JUNE, 1935
INDUSTRIAL AND ENGIKEERING CHEhlISTRY
motion, thus further mixing air, organism, and culture liquid. The progress of the fermentation may be observed through the sight glasses, K . There are three of these in each end, placed opposite one another. Samples are taken under pressure through the pet cock, F , which is also used for emptying the drum. Figure 2 is a photograph of the first drum, set u p for operation. The solution t o be used for fermentation is sterilized in a glass flask in an autoclave. This flask is equipped with two outlets. When charging the drum, one outlet, which extends only through the rubber stopper, is connected by a sterile rubber tube to inlet G which has been previously protected by a piece of sterile cotton. The flask is then inverted. Filtered air enters through the second outlet, which extends to the bottom of the flask, and the contents of the flask flow into the drum without contamination. Inoculum is added to the flask under sterile conditions before emptying the contents of the latter into the drum. The inoculum used is made up of the spores of the desired mold, which have been previously germinated to a point at which they are ready t o act on the culture liquid, thereby diminishing the “lag” period, so characteristic of fermentations 11 hich start from untreated spores. For the purpose of testing the apparatus, the culture medium and organism described in the previous paper (4) on this work were used. While much has been done on the production of gluconic acid, it seemed best to start the work with a n organism and reaction concerning which detailed comparative data were a t hand. Using the optimum conditions of temperature and pressure, as previously determined, it was possible to shorn marked improvement in time, and yields nearly as good as any obtained in the laboratory work in zlitro. For instance, under 30 pounds per square inch (2.1 kg. per sq. em.) of air pressure, yields of gluconic acid as high as 86 per cent of f heory were
683
obtained in glass in an 8-day culture period, whereas, with the drum, yields approaching 80 per cent of theory have been obtained in 56 hours. The curves in Figure 3 illustrate the production of acid and consumption of sugar in a selected run, and are based on determinations of acid and sugar made every 4 hours during this run. As a result of the success of this first drum, five others have been built and are now in operation, as shown in Figure 4. It is not intended to present, the experirnent’al details of this work a t this time. It is still being carried on, and the results will be reported later. Fermentations under pressure in a rotating drum offer a n entirely new set of experimental conditions which, when applied to various organisms and biochemical reactions, may afford unusual and satisfactory results. Acknowledgment The authors are indebted to I. Bauserman, of the Chemical Engineering Division of this bureau, for drawing Figure 1, Literature Cited (1) Calmette, A., German P a t e n t 129,164 (1902). (2) Currie, J. N., Kane, J. H., a n d Finlay, A,, U. S. P a t e n t , 1,893,819 (1933). (3) Fernbach, A,, Compt. rend., 131, 1214 (1900). (4) M a y , 0. E., Herrick, H. T., Moyer, A. J., and Wells, P. 8., IXD. EXG.CHEM., 26,578 (1934). (5) Muller, D., Biochem, Z . , 199, 136 (1928); 205, 11 (1929). (6) Pottevin, H., Compt. rend., 131, 1215 (1900). (7) Schreyer, R., Biochem. Z . , 202,131-56 (1928) (8) Takamine, J., J. IND. ENO.CHEivf., 6, 824 (1914). (9) Thies, W., Centr. Baht., Parasitenk., 11,82, 321-47 (1930). (10) Wehmer, C., in Lafar’s Handbuch der technischen Mycologie, Vol. 5 , pp. 319-42, Jena, 1905-14.
RECEIVED March 14, 1935. Presented before the Division of Industrial and Engineering Chemistry a t the 89th Meeting of the Americitn Chemical Society, S e w York, N. Y . , .Ipril22 to 2 6 , 1935. This paper is Contribution 251 from the Color and Farm \Taste Division.
Activated Sludge as a Biozeolite EJXERY J. THERI-IULT, Zatiotial Institute of Health, Washington, I). C.
This is the second of a series of papers (10) in which the theory is developed that the removal of organic matter from sewage by the biological slimes or activated sludges is basically the same as the process of removing hardness and other objectionable constituents in a widel?- used process of
EP
REVIOUS cornmunications ( I O ) have indicated that the adsorbent principle in activated sludge may be regarded as a base-exchanging substance chemically the same as the zeolites of water purification. I n the present paper it is proposed to submit some of the purely chemical evidence leading to this conclusion. The physical properties of this base-exchanging gel mill be more fully considered in the next paper of this series. The possibility that silica was of considerable importance in purification processes depending on the presence of biological slimes n.as suggested to the writer several years ago on the basis of certain analogies with the action of siliceous filters (6). As a very general rule, however, no beneficial
water purification. O n the basis of proximate analysis, the adsorbent principle in the gelatinous matrix of activated sludge is shown to be a zeolite. The clotting enzyme or sewage colloid of the earlier chemists in all probability is identical with the sludge zeolite.
action ha. ever been ascribed to silica in either mater purification or sewage treatment. On the contrary, the adverse effect of silica on the coagulation of certain types of water by alum and its scale-forming properties in boiler maters are well known to the chemist. The Schmutzdeckr?of the slow sand filters is nevertheless known t o contain appreciable amounts of silica, and the silica content of actirateti sludge, as indicated by rare analyses made in connection with its use as a fertilizer, is correspondingly high. These relatively high figures for silica could, no doubt erroneously, be attributed to the inclusion of sand in the samples. In any event, it does not appear that any particular significance was ever attached to the findings.
VOL. 27, NO. 6
INDUSTRIAL AND ENGINEERING CHEiMISTRY
684
TABLEI. COMPOSITION OF ACTIVATED SLVDGE Basis of Computation
Si01
AlzOa
FezOa
MnnOs
CaO
MgO
NazO
KzO
SOa
4.44
2.96
0.30
8.39
98.85
1.15
7.88 6.23 1.85
0.80 0.59 0.21
22.35 (22.35)
263.32 39.79
3.06 0.24
0.02
..
...
....
..
PlOS
Sum
Undetd.
Percentages (Uncorrected) Total ash
45.77
24.96
1.08
0.83
7.58
2.54
Milligrams per Liter (Total = 266.35) Total ash Bacterial ash Difference
121.92
66.49
121.92
66:49
....
2.88 1.33 1.55
2.21 2:il
20.19 5.51 14.68
6.77 2.37 4.40
11.83 1.41 10.42
...
....
..
hlillimoles per Liter (hlatrix) Corrected ash
2.01
0.65
0.01
0.01
Computation:
0.26
0.11
0.17
0.26 0.11 0.17 0.02
CaO htg0 NazO Kz0
0.65 0.01 0.01
A1201
FenOa RlnzOs
2.01
Si02
0.56
Mz0
0.67
R~OJ
2.01
Si01
0.84
Mz0. R10a.3Si02
Preliminary Experiments Interest in the possible role of silica in sewage treatment was revived when, in the course of studies on the absorptive capacity of activated sludge, it became necessary t o obtain further information regarding the nature of the active or clotting agent in the sludge. The absorbent properties of the silica gels are well known, although numerous experiments with prepared gels have given uniformly negative results in regard t o the duplication of the phenomena observed in sewage treatment. In fact, it is now probably safe to say that silica gels, as such, do not exist in the sludge. The presence in the sludge of the more complex ferro- or aluminosilicate or zeolites was a more or less obvious possibility although this theory could not very well be confirmed by such random analyses of activated sludge as were available a t the start of these studies. Methods of Analysis
As the next step it appeared necessary to obtain several reasonable complete and accurately performed mineral analyses on representative samples of activated sludge. The procedure followed in these examinations was essentially that given by Hillebrand (6) for the analysis of silicate and carbonate rocks. Starting with the sludge ash, it was imperative t o fuse the material with sodium carbonate in order to break up the complex of ferrosilicates which the sludge undoubtedly contains. The fused material could then be readily decomposed by hydrochloric acid. The silica was volatilized with hydrofluoric acid in the usual manner and the residue, if appreciable, was subjected to further treatment. Redistilled ammonia was used for the precipitation of the combined oxides, and platinum ware was used throughout. Calcium was precipitated twice as oxalate and ignited to calcium oxide. A small amount of calcium was invariably found on the examination of the second magnesian precipitate. Phosphorus, sodium, and potassium were determined on separate samples, using nitric acid for the decomposition of the ash. Purification of Activated Sludge A miscellany of undigested food particles together with unclassified debris is inevitably present in activated sludge, even though the active material is properly defined as a zoogleal aggregate. While the complication introduced into a mineral analysis is probably not so serious as might appear a t first thought, this difficulty should neverthless be largely avoided by working with a nitrifying sludge. Starting with ordinary activated sludge secured from an experimental plant, the sludge was aerated for several days without the addition of fresh sewage until the carbonaceous stage of oxidation was definitely passed, Considerable quantities of ammonia are
released during the first stage of oxidation to be utilized later when the nitrifying organisms become definitely established. The ammonia content was watched, and more ammonia was added daily a t the rate of 10 to 20 mg. as nitrogen per liter of aerated mixture. The sludge settled readily to a volume of 100 ml. or less when the total solids a t 110” C. were in the neighborhood of 1000 mg. per liter, and in other respects it manifested all the properties of a “good” sludge, the limiting factor in its oxidizing capacity being the pH value of the mixture. As the addition of phosphates or similar remedial agents was contra-indicated, reliance was placed on the daily renewal of the supernatant liquor with tap water containing the desired quantity of ammonium hydroxide which was preferred to ammonium chloride for the purpose a t hand. In this way the sludge was gradually purified to the point where, on the basis of procedure used, it should only have contained actively nitrifying organisms, together with the plankton introduced with the sludge as drawn from the plant. ST’hile more reliance may be placed on the representative analysis given in Table I than on available analyses from other sources, it has not appeared that the results were significantly different when the samples were not subjected to a preliminary purification as in the case of nitrifying sludge. Other sources of material have been sludge as drawn from the small experimental unit a t the Stream Pollution Investigation Station of the U. S.Public Health Service in Cincinnati. Partial analyses were also made of a synthetic sludge developed by Butterfield in the same laboratory from pure cultures on aoogleal organisms. Confirmatory evidence of the essential sameness of activated sludge from various sources was also afforded by a reexamination of the Houston sludge already reported by Wagenhals, Theriault, and Hommon (11). Originally the silica content of this sludge was simply lumped with the HC1-insoluble residue and the result reported as “silica plus HC1-insoluble matter.” Fortunately, the Houston samples had been preserved and were therefore available in connection with the present study. On reexamination the silica content of the sludge collected from the drier was found to be 67.08 per cent in terms of the sludge ash. The “HC1-insoluble residue” was found to consist largely of undecomposed aluminum or iron silicates. The corrected figure for combined oxides leads to the empirical formula R20s.4Si02, instead of R203.3Si02as will presently be shown for the data of Table I. A search of the scattered literature of sewage treatment has revealed very few complete mineral analyses of activated sludge, although such data have no doubt frequently been obtained in the course of plant investigations. The mineral
JUNE, 1935
INDUSTRIAL AKD ENGINEERING CHEMISTRY
analybis of sludge given by Ilienert (3) points to the enipirical formula RzO3.7Si02. Since the methods of analysis are not stated, there is some uncertainty regarding the exact significance of the figure for silice. A slight error in this figure exerts a disproportionate leverage effect on the ratio of silica t o combined oxides. Considering the analysis of the partly purified sludge given in Table I, the figure for total inorganic solids (266.38 mg. per liter) is exceedingly low. In part this is due t o the use of a sludge which contained only 1008 mg. of suspended matter per liter when filtered through a Gooch crucible and dried a t 110” C. The high figure for phosphorus and the deficiency of other acidic radicals is noteworthy. No examinations, however, were made for chlorine as chloride, although on the basis of the earlier examinations of the Houston sludges and, also, of the present samples, this constituent was certainly not present in excess of 1 per cent of the total ash. Larger amounts of acidic constituents might be expected in a material suspended in sewage (or tap water),. particularly since the water content of the settled sludge, prior t o centrifuging, was well over 99 per cent. The presence of phosphorus in relatively large amount is explainable as a normal constituent of the bacteria embedded in the gelatinous matrix. The practical absence of other acidic radicals is reassuring when the matrix itself is regarded as a zeolite.
685
pentoxide, 55.23 per cent; calcium oxide 12.84; magnesium oxide, 11.55; sodium oxide 13.62; and potassium oxide, 6.35 per cent. The phosphorus content of 55.83 given by Guillemin and Larson (4) for E. coli ash corresponds to the highest value given by Waksman (12) for bacteria. Much lower values for this constituent should probably be credited to dilution by salt inclusions from the nutrient medium. Other values for phosphorus, given by Waksnian (I%’), are for fungi, 44.8 to 59.4 per cent; for yeast, 51.0 to 59; and for higher fungi, 40.0 (see also Buchanan and Fulmer, 2). On the whole, it has appeared that the analysis of E. coli ash given by Guillemin and Larson could be taken as reasonably representative of the general composition of the sewage bacteria and other microorganisms present in the sludge.
Correction for Bacterial Ash The primary assumption in applying a correction to the sludge ash for the mineral components of the bacteria and other microorganisms has been that nearly all of the phosphorus was concentrated in the bacterial cells. It should be remembered in this connection that the nutrient furnished t o the bacteria was ammonia in tap water and that no important source of phosphorus existed to satisfy the requirements of the bacteria for this necessary ingredient except the phosphorus which might have been present, in the geMineral Composition of Bacteria latinous matrix a t the start of the experiment. With this assumption the proportion of the total ash which could be As a basis of correction for bacterial ash, use will be made of credited to the bacteria was computed, as shown in Table 11, the complete analysis of a colon organism reported by using the factor 8.39/55.83, to proportion the other mineral Guillemin and Larson (4). As given in Table 11, the values constituents of the bacteria to the same scale as the phosoriginally submitted by Guillemin and Larson for sodium, phorus. These percentage values were then used to calculate potassium, and sulfate have been recalculated, respectively, the milligrams per liter of the various constituents in the in terms of sodium oxide, potassium oxide, and sulfur tribacterial ash when referred to a sludge containing approxioxide to accord with the analyses given in Table I. The mately 1000 mg. of dried solids per liter. absence of aluminum and silica from this analysis of bacThe bacterial ash amounts to only 15 per cent of the total terial ash is of obvious significance. At most, the bacterial mineral ingredients of the sludge ash. As shown in Table I ash could not have contained more of these constituents the bulk of the potassium and a corresponding amount of than the small undetermined fraction of 3.79 per cent, as the sulfur are accounted for as normal constituents of the originally computed by Guillemin and Larson, or 0.54 per bacterial ash. The calculation, therefore, could very well cent as recalculated in Table 11. have been based on these ingredients rather than the phosphorus. There is an obvious advantage, TABLE11. DERIVATION OF CORRECTION FOR BACTERIAL ASH nevertheless, inusing the most prominent constituSi02 AliOa FenOa CaO MgO Nan0 Kz0 SOa PnOa Sum Undetd. ent of the bacterial ash as a basis of correction. The reasonableness of the correction deduced Bacterial Ash (Percentage Values by Guillemin and Larson) .. .. 3 . 3 5 13.77 5 . 9 2 3 . 5 2 1 5 . 5 9 1 . 4 8 5 5 . 8 3 9 9 . 4 6 0 . 5 4 for the bacterial ash may be checked by a rough calculation of the indicated numbers of bacteria Percentage in Sludge Ash (PlOb = 8.39) .. . . 0 . 5 0 2 . 0 7 0.89 0 . 5 3 2 . 3 4 0 . 2 2 (8.39) 14.94 0.09 in the sludge. Guillemin and Larson (4) state that the ash constituted 12.75 per cent of the dry >Iilligrams per Liter (Total Ash = 266.38 P. P. M.) .. . . 1 . 3 3 5 . 5 1 2 . 3 7 1.41 6 . 2 3 0 . 6 9 (22.35) 3 9 . 8 0 0 . 2 4 weight of the colon organisms taken for the ___anaiysis given in Table 11: For the case a t hand, the dry weight of the bacteria themselves when Naksiiiaii (1.2) omits alumina from a tabulation of the expressed in terms of E. coli should have been 40.04/0.1275 mineral composition of various microorganisms. He gives a or 314 mg. per liter. Rahn (7) deduces a figure of 4.5 X l o 9 range of 0.5 to 7.8 per cent for their silica content but quesas the number of colon organisms in 1 mg. of dry bacterial tions the higher figure. Reference to the original literature substance. Using these data, a simple calculation leads to an indicates that both analyses were performed on the same estimate of 1,400,000,000bacteria per cc. in a sludge mixture zoogleal organisms-namely, the acetic acid ferment. The containing about 1000 mg. of suspended solids per liter. The low value of 0.5 per cent was reported by Alilaire ( I ) and is order of magnitude of this figure is consistent with the known based on analyses of “degreased” bacterial cells, presumably close-packing of the bacteria in the zoogleal formations which freed from the gelatinous matrix. In other respects the constitute the sludge. mineral analysis of acetobacter given by Alilaire (1) accords Proximate Composition of Activated Sludge reasonably well with the more recent analysis of E. coli given by Guillemin and Larson. The higher value of 7.8 per cent It should now be possible t o separate the bacterial ash from for silica given by Romegiallo (8) may refer to the entire the total ash so as to obtain corrected figures for the mineral mycoderm rather than to the bacteria. Schweinitz and constituents of the gelatinous matrix in which the bacteria Dorset (9) report 0.57 per cent for the combined value of the are embedded and on which the protozoa feed. When this carbon and silica in the ash of the tubercle bacillus. Other is done, as shown in Table I, a residue is obtained which may values given by them for the same organism are: phosphorus be represented by the empirical formula, 0.84 MzO.R9O3-
INDUSTRIAL AND ENGINEERING CHEMISTRY
686
3SiO2. I n this forniula the term “U20” is used to represent exchangeable items, such as SarO, K20, CaO, or MgO, and the term “R203” is taken to represent either AI2O3or Fez03. With allowance for the loss of absorbed nitrogen in the ashing process, thereby creating a deficiency in the basic elements. this is unmistakably the formula for a zeolite. This view of the composition of the matrix in biological slimes is strengthened by the circumstance that the centrifuged or partly dried material is certainly gelatinous. I t would be most extraordinary for the elements in question to coexist in the alndge without forming a zeolite. The pertinent question as to x-hether tbe sludge does actually possess the physical properties of a zeolite has already been partly an.aered in the first paper of this series on tlie rate of clarification ( I O ) . Parson’s work on the equilibrium which obtains betn-een carbonaceous matters in sewage, as defined by the oxygen consumed test, and suspended sludge particles is very much in point. Evidence to the effect that the sludge will also adsorb ammonia readily from solution and that it can be repeatedly regenerated with sodium chloride in the same manner as the commercial zeolites will be given in the next paper of thiq series. Properly dried sludge should be a good source of zeolitic material for use in sewage purifica-
tion, with the possible advantage that carbonaceous matters may be removed, together with the nitrogenous compounds. Literature Cited Alilaire, E., Compt. rend., 143, 176-8 (1908). Buchanan, R . E., and Fulmer, E. I., “Physiology a n d Biochemistry of Bacteria,” p. 72, 1928. , 113-66 (1922). Dienert, F., Rec. h y ~ .44, Guillemin, M., and Larson, K. P., J. Infectious Diseaees, 31, 349-55 (1922). ( 5 ) Hillebrand, W. F., z‘.S. Geol. Survey, Bull. 700 (1919). (6) K r a m e r , S. P., J . Gen. Physxol., 9,811-12 (1926). (7) R a h n , O., “Physiology of Bacteria,” p. 397, 1932. (8) Romcaiallo, A . , Riv. vitic. ital., 7, 307-14, 359-70 (1853); cited by Buchanan and Fulmer ( 2 ) . (9) Schweinits, E. A. de, and Dorset, 31.,Centr. Bakt., Parasitenk., 2 3 , 993-5 (1898). (10) Theriault, E. J., Pub. Health Repts., in press; see also 50, 143-4 (Feb. 1, 1935). (11) TI-agenhals, H. H . , Theriault, E. J., and Homnion, H. B., Pub. Health Bull. 132 (1923). (12) Waksnian, 9. -4.., “Principles of Soil Microbiology,” p. 381, 1927. R E C E I V EMay D 2 , 1933. Presented before the Division of Water, Sewage, and Sanitation Chemistry a t the 89th Meeting of the American Chemical Society, New York, N. Y . , April 22 to 26, 1936.
Phase Equilibria in Hydrocarbon Systems VIII.
Methane-Crystal Oil System N PART
I
111 of this series there was described the physical behavior of a twocomponent hydrocarbon system consisting of methane and propane and representing a simplified case of a [‘dry” natural gas and a very volatile oil. For comparison with these results, it is of interest to know how the equilibrium relations of pressure, volume, and temperature mould be altered by the substitution of a relatively nonvolatile, highmolecular-weight oil for the propane. With this point in view, a study was made of the system consisting of methane and crystal oil. The temperature range investigated was from 70” to 220’ F., and pressures m-ere varied from vapor pressure of crystal oil to 3000 pounds per square inch absolute. The mixtures used ranged systematically from pure crystal oil to a mixture containing over 50 mass per cent of methane. Measurements were made of the total volume of the system of known mass as a function of pressure, temperature, and composition.
VOL. 27, NO. 6
B. H. SAGE, H. S . BACKUS,
AND W. N. LACEY California Institute of Technology, Pasadena, Calif.
range of molecular weights, with the accompanying characteristics of low volatility, narrow boiling range, and moderately high viscosity. The sample used in this work was refined from a western, nonwaxy, asphalt crude oil. Its molecular weight, as determined by the freezing-point lowering
3Iaterials Crystal oil mas chosen for this and for subsequent studies to meet the requirements of a stable liquid hydrocarbon material which had high molecular weight combined with small 1 Part I appeared on pages 103-6, January, 1934; Part 11, pages 214-17 February, 1934; Part 111. pages 652-4, June, 1934; Part I V , pages 874-7, August, 1934; Part V, pages 1218-24, November, 1934: Part V I , pages 48-50, January, 1935; Part V I I , pages 162-5, February, 1935.
)
PRESSURE
LBS. P E R
2500
2000 SQ.
IN.
FIGURE1. REPRESENTATIVE EXPERIMENTAL CURVES
