for such "negative" values were not included in the figures as they would have fallen below the abscissa. Such results are interpreted as indicating inhibition of en­ dogenous respiration of the sludge; thus, the amount of this negative difference was reported as mg/liter of "toxicity” of that compound. A high toxicity value (TV) indicates a marked toxic effect on the sludge by that particular compound.

Calculations

Theoretical 02 Uptake. Figures in the tables representing the theoretical 0 2 uptake were based on the oxidation of the molecule to carbon dioxide and water plus nitrate or sulfate when nitrogen or 21 22

sulfur are present in the molecule. The equation for substrate oxida­ tion was balanced, and the number of oxygen atoms required was multiplied by 16 to obtain the weight in grams required to oxidize completely a gram-mole of substrate. A formula was devised to facilitate the remain­ ing calculations: X * G F / M, where X = grams O2 required to oxidize the amount of substrate in the Warburg flask, G = grams of O2 reouired to oxidize one gram-mole of substrate, F = 0.01 gram, i.e., the weight of substrate in each flask, M * gram-molecular weight of the substrate.

X multiplied by 50,000 gave T, the theoretical 0j uptake in mgAiter of

O2 required to oxidize completely the 500 mg/liter concentration of sub­ strate in the Warburg flask.

Percentage of Theoretical O2 Uptake. The formula, D (100) / T

» Y, was used to calculate the "Percentage of Theoretical O2 Uptake" values in the tables, where D * the difference in mg/liter of O 2 uptake between substrate and endogenous control flasks, T = theoretical O2 uptake calculated above, and Y = the percentage value.

Groups of Compounds

Alkanes

Based on the percentage of theoretical oxygen uptake required for complete oxidation to carbon dioxide and water, the alkanes were poorly oxidized (Fig. I; Table 1). n-Pentane was slightly oxidized, while hexane, heptane, and octane (the 6 -, 7_ and ^-carbon compounds, respectively) were toxic to the activated sludges. UPTAKE,Mg/L 400 200 300 500r 0 0 1 BR00KSIDE Tatrodacona> lodacona Dodacana n-P anto no anto n-P -A C h f at e ;n n; ne Panto Figure 1. - Alkane Oxidation by Activated Sludge by Activated Oxidation Alkane - 1. Figure Dacana EGH F ABR RUN,Hr. WARBURG OF LENGTH 4 6 2 8 4 0 24 18 12 6 0 24 Nonana COLUMBUS HILLIARD i i 1 i i i lI I M TABL6 1. - Alkane Oxidation by Activate'

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula O2 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 br Brookside 0.4 0 .1 Pentane ch3(ch2)3ch5 1774 Columbus 0 . 6 1 . 2 0.7 Hilliard 1 . 1 0 .1 1.5

Brookside (-)* 0.4 3.6 Hexane 0 1 3(0 1 2 ) 4 0 1 3 1764 Columbus (-) (-) (-) Hilliard (-) (-) (-)

Brookside (-) (-) (-)

Heptane ch j(ch2)5 chj 1756 Columbus (-) (-) (-)

Hilliard 0 .1 (-) (-)

Brookside (-) (-) (-> Octane CH,(CH,)rtCH, 1751 Columbus 0 .1 0 .1 (-) J fc v J Hilliard (-) (-) (-)

Brookside 0.2 0.1 0.3 Nonane CH3(CH2)7CH3 1747 Columbus 0.3 0.9 1.3 Hilliard (-) 0.3 1.7

Brookside 1.6 2.7 4.9 Decane 01,(01, )flCH, 1743 Columbus 0.7 1.5 3.0 J A O J Hilliard 1.6 3.6 6.1 TABLE 1. - Continued.

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula £>3 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 1 . 2 2.9 4.3 Dodecane CHjCCHjl^gCHj 1738 Columbus 1 . 2 2.4 5.6 Hilliard 2.5 7.0 1 2 . 2

Brookside 1 . 0 2.4 4.0 Tetradecane CH312CH3 1734 Columbus 1.3 2 . 6 6 . 2 Hilliard 2 . 2 5.2 10.7

Brookside 0 . 8 1 . 0 2 . 8 Octadecane CH3(CH2)16CH3 1729 Columbus (-) (-) 0 . 6 Hilliard 0.7 2.3 6 . 1

1t Hereafter negative sign indicates that the compound was toxic to the sludge,i.e. .0 uptake was less than the endogenous control, and hence does not appear above the abscissa on a corrected graph.

NJ 'Jl 26

The toxicity values of these three compounds at 24 hr were as

follows:

Toxicity Values

Substrate Brookside Columbus Hil1iard

n-hexane 63 19 29 n-heptane 36 11 10 n-octane 4 2 16

A decreased toxicity was observed as molecular weight of the compounds

increased. Results for the 9- to 14-carbon alkanes suggested that per­ centage of oxidation was directly proportional to molecular weight.

This trend, however, was not pronounced enough to warrant generaliza­

tions. Vhen the 18-carbon octadecane was used, the sludges showed

slightly lower oxygen uptake than in the case of the 9- to 14-carbon compounds. This suggests that an increase in chain length tended to re­ duce the oxidation of the compound.

Hilliard sludge oxidized the susceptible alkanes more readily than did the Brookside or Columbus sludge. Oxidation of the non-toxic compounds by all three sludges was sufficient to suggest thct the con­ centration of these alkanes could be significantly reduced by oxidation

in an aeration period longer than 24 hr.

Olefins

The unsaturated aliphatic hydrocarbons were oxidized to some extent by one or more of the activated sludges (Fig. 2,3; Table 2,3).

Brookside sludge showed an unusual pattern in the oxidation of the

5-carbon olefins. A toxic effect lasted until the 10th hr when the pentenes and methyIbutenes (5-carbon compounds) were rapidly oxidized for approximately 3 hr, after which there was an abrupt cessation of 27

oxidation. These compounds all possessed one double bond and a boiling point range approximately 30 C below the olefins of higher molecular weight. Volatility may in part explain the peculiar results, but the data from chemical oxidation studies indicated that the temperature of the water-bath was sufficiently below the boiling points of these com­ pounds to eliminate volatility as the reason for the results.

Several olefins were toxic to one or more sludges throughout the experiment. The 24-hr toxicity values were as follows: Toxicity Toxicity Values Values Substrate Columbus Hilliard Substrate Brookside

1 -pentene 29 43 1 -hexene 18 2-pentene 17 43 4-methyl-2-pentene 11 2-methyl-l-butene 26 40 octene- 1 and - 2 4 2-methy1 - 2-butene 14 28 2,4,4-tr imethyl-1-pentene 29 2,4 ,4-trimethyl-2-pentene 30 2,6 -dimethyl-3-heptene 13

From the experimental results, it could not be stated with certainty that branching in the olefin molecule hindered oxidation since the least branched compounds were toxic to two sludges whereas those with the most branching (the tri-methyl pentenes), while not toxic, did not stimulate a significant oxygen uptake by two sludges. Brookside sludge oxidized the 6 -, 8 -, and 9-carbon olefins to the least extent, and the

5-carbon olefins to the greatest extent. No pattern of oxidation based on the position of branching or of unsaturation within the olefin mole­ cule was evident. Since all the olefins were slightly oxidized by at least one of the sludges, it appears that organisms existed in unaccli- matized sludges capable of attacking these compounds. The point of initial attack was probably the double bond in the molecule. t\J UPTAKE, Mg / L 0 0 4 500 300 200 100 -Methyl-1- 4 - ethyh2-butene 2-M Pentene BROOKSIDE

2-Pe«tene 2-Methyl-1 - butene - 2-Methyl-1 18 Figure 2. - Olefin Oxidation by Activated Sludge. byActivated Oxidation Olefin - 2. Figure

LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 24

0

6

COLUMBUS 2 - Methyl-)- pentene, Methyl-)- - 2 12

18

24

0 HILLIARD - ethyl-2-P*ntene 4-M \ 12

18

24 TABLE 2. - Olefin Oxidation by Activated Sludge

Theoretical treatment Percentage of Theoretical O2 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 2.3 1 .6 1-Pentene CH3(CH2 }2CHiCH2 1711 Columbus (-) <-) (-) Hilliard (-) (-) (-)

Brookside 1.9 1.9 2-Pentene CHjCH: GHCH2CH3 1711 Columbus Uf I (-) (-) Hilliard (-) (-) (-)

Brookside t « 1 2.5 1 . 8 2-Methyl-l CHjCHj CCHj JCzCHj 1711 Columbus (-) (-) (-) Butene Hilliard (-) (-) (-)

Brookside 3.3 2.7 2-Methyl-2 (GH3)2C:CHCH3 1711 Columbus V 0 . 1 (-) Butene Hilliard (-) (-) (-)

Brookside <-) (-) 1-Hexene CH3(CH2)3CH:CH2 1672 Columbus 0.7 0.7 Hilliard if y 0.7 1.3

Brookside 0.7 0 .R 0.5 2-Hethyl- H 2C:CCH3(CH2)2CH 1672 Columbus 0 . 8 1 .1 1.5 1-Pentene Hilliard 1.3 1.3 3.1 TABLE 2. - Continued.

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula O2 Uptake Facility Uptake______(mg/liter) 6 hr 12 hr 24 hr

Brookside 0.5 0.4 0 4-Methyl-l- C H :CHCHjCHCHj CHj 1672 Columbus 0.7 0.9 1.4 Pentene Hilliard 1 . 6 1 .1 2.9

Brookside 0 . 1 0 . 2 (-) 4-Methyl-2- CH-CH:CHCHCH*CH- 1672 Columbus 0.3 0.7 1.3 Pentene Hilliard 1.3 0.3 2.5 JN UPTAKE,Mg/ L 400 500 300 200 100 11I■ 1 1 1I ■ .— 6 BROOKSIDE

Octen* 12

18 iue . lfnOiain yAtvtd Sludge, Activated by Oxidation 3. -Olefin Figure

LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 24

0 ,,- i t l lPentene -l-P yl eth rim 2,4,4-T 2 ,4 ,4 - Trimettryl-2 pentene - - Trimettryl-2 ,4 ,4 2 6

COLUMBUS 12

18

24

0 -D im eth yl-3 hepteae yl-3 eth im -D HILLIARD TABLE 3. - Olefin Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical Oj Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 0.4 (-) (-) Octene- 1 and - 2 CH3(CH2)5CH:CH2 & 1711 Columbus 0 . 6 1.3 1 .1 (mixture) CH3(CH2)4GH:CHCH3 Hilliard 0.9 0 . 6 1.7

Brookside (-) (-) (-) 2,4,4-Trimethy1 - CHjtCCHjCHjCfCHjJj 1711 Columbus 0 . 6 1 . 1 1.4 l-Pentene Hilliard 0 . 8 0.4 1.5

Brookside <-) (-) (-) 2,4,4-Tr imethy1- CH3CCH3:CHC(CH3 ) 3 1711 Columbus 0.5 0 .6 0.4 2-Pentene Hilliard 0 . 8 0.4 1 . 6

Brookside (-) (-) C-) 2,6 -Dimethy1- CHtCHCH,CH:CHCH_CH(CH- ) 0 1711 Columbus 1 . 1 1 . 6 2.9 3-Heptene Hilliard 1 .1 1 .1 1 . 6 Chloroalkanes

All the chloro-substituted alkanes (Fig. 4; Table 4) were oxi­

dized to the same extent as or to a greater extent than the corresponding

non-substituted alkanes.

Toxicity was encountered in the low molecular weight compounds.

The toxicity of these compounds to the Brookside sludge was as follows:

Substrate Toxicity Values

l-chloropropane 39 1-chlorobutane 19 1-chloropentane 4

Toxicity of these compounds was inversely proportional to their molecu­

lar weight. This trend was observed with the non-toxic compounds

(Fig. 4). Oxygen uptake was directly proportional to molecular weight

of the substrate. The results with the Brookside sludge were the best

examples of this trend. In general, compounds with the longer carbon

chains exerted the greatest O2 demands.

Chloroheptane was oxidized in an unusual manner which was most

pronounced with Hilliard sludge: a toxic lag period of 9 hr occurred,

followed by a period of rapid O2 uptake until the 1 2th hr, after which

the oxidation ceased abruptly.

Witroalkanes

Two nitro-substituted alkanes, nitroethane and 1-nitropropane, were toxic to the three sludges (Fig. 4; Table 4). Nitromethane was

slightly oxidized by Hilliard sludge after an extended lag period. The UPTAKE,Mg/L 400 200 300 500 100 0

I Chlorohtpton* - 6 ROSD COLUMBUS BROOKSIDE Figure 4. - Chloro- and Nitro-Substituted Alkane Oxidation by Activated Sludge. Activated by Oxidation Alkane Nitro-Substituted and Chloro- - 4. Figure

12 l-Chlorgpantont

I CNorododtcon* - ZHrisfcJ-L-UL 18 1~ChlorolMxone l-Chlorodtccm*

LENGTH OF WARBURG RUN , RUNHr. WARBURG OF LENGTH 24

0

l-Chlorotnrton* 6

12

18

24

0

6

HILLIARD 12

18

24 TABLE 4. - Chioro- and Nitro-Substituted Alkane Oxidation by Activated Sludge

— ™"™""""” ^ffieOTelt?ca'ra^^eatoen?"^^^,"l^ercentagel^?T5 e o r e t T c a ^ 5^ Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside (-) V (-) (-) 1-Chloropropane CHjCHjCI^Cl 1171 Columbus 0.5 0 . 1 0 Hilliard 1.7 2.4 5.7

Brookside (-) (-) (-) Columbus 0.7 0 . 6 1-Chlorobutane CH,(OU),ClJ A J 1253 1 . 1 Hilliard 2 . 0 3.3 6 . 8

Brookside 1.4 0 . 8 (-) 1-Chloropentane CH 5 (CH2)3CH2C1 1313 Columbus 1 .1 1 . 6 2.3 Hilliard 1.9 3.0 6 . 2

Brookside 3.0 3.1 3.4 1-Ch1orohexane GH3(CH2)4CH2Cl 1360 Columbus 3.2 4.6 5.2 Hilliard 5.6 6 . 8 1 0 . 6

Brookside 2 . 0 3.7 5.3 1396 Columbus 0 . 8 1 . 2 0 . 6 1-Chloroheptane CH3(CH2)5CH2C1 Hilliard (-) 8 .1 7.4

Brookside 1 . 1 3.1 6 . 6 1-Chlorodecane CH,

Theoretical Treatment Percentage of Theoretical Oj Compound Structural Formula O2 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 4.2 6 . 8 1 1 . 8 1-Chlorododecane CH3(ai2 H o CH2cl 1504 Columbus 0.9 1.5 3.4 Hilliard 5.5 10,4 2 0 .1

Brookside (-) (“ ) (-) Nitromethane CH3NO2 590 Columbus 0 .1 ( - ) (-) Hilliard (-) (-) 0.3

Brookside (-) f ™ 1 (-) Nitroethene CHjCHjNOj 799 Columbus (-) (-) (-) Hilliard (-) (-) (-)

Brookside (-) (.) (-) 1 -N i tr opr opane CH3CH2CH2NO2 943 Columbus (-) (-) (-)

Hilliard (-) \ * / (-)

c\ 37

O2 uptake, however, stopped within 2 hr. Toxicities of the nitro- alkanes for the sludges were as follows:

Toxicity Values Substrate Brookside ColumEus Hilliard

nitromethane 220 3 nitroethane 128 24 50 1-nitropropane 10 74 46

Alcohols

No aliphatic alcohol studied was completely toxic to the three sludges (Figs. 5-9; Tables 5-9). Results in most cases showed either very rapid rates of oxygen uptake or a suggestion of adaptation.

Primary aliphatic alcohols. Alcohols having 2, 3, 4, 5, and

7 carbon atoms were oxidized at a very rapid rate (Fig. 5, 6; Table 5,

6). The results for ethanol, propanol, butanol, pentanol and heptanol revealed an immediate, initial rise in accumulative oxygen uptake, followed by a reduction in the rate of oxidation between 8 and 16 hr of aeration. Results for methanol indicated that it was oxidized at a steady rate for 24 hr.

Nonanol-1 was toxic to all of the sludges tested, but after

11-23 hr the sludges adapted to this compound and showed a rapid rate of

(>2 uptake. Decanol-1 with Brookside sludge also showed a toxic lag period, but only a resistant lag period was observed with Columbus and

Hilliard sludges. Lag periods for decanol were not as long as the lag for nonanol. After the lag period, rates of (>2 uptake comparable to the

2-7 carbon series were observed. Dodecanol (Fig. 6) was oxidized to a lesser degree than compounds having fewer than 12 carbon atoms. jN 400 UPTAKE,Mg/L 500 0 0 3 200 - 0 0 1 - - BROOKSIDE Butanol*! Ethanol Propanol 18 Figure 5. - Alcohol Oxidation by Activated Sludge, byActivated Oxidation -Alcohol 5. Figure

LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 24

0

'i ' I I II ■I I - J I 1 11 1 1 1 1 1 '‘-1 1 6

COLUMBUS 12

18

24

0 a 4 2 at 5 5 6 ■ i ■i I *■ ‘ 11 1 1 1 ‘ ‘ 1 1 1 1 1 * Hr 6 HILLIARD

12

18 1

TABLE 5. - Alcohol Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0j Compound Structural Formula (>2 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 3.5 6.1 13.2 Methanol CHjOH 749 Columbus 5.1 11.5 23.4 Hilliard 7.2 13.8 26.5

Brookside 3.2 19.1 33.6 Ethanol c h 3c h 2o h 1042 Columbus 14.1 20.7 34.8 Hilliard 16.4 38.0 43.4

• Brookside 9.8 20.1 34.4 CH,CH-,CH^OH 1198 Columbus 13.8 26.4 35.2 Propanol-1 ^ A Hilliard 18.2 34.0 41.2

Brookside 11.7 20.9 27.3 Butanol-1 CH3(CH2)3CH 1309 Columbus 14.2 27.6 30.5 Hilliard 23.5 43.8 50.6

Brookside 13.4 21.1 22.2 a 1361 Columbus 14.2 22.6 26.4 Pentanol-1 CH,(CH5)^ & *v0H Hilliard 21.5 32.5 35.5 UPTAKE, Mg / L 400 200 300 500 100 ROSD CLMU HILLIARD COLUMBUS BROOKSIDE Octodaconol-1 •\ iue . loo Oiain yAtvtd lde o Sludge. byActivated Oxidation -Alcohol 6. Figure T conol-I o n o tc O 5 5 0 ot 24hr ot 0 5 5 LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 24

0 12 6 24 0 6 12 60S at60S 24 hr 24 TABLE 6. - Alcohol Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 9.8 24.6 27.7 Heptanol-1 ® 3 ( C H 2)60H 1446 Columbus 4.4 7.5 17.6 Hilliard 9.5 29.2 41.6

Brookside C-) (-) 1.8 1497 11.5 Nonanol-1 CH,(CH-,)nOHJ a O Columbus (-) (-) Hilliard (-) 2.8 16.6

Brookside (-) 15.2 36.3 Decanol-1 c h ,(c h 9)q oh 1516 Columbus 0.1 2.2 22.0 J & 7 Hilliard 2.8 10.1 29.7

Brookside 5.2 14.8 15.6 Dodecanol-1 CH,(CH_), .OH 1546 Columbus 0.6 1.0 1.1 J a ii Hilliard 7.8 14.4 23.4

Brookside (-) (-) (-) Hexadecanol-1 CH 3(CH2)i50H 1584 Columbus (-) (-) (-) Hilliard 0.9 1.1 (-)

Brookside 0.1 0.3 1.0 Octadecanol-1 CH3(CH2)i70H 1597 Columbus (-) (-) (-) Hilliard 0.9 1.1 (•) 42

Hexadecanol and octadecanol vere resistant to oxidation by all three sludges, but these compounds were not highly toxic as shown by the folloving values:

Toxicity Values Substrate Brookside Columbus Hilliard

hexadecanol 21 4 8 octadecanol 4 9

The oxygen uptake by the activated sludges from the three treat­ ment plants were similar for all the primary aliphatic alcohols, although

Hilliard sludge was the most active.

Secondary aliphatic alcohols. Secondary alcohols (Fig. 7, 9 ;

Table 7, 8) are alcohols having the hydroxyl group attached to a carbon atom that is attached to two other carbon atoms (8). The percentage of oxidation of the primary alcohols was greater than the percentage of oxidation of the secondary alcohols. Propanol-2 and butanol-2 were nearly equal in percentage of oxidation (7 to 13 per cent). Hexsnol-2, hexanol-3, octanol-2, and octanol-4 were not oxidized to significant < percentage. Octanol-2 was toxic to Brookside sludge. A mixture of undecanol-1 and undecanol-2 was readily oxidized.

The secondary hydroxyl group in hexanol-3 and octanol-4 seemed to depress oxidation. These two compounds were the least readily oxi­ dized of the secondary alcohols.

Tertiary aliphatic alcohol a. Tertiary alcohols (Fig. 7; Table 7) are alcohols with the hydroxyl group attached to a carbon atom that is attached to three other carbon atoms (8). 2-Methyl propanol-2 (tert- butyl alcohol), and 2-methyl butanol-2 (tert-amyl alcohol) were the least oxidized of all alcohols studied. The tertiary grouping therefore UPTAKE,Mg/ L 400 200 300 500 100 —■— 0 " ‘'’T M t t t y l proponpi-2 proponpi-2 l y t t t M ‘'’T " 0 —■— l i l^rlM I I I B VI I 1 I I I iue . ScnayTrir n Bace-hi loo Oiain yAtvtdSug. £ Sludge. Activated by Oxidation Alcohol Branched-Chain and Secondary,Tertiary - 7. Figure 6 ROSD CLMU HILLIARD COLUMBUS BROOKSIDE

Mty proponol-l Mathyl 2 12

utonol-2 B 2 at butanol-1 I Matty 2 Mty V -Mathyl 18

LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 24

0 A 12 6 0 6 6 f24hr 4 2 of 565 ot 4 t2 lo 3 3 24 TABLE 7. - Secondary, Tertiary and Branched-Chain Alcohol Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 0^ Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 2 .1 4.6 8.9 Propanol-2 (c h 3)2c h o h 1198 Columbus 5.6 9.3 9.8 Hilliard 6.3 1 1 . 0 1 2 .6

Brookside 9.3 20.3 34.9 2-Methyl (ch3)2chch2oh 1295 Columbus 8.7 15.8 21.5 Propanol-1 Hilliard 6 . 8 18.1 41.0

Brookside 2 .1 4.5 6 . 8 Butanol-2 CH,CHOHCH0CH, 1295 Columbus 4.6 7.3 1 1 . 0 J a J Hilliard 5.8 7.7 1 0 . 2

Brookside (-) 0 . 2 0 . 2 2-Methyl (CH3)3COH 1295 Columbus (-> (-) 0.4 Propanol-2 Hilliard 1.5 1.9 1 . 8

Brookside 8.4 16.5 26.: 2-Methyl (aUKCHCH^CHnOH 1361 Columbus 7.0 14.0 21.7 ^ a & & Butanol-4 Hilliard 15.3 30.7 41.5

Brookside 1 . 0 1.4 4.4 2-Methyl (CH5 )2COHCH2CH3 1361 Columbus 0.9 1 .1 3.0 Butanol-2 Hilliard 2 .1 2.7 3.6 UPTAKE t Mg / L 400 200 300 500 100 - ROSD CLMU HILLIARD COLUMBUS BROOKSIDE xnol-3 o txan H Figure Figure Hcxonol- 8 - eodr loo Oiainb ciae Sludge. Activated by Oxidation Alcohol . Secondary - EGH F ABR RUN,Hr. WARBURG OF LENGTH 0 6 12 18 0 6 12 Octonol -4 24 TABLE 8. - Secondary Alcohol Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 1.5 1 . 8 2 . 6 Hexanol-2 CH5CHOH(CH2)3CH3 1409 Columbus 2 .1 2 . 6 3.3 Hilliard 3.5 4.4 4.8

Brookside 1.3 1.3 1 . 8 Hexanol-3 CH3CH2CHOH(CH2 ) 2 CH3 1409 Columbus 1.9 2 .1 3.0 Hilliard 3.4 3.3 3.4

Brookside (-) (-) (-) Octanol-2 CHtCH0H(CH5 )eCH, 1474 Columbus 1.5 3.3 5.6 Hilliard 1.3 2.4 2.5

Brookside (-) <-) (-) Octanol-4 CH3 ( CH2 ) 2CHOH ( CH2 ) 3CH 3 1474 Columbus 0.5 l.R 3.5 Hilliard 2.4 2 . 6 0.7

(Mixture) Brookside 1.4 14.4 31.3 Undecanol-1 , ai5 (CH2)qCH2OH 1532 Columbus 1 . 0 4.3 1 2 . 6 Undecanol-2 CHj CCHj JqCHOHCHj Hilliard 2 .8 17.6 31.9 47

appeared to render the alcohol resistant to oxidation by activated

sludge.

Primary alcohols vlth branching. When the 4- and 5-cerbon

branched primary alcohols were studied (Fig. 7; Table 7), the following

relationships between structure of the molecule and extent of oxidation

was noted:

Number of Average Percentage Alcohol Carbon Atoms Type of Chain Oxidation At 24 hr

2-methyl propanol-1 branched 31.5 4 butanol-1 unbranched 36.1

2-raethyl butanol-4 branched 29.8 5 pentanol- 1 unbranched 2fi, 0

The unbranched butanol-1 was oxidized to a greater extent than the

branched 2-methyl propanol-1, while the branched 2-methyl butanol-4 was

oxidized slightly more than pentanol-l. Since these results showed no

significant differences in percentage of oxidation it cannot be inferred

that branching either enhanced or depressed total oxidation in 24 hr.

However, considering the rate of oxidation (comparison of 6 hr columns

in Tables 5 and 7, and Fig. 5 with Fig. 7 ) it appeared that the un­

branched compounds were oxidized initially at the most rapid rate, since

the 6 -hr percentage of theoretical values were higher for the unbranched

compounds than the 6 -hr values for the branched compounds.

Polyhydric alcohols. Alcohols having two hydroxyl groups per molecule are glycols (R). Three glycols were studied: ethylene glycol, diethylene glycol, and triethylene glycol (Fig. 4; Table 9). Oxidation UPTAKE,Mg/L 400 200 300 500 100 ROSD CLMU HILLIARD COLUMBUS BROOKSIDE Figure 0. - Tolyhydric Alcohol Oxidation by Activated Sludge. byActivated Oxidation Alcohol Tolyhydric - 0. Figure rohln glycol Trlothylono EGH F ABR RUN,Hr. WARBURG OF LENGTH 4 0 24 6 12 24 0 12 6 ityo# glyoolj- Diithylon# Triothyteno glycol ond 24

TABLE 9. - Polyhydric Alcohol Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 5.7 17.7 42.2 Ethylene Glycol 644 Columbus 19.3 31.1 36.5 Hilliard 5.9 16.6 41.8

Brookside <-) 2.9 4.6 Diethylene Glycol (hoch2ch2-)2o 754 Columbus 4.8 9.3 15.5 Hilliard 2.4 5.0 9.8

Brookside 0 .1 2 . 8 6 . 6 Triethylene Glycol (hoch2ch2-o-ch- ) 2 799 Columbus 9.0 13.8 17.5 Hilliard 2.4 4.5 9.0

o 50

of glycerol, a trihydroxy alcohol, and the sugar alcohols (hexahydroxy alcohols) was considered with the carbohydrates (Fig. 45).

Ethylene glycol was oxidized to a greater extent than the other polyhydric alcohols, including the sugar alcohols. Glycerol was less readily oxidized than ethylene glycol, but was more susceptible to oxi­ dation than the other glycols. Triethylene and diethylene glycol showed an initial resistance to oxidation by Brookside sludge. These two com­ pounds were approximately equal in percentage of theoretical total oxidation (Table 5).

Aldehydes

With the exception of methanal, the aldehydes having 1 to 12 carbon atoms were readily oxidized by the activated sludges (Fig. 10,

11; Tables 10, 11).

Toxicity values for methanal at various time intervals were as follows:

Treatment Toxicity Values Plant 6 hr 12 hr 24 hr

Brookside 55 100 215 Columbus 10 16 42 Hilliard 57 0 4 25fl

Toxicity values of methanal were the least for the Columbus activated sludge and in aeration periods longer than 24 hr the Columbus sludge might be the ouickest to overcome this toxicity.

Results for the aldehydes were similar to those obtained with the alcohols (Figs. 5-"). in most experiments with the alcohols and the aldehydes, there was a rapid rate of oxygen upta e during the first h*lf of the Warburg analysis followed bv an abrupt cessation of oxygen uptake, JN UPTAKE,Mg/L 400 200 500 300 0 0 1 q 6 2 8 4 6 2 8 4 6 2 8 24 18 12 6 0 24 18 12 6 0 24 18 12 6 0 r,i i I i i i i > I i i i Proponol ROSD COLUMBUS BROOKSIDE Glycorol L D Motfcyl propanol 2 11 i i i i Iri. i I i i 1 > i I i i I i I i i i i I i i i I i i i i i I ^ i i > i 1 i i i i i I i i i-.i Irr j i i i i i 1 i i Ethanol / gure 10. dehyde Oxidation by y b n o i t a d i x O e d y h e ld A - . 0 1 e r u ig F Pontanal I Butanol LENGTH OF WARBURG RUN , RUN Hr. WARBURG OF LENGTH

Activated Activated udge. e g d lu S HILLIARD t

1 i i j i .. i | j i i i.i. i j i i i i 1 VT TABLE 10. - Aldehyde Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula O2 Uptake Facility Uptake______(mg/liter) 6 hr 12 hr 24 hr Brookside " c~) ' ' " 7 : r - " (-) Methanal HCHO 533 Columbus (-) (-) (-) Hilliard ( - ) (-) (-)

Brookside 9.9 2 0 . 6 26.4 Ethanal* CH3CHO 908 Columbus 8 . 0 12.4 17.6 Hilliard 15.2 31.6 38.7

Brookside (-) 2 .1 9.0 DL Glyceraldehyde CH2OHCHOHCHO 533 Columbus 7.3 12.9 2 2 .1 Hilliard 7.3 13.3 29.3

Brookside 15.2 25.9 29.5 Propanal* CH3CH2CHO 1102 Columbus 8 . 0 1 1 . 2 16.5 Hilliard 2 0 . 0 37.7 40.4

Brookside 15.7 19.0 18.8 Butanal CH 7(CH,)0CHO 1221 Columbus 10.4 17.4 20.5 J £ £ Hilliard 16.5 28.7 29.1

Brookside 7.9 16.5 28.7 2-Methyl Propanal (CHj^CHCHO 1221 Columbus 8 . 0 10.4 17.8 Hilliard 10.3 19.2 26.4

Brookside 15.2 17.8 17.2 Pentanal CHt(CH2)3CHC 1300 Columbus 9.1 16.7 2 2 . 6 Hilliard 13.7 15.0 13.6

*Note: Due probably to its volatility, substrate formed a precipitate with KOH in the center-well. 53

Comparisons were made of the oxidation of aldehydes possessing the same number of carbon atoms but with differences in functional groups or in branching. Three such comparisons were possible: (1) butanal was compared with 2-methyl propanal (Fig. 10); (2) pentanal was compared with 2-methyl-4-butanal (Fig. 11); and (3) propanal was compared with glyceraldehyde (Fig. 10). In comparisons 1 and 2, it was observed that branching apparently reduced the initial rate of oxidation, but not the percentage of oxidation in 24 hr. In comparison 5, it was seen that the -OH group apparently reduced both the rate and the percentage of oxidation in 24 hr. These findings were consistent with those observed for the alcohols: branching depressed the rate of oxidation but not the percentage of oxidation in 24 hr, and the hydroxyl group in a secondary position depressed both the rate of 0 2 uptake and the 24 hr percentage of oxidation of the alcohol.

Results for all sludges using glyceraldehyde as substrate

(Fig. 10) suggested an oxidation pattern which was different from that of the non-hydroxy aldehydes. Glyceraldehyde was oxidized at a slow, but steady rate during the Warburg experiment.

Hilliard sludge was the most active in oxidizing the aldehydes.

Results for the Columbus sludge in the presence of the lower aldehydes indicated a progressive oxidation that was not observed with the other sludges or with the higher aldehydes.

Ketones

Ketones are the oxidation products of secondary alcohols, and are more resistant to chemical oxidation than are the aldehydes (R).

Results from this study indicated that this generalization is also JN UPTAKE,Mg/L 200 400 300 500 0 0 1 6 2 8 4 6 2 8 4 6 2 8 24 18 12 6 0 24 18 12 6 0 24 18 12 6 0 BROOKSIDE ndacanal Nononal Dodecanal Htpfonol iue 1 -Adhd xdto yAtvtd Sludge. byActivated Oxidation Aldehyde - 11. Figure 4 - butanol - 4 2-Mathyl- EGH F ABR RUN, Hr. WARBURG OF LENGTH

COLUMBUS Octonal — i i ii 1 ii HILLIARD ii i I ii i i i 1i i ii i I 0 t hr 4 2 at 5 5 5

TABLE 11. - Aldehyde Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr Brookside 8.0 14.0 16.9 2-Methyl-4 ( c h 3 )2c h c h 2c h o 1300 Columbus 5.4 8.2 14.2 Butanal Hilliard 14.3 20.3 17.1

Brookside 1.3 5.4 21.6 Heptanal CH3(CH2)5CHO 1401 Columbus 1.6 2.4 10.9 Hilliard 12.0 13.1 11.6

Brookside (-) (-) (-) Octanal* CH3(CH2 )6CHO 1435 Columbus 0.B 0.5 7.6 Hilliard 7.2 8.2 7.0

Brookside 6.8 18.7 19.8 Columbus 0.8 1.6 23.5 Nonanal c h J,< c h fc,)7 /c h o 1462 Hilliard 17.6 20.2 20.0

Brookside 8.9 17.4 18.8 Undecanal cH3(ai2)9aio 1503 Columbus 6.1 10.0 13.4 Hilliard 28.4 34.1 32.0

Brookside 7.6 19.2 25.5 Columbus 3.8 8.9 15.2 Dodecanal CH3(CH2>ioc h o 1510 Hilliard 12.6 15.7 18.9

* Note: Due probably to its volatility, substrate formed a precipitate with KOH in the center-we11. 56

applicable to biological oxidation. The ketones studied ranged in length from 2 to 13 carbon atoms (Figs. 12-14; Tables 12-14).

Two ketones, 2 ,3-pentanedione and 2,4-pentanedione, were toxic to all activated sludges. These molecules shared two characteristics: a 5-carbon chain and two carbonyl groups. Toxicity values at 24 hr were as follows:

Toxicity Values Substrate Brookside Columbus 11 il 1 iard

2.3-pentanedione 91* 1C 25R 2.4-pentanedione 137 61 156

The toxic effect was least for the Columbus sludge. Hilliard sludge was, in general, the least active toward the ketones, being unable to oxi­ dize 14 of the 1C ketones tested. Brookside sludge was relatively inactive toward this group of compounds, oxidizing only " of 14 ketones.

Most readily oxidized of the ketones were the biologically important 3-hydroxy-2-butanone (acetoin) and the two largest nolecub's studied, 2-undecanone and 2-tridecanone. The only ketones not completely toxic to at least one sludge were acetoir., 5-nonanone, 2-undecanone, and

2-tridecanone.

There was no apparent relationship between oxidation and carbon chain length of the ketone. Several comparisons were made, however, regarding other structural features. Of the two ketones with 4 carbons,

2-butanone and acetoin, the latter was the most readily oxidized. The increased susceptibility may result from the secondary alcohol group on the acetoin molecule.

Five ketones with 5-carbon chains were studied (Table 12). Com­ parison of the oxidation of 2-pentanone and 3-pentanone with that of UPTAKE,Mg/L 400 200 300 500 100 3-Ptntanant BROOKSIDE *yr*--uoo* ►[ — 3*Hydro*y-2-butonon* 8 4 6 2 8 4 0 24 18 12 6 0 24 18 Figure 12. - Ketone Oxidation by Activated Sludge. Activated by Oxidation -Ketone 12. Figure Ptntonone EGH F ABR RUN,Hr WARBURG OF LENGTH COLUMBUS 3 - M tthyh 2 tthyh M - 3 111 Proponon* tanon* j i 1 i i ■i i 2 -but on -but 2

11 ■ ,I 11 1 1I I I . l/l 1 I 1 I 1 iI HILLIARD 2 8 24 18 12 Ui 'sJ TABLE 12. - Ketone Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr Brookside (-) (-) <-) Propanone CH3COCH3 1102 Columbus 0.4 0 .1 (-) (Acetone) Hilliard (-) (-) (-)

Brookside (-) <-) (-) 2-Butanone CH3COGH2CH5 1221 Columbus 1 . 2 22.9 9.4 Hilliard (-) (-) (-)

Brookside 0.4 2 .1 8.8 3-Hydroxy-2 ClKCOaiOHCH, 908 Columbus 7.4 10.9 23.7 Butanone Hilliard (-) (-) 8.9 (Acetoin) Brookside 0 .1 0.7 0 . 8 2-Pentanone ch3coch2ch2ch3 1300 Colutnbus 1 . 0 1.7 4.5 Hilliard (-) (-) (-)

Brookside 0.7 1 . 0 1.4 3-Pentanone ch 3ch2coch2ch3 1300 Columbus (-) (-) 0 .1 Hilliard (-) (-) (-)

Brookside (-) (-) (-) 3-Methyl-2- CH,COCH(CH ) 1300 Columbus 3.5 3.8 6.6 5 j Butanone Hilliard (-) (-) (-) TABLE 12. - Continued

^nJeor^tTcaT^TreatoenT ^e^entag^TF?heoret?can^ Compound Structural Formula O2 Uptake Facility Uptake (mp/liter) 6 hr 12 hr 24 hr Brookside <-> - " < ! ) ----

Brookside (-) (-) (-) 2 f4-Pentanedione CHtCOCH0COCHt 959 Columbus P / J (-) (*) (-) Hilliard (-) (-) (-) 02 UPTAKE, Mg/L 400 200 500 300 100 Figure 13. - Ketone Oxidation by Activated Sludge. Activated by Oxidation -Ketone 13. Figure BROOKSIDE 0

6 COLUMBUS

3 -Octonon* 12

18 2

-Htptonoo# 24 TABLE 13. - Ketone Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr Brookside (-> <-) 4-Methyl-2- CH3COCH2CH(CH3)2 1358 Columbus 4.6 5.4 9.1 Pentanone Hilliard (-) (-) (-)

Brookside 0.2 0.9 1.9 2-Heptanone CHjC O C C H ^ C H j 1401 Columbus 4.1 6.4 12.5 Hilliard (-) (-) (-)

Brookside 0 0.7 1.8 3-Heptanone c h 3(c h 2)3c o c h 2c h 5 1401 Columbus 3.6 5.5 9.3 Hilliard <-) (-) (-)

Brookside 0.9 0.9 1.8 4-Heptanone c h 5c h 2c h 2c o c h 2c h 2c h 3 1401 Columbus 3.4 5.6 9.7 Hilliard (-) (-) C-)

Brookside 1.1 1.3 3.0 2-Octanone CH3CO(CH2)5CH3 1435 Columbus 3.2 5.7 10.9 Hilliard (-) (-) (-)

Brookside (-) (-) (-) 3-Octanone CH3CH2C0(CH2)4CH3 1435 Columbus 3.4 6.0 10.3 Hilliard (-) (-) (-) UPTAKE,Mg/L 400 200 300 500 100 2-Tridecanone ROSD COLUMBUS BROOKSIDE Figure 14. - Ketone Oxidation by Activated Sludge byActivated Oxidation Ketone - 14. Figure EG H F WARBURGHr. OF RUN, LENGTH 0 6 12 8 24 18 TABLE 14. - Ketone Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretics Compound Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside (-) (-) (-) 2-Nonanone c h 3c o (c h 2)6c h 3 1462 Columbus 3.5 6.8 13.7 Hilliard (-) (-) (-)

Brookside 0.9 1.7 4.7 5-Nonanone c h 3(c h 2) 3c o (c h 2)3c h 3 1462 Columbus 1.8 4.5 13.3 Hilliard (-) 1.6 5.6

Brookside (-) 2.9 26.9 2-Undecanone CH3CO(GH2)q CH3 1503 Columbus 1.3 2.8 12.3 Hilliard l.a 13.7 26.2

Brookside 2.1 8.2 19.4 2-Tridecanone CH300(CH2)10CH3 1533 Columbus 3.1 7.0 18.4 Hilliard 4.6 9.8 20.7 64

2,3-pentanedione suggests that introduction of a second carbonyl group­

ing increases the resistance or toxicity of the molecule.

Of the three heptanones tested, the molecule with a carbonyl group at the number two carbon atom was oxidized to the greatest per­ centage, while in the 9-carbon ketones the molecule with a carbonyl group at the number five carbon atom was most completely oxidized. Thus no definite conclusion is possible regarding the effect of carbonyl position on oxidation of the ketone.

Saturated Fatty Acids

The saturated fatty acids were, in general, readily metabolized by all three sludges (Fig. 15, 16; Table 15, 16).

The fatty acids from 1-5 carbons in length were oxidized immediately and rapidly. In general, the 24-hr accumulative 02 uptake for the acids up to an 8 -carbon chain increased in a stepwise fashion with increasing chain length. In Hilliard sludge the 5-carbon pentanoic acid was not oxidized to as great a percentage as were the other fatty acids, but its rate of 0 2 uptake increased near the end of th*’ experi­ ment. Averages of the 6 , 12, and 24 hr percentage of oxidized data for all sludges were computed. These averages revealed that the order of increasing oxidation by number of carbon atoms in the molecule was:

5, 4, 5, 2, 1. The results showed that, for fatty acids of 1-6 carbon atoms, an inverse relationship existed between molecular weight and per­ centage of oxidation achieved by the sludges. The data also showed, however, that a direct relationship occurred between molecular weight and 24-hr accumulative oxygen uptake. The distinction between "accumu­ lative C>2 uptake" and "percentage oxidized" should be noted. 0-a UPTAKE ,M g /L 400 200 300 500 100 BROOKSIDE rpni ocid Propanoic uooc ocid Butonoic taoc acid Etnanoic taoc acid tthanoic M iue 5 -FtyAi Oiainb ciae Sludge. Activated by Oxidation Acid Fatty - 15. Figure 8 4 6 2 8 24 18 12 6 0 24 18

E GH F WARBURG OF RUNLENGTH , Hr. COLUMBUS 0 6 HILLIARD 12 24 0\ ji TABLE 15. - Fatty Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula O2 Uptake Facility ______Uptake______(mg/liter) 6 hr 12 hr 24 hr Brookside 24.7 39.1 54.6 Methanoic Acid* HCOOH 174 Columbus 51.6 59.2 103.2 Hilliard 23.7 37.9 52.3

Brookside 27.2 30.0 34.3 Ethanoic Acid* CH3COOH 529 Columbus 22.3 30.4 36.7 Hilliard 40.6 44.2 49.1

Brookside 24.2 33.7 35.7 Propanoic Acid* ch2ch2cooh 756 Columbus 3.9 19.4 34. C Hilliard 2 2 .6 40.3 50.7

Brookside 17.3 24.6 27.1 Butanoic Acid* CH,(CHO~COOH 90? Columbus 13.5 21.5 24.B J £ £ Hilliard 21.9 29.5 31.9

Brookside 2 0 .6 25.5 26.7 Pentanoic Acid CH,(CH->),COCH 1018 Columbus 17.5 26.6 30.3 Hilliard 2.7 3.2 9.2

* Added to ilask as the sodium salt, but calculated as the pure fatty acid. 6*? TABLE 16. - Fatty Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 19.4 27.3 33.7 Hexanoic Acid CH,(CH,),COOH 1102 Columbus 13.4 25.5 29.9 (Caproic) Hilliard 5.6 13.1 54,4

Brookside 13.8 27.2 31.6 Heptanoic Acid CH3(CH2)5COOH 1168 Columbus 11.7 26.0 33.6 Hilliard 7.9 23.0 62.7

Brookside 15.2 23.1 35.0 Octanoic Acid CH,(CH,).COOH 1220 Columbus 2.4 3.5 5.0 2 A O (Caprylic) Hilliard 11.8 35.9 50.4

Brookside 13.2 20.5 28.5 Decanoic Acid CH3(CH2)pCOCH 1300 Columbus (-) (-) (-) (Capric) Hilliard 19.5 36.1 41.6

Brookside 13.1 16.8 19.8 Undecanoic Acid CH,(CH0)qCOOH 1331 Columbus 0.9 2.3 13.7 Hilliard 20.7 24.5 29.3

Brookside 4.2 5.4 6.6 Dodecanoic Acid CH,(CR0)lnCOOH 1550 Columbus 6.6 5.4 8.1 / 1 U (Laurie) Hilliard 1.4 2.2 3.5 TABLE 16. - Continued.

TheoreticalTreatment Percentage of Theoretical Oj Compound Structural Formula O2 Uptake Facility Uptake (rag/liter) 6 hr 12 hr 24 hr

Brookside (-) (-) (-) Tetradecanoic Acid 1401 Columbus (-) 0.8 0.7 (Myristic) Hilliard 1.7 4.4 10.3

Brookside (-) (-) (-) Hexadecanoic Acid (^(CI^^COOH 1435 Columbus (-) 0.5 (-) (Palmitic) Hilliard 0.9 2.4 7.5

Brookside (-) (-) (-) Octadecanoic Acid CH^C^JigCOCH 1350 Columbus (-) 0.4 (-) (Stearic) Hilliard 0.5 1.3 3.8 7 0

Fatty acids of more than 5-carbon atoms vere oxidized in a different manner by the sludges. Hilliard sludge vas extremely active.

Averages of the percentage data revealed that hexanoic acid was readily oxidized, but heptanoic acid was oxidized to a greater percentage than hexanoic acid. However, above the 7-carbon acid percentage oxidation of the fatty acids bore an inverse relationship to the increase in molecular weight. This relationship was observed for each compound up to the largest molecule studied, octadecanoic acid.

Unsaturated Fatty Acids

The four unsaturated fatty acids studied appeared to be readily oxidized (Fig. 17; Table 17). Hilliard sludge was the most active toward these compounds. The largest molecule, 13-docosenoic acid, was oxidized to the least extent. The longest lag period occurred with

10-hendecenoic acid. Two compounds had the sane number of carbon atoms in the molecule, ricinoleic acid with unsaturation at positions 9 and 1 2 , and linoleic acid with unsaturation at position 9 r d a hydroxyl gr-.n ; at position 12. The results revealed that: first, the hydroxy compound

(linoleic acid) vas oxidized after a lag period with each sludge; second, the 24-hr percentage of oxidation was not significantly different for the two compounds. The conclusion was that the hydroxyl group only initially depressed the rate of oxidation. This result was also noted with the secondary alcohols.

Dicarboxylic Acids

Erratic oxidative patterns were observed in study of the dicar- boxylic acids (Fig. 18; Table 18). Columbus sludge vras the most active of the sludges and oxidized each acid to some extent. The rate of O2 UPTAKE,Mg/L 400 0 0 5 300 200 100 Octodtcoditnoic 12- 2 ,1 9 ocid ocid ROSD CLMU HILLIARD COLUMBUS BROOKSIDE

2Hdoy9otdcni acid l2-Hydroxy-9-octod»c#noic J

Figure 17. - Unsaturated Fatty Acid Oxidation by ActivatedSludge. 0 tlttoc acid 10- Htfldtctnoic I3-Docownotc acid I3-Docownotc LENGTH OF WARBURG RUN tHr. RUN WARBURG OF LENGTH 24 0 6 2 8 24 18 12 6 0 2 8 24 18 12 TABLE 17. - Unsaturated Fatty Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr Brookside 1.2 10.6 19.0 Undecylenic Acid CH2 :CH(CH2)bCOOH 1302 Columbus (-) (-) 34.6 (10 Hendecenoic Hilliard (-) (-) 32.6 Acid) Brookside 5.1 14.2 21.8 Ricinoleic Acid CH3(GH2)5CHOHCH2CH: 1340 Columbus 1.9 IB.6 36.4 (12-Hydroxy-9- CH(CH2)7C00H Hilliard 9.6 20.1 30.3 Octadecenoic Acid)

Linoleic Acid CH3(CH2)7CH:CHCH2CH: 1426 Brookside 9.4 14.2 19.1 (9,12-Octa- c h (c h 2 )4c o o h Columbus 5.0 8.6 13.4 dccadienoic Acid Hilliard 13.6 20.9 40.2

4.0 4.4 Erucic Acid CH3(CH2)7CH:CH' 1439 Brookside 6.9 (13-Docosenoic (CH2)n COOH Columbus 0.5 1.8 5.0 Acid) Hilliard 8.0 11.3 21.2 JN UPTAKE,Mg/L 00 4 200 300 0 0 5 100 0 Plmelic acid Plmelic 0 BROOKSIDE Ketoglutaric acid Ketoglutaric Glutoricacid uai ocid Subaric xlc ocid Oxalic ooi acid Molonic dpc acid Adipic Figure 1R. -Dicarboxylic Acid Oxidationby Activated Sludge.

18

uei acid Suberic LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 24

0

6

COLUMBUS Pi me aci lie eoltrc cd : > acid Ketoglutaric a 12

Oxalocttic ocid Oxalocttic 18

Glutoric Adipic 24

acid 0 HILLIARD Mucic 12

ucnc ocid Succinic Suberic ocid, i l i U i l„ i I i i 18

Adipicocid i 24

TABLE 18. - Dicarboxylic Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical O2 Compound Structural Formula O2 Uptake Facility Uptake (mg/1 iter) 6 hr 12 hr 24 hr

Brookside (-) (-) <-) Oxalic Acid (-COOH)2 89 Columbus 13.6 1.1 (-) Hilliard (-) (-) (-)

Brookside (-) (-) (-) Malonic Acid ho co c h 2cooh 308 Columbus 3.6 (-) 2.6 Hilliard (-) (-) (-)

Brookside 9.1 32.7 36.1 Succinic Acid hococh2ch2cooh 474 Columbus 24.5 40.3 46.4 Hilliard (-) 8.6 44.7

Brookside (-) (-) (-) Glutaric Acid h o c o (c h 2)3cooh 606 Columbus 2.8 1 . 0 4.1 Hilliard (-) (-) (-)

Brookside (-) (-) 6.5 Adipic Acid HOCO(CH2)aCOOH 712 Columbus 3.8 3.8 9.9 Hilliard (-) (-) 4.8

Brookside (-) (-) (-) Pimtlic Acid h o c o (ch2)5cooh 799 Columbus 3.4 3.1 8.5 Hilliard (-) (-) (-) TABLE 18. - Continued.

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/1 iter) 6 hr 12 hr 24 hr

Brookside (-) (-) 7.2 Suberic Acid h o c o (c h 2)6c o o h 873 Columbus 3.6 4.5 14.9 Hilliard (-) (-) 3.3

Brookside 14.6 36.5 29.7 Mucic Acid h o c o (c h o h )4c o c h 343 Columbus 7.3 9.6 3.8 Hilliard 9.0 14.9 38.5

Brookside 28.7 40.6 31.7 Oxalacetic Acid h o c o c o c h 2c o g h 303 Columbus 30.0 36.3 41.3 Hilliard 39.3 43.6 49.2

Brookside (-) (") (-) t>r Ketoglutaric HOCOCO(CH2)2OOOH 438 Columbus 6.2 9.4 17.8 Ac id Hilliard (-) (-) (-) 76

uptake by sludge using several compounds was rapid for the first 2 hr,

ceased abruptly when less than 25 mg/liter O 2 uptake had been achieved,

then showed a lag period in which the endogenous respiration rate

apparently exceeded the rate of oxidation of the substrate. The lag period lasted as long as 10-20 hr, followed by an adaptation toward the end of the experiment. This series of events was observed in the study of all three sludges. Oxidation of oxalacetic acid, however, differed

in that it did not elicit a narked decline in oxidation rate. Oxal­ acetic acid was rapidly oxidized in the early phases of the experiment.

Succinic acid was not oxidized as rapidly as oxalacetic acid in the first 6 hr. However, succinic acid stimulated the highest 24-hr accumulative O2 uptake and a slightly higher percentage of theoretical oxidation in 24 hr than the other compounds in this family. Oxalacetic, succinic, and ketoglutaric are Krebs cycle intermediates, and, of the three, only ketoglutar ie was not readily oxidized. This compound was toxic to Brookside and Hilliard sludge, and was oxidized by Columbus sludge only after a long lag period. Results suggest that the sludge exposed to ©< ketoglutaric acid may ultimately be able to overcome the initial inhibition. The 24-hr toxicity values were less than the 12-hr values, and were as follows:

Toxicity Values Time HillTard Brookside

6 hr 25 5 3 12 hr 62 115 24 hr 34 102

In general, compounds with two carboxyl groups appeared to be somewhat resistant to oxidation by the activated sludge organisms. 77

However, toxicity values on most of the dicarboxylic acids except oxalic

acid suggested that, given time, activated sludge could develop the

ability to utilize them. Oxalic acid maintained its initial toxicity

throughout the Varburg experiment.

Of the dicarboxylic acids not identified as components of the

Krebs cycle, mucic acid was oxidized to the greatest extent. The pecu­ liarity of this dicarboxylic acid is a structure possessing four adjacent secondary hydroxyl groups. On the other hand, adipic acid, a non-hydroxy 6-carbon analogue of mucic acid, caused an extended lag period. Mucic acid is the oxidation product of galactose (Table 27).

It apparently entered the microbial metabolic system readily, and vas oxidized to a greater extent than was galactose.

The effect of a keto group on the oxidation of dicarboxylic acids was difficult to evaluate since the only two keto-bearing compounds available were Krebs cycle intermediates and as such were presumed to be readily utilized by preexisting enzyme systems. Glutaric acid, a

5-carbon acid, was less readily oxidized than its 5-carbon carbonyl analogue, o

Unsaturated Dibasic Acids

The unsaturated dibasic maleic and fumaric acids (cis- and trans-isomers rf the 4-carbon compound) were oxidized to approximately the same extent by the Columbus sludge, and were toxic to the Brookside and Hilliard sludges (Fig. 19; Table 19). Brookside sludge showed a tendency to overcome the toxicity caused by fumaric acid. Toxicity values for Brookside sludge were as follows:

Toxicity Values Substrate 6 hr 12 hr 24 hr

maleic acid 70 117 120 fumaric acid 70 107 11

Compared with the saturated counterpart, succinic acid, these compounds were not as readily oxidized. Since both succinic acid and fumaric acid are Krebs cycle intermediates, an explanation probably lies in the fact that either significant differences in assimilation existed, or that mechanisms responsible for transport of the compound across cell mem­ branes were not as readily available for fumaric acid. These results suggest that unsaturation in the carbon chain between carboxyl groups does not enhance oxidizability.

Dibasic Hydroxy Acids and Lact ic Ac id

Lact ic acid. Tactic acid (Fig. 19; Table 19) may be considered propanoic acid with a secondary hydroxyl group. Sludge using lactic acid (Fig. 15) exhibited approximately the same initial rate of O2 uptake, as sludge using propanoic acid but the accumulative O2 uptake achieved was significantly lower for lactic acid than for propanoic acid

Dibasic hydroxy acids. Sludges using the dibasic hydroxy acids as substrates were observed to have the same rate of oxidation as sludge using the non-hydroxy dibasic acids (Fig. 19; Table 19). Two of the di­ basic hydroxy acids, citric acid and L malic acid, are citric acid cycle components. The L malic acid was oxidized to a higher percentage than the racemic DL mixture. This was consistent with the fact that the L form of most organic compounds is the biologically active form. In UPTAKE f M g /L - 0 0 4 0 0 3 0 0 5 200 100 f Malic L acid Loctic ocid*. iue1. nauae iai n yrx cdOiainb ciae lde o Sludge. Activated by Oxidation Acid andHydroxy Dibasic Unsaturated 19. - Pigure ROSD CLMU HILLIARD COLUMBUS BROOKSIDE

DLMolicacid LENGTH OF WARBURG RUN, OF Hr. LENGTH WARBURG irc ocid Citric 24 LMalcj id je lic a M DL 24 TABLE 19. - Unsaturated Dibasic- and Hydroxy Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside (-) (-> <-) (cis) Maleic HOCOCH:CHCOOH 414 Columbus 8.2 13.5 8.2 Acid Hilliard (-) (-) (-)

Brookside (-) (-) (-) (trans) Fumaric HOCOCH:CHCOOH 414 Columbus 3.1 0.2 5.1 Acid Hilliard (-) (-) (-)

Brookside 26.6 33.3 36.8 Lactic Acid c h 2c h o h c o o h 533 Columbus 21.8 26.5 29.6 Hilliard 34.2 28.5 33.4

Brookside (-) 9.2 24.3 DL Malic Acid HOCOCHOHCH2COOH 358 Columbus* Hilliard 12.0 (-) 17.3

Brookside (-) 29.9 55.0 L Malic Acid h o c o c h o h c h 2c o o h 358 Columbus 28.8 38.8 46.4 Hilliard (-) (-) 33.0

Brookside (-) (-) (-) Tartaric Acid (-CHOHCOOH)- 267 Columbus 7.9 4.1 2.2 £ Hilliard (-) (-) (-)

Brookside (-) (-) 5.3 Citric Acid H0C(C0CH)(CH^C00H)5 375 Columbus 5.1 2.9 21.6 Hilliard (-) C-) 12.8

* Not run. SI

Brookside and Hilliard sludge, the DL form exerted only half as much 02 demand in 24 hr as the L form:

Percentage of Total Oxidation Brookside Hilliard

L form 55.0% 33.0*. DL form 24.0% 17.5%

Presumably, the O2 used in oxidation of DL malic acid vas used to oxi­ dize the L portion of the racemic mixture.

Tartaric acid was not oxidized within the 24-hr period of the experiment, and may have been toxic to the activated sludge in the con­ centration used. Tartaric acid has two adjacent secondary hydroxyl groups which may have contributed to its resistance. Malic acid is a tartaric acid molecule with one less -OH group. Malic acid was oxidized more readily than was tartaric acid. The configurational types of tartaric acid, D, L, and meso, and the racemic mixture of D and L were not available for study. The configuration of the form used in this investigation was not supplied by the manufacturer, and may have been a resistant form.

Citric acid was poorly oxidized for a compound which is known to be important to aerobic metabolism, but a positive total 02 uptake toward the end of the experiment in all three sludges indicated that it may be utilized in longer periods of aeration.

Glycerides

The following glycerides (Fig. 20; Table 20) were oxidized to nearly the same percentagef and stimulated much the same total accumu­ lative 02 uptake; 1-monopropionin, r.ono-n-butyrin, tri-n-butyrin, tri- hexanoin, and tri-octanoin. The accumulative O 2 uptake for these UPTAKE t M g/ L 0 0 4 300 0 0 5 200 100 h Monopropionin h Triourin BROOKSIDE Figure 20. - Glyceride Oxidation by Activated Sludge. Activated by Oxidation Glyceride - 20. Figure Trfoctonoin LENGTH OF WARBURG RUN , Hr., RUN WARBURG OF LENGTH 24 6 2 8 4 0 24 18 12 6 ) Tri-n-butyrin COLUMBUS Trimyrittin HILLIARD 2 8 24 18 12 t win tw rto TABLE 20. - Glyceride Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake______(ng/liter) 6 hr 12 hr 24 hr

Brookside 14.4 22.5 23.0 1-Monopropionin c h 2o h c h o h c h 2o c o c h 2c h 5 756 Columbus* Hilliard 17.6 25.9 32.9

Brookside 13.5 18.2 18.4 Mono-n-Butyr in c h 2o h c h o h c h 2o c o ( c h 2 ) 2c h 3 839 Columbus 13.8 20.6 26.5 Hilliard 13.7 18.1 24.7

Brookside 19.7 20.3 20.1 Tri-n-Butyrin [c h 5(c h 2)2c o o c h 2J2c h o c o - 979 Columbus 12.6 20.7 25.7 (CH2 )2CH3 Hilliard 20.2 24.8 28.7

Brookside 10.4 14.1 16.1 Tri-Hexanoin c5h1 1 coocH2ai( ococ5h, x ) - 1316 Columbus 8.2 15.3 23.6 CH2OCOC5Hn Hilliard 15.0 18.9 22.4

Brookside* 1-Monostearin CH2OHCHOHCH2OCO(c h 2)L6c h 3 Columbus 0.8 0.7 1.2 Hilliard*

Brookside 8.3 12.7 17.0 Tri-Octanoin CyH! 5CXX)CH2CHOCO( CyHx 5 )- Columbus 6.4 13.8 24.2 CH2OCOC-7H^ 5 Hilliard 6.9 11.4 16.4

\

CO TABLE 20. - Continued.

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 0.2 0.2 C-) Tri-Laurin Cx 23COOCH2CH( OCOCl 1H 23 ^ * 1365 Columbus 1.5 3.1 4.5 CH2OCOC1 ^2 j Hilliard 1.9 2.8 2.6

Brookside (-) 0.2 (-) Tri-Myristin j^yCOOQ^CHC OCOC^ 5 ^ 2 7 ^ *” 1405 Columbus (-) 0.1 l.l CH20C0C15H27 Hilliard 0.4 1.6 1.6

Brookside (-) (-) (-) Tri-Palmitin c r5h31cooch2ch(ococj 5h 3l)- 1437 Columbus (-) 0.3 0.6 CH2OCOCj 5H3l Hilliard 0.7 1.5 1.9

Brookside (-) (-) (-) Tri-Stearin CX7H35COOCH2CH(0C0Cl7H 35)- 1463 Columbus (-) (-) (-)

CH2OOOC^ j j Hilliard (-) 0.3 0.8

* Mot run. compounds was similar to that of the fatty acids (Fig. 15) and of gly­ cerol (Fig. 43).

It was previously observed with the saturated fatty acids that those compounds above the 12-carbon lauric acid were poorly oxidized.

In the glyceride series, the triglyceride with lauric acid was also poorly oxidized. The tri-glycerides with 14-, 16-, and 1-’—carbon fatty acids were still more resistant, and even slightly toxic to some sludges during a 24-hr experiment. This paralleled results with the fatty acids.

There was no evidence of increased resistance to total oxidation re­ sulting from a greater number of ester linkages in the molecule (compare oxidation of mono- and tri-butyrin).

Methyl Esters

The methyl esters were readily oxidized (Fig. 21; Table 21).

Results were similar to those for the fatty acids. Sludges using the large molecules, methyl palmitate and methyl stearate, were depressed in activity at the beginning of the experiment, but there was an increasing

(> 2 demand toward the end of the analysis.

The methyl esters fell into three groups based on the type of oxidation curve. These groups included (1) methyl octanoate,(2) methyl propionate and methyl butyrate, and (3) methyl palmitate and methyl stearate. No explanation was available for this grouping of compounds according to their oxidation curves. Oxidation of methyl propionate and methyl butyrate was similar to that of the glycerides of propionate and butyrate (Fig. 20), but the O2 demand of the methyl ester of octanoate far surpassed the O2 demand of the glyceride of octanoate. n j UPTAKE,Mg/L 0 0 4 0 0 3 0 0 5 200 - 0 0 1 6 2 6 4 6 2 8 4 6 2 8 24 18 12 6 0 24 18 12 6 0 24 16 12 6 0 BROOKSIDE 1 *1* * i1 11 »ii Methyl oetonoate ehl polmitote Methyl Methyl-n-botyrote iue 1 -Mty EtrOiain yAtvtdSug. ^ Sludge. Activated by Oxidation Ester -Methyl 21. Figure ehl propionate Methyl ehl stearate Methyl LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH COLUMBUS i i i ii i M I l 1 i i i i i I > t l t i HILLIARD TABLE 21. - Methyl Ester Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0 2

Compound Structural Formula O2 Uptake F a c ilit y Uptake

(m g /lite r ) 6 hr 12 hr 24 hr

Brookside 1 0 . 1 15.1 17.2

Methyl Propionate CH3 CH2 COOCH3 908 Columbus 1 2 . 8 24.6 30.7 Hilliard 6.3 1 1 . 8 9.9

Brookside 1 2 . 8 15.5 17.7 Methyl-n-Butyrate CH,CH~CH~COOCH, 1018 Columbus 15.5 23.1 27.0 J J H illia r d 1 2 . 0 15.4 15.0

Brookside 1 1 . 2 26.7 35.4 Methyl Octanoate CH3 (CH2) 5 0 0 0 0 1 3 1264 Columbus 4.1 13.0 50.2

H illia r d 2 1 . 0 26.4 27.1

Brookside 0.5 2.5 4.8 Methyl Palmitate CH3(CH2) 14COOCH3 1449 Columbus 0.9 1 . 8 4.1 Hilliard 2.3 4 .3 1 1 . 0

Brookside 1 . 0 2 . 2 4.1 Methyl Stearate CH3(CH2) 16COOCa3 1474 Columbus 1 . 1 2 . 2 4 .7 H illia r d 2 . 0 4 .7 15.3 Acetic Acid Esters

In general the acetic acid estera available vere of lover mole­ cular weight than vere the methyl esters. The largest acetate ester vas the 12-carbon decyl acetate while the largest methyl ester vas the 1

The amount of oxygen consumed was proportional to molecular weight of the compound. Since this was true in general for the fatty acids (Fig. 16). the data suggested that the ester linkage vas broken early, and that the O2 uptake represents oxidation of the fatty acid portion of the molecule.

The results in Table 22 indicated that this percentage increase in the 24-hr O2 uptake of the acetic acid esters was paralleled by an increase in the percentage of theoretical oxidation. An exception vas ethyl acetate which was oxidized to a greater extent than methyl acetate or propyl acetate. This suggested that the split occurred at the ester linkage, and that two 2-carbon radicals were oxidized to a greater ex­ tent than were the 1-carbon plus a 2-carbon unit {derived from methyl acetate), or the 3-carbon plus 2-carbon unit (derived trom propyl ace­ tate). This hypothesis should be tested further since in experiments on the alcohols, aldehydes, and fatty acids, the 3-carbon unit had a greater O 2 demand than did the 2-carbon unit, but the average percentage of theoretical oxidation of the 2- and 3-carbon units was essentially the same. JN UPTAKE,Mg/L 0 0 4 200 0 0 3 500 100 BROOKSIDE -otl ocototo n-Hoptyl r»- Butylocototo oy ocototo Doeyl 749 ot 24 Hr 24 ot 749 iue 2 - Aeae ae Oiain y ciae Sludge. Activated by Oxidation Eater Acetate - 22. Figure Ethyl ocototo Ethyl 8 4 6 2 8 4 0 24 18 12 6 0 24 18 oy ocototo Hoiyl

a — LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH

ocototo ‘ ' ‘ L J M l ot 24Hr ot l M 1 * COLUMBUS 1

1

1 I 1

1

' ‘ 1 I ‘ > i I ‘ I 996 ot 996 24 Hr 24

ot 3 3 6 24 Hr 24 L _ L 1 j I I 1 . 1 1 J 1 I I I I I I I I I I I 659 ot 659 H -*I * - Hr 4 2 6

HILLIARD 12

TABLE 22. - Acetate Eater Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0 2

Compound Structural Formula 02 Uptake F a c ility Uptake

(m g /lite r ) 6 hr 12 hr 24 hr Brookside 3.6 8.5 12.7 Methyl Acetate CHjCOOCHj 756 Columbus 13.5 26.6 32.4 Hilliard 6.5 15.9 16.9

Brookside 17.2 25.1 25.8 Ethyl Acetate CBjCOOCHjCHj 908 Columbus 23.1 25.9 29.2 H illia r d 19.9 26.4 29.1

Brookside 15.1 21.7 23.1 Propyl Acetate CH,OOOCH,CH,CH, 1136 Columbus 19.1 21.7 24.7 y & £ j H illia r d 19.5 21.5 2 1 . 6

Brookside 16.5 21.7 22.3

Butyl Acetate CH,COO(CH-),CH- 1 1 0 2 Columbus 2 0 . 0 2 2 . 1 24.2 5 A J J H illia r d 21.7 23.7 24.6

Brookside 21.5 31.6 32.0 Hexyl Acetate CH,COO(CH,)cCHt 1 2 2 0 Columbus 4.2 10.7 43.9 Hilliard 31.2 36.1 28.4

Brookside (-) >24.0 ★ Heptyl Acetate CH5 COO(CH2 ) 6 GH3 1264 Columbus 3.7 7.9 27.4 H illia r d 11.7 45.8 50.2

Brookside 14.7 31.8 55.2 CH,COO(CH,)qCH, 1358 Columbus 13.1 27.1 41.3 Decyl Acetate J » 7 J H illia r d 30.0 38.4 46.6

* Laboratory accident. The Columbus sludge oxidized the alkyl esters more actively than

did the other aludgea (Pig. 23; Table 23). Sludges utilizing these com­

pounds showed an accumulative O2 uptake that was proportional to the

molecular weight of the compounds, but the percentage of theoretical

oxidation of these compounds had no apparent relationship to molecular weight. Propyl formate, propyl acetate, and propyl propionate were oxi­ dized to approximately the same extent. Results for propyl acetate were

reported with the acetate esters.

A1lyl Esters

The allyl esters are aliphatic esters having a point of unsatura­ tion in the alcohol moiety of the compound. The allyl radical is the unBBtursted counterpart of the propyl radical of the alkyl series.

Although experiments with Brookside and Columbus sludge (Fig. 23;

Table 23) indicated that allyl propionate, allyl acetate, and allyl

formate were oxidized slightly in the earl£ stages, they were toxic at

the end of each experiment. Allyl alcohol was toxic to all sludges with no tendency toward a decrease in toxicity during the experiment. There was a slight decrease in the toxicity value of the allyl compounds with

increasing molecular weight, as seen with the Hilliard sludge after 24 hr

Toxicity Values Substrate Hilliard

allyl alcohol 398 allyl formate 370 allyl acetate 367 allyl propionate 344

Since a constant weight of substrate was used, it is possible that there UPTAKE,Mg/ L 0 0 4 0 0 3 0 0 5 200 100 ly propionats Allyl ROSD CLMU HILLIARD COLUMBUS BROOKSIDE Figure 23. - Alkyl and Allyl Ester Oxidation by Activated Sludge. by Activated Oxidation Ester Allyl and -Alkyl 23. Figure -rpl proptonot* n-Propyl LENGTH OF WARBURG RUN , Hr., RUN WARBURG OF LENGTH 6 0 ly format* Allyl 2 8 24 18 12 0 6 2 8 24 18 12 ^ TABLE 23. - Alkyl and Allyl Eater Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake F a c ility Uptake (m g A ite r) 6 hr 12 hr 24 hr Brookaide 10.1 23.5 35.6 Methyl Formate CHjOCHO 535 Columbus 22.0 41.3 60.6 H illia r d 15.0 27.6 33.8

Brookaide 20.0 25.0 23.1 Propyl Formate ch 3( ch 2) 2ocho 908 Columbus 25.7 27.4 29.7 H illia r d 22.7 24.1 21.6

Brookaide 18.5 25.4 26.5 Propyl Propionate ch 3ch 2cooch 2ch 2ch 3 1102 Columbus 15.5 26.3 30.2 H illia r d 20.5 21.9 20.6

Brookaide (-) C-) (-) A lly l Alcohol CH,:CHCH,OH 1102 Columbus (-) (-) (-) H illia r d C-) (-) (->

Brookaide (-) (-) (-) A lly l Formate Columbus 1.0 HCOOCH-CHrCH-^ A 836 (-) (-) H illia r d (-) (-) (-)

Brookside (-) (-) (-) Allyl Acetate CH tCOOCH.CH:CH. 959 Columbus 1.8 (-) (-) J A A H illia r d (-) (-) (-)

Brookside (-) (-) (-) Allyl Propionate CH3CH2COOCH2CH: CH2 1051 Columbus 2.8 0.6 (-) H illia r d (-) (-) (-) fever molecules of the heavier compounds present to antagonize the

metabolism of the sludge organisms.

Nitrjles

All nitriles except malononitrile, were slightly oxidized in

24 hr by at least one of the activated sludges (Fig. 24; Table 24).

Results indicated that rapid oxidation of the aliphatic nitriles by acti­

vated sludges may be achieved after very short periods of adaptation.

This confirms previous reports that certain nitriles serve as source of

carbon and nitrogen for river water organisms after extended periods of

acclimatization and for activated sludge after short periods of aeration

(47, 48).

Malonic nitrile shoved no decrease in toxicity throughout the

analysis. The toxicity values for this compound follov:

Toxicity Values Time Brookside Columbus Hil1jard

6 hr 45 13 20 12 hr or 25 60 24 hr 217 49 1«7

An intensive attempt to acclimatize activated sludge to this resistant

compound should shov if acclimatization is possible after extended

periods of aeration.

The data revealed that auccinonitrile vith two cyanide groups in

the molecule vas oxidized to a slightly greater extent than butyronitrile vith a single cyano grouping. In the case of the 5- and 7-carbon ni­

triles, the di-cyanide compounds , glutaronitrile and pimelonitrile,

were slightly more resistant than the corresponding mono-cyanide rn?-

logues, valeronitrile and heptanenitrile. Aside from malononitrile. JN UPTAKE,Mg/L 0 0 4 0 0 3 200 0 0 5 100 BROOKSIDE Butyronitrile iue 4 - Ni l iain y tvtd Sludge. ctivated A by xidation O ile r it N - 24. Figure 8 4 1 1 24 0 4 2 18 12 6 0 24 18 EGH F ABR RUN, WARBURGHr. OF LENGTH COLUMBUS Hwonanitrila Glutaronltrile Haptcmanitrlfc HILLIARD Voleroai trite Voleroai '3 TABLE 24. - Ni.trile Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0 2 Compound Structural Formula 0 Uptake F a c ility Uptake

Brookside (-)(-) (-) Malononitrile CNCH,CN 1574 Columbus (-) (-)(-) H illia r d (-) (-) (-)

Brookside 1.5 2 . 0 2.5

Butyronitrile ch 3 (ch2) 2cn 1679 Columbus 0 . 2 0 . 1 (-) H illia r d 1 . 8 2.3 2 . 6

Brookside 1.5 2.3 2 . 0 Succinonitrile ncch 2 ch2o i 1199 Columbus 1 . 0 1.7 2 . 6 H illia r d 2 . 0 3.3 6 . 0

Brookside 2.4 1 2 . 2 4 .0

Valeronitrile ch ,( ch 9) , cn 1684 Columbus 1.5 1 . 8 2.7 H illia r d 2.9 3.7 4.8

Brookside 0.7 1.7 1 . 6 Glutaronitrile NC(CH,),0» 1615 Columbus 1.4 2 . 8 5.6

H illia r d 1 . 1 1.5 1.5

Brookside 1.3 1 . 8 1.4 Hexanenitrile CH,(CH-),CN 1688 Columbus 1.9 3.9 1 1 . 6 H illia r d 2.4 3.0 2.4 TABLE 24. - Continued.

TheoreticalTreatment Percentage of Theoretical Oj

Compound Structural Formula O2 Uptake Facility Uptake

(m g /lite r ) 6 hr 1 2 hr 24 b

Brookside (-) (-) (-> Heptanenitrile CHjCCH^sCN 1691 Columbus 0.3 0.9 3.5 Hilliard 2.4 3.5 1 .7

Brookside 1.9 2.9 3.8 Piroelonitrile NC«H2 ) 5® 1637 Columbus C-) (-) (-) H illia r d < -) (-)(«)

Brookside 0.9 1.3 (-) Bensonitrile C^HcCN Columbus 0.5 0 . 1 v J 1513 (-) H illia r d 0.3 1.9 (-> the only toxicities observed during the entire experiment vere for heptanenitrile and pimelonitrile. The following toxicity values show that Brookside sludge was overcoming the toxicity of heptanenitrile, while pimelonitrile exerted a constant toxicity for Columbus and Hilliarl sludges throughout the experiment.

Toxicity Values Treatment 1’lant Heptanenitrile_____ 6 hr 12 hr 24 hr Brookside 53 84 25

Toxicity Values ______Pimelonitr ile_____ Columbus 1 4 11 Hilliard 74 12^ 255

Amides

The sludges shoved approximately the same Op uptake in 24 hr in the presence of the different amides (Fig. 25; Table 25). There seemed to be no direct relationship between molecular weight and percentage of oxidation of the amides. Most of the amides were oxidized at a steadily increasing rate during the experiment. Stearamide, an 1^-carbon com­ pound, was poorly oxidized by Columbus and Brookside sludges and was slightly toxic to the Hilliard sludge. Formamide was initially toxic to

Brookside sludge, but this sludge gradually overcame the formamide toxi­ city as the experiment progressed. This is shown by the following toxicity values:

Toxicity Values Substrate Brooks^ ide 6 hr 12 hr 24 hr

Formamide 12 16 3 UPTAKE,Mg/L 400 500 200 300 100 BROOKSIDE Propionomldt Figure 25. - Amide Oxidation by Activated Sludge. Activated by Oxidation Amide - 25. Figure LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 6 0 COLUMBUS 2 8 24 18 12 omo idt om Form HILLIARD TABLE 25. - Amide Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical O 2

Compound Structural Formula 0 2 Uptake Facility Uptake

(m g /lite r ) 6 hr 1 2 hr 24 hr

Brookaide (-)(-) ' <->' ' Formamide HCONH 0 977 Columbus 3.9 1 1 . 0 29.5 * H illia r d 0.9 3.1 5.8

Brookside 0.4 0 . 6 7.0

Acetamide CH3CONH2 1151 Columbus 1 . 1 3.0 1 2 . 2

H illia r d 2 . 0 6 . 2 16.8

Brookside 1 .4 3.7 14.2 Propionamide ch 5ch 2 conh 2 1259 Columbus 3.5 8 . 0 22.9 H illia r d 2.4 6.5 17.3

Brookside 1.4 5.9 6 . 6

Butyramide CH3 CCH2 ) 2 OONH2 1324 Columbus 2 . 2 3.9 8 . 0 Hilliard 0.4 1 . 6 4.5

Brookside 3.5 5.0 1 1 . 8

Valeramide ch 3 (ch 2 ) 3conh 2 1384 Columbus 2.9 5.3 12.3 H illia r d 0 . 8 3.8 16.8

Brookside 1 . 0 1 . 1 2.3

Stearamide CH 3(CH2 ) 1 6 CX)NH2 1594 Columbus 0 . 6 1 . 1 1.7 H illia r d (-)(-)(-) TABLE 26. - Amine Oxidation by Activated kludge

Theoretical Treatment 24 Hour Percentage o£

Compound Structural Formula O2 Uptake Facility Toxicity Theoretical

(mg/liter) Values O 2 Uptake (m g /lite r ) In 24 hr Brookaide 272 (-)

Methylamine ch 3nh 2 1932 Columbus 183 (-) H illia r d 543 (-)

Brookaide 256 ( “ ) Ethylamine 1863 Columbus 177 (-) V e * Hilliard 497 (-)

Brookside 2 1 1 Propylamine CH jCH jCH j NHj 1827 Columbus 164 (-) Hilliard 237 (-)

Brookaide 215 (_)

Allylamine CH 2 :CHCH2 NH2 1752 Columbus 123 (-) H illia r d 38 (-)

Brookside 2 1 1 iso-Propylamine ch,chnh 5ch. 1827 Columbus 166 (-) H illia r d 71 (-)

Brookside 2 0 0 {„) Butylamine CH,CH-CH-CH,NH, 1805 Columbus 144 (-) J e & • * \ H illia r d +24 1.3 TABLE 26. - Continued

Theoretical Treatment 24 Hour Percentage of Compound Structural Formula 02 Uptake F a c ilit y T o x ic ity Theoretical

(m g /lite r ) Values 0 2 Uptake (m g /lite r ) In 24 hr Brookside 105 <-> iso-Butylamine (CH j JjCHCH jNHj 1805 Columbus 170 (-) H illia r d 2 * (-)

Brookside 144 (-) sec-Butylamine CH ,CH -CHCH ,NH « 1805 Columbus 176 C-) H illia r d + 2 0 0 . 1

Brookside 69 (-) tert-Butylamine (CH 3 ) 3CNH2 1805 Columbus 153 (-) H illia r d 5* (-)

Brookside 1 2 0 (-) Amylamine ch 3 (ch 2 ) 4nh4 1882 Columbus 179 (-) H illia r d ♦5 0 0.3

Brookside 138 (-) iso-Amylamine (ch 3 ) 2 chch 2 ch 2 nh2 1790 Columbus 181 (-) H illia r d 41 (-)

Brookside ♦560 3.0 Pyridine 1884 Columbus 1 1 (-) C5H5N H illia r d 2 1 (-) TABLE 26. - Continued.

Theoretical Treatment 24 Hour Percentage of Compound Structural Formula 02 Uptake F a c ilit y T o x ic ity Theoretical

(mg/ 1 it e r ) Values 02 Uptake (m g /lite r ) In 24 hr Brookside 218 (-) Hexylamine c h 3(c h 2)5n h 2 1779 Columbus 195 (-) H illia r d 473 (-)

Brookside 209 (-) cyclo-Hexyl amine CH,(CH,),CHNH, 1724 Columbus 190 (-) 1 * *1 * H illia r d 77 (-)

Brookside 280 (-) Octylamine CH 3 (CH2 ) 7 NH2 1764 Columbus 194 (-) H illia r d 497 (-)

Brookside 287 (-) Decylamine CH,(CH-)nCH,NH, 1755 Columbus 194 (-) ^ * O * A H illia r d 525 (-)

Brookside 143 (-) Naphthy1amine 1537 Columbus 89 C10H7NH2 (-) H illia r d 156 (-)

★ During initial part of run, the substrate accumulative 0 ? uptake exceeded control run, but at 24 hr, the accumulative showed the amine to be to x ic .

Accumulative 0^ uptake at 24 hr slightly higher in substrate than in control flask. 104

Amines

Although the amines were toxic to all sludges (Table 26), the results indicated that the sludges had a slight tendency to overcome

the toxicity of a few compounds near the end of the experiment. For most amines, the toxicity values increased steadily.

Amino Acids

The amino acids were oxidized to a variable extent by the three activated sludges (Fig. 26-30; Tables 27-31). Based on the results ob­ served at 24 hr, »«DL alanine was oxidized to the greatest extent (43 per cent) and DL methionine was least oxidized (2.6 per cent).

Each 3-carbon amino acid (Fig. 26) had a different functional group on the end of the chain opposite the acid radical: methyl group

in alanine, amine group in ytfalanine, hydroxyl group in L serine, and

sulfhydryl group in cysteine. Listed in order of increasing percentage of oxidation, they were: cysteine, alanine, L serine, and ©< alanine.

Vith each sludge, cysteine had a more rapid initial rate of oyidation than the other three compounds. Although the primary alcohol group on

L serine was expected to increase the speed of oxidation of this com­ pound as compared with oc alanine, this did not occur. The primary amine group was previously shown to be toxic; this may account for the fact that ^alanine was oxidized more slowly than «< alanine.

Figure 27 presents the results for the oxidation of the 4-carbon amino acids. Horaoserine, with a primary alcohol group, was less oxidized than DL threonine, which has a secondary alcohol grouping. This was contrary to previous findings in this study which suggested that primary alcohols were more easily oxidized than secondary alcohols. Of the o UPTAKE, MglL 0 0 4 00 30 500 200 100 0 - 6 BROOKSIDE 12 Alanine 0 L-S*rlna 18 iue 6 -AioAi xdto yAtvtd Sludge. Activated by Oxidation Acid Amino - 26. Figure

24 EGH F ABR RNtHr.t RUN WARBURG OF LENGTH 6 COLUMBUS 12

18

24 HILLIARD 12

18 TABLE 27. - Amino Acid Oxidation by Activated Sludge

TheoreticalTreatment Percentage of Theoretical Oj Compound Structural Formula (>2 Uptake* Facility (mg/liter) 6 hr 12 hr 24 hr

Brookside 5.1 8.6 15.9 Glycine CH2n h 2COOH 799 Columbus 4.6 9.8-« 18.9 Hilliard 2.6 5.9 15.8

Brookside 7.5 27.7 39.0 cx -DL-Alanine c h 3c h n h 2c o o h 853 Columbus 7.9 19.2 53.5 Hilliard 19.6 33.1 36.6

Brookside 1.3 2.3 11.6 -Alanine n h 2c h 2c h 2c o o h 943 Columbus 1.3 2.9 6.4 Hilliard 2.4 15.5 30.0

Brookside 4.6 14.4 22.1 L-Serine h o c h 9c h n h ,c o o h 723 Columbus 10.5 23.0 44.8 Hilliard 10.7 25.7 29.0

Brookside 8.3 10.1 13.8 Cysteine h s c h 2c h n h 2c o o h # 957 Columbus 8.7 10.2 11.5 Hilliard 5.5 5.4 8.2

+ Calculated amount of 0- required per liter to oxidize completely the substrate present in a concentration of 500 mg/liter.

Added to flask as cysteine hydrochloride, but calculated as cysteine. O

CVI UPTAKE ,MglL 0 0 4 0 0 5 0 0 3 200 100 0 6 BROOKSIDE 12 L o MflM oM Hom DL Figure 27. - Amino Acid Oxidation by Activated Sludge. Activated by Oxidation Acid Amino - 27. Figure L sote Acid Asportte DL 8 4 0 24 18 L Thrconlnt DL EGH F ABR RUN,Hr. WARBURG OF LENGTH 6 COLUMBUS 12 8 24 18 0 6 HILLIARD 12

8 24 18 107 TABLE 28. - Amino Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0 2

Compound Structural Formula 0 2 Uptake F a c ilit y Uptake

(m g /lite r ) 6 hr 1 2 hr 24 hr

Brookside 2.5 5 .7 12.4 DL Threonine ch 3chohghnh2cooh 839 Columbus 5.2 10.7 2 0 . 1 Hilliard 3.9 8.3 16.1

Brookside 0 . 8 1.4 2.5

DL Homo serine 1590 Columbus 1 . 2 2 . 0 3.5 HOCH2c *12CHNH2COOH H illia r d 1.3 3 .6 7.2

Brookside 8 . 1 12.4 2 2 . 8

DL Aspartic Acid hoocch 2 chnh2cooh 631 Columbus 13.5 24.9 42.2 H illia r d 5.2 11.4 21.4

Brookside 2 . 6 11.3 14.4 L ( ♦ ) Asparagine H-NOCCH-CHMH-COOH 908 Columbus 1 0 . 0 26.7 37.7 A A * H illia r d 18.4 2 0 . 6 2 2 . 1

Brookside 4 .7 13.0 19.9

L Glutamine H,NOCCH-CH-CHNH-COOH 985 Columbus 1 2 . 8 25.6 52.0 H illia r d 1 0 . 0 10.9 20.3 O UPTAKE , MalL 0 0 4 200 0 0 3 0 0 5 100 0 tlrol Acid LtGlirtomlc 6 BROOKSIDE ^ DL Methionine DL ^ yre L-Prollne Hydroey L Pro) Pro) Inc L L Norlcuclnc DL Figure 28. - Amino Acid Oxidation by Activated Sludge. by Activated Oxidation Acid -Amino 28. Figure 4 0 24 EGH F ABR RUN,Hr. WARBURG OF LENGTH

12126 COLUMBUS 6 HILLIARD 8 24 18

601 TABLE 29. - Amino Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Uptake (mg/liter) 6 hr 12 hr 24 hi

Brookside 10.2 21.5 31.9 L (♦) Glutamic Acid HOOCCH2CH2CHNH2COOH 734 Columbus 12.3 25.6 45.5 Hilliard 19.5 28.3 30.1

Brookside 2.3 3.3 4.8 DL Methionine CH , SCH ,CH 0CHNH0COOH 1099 Columbus 0.2 1.0 1.7 ^ * A £ Hilliard 2.0 3.0 1.2

Brookside 2.6 4.3 8.4 DL Valine CH tCHCH , CHNH -COOH 1127 Columbus 2.1 4.4 9.2 ** J £ Hilliard 1.7 4.3 10.6

Brookside 2.1 7.4 25.9 L Proline CH ~CH ~CH -CNHCOOH 1042 Columbus 2.2 6.2 21.9 I 2 _ . 2 2 1 Hilliard 10.1 25.6 28.7

Brookside 0.8 1.8 13.5 Hydroxy-L- CH ^CHCHCH -jCNHCOOH 854 Columbus 1.2 2.8 8.8

Proline T . i J Hilliard 0.9 4.1 32.3

Brookside 3.1 5.6 11.9 DL Norleucine CH,(CH„),CHNH_COOH 1189 Columbus 1.5 4.4 9.3 j 2 t> 2 Hilliard 2.2 5.6 17.5 UPTAKE,Mg/L 0 0 4 0 0 3 200 0 0 5 100 Cyatlnc ) - ( L ROSD CLMU HILLIARD COLUMBUS BROOKSIDE L + Arginlnt Arginlnt L + Figure 29. - Amino Acid Oxidation by Activated Sludge. Activated by Oxidation Acid Amino - 29. Figure LENGTH OF WARBURG R U N , Hr. , N U R WARBURG OF LENGTH c 0 6 1 12 8 24 18 0 6 12

8 4 2 18 111 TABLE 30. - Amino Acid Ocidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 0^ Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 1.0 1.6 4.5

L (*) Lysine nh2 (ch 2 ) 4 c h n h 2 cocjh# 1259 Columbus 1.6 3.5 7.1 Hilliard 2.9 8.4 30.6

Brookside 0.7 3.4 15.5 L (♦) Arginine Columbus 2.3 5.9 19.1 Hilliard 3.4 13.7 14.9

Brookside 1.7 3.6 8.8 L Leucine (CH3)2CHCH2CHNH2COOH 1311 Columbus 1.3 3.6 8.9 Hilliard 1.3 3.7 12.0

Brookside 1.1 3.2 7.9 L Isoleucine CH5CH2CH(CH3)CHNH2a)OH 1189 Columbus 1.3 3.5 8.6 Hilliard 3.8 9.3 27.9

Brookside 2.1 3.5 6.9 L (-) Cystine HOCOCHNHjCHj S-i 932 Columbus 1.4 2.7 4.8 h o c o c h h h 2c h 2s J Hilliard 0.9 1.0 2.3

Added to flask as L (*) lysine monohydrochloride, but calculated as lysine. ★ Added to flask as L (+) arginine monohydrochloride, but calculated as arginine. o

UPTAKE Mg/|_ 0 0 4 200 0 0 5 0 0 3 100 BROOKSIDE iue 0 -AioAi xdto yAtvtd Sludge. Activated by Oxidation Acid - Amino 30. Figure DL Tyrortnt Tyrortnt DL EGH F ABR RN, Hr. , RUN WARBURG OF LENGTH c 0 6 COLUMBUS 12 8 24 18 0 6 HILLIARD 2 8 24 18 12 TABLE 31. - Amino Acid Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (rag/liter) 6 hr 12 hr 24 hr

Brookside 4.6 6.4 10.6 L Histidine NHCH: NCH: COUCHNH ->COOH 1212 Columbus 2.2 4.5 18.3 1 1 Hilliard 1.7 7.9 20.5

Brookside 2.2 3.9 9.3 DL Phenyl­ C6H6CH2CHNH 2COCW 1186 Columbus 1.0 2.8 7.0 alanine Hilliard 3.4 10.1 32.9

Brookside 2.8 8.3 22.4 DL Tyrosine h o c 6h 6c h 2c h n h 2c o o h 1038 Columbus 1.8 3.8 13.7 Hilliard 5.5 16.1 31.7

Brookside 0.6 1.3 4.3 DL Tryptophane C6H4NHCH:CCH2CHNH2COOH 2507 Columbus 0.4 0.9 2.8 1------1 Hilliard 0.7 2.0 6.0

Brookside 0.5 11.3 18.4 Acetylglycine CH .CONHCH oC00H 786 Columbus 18.4 18.1 34.7 Hilliard 0.9 0.5 2.5

Brookside 1.4 3.9 23.6 Glutathione h o c o c h n h 2(c h 2)2c o n h c h - 924 Columbus 7.5 11.3 26.2 ( CH 2StH ) CONHCH 2COOH Hilliard 6.5 9.4 16.3 115

4-carbon acids, DL aspartic acid was oxidized to the greatest extent in

24 hr. This was no surprise since aspartic acid is a dicarboxylic acid

which differs in structure from succinic acid only in the possession of

an amino group. It will be recalled that of the dicarboxylic acids

succinic acid was oxidized to the greatest extent (Fig. 18). Consider­

ing averages of the 24-hr oxidation percentage data for the three

sludges, succinic acid was oxidized to a greater degree than was DL

aspartic acid. L(+) asparagine, which has an amide group, was oxidized

more readily than butyramide (Fig. 25) which has an amide, but no car­

boxyl group.

Six amino acids having 5 carbon atoms each were tested (Fig. 28).

This made several comparisons possible. The compound in this series which exerted the greatest 0^ uptake was glutamic acid. «*< Alanine

(Fig. 26) was the only amino acid which exerted a greater 0^ uptake than

glutamic acid. The relationship of these tvo compounds to the Krebs

cycle may explain their ready utilization by an aerobic system. The

results for glutamic acid were almost identical with the results for

glutamine.

The grouping CH 3SCH2-R was encountered for the first time in DL

methionine, and was apparently resistant to oxidation. Of the amino

acids, this compound exerted the lowest 24-hr oxygen demand.

Vhen oxidation of the cyclic compounds proline and hydroxyproline

were compared, it was found that with hydroxyproline the secondary -OH

grouping appeared to cause a significant lag period.

Seven amino acids of 6 -carbons each were studied. Brookside and

Columbus sludges oxidized the straight-chain DL-norleucine (Fig. 2^) to 116 a slightly greater extent than the branched L leucine and L iaoleucine

(Pig. 29). However, the percentage of oxidation of the straight chain compounds by Hilliard sludge was intermediate between the oxidation of the branched compounds. L leucine is branched at the end of the chain, while L isoleucine has a methyl group on carbon atom 3. The two branched compounds, L leucine and L isoleucine, were oxidized to the same extent by Brookside and Columbus sludges. In the alcohols, too, branching had

little effect on the percentage of oxidation of the compounds. The location of branching also bore no apparent relationship to the extent of oxidation of the molecules, but further study should be carried out to prove this latter point.

The 6-carbon amino acids containing more than one nitrogen atom per molecule (histidine in Fig. 30; lysine and arginine in Fig. 29)

seemed to be readily oxidized. This confirms the conclusions of Pavson and Jenkins (41) that the amino acids with the highest percentage of nitrogen were oxidized to the greatest extent.

The sulfur-containing cystine was the least oxidized of the

6-carbon acids. The aromatic amino acids, phenylalanine and tyrosine,

are constituents of many proteins. Tyrosine was oxidized more rapidly

and to a greater extent than was phenylalanine (Fig. 30). The -OH group

on the aromatic ring of tyrosine may have been responsible for the en­ hanced oxidation.

The data for acetylglycine and glutathione revealed that both were readily oxidized (Fig. 30). 117

Proteins

Since the structural formulae of proteins are not available, an empirical formula baaed on the average percentage elemental composition of proteins was chosen (61). This formula was calculated to be

(C^H7ON)x , where x represented the unknown number of repeating units per molecule. Since the phosphorous and sulfur content of proteins is low, these elements were ignored in the empirical formula. "Approximate

Theoretical 02 Uptake" and "Percentage of Approximate Theoretical 02

Uptake" for proteins were calculated by the methods previously described.

The proteins were rapidly oxidized by the sludges. This suggested that the metabolic pattern of the activated sludges possessed proteoly­ tic characteristics.

Gelatin and the blood proteins (Fig. 31, 32) were the most com­ pletely oxidized of the proteins. The plant proteins, edestin, zein, and gluten (Tables 33 and 34), were oxidized to a variable extent.

Blood albumen was oxidized to a greater extent than was egg albumen.

Casein was oxidized to a greater extent than was lactalburnen. Among the most resistant of the proteins, Keratin which is known to be resistant to degradation, was also quite resistant to oxidation in this study, but was slightly oxidized. Brookside sludge oxidized the proteins nost actively.

Enzymes

Several purified enzymes were tested for oxidation by the three activated sludges (Fig. 34, 35; Tables 35, 36). nata for the enzyme tables were calculated in the same manner as for the proteins. Oxida­ tion of each enzyme by the three sludges resulted in similar results, UPTAKE,Mg/ L 0 0 4 200 0 0 3 0 0 5 100 am globulin Gammo BROOKSIDE oieE lh globulin alpha BovineUE (bovine) / (bovine) Hemoglobin Gtlatin

526 526 Figure 31. - Protein Oxidation by Activated Sludge. byActivated Oxidation Protein 31.- Figure 01 24 hr 4 2 LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH COLUMBUS ea globulin Beta 24 1 1 24 18 12 6

HILLIARD 8 1 1 TABLE 32. - Protein Oxidation by Activated Sludge

Average Empirical Approximate Treatment Percentage o£ Approximate Compound Formula Minus Theoretical Facility Theoretical 0? Uptake Fractional S & P 02 Uptake 6 hr 12 hr 24 hr (mg/liter) Brookside 4.9 7.0 12.9 Hemoglobin (c 4h 7o n )x 1660 Columbus 5.1 11.4 19.3 Hilliard 5.8 10.1 16.5

Brookside 5.0 >9.2 * •(Globulin (C4H70N)x 1660 Columbus 2.9 6.9 13.2 (bovine) Hilliard 4.1 U . l 15.7 Fraction IV Brookside (-) >4.8 * jB Globulin A (bovine) Hilliard 4.7 10.7 15.4 Fraction III Brookside 2.5 4.6 * r1 Globulin (C4H7ON) 1660 Columbus 0.7 0.7 11.6 “V r A Hilliard 2.4 8.3 15.0

Brookside 10.0 21.4 31.7 Gelatin (CHON) 1660 Columbus 7.7 14.7 23.5 *t T A Hilliard 8.0 12.5 15.8

Brookside 6.9 9.2 13.8 Casein (C.H 1660 Columbus 6.7 14.0 21.9 f A Hilliard 10.0 14.6 18.9

Laboratory accident. j

N UPTAKE,Mg/ L 0 0 4 200 0 0 3 500r 0 0 1 oi*(I) Albuman ) I ( Bovin* ROSD COLUMBUS BROOKSIDE I ' I 1 I 1 * -L-L-L- 1 I I i 1 ‘ I 1 ' i I 1 Lactalbumm Figure 32. - Protein Oxidation by Activated Sludge. Activated by Oxidation Protein - 32. Figure 8 4 1 1 2 0 24 18 12 6 0 24 18 Fibrinogan Edaatin Albumin LENGTH OF WARBURG WARBURG OF LENGTH Egg RUN, Hr. RUN, HILLIARD ‘ ■i 1 1 '1 > 1 1 I* ‘ ‘ ‘i I

2 8 24 18 12 120 TABLE 33. - Protein Oxidation by Activated Sludge

Average Empirical Approximate Treatment Percentage of Approximate Compound Formula Minus Theoretical Facility Theoretical 0, Uptake Fractional S & P O2 Uptake 6 hr 12 hr 24 hr (mg/liter) Brookside 2.4 5.6 6.0 Egg Albumen (c 4h 7o n )x 1660 Columbus 2.5 6.4 16.0 Hilliard 1.7 7.1 12.4

Brookside 3.7 >14.2 * Bovine Albumen (CAHyON).. 1660 Columbus 3.7 9.3 21.8 “ r A (fraction V) Hilliard 0.5 4.3 12.2

Brookside 0.8 2.0 7.2 Lactalbumen (C4H7CM) 1660 Columbus 1.6 2.5 3.2 “ ' A Hilliard 1.3 2.0 4.3

Brookside 0.1 2.6 9.5 Edeatin 1660 Columbus 0.5 0 3.6

Brookside 2.5 6.1 12.E Bovine Fibrinogen (c 4h 7o n )x 1660 Columbus 2.0 3.9 7.4 (fraction I) Hilliard 4.0 1.7 7.5

Brookside 4.5 7.7 12.4 Egg Yolk (C4h ,°N) 1660 Columbus 4.6 10.2 15.0 *♦ I A Hilliard 7.9 10.9 14.9

★ Laboratory accident. UPTAKE , Mg / L 400 300 0 0 5 100 Glutan Globin, ROSD CLMU HILLIARD COLUMBUS BROOKSIDE Figure 33. - i’rotein Oxidation by Activated Sludge. -i’rotein 33.Activated by Figure Oxidation LENGTH OF WARBURG R U N , Hr. , N U R WARBURG OF LENGTH 0 6 12 18

24 0 6 12 Zain

24 122 TABLE 34. - Protein Oxidation by Activated Sludge

Average Empirical Approximate Treatment Percentage of Approximate Compound Formula Minus Theoretical Facility Theoretical 02 Uptake Fractional S & P Op Uptake 6 hr 12 hr 24 hr (mg/1 iter)

Brookside 2.0 3.7 5.3 Keratin (CHON) 1660 Columbus 2.2 3.5 5.1 ■+ / A Hilliard 1.9 3.3 4.6

Brookside 3.1 6.6 14.8 1.9 3.7 6.4 Glutenin (C,H?ON)„*t / A, 1660 Columbus Hilliard 3.6 7.5 14.5

Brookside 4.1 6.1 11.6 Gluten (C4H 7ON)x 1660 Columbus 5.1 6.1 9.5 Hilliard 4.2 6.1 9.6

Brookside 4.9 6.9 11.5 Globin (c4h 70N) 1660 Columbus 4.3 7.5 10.2 H f X Hilliard 5.1 6.9 10.8

Brookside *** * Zein (c 4h 7o n )x 1660 Columbus * * Hilliard 1.1 1.7 5.1

*Not run. 123 UPTAKE,Mg/L 400 200 0 0 5 100 BROOKSIDE A my lot < Figure 34. - Enzyme Oxidation by Activated Sludge. byActivated Oxidation Enzyme 34.- Figure patt a ip L EGH F ABR RUN,Hr , N U R WARBURG OF LENGTH 0 6 COLUMBUS 12 8 24 18 0 6 HILLIARD 12 8 24 18

*.-Cl TABLE 35. - Enzyme Oxidation by Activated Sludge

Average Empirical Approximate Treatment Percentage of Approximate Compound Formula Minus Theoretical Facility Theoretical Oj Uptake Fractional 3 & P O2 Uptake 6 hr 12 hr 24 hr (mg/liter)

Brookside 0.9 0.9 1 .3 Amylase (alpha) (C^HyON^ 1660 Columbus 9.7 1.2 1 .6 Hilliard 0.7 1.2 1 .8

Brookside 3.1 4.0 4 .2 Invertase (C4H 70H)x 1660 Columbus 3.0 5.1 6.6 Hilliard 3.7 7.1 10.6

Brookside 10.7 15.5 20.9 (C.H,ON) 1660 Columbus 8.2 12.8 19.9 Lipase “ < A Hilliard 9.9 13.9 17.9

Brookside 5.3 7.7 8.7 Proteinase (C.H <*> 1660 Columbus 4.6 7.5 9.7

H / A CO Hilliard 6.2 * 9.3

Brookside 3.3 6 .2 10.8 Urease ((LH7ON)v 1660 2.7 4 .0 6.5 ** / A Columbus Hilliard 3.0 5 .7 9.9 UPTAKE,Mg/L 0 0 4 200 0 0 3 0 0 5 100 ROSD COLUMBUS BROOKSIDE Figure 35. - Enzyme Oxidation by Activated Sludge. Activated by Oxidation Enzyme - 35. Figure LENGTH OF WARBURG RUN f Hr.f RUN WARBURG OF LENGTH 0 6 2 8 24 18 12 HILLIARD TABLE 36. - Enzyme Oxidation by Activated Sludge

Average Empirical Approximate Treatment Percentage of Approximate Compound Formula Minus Theoretical Facility Theoretical 0? Uptake Fractional S & P O2 Uptake 6 hr 12 hr 24 hr (mg/liter) \ Brookside 2.4 4.6 7.7 Cellulase (C4H70N)x 1660 Columbus 3.7 7.0 8.9 Hilliard 3.1 5.8 9.3

Brookside 2.6 2.1 2.1 Catalase (c 4h 7o w )x 1660 Columbus 2.4 2.7 2.9 1 Hilliard 0.2 1.7 3.6 i Brookside 3.6 5.1 6.5 Takadiastase (C4H7°K)x 1660 Columbus 2.8 6.1 9.2 Hilliard 6.2 10.a 13.5

Brookside 4.5 8.6 20.6 Peroxidase (C4H 70K)x 1660 Columbus 3.6 5.9 10.8 (horseradish) Hilliard 3.8 6.0 9.6 127 1 2P indicating that an. activated sludge, regardless of source, may possess the same mechanism for degrading these enzymes.

Carbohydrates

Hexoses. The hexoses were not oxidized to the same extent by the three activated sludges (Fig. 36; Table 37). The only general observation that could be made from the results vas that the hexoses as a group were not oxidized to as great an extent as might have been pre­ dicted. Glucose is one of the best sources of carbon and energy for most organisms. During conventional detention periods in domestic acti­ vated sludge, however, glucose is probably a better source of carbon than of energy (34, 52, 53, 54, 55). The idea has been often postulated that carbohydrates are rapidly assimilated but are not oxidized to com­ pletion in periods less than 24 hr (34, 53).

Results for Brookside sludge (Fig. 3^) gave several unusual oxidation curves. The most striking inconsistency occurred in the oxi­ dation of glucose and levulose. The sharp cessation of oxidation of these two sugars between P and 1 C hr was not observed with any other compound nor with any other sludge. « n methyl glucoside was oxidized by Brookside sludge at a faster rate and to a greater extent than vas glucose, but the reverse was true for the Columbus and Hilliard sludges.

Glucose was oxidized at a slightly more rapid rate and to a

slightly greater extent than levulose by Brookside and Hilliard sludges.

However, in Columbus sludge levulose was oxidized at a more rapid rate and to a greater extent than was any other hexose. Columbus and Hilllard

sludges oxidized most of the sugars only after a 4-6 hr lac period.

Glucose was oxidized after a lag period of 23 hr by Columbus sludge. N UPTAKE tMg/L 0 0 4 200 0 0 3 0 0 5 100 ROSD COLUMBUS BROOKSIDE Mty Glucotide Methyl D a O+Mannote Figure 36. - Hcxose Oxidation by Activated Sludge. Activated by Oxidation - Hcxose 36. Figure LENGTH OF WARBURG RUN , Hr., RUN WARBURG OF LENGTH 2 8 4 0 24 18 12

‘ 12 HILLIARD 2 8 4 2 18 12 " m

i I i lil l>T ^

SorfeoM 129 TABLE 37. - Hexose Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr TTTIr 24 hr

Brookside 4.1 8.8 11.1 D (♦) Galactose 2OHCH(GHOH)3CHOHO 533 Columbus 0.4 3.9 15.9 Hilliard 4.7 16.7 39.0

Brookside 7.5 16.9 21.4 D (♦) Mannose CH2OHCH(CKOH>3CHOHO 533 Columbus 0.4 1.3 12.9 Hilliard 1.7 10.3 27.2

Brookside (-) (-) (-) (♦) Rhamnose CH3(jM(CHOH)3CHOHO*H20 634 Columbus 0.8 1.7 7.7 Hilliard 0.2 (-) 12.1

Brookside 10.2 11.6 7.7 D (♦) Dextrose CH2OH

Brookside 8.6 9.6 4.7 D (-) Levulose yCH2( CHOH) 3(jOHCH2OH 533 Columbus 3.7 10.5 20.6 Hilliard 6.0 15.6 36.6

Brookside 2.1 2.6 6.4 533 Columbus 1.9 3.8 L (-) Sorbose < £ h (CHOH)3COHCH2OH l.l Hilliard (-) (-) 1.1

Brookside 13.1 23.8 45.6 «* D Methyl CH,OHCH(CMOH),CH(OCU,)0 618 Columbus 0.3 1.6 5.7 d | 3 3 i Glucoside Hilliard 2.3 4.0 12.0

jr Added to flask as the monohydrate, but calculated as pure rhamnose. 131

Given the specific substrate, adaptive enzymes are normally produced in shorter intervals of time than the lag periods observed with the hexoses

(62, 63). Bacterial enzymes for dextrose and levulose degradation are considered by most workers to be constitutive; therefore, adaptive enzyme formation for glucose metabolism was eliminated from considera­ tion. It has been suggested (64) that nitrogen could have become deficient in the Brookside sludge after aeration for 8 hr, or was ini­ tially deficient in the other sludges. This conclusion seemed unjustified since the results for mannose indicated that there was nearly the same initial rate of O2 uptake as for glucose and levulose. For mannose the rate did not cease sharply after 8 hr, and in 24 hr this sugar vas oxi­ dized to a greater extent than was glucose or levulose.

L sorbose and L rhamnose were among the least readily oxidized hexoses. With the exception of methyl glucoside in Brookside sludge, the Hilliard sludge was the most active of the three sludges in oxidizing the hexose sugars.

Pentoses. The pentose sugars were poorly oxidized when compared with the hexoses (Fig. 37; Table 3P). Like the hexoses, several pen­ toses induced extended lag periods. Arabinose, the only pentose with an

L configuration, was the most readily oxidized of the pentoses. Brookside and Hilliard sludges oxidized the pentoses most actively, while the

Columbus sludge was the least active toward these compounds.

Disaccharides. Although the disaccharides composed of hexose sugars were not readily oxidized, they were oxidized to a greater extent than were the pentose sugars (Fig. 3?, 40; Tables Tf)-41). The Hilliard sludge was the most active of the sludges in oxidizing the disaccharides. UPTAKE, Mg / L 200 0 0 4 00 3 0 0 5 100 - ROSD COLUMBUS BROOKSIDE +Arobinot* A L+ Figure 37. - Pentose Oxidation by Activated Sludge. Activated by Oxidation - 37. Pentose Figure LENGTH OF WARBURG R U N , Hr. , N U R WARBURG OF LENGTH 0 6 24 HILLIARD

2 8 24 18 12 132 TABLE 38. - Pentose Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical CL Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside (-) (-) 2.6 D (♦) Xylose 0CH5(CH0H),CH0H 533 Columbus (-) 0.2 6.0 I 2 Hilliard 3.0 5.6 22.0

Brookside 3.6 6.2 10.1 D (-) Arabinose CH2(CHOH)3CHOHO 533 Columbus 2.1 4.5 10.7 Hilliard (-) 0.2 1.5

Brookside 1.7 4.1 8.3 D (-) Ribose CH,(CHOH),CHOHO 533 Columbus 1.1 2.1 8.6 1 2_ . ___I ---- 1 Hilliard 3.2 7.1 22.7

Brookside C-) (-) 0.8 D (-) Lyxose CH2(CHOH)3CHOH 533 Columbus 1.5 3.0 8.1 Hilliard 2.1 2.3 6.2

Brookside 4.5 10.3 21.4 L (♦) Arabinose CH - ( CHOH ) ,CHOHO 533 Columbus (-) 0.2 10.1 i.2 ..... J b Hilliard 0.2 8.4 25.9 O l UPTAKE #M g /L 0 0 4 200 0 0 3 0 0 5 OO}- MalibioM - } O lO alboa £-LoctOM £ ' Callobiosa * 0 BROOKSIDE Figure 33. - Hexose Disaccharide Oxidation by Activated Sludge. by Activated Oxidation Disaccharide - 33.Hexose Figure 8 4 1 1 2 0 6 0 24 18 12 6 0 24 18 LENGTH OF WARBURG RUN , Hr. , RUN WARBURG OF LENGTH COLUMBUS i I i iI i i i < HILLIARD i i » I 11 I * 1

2 8 24 18 12 134 TABLE 39. - Disaccharide Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 0, Compound Structural Formula O2 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 5.9 10.2 11.4 Sucrose Columbus 1.4 6.2 16.9 C12H 22°11 561 Hilliard 6.6 15.2 33.9

Brookside 4.1 6.2 10.7 otLactose 0.4 2.6 15.0 C12H 22°11*H2° 533 Columbus Hilliard 3.6 4.7 25.9

Brookside 5.6 8.8 13.5 Lactose Columbus 1.5 3.8 11.4 C12H22°11*H 2° 553 Hilliard 4.9 15.8 39.0

Brookside 1.2 2.3 6.8 D (♦) Cellobiose Columbus 6.8 3.6 12.1 C12H22°11 561 Hilliard 1.6 10.3 26.6

Brookside 0 0.9 3.9 D Melibiose ^12^22^11*^2® 533 Columbus (-) (-) 3.6 Hilliard 7.9 14.4 25.3

Brooksi-~\c. 5.1 7.6 12.8 D (♦) Maltose Columbus 0.2 5.6 12.2 C12H 22°11*H 2° 533 Hilliard 6.2 15.9 37.0 136

Amygdalin Is composed of two glucose units linked as gentiobiose

to D mandelonitrile. The latter is a compound of hydrocyanic acid and

benzaldehyde. Araygdalin was oxidized by Hilliard sludge but not by the

Brookside and Columbus sludges (Fig. 39).

Triaaccharides. The two trisaccharides studied were melezitose,

composed of two glucose units and one fructose unit, and raffinose, com­

posed of fructose, glucose, and galactose (Fig. 30; Table AO). Neither

trisaccharide was oxidized to the same extent as any one of its com­

ponents alone (see Tables 37 and 40). All sludges acclimated to these

trisaccharides after extended lag periods.

Polysaccharides. Figure 40 and Table 41 present the results of

the oxidation of the polysaccharides, inulin, starch, dextrin, end

glycogen. The "Approximate Theoretical C>2 Uptake" values for these

compounds were calculated from the estimated molecular weights in the

same manner as for the proteins and enzymes.

Inulin is composed of methyl fructofuranose units, may be

branched, and has a molecular weight of approximately 5000. It is not

utilized in animal metabolism (61),but appeared to be oxidized by acti­

vated sludge after an extended lag period. Starch appeared to be the

least readily oxidized of the polysaccharides. This was an unexpected

finding, since it has been shown that activated sludge possesses amylase

activity (59).

Dextrins are products of the hydrolysis of starches. Glycogen

is like starch and dextrin in that it possesses glucose units joined by

o* 1-4 linkages. However, the glycogen molecule shows more branching OL UPTAKE ,M g /L - 0 0 4 200 0 0 3 0 0 5 100 ROSD CLMU HILLIARD COLUMBUS BROOKSIDE Figure 39. - Di- end Trisaccharide Oxidation by Activated Sludge. Activated by Oxidation endTrisaccharide Di- - 39. Figure LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH l I I I I 0 6 12 24 TABLE 40. - Di- and Trisaccharide Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 05 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 6.2 9.3 16.9 561 Columbus 2.9 6.4 20.7 Trehalose ^12^22^11 * ^*2^ Hilliard 10.9 20.1 24.4

Brookside <-) (-) 1.9 Amygdalin CgHcCHCjN 796 Columbus (-) (-) (-) 1 Hilliard 2.4 5.2 7.9 0C12H21°10 Brookside 0.4 3.0 7.2 571 5.3 D (*) Melezitose C 18H 32°16 Columbus (-) (-) Hilliard 1.8 13.8 33.1

Brookside l.l 1.4 2.6 571 7.9 D (♦) Raffinose CieP32°l6 Columbus (-) (-) Hilliard 6.7 13.3 23.8 UPTAKE,Mg/L 0 0 4 200 0 0 3 500r 0 0 1 6lycog«n BROOKSIDE Figure 40. - Maltose and Polysaccharide Oxidation by Activated Sludge. Activated by Oxidation Polysaccharide and -Maltose 40. Figure D+MOltOM LENGTH OF WARBURG R U N , Hr. , N U R WARBURG OF LENGTH COLUMBUS 4 0 24 6 HILLIARD

2 8 24 18 12 9 3 1 TABLE 41. - Polysaccharide Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Empirical Formula 0. Uptake Facil ity Uptake (rag/liter)* 6 hr 12 hr 24 hr

Brookside 0.5 0.7 5.1 Inulin 592 Columbus 0.7 1.4 6.9 U 6*10°S>xV 1 V m f A Hilliard 1.4 4.7 15.0

Brookside (-) 1.7 2.2 Starch 592 Columbus 0.5 0.7 0.8 (C6H1 0 V x Hilliard 1.2 (-) 1.0

Brookside 2.9 5.6 7.8 592 Columbus 0.8 4.1 12.0 Dextr in ^6®10®5^x Hilliard 14.0 23.5 29.9

Brookside 3.9 7.3 9.9 Glycogen x 592 Columbus (-) 1.2 4.4 Hilliard 13.3 19.6 26.0

* Approximate values for theoretical O2 uptake, and for per cent of theoretical 0^ uptake are given for the polysaccharides since accurate molecular weights are not available. CL UPTAKE , M g /L 4 200 0 0 4 0 0 3 0 0 5 100 - BROOKS BROOKS IDE Figure 41. - Polysaccharide Oxidation by Activated Sludge. byActivated Oxidation Polysaccharide - 41. Figure ahd Aaar Washed 8 24 18 Poctin EGH F ABR RUN,Hr. WARBURG OF LENGTH 0 12 6 COLUMBUS 24

HILLIARD 141 TABLE 42. - Polysaccharide Oxidation by Activated Sludge

Hydrolysis Products Approximate Treatment Percentage of Approximate Compound and Approximate Theoretical Facility Theoretical 0o Uptake Empirical Formula 02 Uptake

Brookside 2.5 3.4 4.5 Agar Agar galactose, H2SO^ 444 Columbus (-) 0 0.2 (Difco) * Hilliard (-) (-) (-)

galacturonic acid, CHj., Brookside 4.9 11.0 21.9 Pectin galactose, arabinose#. 547 Columbus 5.9 7.1 9.5 Hilliard 1.8 6.7 18.5 (C7H10°6^x

Brookside (-) (-) 0 Xylan xylose*. (C5H^q°4^x 606 Columbus (-) 7.4 12.2 Hilliard 0.5 2.5 6.3

galactose, arabinose, Brookside (-) (-) (-) Gum Arabic rhamnose*, glucuronic 592 Columbus (-) (-) (-) •cid. (c 6h 10o 5)x Hilliard 1.0 3.0 5.2

Brookside 1.4 2.0 2.7 Gum Tragacanth bassorin®, pectin, 592 Columbus 0.8 2.2 4.9 starch. (C6H10O5>x Hilliard 1.4 3.9 8.8

^ 1P.J1 Given in Table 37. Given in Table 38. ^ ^6H 10°5 " a vegetable mucilage. 143 than the starch molecule. Neither glycogen nor dextrin vas oxidized to any great extent, but both appeared to be more susceptible to oxidation than was starch.

Figure 41 and Table 42 present data for pectin, agar, xylan, gum arable, and gum tragacanth. All except pectin were poorly oxidized.

Pectin is a methyl ester of pectic acid, which is a galacturonic acid polysaccharide. Pectin was most readily oxidized by Brookside sludge.

Xylan, a hemlcellulose composed of xylose units, caused toxic lags with

Brookside and Columbus sludge, but all three sludges gave an indication of potential adaptation to xylan. Gum arable and gum tragacanth were resistant to oxidation during the 24-hr aeration period. Agar-agar from a commercial source (Difco) and agar washed several times with acetone and distilled water were relatively resistant to oxidation. Accumula­ tive C>2 uptake for the polysaccharides was less than for the ol igosaccharides.

Sugar Alcohols

Sugar alcohols are distinguished by having one hydroxyl group attached to each carbon atom in the molecule. Inositol which is a cyclic compound, is a growth factor for rats and possibly for other animals

(61). In 24 hr inositol was poorly oxidized, but the date for Columbus and Hilliard sludges indicated an increased susceptibility after an ex­ tended lag period (Fig. 42; Table 43).

Glycerol was the most readily oxidized sugar alcohol, due probably to its common occurrence in nature as a component of fats, and to its position in the glycolytic pathway. The results for the oxida­ tion of glycerol were discussed with the polyhydric alcohols and with JN UPTAKE,Mg/L 0 0 4 0 0 3 200 0 0 5 100 BROOKSIDE i i i i I i ■ « ■ i I i j * i » 1 ■rythritol^ FiTvre 42. - Sugar Alcohol Oxidation by Activated Sludge. by Activated Oxidation Alcohol Sugar - 42. FiTvre ^Oulcitol Glyctrol 8 4 6 2 8 24 18 12 6 0 24 18

EGH F ABR RUN,Hr. WARBURG OF LENGTH Sorbitol COLUMBUS HILLIARD 12

24 144 TABLE A3. - Sugar Alcohol Oxidation by Activated Sludge

Theoretical Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 12.2 18.4 25.5 Glycerol OUOHCHOHCH-OH 608 Columbus 9.4 18.4 29.1 Hilliard 9.9 18.8 31.6

Brookside 3.1 4.2 6.9 Erythritol GH2OH(CHOH)2CH2OH 590 Columbus 0.8 1.9 9.2 Hilliard 1.2 3.1 15.4

Brookside 6.0 10.4 16.7 D Mann it ol CH.,OH(CHCH), CH-OH 60A Columbus 0.2 2.8 13.2 Hilliard 8.3 15.6 28.5

Brookside 5.1 8.8 16.9 D Sorbitol CH-,OH(CHOH),CH0OH 60A Columbus 0.5 2.3 13.4 a *» a. Hilliard 7.3 15.1 23.5

Brookside 2.8 3.8 6.6 Dulcitol CH,OH(CHOH),CH.,OH 60A Columbus 0 0.3 2.8 a H a Hilliard (-) (-) 5.9

Brookside 1.5 4.6 8.8 Inositol CHOII(CHOH)aCHOH 533 Columbus (-) 0.6 9.4 1------±J Hilliard (-) (-) 14.6 146 the glycerides. The difference in molecular configuration between manni- tol and sorbitol was apparently not great enough to lead to any significant difference in oxidation. Dulcitol, however, showed con­ siderable resistance in the first half of the V'arburg experiments.

Erythritol, a 4-carbon sugar alcohol, was oxidized at about the same rate as was dulcitol and inositol.

Hilliard sludge was the most active of the sludges in oxidizing the sugar alcohols as it was in oxidizing most of the carbohydrates.

Thiols

The thiols were poorly oxidized, and results for all but one thiol showed toxicity values at 24 hr for at least one of the activated sludges (Fig. 43; Table 44). The data indicated that the 5-carbon pentanethiol was oxidized to the greatest extent. Next in extent of oxidation was the 12-carbon dodecanethiol, but this compound was toxic to Hilliard sludge. The 6-, 7-, and 9-carbon thiols were toxic at 24 hr to each sludge as the following, toxicity values indicated:

Toxicity Values Substrate BrooVside Columbus Hilliard

hexanethiol 50 24 75 heptanethiol 49 35 129 octanethiol 46 2R 71 dodecanethiol 6

The data for thiols with more than 7 carbon atoms indicated decreased toxicity. This suggested the possibility that during extended aeration periods, acclimatization might occur. UPTAKE t Mg / L 400 200 300 500 100 I- Bu I- ton* thiol ROSD CLMU HILLIARD COLUMBUS BROOKSIDE l-Dod«can«thtol iue4. ho Oicinb ciae Sludge. by Activated Oxidction -Thiol 43. Figure LENGTH OF WARBURG RUN, Hr. RUN, WARBURG OF LENGTH 0 6 0 6

24 147 TABLE 44. - Thiol Oxidation by Activated Sludge

Theoretical ‘Treatment Percentage of Theoretical 02 Compound Structural Formula 02 Uptake Facility Uptake (mg/liter) 6 hr 12 hr 24 hr

Brookside 1.3 1.0 (-) 1-Propanethiol c h 3c h 2c h 2sh 1470 Columbus 0.1 0.4 (-) Hilliard 3.5 3.5 2.7

Brookside 1.4 0.8 C-) 1-Butanethiol 1510 Columbus 0 CH3(CH2)3SH (-) (-) Hilliard 1.7 2.1 1.3

Brookside 3.1 3.7 2.9 1-Pentanethiol OU(CH-),SH 1535 Columbus 2.8 3.1 2.5 ^ Z *4 Hilliard 2.2 2.2 1.2

Brookside 0.3 0.5 (-) 1-Hexanethiol CH,(CH0),SH 1556 Columbus (-) 0.1 (-) Hilliard (-) (-) (-)

Brookside (-) (-) (-) i-Heptanethiol a i3(CH2)6SH 1573 Columbus (-) (-) (-) Hilliard (-) (-) (-)

Brookside 0.4 0.1 (-) 1-Octanethiol CH3(CH2 )7SH 1586 Columbus (-) (-) (-) Hilliard (-) <-) (-)

Brookside 0.9 1.8 4.2 1-Dodecanethiol CH,(CH_). - SH 1700 Columbus 0.4 1.2 1.5 j Z IL Hilliard (-) (-) (-) 149

Constant Weight Versus Constant Molarity and Substrate Oxidation

Sanitary engineers orginarily express concentrations as weight of chemical per unit volume of solvent or licuid phase of the waste.

The most common expression is *'mg/liter.** This was the primary reason

for adjusting flask concentrations to 500 mg substrate per liter of reaction mixture. Unfortunately when this constant initial weight method was used, constancy in initial number of molecules present was sacri­

ficed. The method whereby numbers of molecules are held constant (i.e., substrates present in the flask on a molar basis) offers a means of comparing oxidation of organic compounds molecule for molecule. The latter method may be more effective in pointing out differences in oxi­ dative metabolism based on structure than the constant weight method.

Of course, it is not possible to add the same number of molecules when exact molecular weights are not known, as was true with the poly­ saccharides, proteins, and enzymes. Furthermore, if the number of molecules ore held constant and two compounds, with different molecular weights are being compared, it is obvious that the flask containing the

lighter compound contains less carbon and therefore less total bond energy than the heavier compound. On the other hand, when the substrate weight is held constant, the compound having the highest molecular weight will have fewer molecules in the flask.

To study the influence of these two methods of attaining ini­

tial concentration upon oxidation, a group of compounds was chosen whose

24-hr oxygen uptake was proportional to molecular weight, i.e., the fatty

acid group ranging in molecular weight from 46.02 (methanoic acid) to

172.26 (decanoic acid). Flasks were prepared in parallel using the same 150 activated sludge in the presence of both 500 mg/liter and 0.1 raTole of the sane substrate. Sludge concentration vas adjusted aa in previous experiments. Three analyses were made, using Brookside, Columbus, and

Hilliard sludges. Results are shown in Fig. 44-46. Table 45 lists the absolute weight of substrate added to the respective flasks.

The oxidation of the substrates in the flasks at 0.1 raM con­ centration was erratic compared with the oxidation of the same compounds at concentrations of 500 ragAiter. Unexplainable increases in rate and stoppages of oxidation were observed. The data in Table 45 revealed that the weight per flask of each of the first three acids was greater at 500 mg/liter than at 0.1 mMole. In each case rate and total 0^ uptake were greater with a concentration of 500 mg/liter. However, there was a proportional relationship between the weight of substrate and oxidation, indicating that a progressive increase in demand occurred.

TABLE 45. - V.'eight Versus Molecular Basis of Comparing substrates

Grams/fTask Grams/flask Chemical (500 mg/liter) (0.1 nMole) Methanoic acid 0.01 0.006.?

Ethanoic acid 0.01 0.00^2

Propanoic acid 0.01 0.0046

Butanoic acid 0.01 0.0110

Pentanoic acid 0.01 0.0102

Hexanoic acid 0.01 0.0116

Heptanoic acid 0.01 0.0130

Octanoic acid 0.01 0.0158

Decanoic acid 0.01 0.0172 CM UPTAKE, Mg/L 0 0 4 0 f/ SUBSTRATES 500Mfl/L 0 0 6 200 500 300 100 1 1 24 2 18 12 6 0 u C\ 1x* 1* 11 11 1■ 1* 1 |1 1 1 1L* 1 11 1_l gur - Wegt ess ecul Bss Comparing f o Basis r la u c le o M Versus eight W - . 4 4 re u ig F - t cd daton b Bokie i ed Sludge. d te a tiv c A Brookside by n tio a id x O Acid tty a P EGH F ABR RN Hr. RUN, WARBURG OF LENGTH Ethanoic Octonolc BROOKSIDE

. ML SUBSTRATES 0.1 -MOLE m Dtconoic i i / / t . .1 i. Butanolc i i i t Heptonoie

151 COLUMBUS

500 Mg/L SUBSTRATES O.lm-MOLE SUBSTRATES 6 0 0

5 0 0 r

Ethanoic C3

Hcpfanotc

Hcxonoic - W. I . 1 1 ... I t 2 4 O § 1 2 1 8 LENGTH OF WARBURG RUN, Hr. Figure 45. - Weight Versus Moleculsr Bssis of Comparing Fatty Acid Oxidation by Col inn bus Activated Sludge. 153

HILLIARD

6 0 0 r 500 Mg/L SUBSTRATES 0.1 m-MOLE SUBSTRATES

Hcptonotc

5 0 0

Psntonoic o»400

UJ Propionic

< Otconolc I- Q_ 300 Z > 0J Butonolc O

M sthanoic 200

1 0 0 -

Ethanoic

1 1 ■ 1 ■ * 1 * ■ ■ ■ 1 * * ■ ■ ■ *1 . 6 12 18 24 0 6 12 LENGTH OF WARBURG RUN, Hr. Figure 46. - Weight Versus Molecular Basis of Comparing Fatty Acid Oxidation by Hilliard Activated Sludge. 154

The results for the 4, 5, and 6-carbon fatty acids were similar since

the weight of substrate per flask was nearly the rame regardless of the basis on which the substrate was added. Prom heptanoic acid to decanoic

acid, however, total weight of substrate per flask was greater at 0.1 oiMole concentration than at a concentration of 500 mg/liter. Results

for the Columbus sludge indicated that four of the compounds which were

toxic in a concentration of 0.1 mMole were not toxic in a concentration

of 500 mg/liter. These four were in higher concentration in the 0.1 mMole flasks. The sludges in flasks with the heaviest weight of sub­

strate usually showed the greatest accumulative oxygen uptake. The results in Hilliard sludge for heptanoic acid and octanoic acid in con­ centrations of 0.1 mMole revealed an exception. The lag period for

these compounds lasted 18-20 hr suggesting that the small increase in

total weight of substrate put the concentration in the toxic range for

this sludge.

It was concluded from these results that the constant weight method gave essentially the same oualitative results as the method main­

taining a constant number of molecules. However, It would be desirable

to repeat the entire study using the molar basis for ««idding substrates

in order to compare the two methods more adequately. DISCUSSION

Effect of Functional Groups upon Oxidat ion

All the compounds studied were placed in groups according to the number of carbon atoms in the molecule. Members of each group were arranged in order of the increasing average percentage of theoretical oxygen uptake at 24 hr. Compounds that were toxic to all sludges were arranged in order of decreasing toxicity values.

Table 46 lists the compound of each carbon chain length which was oxidized to the greatest extent in 24 hr. More than half of these compounds contain the carboxyl grouping. The compounds in each group which ranked second in percentage of oxidation were primarily acids and esters, but included alcohols, aldehydes, and glutamine, an amino acid amide. The alkane, tetradecane, was compared with only one other 14- carbon compound, tetradecanoic acid.

In the 5- and 6-carbon series, the sugars were not oxidized to a great extent. In the same series, the substituted alkanes ve.re oxi­ dized to a greater extent than either the saturated or unsaturated hydrocarbons. In general, unsaturation in the smaller molecules did not increase susceptibility, and in two instances seemed to increase resistance.

The ketones and the thiols were, in general, not readily oxi­ dized. The most toxic functional group, regardless of carbon chain length, was the amine structure, R-Cll^Nl^. an<* 5-carbon compounds, the allyl compounds were the most toxic. 156

Oxidation o£ Enzymes by Activated Sludge

There were several possible explanations for oxygen uptake by sludges in the presence of enzymes. First, the enzyme ss a protein may have been hydrolyzed to its constituent amino acids, with subseouent oxidation of the araino acids. Secondly, the specific substrate of the enzyme was present in the unwashed sludges, and, consequently, was hydrolyzed by the added enzyme. The hydrolysis products thus became available for oxidation by the sludge. Thirdly, both of these phenomena could have occurred simultaneously. It is interesting to note that the enzyme most slowly oxidized was amylase, whose natural substrates are glycogen, dextrin, and the starches. It has been shown that amylase activity is present In activated sludge (59). The amylase substrates for amylase were poorly oxidized. Results for the oxidation of these substrates gave 02 uptake curves similar to those for amylase (Fig. 41).

Of the enzymes, lipase stimulated the greatest 02 uptake by the sludges.

It will be recalled that the substrates for lipase, the glycerides, gave results quite similar to those shown for lipase.. Sludges are constantly exposed to fatty materials and it has been reported that glycerides are removed quickly during aerobic treatment (46); therefore, lipase acti­ vity should be present in activated sludges. Many of the results for the proteins (Figs. 31-53) were similar to results for proteinase. It seems contrary to the economy of nature for enzymes to be degraded by the same system which produces them. However, an enzyme produced by one organism in an activated sludge need not be degraded by the same organ­ ism, since in a dynamic system such as activated sludge there may be a thousand different systems at work, each with individual requirements 157

TABLE 46. - Compound of Bach Carbon Chain Length Showing the Highest Percentage of Theoretical Oxidation in 24 hr

M uaW r o f Name o f Compound Percentage of theore­ Carbon Atoms Most Completely tical Uptake, 24-hr In Chain Oxidised in 24 hr Average for A ll Sludges

1 sMthanoic acid 70.0

2 methyl formate 4 3 .3

3 oc a la n in e 4 3 .0

4 L sialic acid 44.8

5 L glutamic acid 3 5 .8

6 hexanolc acid 3 9 .0

7 heptanoic acid 42.7 > CD 8 hexyl acetate •

9 heptyl acetate 4 0 .8

1 0 decanol - 1 2 9 .6

1 1 1 0 -hendecenolc acid 28.7

1 2 decyl acetate 4 7 .7

14 tetradecane 6 .9

16 hexadecanoic acid 2 .5

18 l2-hydroxy-9-octa- 3 3 .0 decenoic acid

2 1 tri-hexanoin 2 0 .7

and ab ilities. Also, the commercial product was obtained from an animal or plant source. Although it has the same enzyme capacity as the bac- terial enzyme, it may, nevertheless, differ sufficiently in chemical structure to make it foreign to the bacterial system and thus susceptible to oxidation. 158

Solubility of Substrates

Consideration of the relationship of the solubilities of the sub­

strates in water to the extent of oxidation by activated sludge revealed

that eaae of solubility alone did not predetermine the rate of oxidation.

In aeveral chemical groups the least soluble member was the moat readily

oxidised. The converse also occurred, i.e ., the most soluble compound was the least oxidised of the chemical family.

Won-branched alcohols. Alcohols with 9-18 carbons are insoluble

In water (65, 6 6 ), yet the 10- and 12-carbon compounds were oxidised by

the three sludges.

Tertiary alcohols. Of the tertiary alcohols, the completely

so lu b le compound, 2 -methyl propanol- 2 , was oxidised less readily than

the slightly soluble 2 -methyl propanol- 2 .

Ketones. Several soluble ketones were not oxidised to any great

extent, while several slightly soluble ketones were readily oxidised.

Dicarboxylie acids. Oxalacetic acid was among the most readily

oxidised of these compounds, but it was also the least soluble.

M ethyl e s te rs . S o lu b ilit y o f these compounds d id not e ffe c t

oxidation. The three heaviest are insoluble, yet one of these esters,

methyl octanoate, was oxidised to a greater degree than any of the more

aoluble methyl esters.

W itriles. In the two 4- and the two 5-carbon compounda, the

di-cyanide compound of each pair is more soluble than the member with a

single cyanide group yet the di-cyanide was the least readily oxidized.

Thiols. Although the solubility of the thiols decreases steadily

with increasing molecular weight, the thiols having more than 7 carbon 159

•tana In the molecule exhibited a trend to decreased toxicity and a greater rate of oxidation than the thiola below 7 carbon atoms In length.

Comparative A ctivity of Sludgea

Certain chenical groups were oxidized beat by one aludge, but poorly oxidized by another aludge. It was d ifficu lt to judge which of the activated sludges investigated was moat active since oxidation aeemed to depend prim arily on the chemical family used as substrate.

Unusual Oxidation Curvea

The o x id a tio n o f s e v e ra l compounds fo llo w ed a r a th e r unusual pattern. For example, the results for n-pentane (Fig. 1) indicated that this compound was oxidized to a certain level of accumulative 0 2 u p ta k e , followed by an actual cessation of accumulative uptake. Some curves actually revealed an apparent decrease in accumulative 02 uptake. This was obvious in the endogenous-corrected curves but would not have been revealed as clearly in uncorrected curves. The corrected curves were a plot of the difference between the control results and the substrate results. When the rate of increase in accumulative 02 uptake in the substrate flask exceeded the rate of increase in accumulative 0 2 uptake in the control flask, the curve revealed a net increase in the rate of oxidation. When the uncorrected rates became the same, the corrected curve revealed a cessation in net 0 2 uptake. When the control rate ex­ ceeded that of the substrate, the corrected accumulative 0 2 uptake curve fell to a lower value. Volatility of the substrate could possibly ex­ plain the drop in accumulative uptake due to an increased pressure occurring progressively during the experiment. However, vo latility is a physical constant and should not be expected to increase during the 160 experiment. Adequate period* of equilibration were uaed at the begin­ ning of each experiment. The*e equilibration period* were long enough to allow any volatile compound to come into equilibrium with it* vapor phase. Another possibility was that denitrification occurred late in the experiment with the evolution of gaseous nitrogen. This seems un­ likely, however, since 0 2 uptake was often seen to increase again later in the experiment, an occurrence not to be expected if denitrification was increasing. There is much evidence against denitrification occurring in a highly aerobic system (62, 67). Another possible explanation in­ volves a change in permeability of the organisms during the experiment.

Such a change could have led to an increased absorption of a toxic com­ pound — le a d in g to a c e s s a tio n o f 0 2 uptake, or to a decreased absorption of a readily degraded compound — also leading to a cessation

o f 0 2 uptake. Although it is highly improbable, s till another explana­ tion is possible. A gas in addition to C0 2 could have been given off,

thus leading to falsely low or negative accumulative 0 2 uptake values. NUMMARY

Approximately three hundred organic aliphatic compounds were

examined for susceptibility to oxidation by activated sludges taken from

three domestic waste treatment plants. Oxidation of these substrates waa determined in the Warburg Constant Temperature Respirometer using

oxygen uptake at 20 C as the criterion. Flask concentrations employed

throughout the study were 500 mg/liter for substrates and 2500 mg/liter

for mixed liquor suspended solids. Results showing mg/liter 02 uptake,

corrected for endogenous respiration, were plotted for each substrate

with the exception of a few toxic chemicals. Tables were presented

which included the structural formulae, calculated theoretical total

O2 demand, and the extent of oxidation of the substrates at 6, 12, and

24 hr reported as percentage of theoretical 02 demand.

Alcohols, aldehydes, fatty acids, glycerides, and esters were

in general oxidized rapidly.

Results for the lower fatty acids, including octanoic acid,

indicated a progressive increase in 24-hr oxygen uptake with increasing

molecular weight of substrate, but based on percentage of oxidation the

reverse order was seen with the smallest molecule being oxidized to the

greatest degree by the sludges. Fatty acids above 3 carbons were re­

duced in percentage of oxidation.

The amide group proved to be susceptible to oxidation, as did

the amino acid grouping. The amino acids exhibited a wide range of

susceptibility to oxidation.

161 162

The amine and allyl configurations were highly toxic functional groups. Compounds showing significant resistance to oxidation included a thiols, nitrites, the longer glycerides, certain dicarboxylic acids, several dibasic hydroxy acids, certain ketones, most olefins, and the alkanes. The few low molecular weight nitro-substituted alkanes studied indicated that the nitro group may be toxic to sludges. The chloro- alkanes above 5 carbons in length showed partial oxidation in 24 hr.

The carbohydrates were, in general, not readily oxidized in

24 hr. Among the sugars, there was no apparent correlation between biodegradability and configuration. The hexose sugars and many oli­ gosaccharides were more readily oxidized than were the pentoses, "ost sugar alcohols were oxidized to the same extent as were the hexoses.

Oxidation results suggested that in the low molecular weight molecules, unsaturation did not enhance degradability but actually con­ tributed to resistance. In long chain fatty acids, however, unsaturation seamed to enhance susceptibility to oxidation.

Branching in the alcohols and aldehydes tended to reduce the

initial rate of oxidation of a compound, but did not greatly interfere with the 24 hr percentage of oxidation.

The data suggest that solubility of an organic compound in water did not predetermine its ease of oxidation. Many insoluble com­ pounds were easily oxidized, and many easily soluble compounds were poorly oxidized.

So many individual differences were observed that it cannot be stated with certainty that any one of the sludges was most active 163 biochemically in oxidizing all compounds; therefore, no correlation was established between oxidative activity and plant design. However, one of the sludges was often much more active than the others in oxi­ dizing the members of a given chemical group. On the latter basis,

Hilliard sludge seemed to be more active than Oolutnbus or Brookside sludge.

The results disclosed that the activated sludges from three domestic treatment plants possessed the capacity to oxidize a vide range of aliphatic compounds. lw igas f ramn Plants Treatment of Diagrams Flow

BLOWERS COMPRESSED AIR

MOTOR RETURN SLUDGE

SEWAGE BAFFLE APPENDIX INLET- I-* tin n WEIR o

-AIR LIFTS- COMMUNITOR

SETTLED SLUDGE TREATEC ummaniiinaiinminnuiifff / SEWAGE

AERATION TANK SETTLING TANK

Figure 47. - Flow Diagram of the Brookside Estates Sewage Treatment Plant. PiiRp o-id ttow or Station

I ! Pump Btwer i f v ' T * r r L . L J n 1. »» Ml m r J-'n9r*» * SMf SlHlRf Pi** hhw :: I *■**' | Sf*o* Cwwwber , f-t _ i1 Jj; _... '. JXa . OulMt ' i -. ^il - r ■ - &*j T J 1 St

CLARIFICATION ANO PURIFICATION OF SEWAGE > Cot Faqiao J GonofflHv Stoi* \ Cm A . - -- JT\ '^(SpwoM^ " tq . *— fi ^ Djmp' 9l*d«o 1— H...- j: Get Mo f l»oan T0rfc J 5li*d«* OipfltiOA Too*

DISPOSAL OF SEWAGE SOLIDS

Figure A8. - Generalized Flow diagram of the Columbus, Ohio Sewage Treatment Plant. 165 166

CRUDE SEWAGE

1r \C COLE RI ■Ground Screenings I r z n [gas ENGINE " Z I ..n SCREENS Screenings —W—|gr i n o e rU- ■^[puM Phw—l GENERA TORS [ j [p u m p } “ f1 A T " ' 7 - 4 - j t i |GRIT CHAMBER)— ►— Grit -Tq u m p I r I ?WE. L1 ~T , | p u m p s ] A

S * Overflow i. A. 4 J p RE AERATION I SLUDGE I i A' GAS * TANKS jCQNSENTRATORSn. 4 ■ HOLDER r* 0 r~ I - - J T I j Thickened Cooi Sludge Water I Hot Water

'A PRELIMINARY -Grease and Scum —- e j e c t o r } • T SLUDGF ' r ljC* *" SEDIMENTATION ^ M ixed _j DIGESTION 1 ; :,.er TANKS ►—Raw Sludge - PUMPS r^Siudge TANKS |■ Cj* ^ ~v Settled Sewoge Di ges'ed Sludge

F e m e .. ... f ...... C hi or de ““I CONDiT'ONiNU 1 7 * Return Excess I , TA\»S • ♦ - Sludge Sludge r rli£"iAERATION j TANKS J t t . Cor d.tinned I DISTRIBUTION S>udgt [_ WELL

M ixed j VACUUM [_ r ,M, Liquor j filT E RS : 1 PUMPS I Filter T Cane Activated 1 .. Sludge f 4 FINAL J SETTLING INCINERATOR . TANKS I— , J COLUMBUS, OHIO I ' 4 ♦ SEWAGE TREATMENT WORKS Final Ash Eft luem * t River jo M *

Figure 49. - Block Flow Diagram of the Columbus, Ohio Sewage Treatment Plant. BLOWER

INLET

PRIMARY SLUDGE PUMP ACTIVATED SLUDGE PUMP RAW SEWAGE SETTLING LIFT STATION AERATION TANK FINAL SETTLING

WASTE GAS BURNER

SLUDGE DIGESTER Sludge drying beds TREATED SEWAGE ^

Figure 50. - Flow Diagram of the Hilliard, Ohio Sewage Treatment Plant. 167 BIBLIOGRAPHY

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I, Robert Murray Gerhold, was born in Beckley, West Virginia,

October 19, 1931. I received my secondary-school education in the

public schools of Huntington, West Virginia, and my undergraduate training at Marietta College, Marietta, Ohio, which granted me the

Bachelor of Arts degree in 1953. From Marshall University of

Huntington, West Virginia, I received the Master of Science degree

in 1957. In October, 1957, I was appointed graduate assistant in

the Department of Microbiology at The Ohio State University. In

1959 I was appointed Research Assistant to Professor George .

Malaney while completing the requirements for the Doctor of

Philosophy degree.

174