INDUSTRIAL AND ENGINEERING CHEMISTRY
1276
article.2 The value of anhydrous ammonia, used chiefly in refrigeration, has doubled since prewar years, and in 1923 amounted to $6,415,000, the total manufacture being 23,966,000 pounds. OTHER GASES-of the many other gases which have attained a position on the country's marketplace, few have failed to feel the quickening influence of the great commercial and industrial activity of the years since the war. Chlorine, now used widely as a purifier of city water in addition to its established use in bleaching compounds, was produced to
* THISJ O U R N A L ,
18, 4 0 1 (1926).
Vol. 18, No. 12
the extent of 125,000,000 pounds in 1923, a gain in two years of more than GO per cent. Sulfur dioxide, another bleaching agent and disinfectant, is finding new fields, especially in medicine. The 1923 output was 6,576,000 pounds, eight times as much as in 1919. Of carbonic acid some 51,000,000 pounds were manufactured for the soda fountains and bottlers of soft drinks in 1923, the value being almost $5,000,000. Argon and neon, in electric lighting, and ozone, in water purification, afford interesting examples of the utilization of less common gases, but they have not yet reached a stage of large commercial importance.
Further Studies on the Biochemical Oxygen Demand Test' By R. E. Greenfield, A. L. Elder, and R. E. McMurray ILLINOIS S T A T E W A T E R S U R V E Y LABORATORY, U R B A N A # ILL.
Continuation o f previous investigation on the effect o f low temperature on this test to discover possible causes lag phase in the rate of consumption of dissolved oxygen
T
HE growing use by sanitarians of the biochemical oxygen demand test as a measure of the "strength" of organic trade wastes and sewage, and also as a measure of the per cent of purification accomplished by sewage-disposal plants, makes it of utmost importance that as much information as possible be collected concerning the nature and significance of this test. I n a previous paper2 some data on the effect of low temperature on this test were presented. It was deemed advisable to continue this investigation in order to discover possible causes of the lag phase in the rate of consumption of dissolved oxygen. Certain distinct differences were also noticed in the shape of the deoxygenation curves of river water and diluted sewage. Further work on this point has led to an investigation of the effect of inorganic salts and partial sterilization on such deoxygenation curves. Experiments with Diluted Sewage a t Low Temperatures
I n previous work on the deoxygenation of diluted sewage a t low temperatures a distinct lag phase occurred, followed by a more rapid utilization of oxygen. This lag phase was apparent a t temperatures from 2" to 6" C., while above those temperatures the lag was not so apparent. This lag was thought to be due to a lack of sufficient numbers of low-temperature tolerant organisms in the original dilutions and represented the time necessary to develop a sufficient number of organisms for carrying on the deoxygenation a t the normal rate. If this be true, then bacterial counts of organisms growing a t low temperatures should correlate with the deoxygenation curves. Counts of bacteria growing on agar plates at 8" C. were made from a series of bottles containing 2 per cent domestic sewage diluted with fully aerated distilled water. These bottles were incubated submerged in water at 2" C. Bacterial counts and dissolved-oxygen determinations were made a t regular intervals for 35 days. The methods used were the same as those described in the previous paper. The results of a typical series of these experiments are given in Figure 1, showing that the bacterial count increases rather slowly a t first and during that period little oxygen is used up. After the third day there commences a more rapid increase in bac1 2
Received June 19, 1928. Greenfield and Elder, THISJ O U R N A L . 18, 291 (1926)
of the
terial count and, a t the same time, a more rapid utilization of oxygen. After the period of most rapid utilization of oxygen is past the bacterial count decreases, but during the time of the experiment it never becomes as low as the initial count. This experiment supports the idea that the lag phase is due to an initial low concentration of these low-temperature tolerant organisms. As a further proof of this point, a series of experiments was set up using a 1 per cent sewage dilution, heavily inoculated with two cultures of these low-temperature organisms which had been isolated from the plates in the previous experiment. As a control on this experiment another series of bottles was set up containing the same 1 per cent sewage dilution in aerated distilled water inoculated with the same number of organisms, but in this case the organisms were killed by heat before the inoculation into the sewage dilution. The results of this experiment (Figure 2) show that the experiment in which the inoculation with living organisms was made has no lag phase, while the control experiment containing an equal
I Deory$enation curve L B a c t e d comt
U "
- u
0
a
lb
Days
zc
Je
40
Figure 1-Comparison of Bacterial Count with Rate of Deoxggenation of Diluted Sewage a t 2 O C.
number of organisms which had been killed has the same lag phase as was observed whenever inoculation from sewage alone was made. The original bacterial count of curve 3 with lowtemperature organisms was 200 million per cubic centimeter. The count changed but little during the first 10 days, after which the count gradually dropped off to 10 million by the
INDUSTRIAL AND ENGINEERING CHEMISTRY
December, 1926
twenty-sixth day. The original bacterial count for curve 4 was 300,000 per cubic centimeter and this series had reached 26 million per cubic centimeter by the fourth day; by the eighth day the count had reached 1 billion per cubic centimeter after which the count dropped off to 40 million a t the twenty-sixth day. This, together with results of two other similar experiments, iq rather conclusive proof that the lag phase is due to a lack in sufficient number of low-temperature tolerant organisms of the sewage. It is to be noted that this sewage was collected in the winter time. A cold-water stream receiving I
I
0
8
I6
24
32
Days Figure 2-Effect of Initial Seeding with Lou -Temperature Organisms o n t h e Rate of Deoxygenation of Diluted Sewage at 2°C.
sewage would therefore probably not receive from the sewage a sufficient number of organisms to cause a normal rate of deoxygenation immediately. It would therefore not be safe, in calculating the probable dissolved oxygen in a stream, to assume that such lag phases would not be encountered under stream conditions. The fact that these bacteria multiplied (comparatively rapidly a t these low temperatures in sewage dilutions is apparent. Similar multiplication of bacteria under low-temperature conditions has been noted by Muller.3 Pennington4 showed that meat, milk, etc., usually contained organisms which were capable of increasing in number a t low temperatures. Ravenel, Hastings, and Hammar6 showed that milk kept at 0" C. underwent a quiescent stage of 6 days, after which a progressively increasing count was noted followed by a decrease. With but few exceptions, no attempt has been made to study these low-temperature organisms in pure cultures. I n the course of this investigation sixty-four psychrotolerant and facultative psychrophilic cultures were isolated and studied. Although this study of isolated cultures was not carried to a sufficient degree of completion to warrant a detailed description of the cultures, the following points of general interest concerning the group of cultures isolated are worth noting: Rod forms 90 per cent; chromogens 50 per cent; forty of the sixty-four cultures were examined for spores and none were found; none produced gas from dextrose; 50 per cent produced acid from dextrose; 18 per cent peptized milk; 32 per cent produced acid in litmus milk; 60 per cent liquefied gelatin; 56 per cent produced ammonia from peptone; none produced hydrogen sulfide from peptone; 8 per cent produced nitrogen from nitrate; 23 per cent produced nitrites from nitrates; 10 per cent produced nitrite from ammonia; and 50 per cent peptized egg albumin. These organisms all grew on nutrient agar a t 2" C., although most of them grew 3 4
5
4rch H y g . , 47, 127 (1903). J. B w l Chem , 4, 353 (1908), 16, 331 (1913), 29, 31 J Infeclzous Dtseases. 7 , 38 (1910)
.1917).
1277
slowly. The most rapid growth was obtained from 15" to 20" C., and they fairly uniformly refused to grow a t 37" C. Although the growth was more rapid a t 15" to 20" C., with a few cultures the final growth on agar appeared to be more luxuriant a t 2" C. than a t the higher temperatures. Since the nature of the psychrophilic organisms has not been well studied, it would seem desirable that the cultural characteristics and growth habits of this group of organisms be further investigated. Effect of Death of Plankton Organisms on Rate of Deoxygenation of Polluted Stream Water
I n the previous paper2 it was pointed out that deoxygenation data obtained from experiments with diluted sewage gave smooth velocity curves, while similar experiments on Illinois River water gave irregular curves with a second period of acceleration coming between the tenth and twentieth days of incubation. It was suggested then that this might be due to the presence of living plankton organisms, which persisted alive in the bottles for some time and which upon death caused a second period of oxygen demand. I n order to test this hypothesis, experiments were made in which polluted stream water was heated to kill the plankton and then reinoculated with 0.25 per cent sewage in order to furnish a bacterial flora. Different experiments made by this method did not always yield identical results. A fairly typical set is shown in Figure 3. Curve 11 represents a 50 per cent dilution of stream water which had been boiled 10 minutes, reaerated by shaking, and inoculated with 0.25 per cent raw sewage. Curve 12 is for a similar experiment in which pasteurization a t 60" C. for 30 minutes was substituted for boiling. Curve 13 is for a similar experiment in which no sterilization was used. Curve 14 is for a 50 per cent dilution of stream
-I
0
4
8
12
Da y ~ Figure 3-Effect
Ib
20
of Heat o n t h e Rate of Deoxygenation of Polluted Water a t 20' C.
\ d e r without sewage inoculation. It is apparent in this set of experiments that the killing of plankton by heat did not eliminate the second period of acceleration in the deoxygenation curve and that the sterilization by heat decreased the total biochemical oxygen demand for the 20-day period of the experiment, and that the more intense the heat used in sterilization the more apparent is this difference. Other experiments run in a similar manner, while not giving identical results, tend to confirm these inferences. Another experiment was run in which large plankton forms were strained from a reservoir by means of a plankton net and a portion of this plankton catch was added to a series of bottles containing a 1 per cent sewage dilution. To another series of
Vol. 18, No. 12
INDUSTRIAL AND ENGZNEERZNG CHEMZXTRY
1278 Table I-Biochemical
Oxygen Demand" of Sewage Dlluted with Distilled Water, a8 Affected by Varying Concentrations of Inorganic Salts (Temperature, 20' C; sewage concentration, 2 per cent) Series A Series B Series C Series D Series E Series F Series G With CaCli 165,MgSO4:Period of 7Hz0 285, KC1 With all salts With twice the incubation h'o 10, NaHC03 With NaHCOs except bicarsalt content of Same as B, Same as B, Days salts added 336 p. p. m. 336 p. p. m. bonates B original p H 6.4 original p H 8.1 5 3.2 3.3 31 2 3.3 3.5 3.1 3.7 3.7 4.8 4.2 5.5 10 4.0 5.2 4.7 7.01, 7.0b 6.7 7.0b 4.1 15 4.6 7.0b 7.0b 7.0b 4.1 7.0b 20 4.6 7.0b 7.0b P. p. m. of dissolved oxygen used up in the period designated. 6 All the available oxygen in the bottle used up.
bottles containing the same 1 per cent sewage dilution the same amount of the plankton catch was added, but the plankton were in this case killed by about 2 to 5 minutes' heating to 60" C. It is to be noted from Figure 4 that the effect of lowtemperature heat sterilization on the plankton themselves is
0
4
8
12
I6
D ays Figure &Rate of Deoxygenation a t 20° C. of One Per cent Sewage, Inoculated with Living and Dead Plankton
not noticeable in the deoxygenation curves for the first 15 days. These experiments, while tending to show that the living or dying of plankton does not explain the second period of acceleration in deoxygenation curves, do show a rather marked effect of heat sterilization in certain cases. Inasmuch as many organic trade wastes have been subjected t o varying degrees of heat treatment, the question of the proper methods of determining the oxygen demand of such wastes, and the amount and character of inoculation of them should be carefully studied for each individual waste before the results of such determinations are compared with those of other wastes or sewages.
It is to be noted that in the series to which no inorganic salts were added a single curve was obtained, while in the series to which the salts were added a very definite two-stage curve was obtained. I n every experiment made with addition of this group of inorganic salts, results comparable with those expressed in Figure 5 were obtained. It was suggested by Theriault' that these two-stage curves were best explained by the work of Adeney8 in which he showed that in the oxidation of organic matter the first stage was one of carbonaceous oxidation, which was followed by a stage of nitrification of nitrogenous compounds. I n order to confirm this, nitrite determinations were made a t frequent intervals in the series of experiments. The concentration of nitrites in the two series is also shown in Figure 5 . Although nitrification takes place to some extent in all bottles a t all times during the incubation period, it proceeds rapidly and extensively only during the latter part of the incubation period, and then only in those bottles to which inorganic salts have been added. The results seem to be a confirmation of Adeney's theory of two-stage oxidation. The effects of different salts of different concentrations of salts, and of different initial hydrogen-ion concentrations, are shown to some extent in the results in Table I. The original p H of all but series E' and G was about 7.7. I n series F hydrochloric acid was added to make initial p H 6.4, and in series G sodium hydroxide was added to make initial pH 8.1. The experiments indicate that a further increase in inorganic-salt concentration increases the biochemical oxygen I
I
7 Deoqrgenation curve-No salts added 0 " -Salts added 9 Nitritatim curve -No salts added I'
0
10
"
- Salt3 added
_-e--*--
Effect of Inorganic Salts on Deoxygenation of Diluted Sewage
I n the previous paper only regular one-stage deoxygenation curves were obtained on diluted sewage diluted with aerated distilled water. The writers were advised by F. TV. Mohlman6 that he obtained two-stage curves using aerated Lake Michigan water for dilution purposes, and that by the addition of inorganic salts to distilled water used for dilution purposes he obtained higher biochemical oxygen demand figures than when using distilled water alone. Several experiments were therefore made in which inorganic salts were added to the distilled-water dilution, in each case one series without the salts being run as a check. The results of one series of typical results are given in Figure 5 . I n this experiment the following concentration of salts was used : P. p. m.
P. p. m. CaClz MgSOa.7HaO
165 285
KCI NaHCOa
10
336
c
Private communication.
2
16
20
3 ays Figure &Effect of Salts on the Rate of Deoxygenation and Nitrate Formation of Diluted Sewage a t 20' C .
demand, even when the initial concentration of inorganic salts was such as would be considered characteristic of a moderately to heavily mineralized water. Bicarbonate alone 7
8
8
U. S. Pub. Health Service, Pub. Health Refits., 41 (1926). Tvans. R o y . Dublin Soc., N . S.,6, Pt. 6 539 (1895).
IXDUSTRIAL AND ENGIA'EERIXG CHEMISTRY
December, 1926
produces a marked effect, but the addition of noncarbonate magnesium, calcium, and potassium salts increases this effect. No experiments were made to determine the effect of increasing the concentration of bicarbonate alone. High initial hydrogen-ion concentration decreases the biochemical oxygen demand, but moderately low hydrogen-ion concentration seems to be without effect. These results are of considerable importance when it is considered that the biochemical oxygen demand test is being used in various laboratories and that the results of the various laboratories are being compared. Up to 5 dags the results using distilled water for dilution are quite similar to those to which inorganic salts have been added; if differing a t all they are slightly lower. For longer periods of' incubation dilution water containing added salts gives distinctly higher results than those with distilled water alone. The second stage of deoxygenation is apparently eliminated when distilled water is used for dilution purposes. It is, therefore, doubtful that the results from various laboratories will be comparable if simply tap water is specified, because the mineral contents of different tap waters differ considerably. I n the case of trade wastes containing acid or alkaline substances the adjustment of the initial pH is very important. If this test is to be used as a standard test to determine the strength of sewage and the results from one laboratory are to be compared to those from another, it would be advisable that further work be done along these lines with the view of developing a standard dilution water which could be duplicated in various laboratories. The present specifications for dilution water given in Standard Methods of JT:iter Analysis cannot well be met in many laboratories, because dilution water is specified which shall not contain more than a specific maximum of iron or nitrogenous compounds. I n many laboratories such tap water is not available, and if the test is run with tap water of different salt concentrations the total biochemical oxygen demands of sewages of similar strength may be found t o be quite different in the various laboratories because of the effect of inorganic salts.
1279
Conclusions
It is desirable that a standard dilution water for biochemical oxygen demand tests of sewage be developed which would give comparable results and which could be duplicated in various laboratories. The present specifications for dilution water given in Standard Methods of Water Analysis of the American Public Health Association cannot be met in many laboratories, and these specifications do not take into consideration the effects of the different inorganic salt concentrations of different waters now used as diluents in various laboratories. Addition of inorganic salts to the dilution water used in the deoxygenation experiments produced two-stage deoxygenation curves. (Dilute sewage made up with distilled water, without the addition of such salts, uniformly gave single-stage curves.) The second stage seems to be primarily nitrification. The total biochemical oxygen demand is increased by increasing the concentration of inorganic salts. Especially for longer periods of incubation is this noticeable, even when the mineralization reached would be considered moderate or high. The bicarbonate salts alone, while producing a marked effect, do not produce so great an effect as with the addition of magnesium, calcium, and potassium salts. That the lag phase in the rate of deoxygenation of diluted sewage a t low temperatures is due to the lack of a sufficient number of low-temperature tolerant organisms capable of carrying on the deoxygenation a t a normal rate has been confirmed. Cultural studies on bacteria isolated from sewage dilutions incubated a t low temperatures show a preponderance of rod forms, a large percentage of chromogens, and a large percentage of organisms capable of carrying on proteolytic reactions. The killing of plankton by heat does not in all cases prevent the production of two-stage curves for deoxygenation of polluted stream water. Intense heat seems to decrease the total biochemical oxygen demand of such polluted stream water. (This effect was not obtained to the same degree in all experiments.)
~
Lubricants for Ground-Glass Joints' By M. J. Bradley and H. E. Wilson L'NIVERSITY OF ILLISOIS, URBAKA, ILL.
T
HE ideal lubricant, one suitable for all the various conditions under which ground-glass joints are used, is not a commercial commodity. There are two or three mixtures obtainable under the trade name of "stopcock grease" which are fairly satisfactory for pressure or vacuum joints but they are readily attacked by strong chemical reagents. During an investigation on decomposition processes of certain products of coal carbonization,2 it was found that the usual lubricants were destroyed by the strong chemical reagents used as absorption solutions in the Orsat gas apparatus. After considerable experimentation several lubricants were compounded which were satisfactory not only on the Orsat machine but also on ordinary stopcocks with gas under 10 inches of water pressure and also a t reduced pressures down to 5 or 6 mm., and also on a yacuiim desiccator !id. Heavy liquid paraffin oil (Stanolind) was found t o be much superior to petroleum jelly as a base material 1
Received April 8, 1926.
2
Bradley and Parr, Chem. M e t . Eng , 27, 737 (1922).
in the lubricants. The texture of the semisolid lubricant was greatly improved by vigorous stirring while heating on the steam bath and also by incorporating on a glass slab after congealing. Inert Lubricants
The following mixture was found to be exceedingly stable and otherwise satisfactory in contact with 40 per cent potassium hydroxide solution, saturated bromine water, fuming sulfuric acid, and potassium pyrogallate: (1) Heavy liquid paraffin oil (Stanolind) and Parowax ( 5 :6). Melt the Parowax over a steam bath and take into solution in low-boiling petroleum ether, add the paraffin oil, and heat the mixture on the steam bath with constant stirring until the solvent is removed. The smoothness and body of the lubricant can be materially changed by varying the proportions of the constituents or leaving a small trace of solvent in the mixture. The Parowax may be replaced by ceresin, giving a somewhat more greasy lubricant but one slightly less stable in the presence of fuming sulfuric acid.
