Pz'..
FIL Hp MACH SCREWS
- 22 - ,
FIGURE1. DIAGRAM OF APPARATUS
Apparatus for the Application of Submerged Mold Fermentations under Pressure 11. T. HERRICK, R. HELLBACH, .4ND 0. E. RIAY
Color and Farm Waste Division,
A new apparatus for the application of submerged mold fermentations under pressure is described. It consists essentially of a revolving drum equipped with internal buckets and baffles and provided with means for the introduction and rernoval of air under pressure.
Bureau of Chemistry and Soils, Washington, D. C.
ROCESSES for the production of ethyl alcohol by the action of yeasts on sugar, and for the manufacture of butyl alcohol and acetone through the fermentation of starch by bacteria of the Clostridium group are widely known. The process for the manufacture of citric acid by the action of a species of Aspergillus niger on sucrose solutions, a more recent development, has also received much attention. In the cases mentioned, organisms of three types-yeasts, bacteria, and molds-are utilized successfully in industrial processes, but with a marked difference. The yeasts and bacteria grow submerged in large vats and are disseminated throughout the solution; all of the solution is thus accessible to their action. The molds, on the other hand, are grown as mycelial felts on the surface of the solution, which must be contained in shallow pans in order that every part of the solution may be subjected to the action of the organisms. Little need be said about the obvious technical advantages of a vat fermenter over the shallow pan type. I n recognition of the disadvantages of surface fermentations, work has been carried on for a number of years dealing with the problem of producing a submerged growth of mold which would retain some or all of the fermentative activity of the surface mycelium of the same organism. Schreyer (7) in 1928 and Thies (9)in 1930 reported experiments in which
oxygen was bubbled through submerged cultures of A4q)ergiUus fumaricus, with consequent increase in the production of gluconic acid by that organism. The essential feature of a patent granted to Currie, Kane, and Finlay (2) in 1933 is a mold fermentation carried on in the foam of an aerated liquid, stirred with a high-speed agitator. Results have been reported recently (4) showing the effects of increased air pressure on submerged mold growths. Gas washing bottles (500-cc.) constructed with false bottoms of sintered glass were used as culture vessels. Air was passed through the sintered glass bottoms which broke up the air stream into fine bubbles, thus aerating and agitating the culture solution. The whole apparatus was kept under pressure in an autoclave. The apparatus to be described in this paper is an attempt to apply this principle t o large-scale fermentations. Submerged mold growths have been used for some time in industrial production. The amylo process for the manufacture of ethyl alcohol from starch (IO) utilizes the amylolytic 681
682
INDUSTRIlL AND ENGINEERING CHEMISTRY
FIGURE 2. THE FIRSTDRUMSET UP
FOR
OPER4TION
action of a variety of Mucor to hydrolyze starch for the production of alcohol by yeast. The manufacture of gallic acid from tannin is carried out by the submerged activity of a variety of Aspergillus (1). Both these processes are, however, hydrolytic, and one, at least, is extracellular. The Aspergillus used in the gallic acid fermentation produces the enzyme, tannase, in the t a n n i n solut i o n , a n d i t has LO been shown (3, 6) 18 t h a t this enzyme 16 .+will c o n t i n u e t o c IQ eL 12 produce gallic acid d: 10 even after the 8 organism itself has 6 been removed 4 from the solution. 2 I n the oxidizing reactions, of which Hours the production of FIGURE 3. CURVESOBTAINED BY THE gluconic acid from ANALYSISOF 10-Cc. SAMPLESTAKEN glucose is a n exEVERY 4 HOURS DURING A TYPICAL ample, a different Rux problem is inT h e results mere calculated in terms of the volved. Here air actual amounts of sugar and gluconic acid present in these samples. and glucose meet inside the cell, presumably in t h e presence of a n enzyme such as the “glucose-oxidase” described by Xuller ( 5 ) , to form gluconic acid which then passes back t o the solution. Thus oxygen, glucose, and mold cell, the essentials to a completion of the reaction, must be brought together under the proper conditions to give t h e desired results. Application to Industrial Scale Following the laboratory work cited (4) in which the procedure utilizing the washing bottle with fritted-glass diaphragm Eas employed, a n attempt was made t o translate this v o r k to an industrial scale A piece of 10-inch (25.4-cm.) wrought-iron pipe was used for the apparatus. This was coated inside with S. D. O., a n acid-resisting paint which had previously been tested for its toxicity to molds and found harmless. Air was introduced through a piece of No. 2 Filtros plate, and the whole machine was designed, as far as possible, to duplicate the original glass apparatus. For some undiscovered reason, satisfactory results were not obtained with this equipment, and i t was abandoned after a great deal of time and energy had been spent on it. One thing was apparent. It would be ex-
VOL. 27, NO. 6
tremely difficult to construct an air-distributing apparatus which would function satisfactorily on a large scale. For this reason, some substitute was sought for the perforated plate which had first been used. After some consideration, i t seemed that a revolving drum offered the best solution for the difficulties encountered. Such a drum could be equipped with baffles and buckets on the inside of the shell, and air could be introduced under pressure through one t’runnionand released through the other. The drum was constructed from aluminum, since it had been shown that certain grades of this metal are not toxic to the microorganisms used and are not seriously attacked by the organic acids formed in the course of the fermentation. The specifications call for less than 0.1 per cent of manganese and copper, but place no limitations on other components of the metal. The drum and heads were of sufficient thickness to withstand 7 5 pounds per square inch (5.3 kg. per sq. cm.) pressure. It was felt that if this drum were revolved at the proper speed and the contents kept at the necessary pressures, the solution would be sufficiently saturated with oxygen t o furnish the amount of this element required by the organism t o carry out the reaction efficient,ly. The idea of using a revolving drum in certain microbiological processes is not new. Such an apparatus was used by Takamine (8) twenty years ago for the preparation of the diastatic enzyme of Aspergillus oryzae, with the difference, however, that the object of these experiments was the production of an abundant mycelial growth on semi-solid material. Moreover, the Takamine drum also differs from the drum reported here in a variety of details which may be seen by a reference to the description of the former. One of these drums, as shown in Figure 1, was constructed for experimental purposes. It is operated as follows: After complete sterilization of the apparatus by steam under pressure, introduced through the air inlet, B , the solution and inoculum are introduced into the drum, A , through the charging inlet, G. The drum is then rotated by means of belts applied to t,he outside of one or both flanges, and air filtered through sterile cotton is introduced under pressure through air inlet B. \\-hen the air in the drum reaches the desired pressure, it is nllmved to flow at a measured and predetermined rate through the air outlet, C (fitted nith a shield to prevent the entrainment of liquid dropping from above), into the liquid trap, H . The air flow is controlled by the needle valve, 7, and measured by a flon-meter. The drum is set up in a constant-temperature room. \Then the drum is rotated, the liquid is lifted in the buckets, D, and dropped from the height of the drum to the bottom. This serves to saturate the culture liquid with the oxygen in the air contained under pressure in the drum. The hnffles, E , between the buckets are so designed as t o give the liquid a back-and-forth
FIGURE 4. BATTERY OF DRUMS IN OPERATION
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 u p of the spores of the desired mold, which have been previously germinated to a point at which they are ready t o act on t h e 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 Patent 129,164 (1902). (2) Currie, J. N., Kane, J. H., and Finlay, A,, U. S. Patent, 1,893,819 (1933). (3) Fernbach, A,, Compt. rend., 131, 1214 (1900). (4) May, 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 t o 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 i t 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 a n y event, it does not appear that any particular significance was ever attached to the findings.
