Studies on the Mass Culture of Various Algae in Carboys and Deep-Tank Fermentations Louis PRUESS, PETER ARNOW, LAWRENCE WOLCOTT, NESTOR BOHONOS, J. J. OLESON, AND J. H. WILLIAMS Chemical and Biological Research Section, Lederle Laboratories Division, American Cyanamid Co., Pearl River, New York Received for publication November 25, 1953 Von Witschl (1948) has grown Chlorella in glass cylinders, achieving yields of approximately 5.0 g of cells in dry weight per liter in 20 days. Spoehr et al. (1949) employed 5-gallon carboys and achieved yields of approximately 4.0 g of cells in dry weight per liter in 30 to 50 days. Ketchum, Lillick and Redfield (1949) studied the growth of various algal cultures in a bottle illuminated by a jacketed neon tube immersed inside the vessel. These workers found that Chlorella pyre- noidosa produced the highest yield of cells, approxi- mately 0.74 g per liter per day maximum density and 0.065 g per liter per day maximum growth rate. Myers et al. (1951) found that Chlorella pyrenoidosa when grown in a modified Knops medium in a 6-mm annulus exposed to full sunlight achieved yields of 2.45 g per liter per day and a maximum population density of 54.9 g per liter in 20 days. Cook (1951) describes an apparatus designed for the large-scale culture of algae by a con- tinuous-culture technique with which he obtained maximum yields of 0.48 g of cells in dry weight per liter each day. Myers and Johnston (1949) applied the continuous-culture technique for the large-scale culture of algae to the problem of carbon and nitrogen balance of Chlorella. The apparatus used in that study had been previously described (Myers and Clark, 1944). Gotaas et al. (1951) have reported on the large-scale culture by continuous-culture technique of algal-bacterial sym- bionts for the purpose of stabilizing domestic sewage and organic industrial wastes in oxidation ponds. These workers used Euglena gracilis and Chlorella pyrenoidosa in their experiments. The large-scale culture of algae has become an engi- neering problem in an attempt to achieve optimum conditions and maximum growth in apparatus espe- cially designed for the purpose. Some of the problems which await solution are the nitrogen source, pH stabilization, light intensity, and the disposal of toxic products. Furthermore, there is no assurance that the organisms used to date are the most desirable form of algae for large-scale culture. The choice of algal strains has been the particular concern of the experiments I The recent Carnegie Institution of Washington Publica- tion 600 (1953) contains an extensive bibliography on algal culture. The material mentioned in the following references is pertinent to our work. reported in this paper. Various algal cultures have been grown in flasks, carboys, and deep-tank fer- mentors in an attempt to determine whether, under these conditions, any of the strains tested were superior to the others in terms of mass yield. EXPERIMENTAL RESULTS The Growth of Various Algal Cultures in Flasks To determine the yields of various algal cultures in a simple medium,2 the serial transfer technique was employed. Pure cultures were streaked on agar slants (basal medium with 1.5 per cent agar) and incubated 7 days at 23 C over four fluorescent lamps having a light intensity of 175 foot candles. The growth on the slants was scraped off and washed into 25 ml of basal medium contained in 250-ml Erlenmeyer flasks. The flasks were incubated for 7 days under conditions as described above. During the incubation period, the flasks were shaken about 5 minutes by hand twice daily. At the end of the incubation period, 0.5 ml of the growth in the flasks was transferred to 25 ml of fresh basal medium in 250-ml Erlenmeyer flasks. Incubation then proceeded for 7 days as above. Three serial trans- fers were made as described. Sterile technique was observed throughout the process. Turbidimetric read- ings were obtained on the growth in the flask after each transfer. An Evelyn turbidimeter with a 540-mu filter was employed. The instrument was set with the uninoculated medium as a blank. The data in table 1 reveal that of the 47 cultures tested, 6 did not survive to the third transfer, and among the survivors some appear to be declining and possibly would not survive a fourth transfer. The latter observation indicates that the minimal require- ments of some of the algae were not satisfied by the basal medium. It will be noted that most of the or- ganisms tested are in the primitive class, Chlorophyceae. To determine yields in dry weight three serial trans- fers of various algal cultures were carried out as above, except that the volume of the basal medium was in- 2 The composition of the basal medium is as follows: NH4NO3, 100 mg; MgSO 7H20, 40 mg; KH2PO4, 40 mg; FeSO- 7H20, 0.4 mg; CaCI2 anhydrous, 20 mg; sodium acetate, 1 g; dextrose, 5 g. Distilled water was added to make a liter. The pH of the medium was unadjusted. 125 on February 29, 2020 by guest http://aem.asm.org/ Downloaded from
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Studies on the Mass Culture of Various Algae inCarboys and Deep-Tank Fermentations
Louis PRUESS, PETER ARNOW, LAWRENCE WOLCOTT, NESTOR BOHONOS, J. J. OLESON, AND J. H. WILLIAMS
Chemical and Biological Research Section, Lederle Laboratories Division, American Cyanamid Co., Pearl River, New York
Received for publication November 25, 1953
Von Witschl (1948) has grown Chlorella in glasscylinders, achieving yields of approximately 5.0 g ofcells in dry weight per liter in 20 days. Spoehr et al.(1949) employed 5-gallon carboys and achieved yieldsof approximately 4.0 g of cells in dry weight per literin 30 to 50 days. Ketchum, Lillick and Redfield (1949)studied the growth of various algal cultures in a bottleilluminated by a jacketed neon tube immersed insidethe vessel. These workers found that Chlorella pyre-noidosa produced the highest yield of cells, approxi-mately 0.74 g per liter per day maximum density and0.065 g per liter per day maximum growth rate. Myerset al. (1951) found that Chlorella pyrenoidosa when grownin a modified Knops medium in a 6-mm annulus exposedto full sunlight achieved yields of 2.45 g per liter perday and a maximum population density of 54.9 g perliter in 20 days. Cook (1951) describes an apparatusdesigned for the large-scale culture of algae by a con-tinuous-culture technique with which he obtainedmaximum yields of 0.48 g of cells in dry weight perliter each day. Myers and Johnston (1949) applied thecontinuous-culture technique for the large-scale cultureof algae to the problem of carbon and nitrogen balanceof Chlorella. The apparatus used in that study had beenpreviously described (Myers and Clark, 1944). Gotaaset al. (1951) have reported on the large-scale culture bycontinuous-culture technique of algal-bacterial sym-bionts for the purpose of stabilizing domestic sewageand organic industrial wastes in oxidation ponds.These workers used Euglena gracilis and Chlorellapyrenoidosa in their experiments.The large-scale culture of algae has become an engi-
neering problem in an attempt to achieve optimumconditions and maximum growth in apparatus espe-cially designed for the purpose. Some of the problemswhich await solution are the nitrogen source, pHstabilization, light intensity, and the disposal of toxicproducts. Furthermore, there is no assurance that theorganisms used to date are the most desirable form ofalgae for large-scale culture. The choice of algal strainshas been the particular concern of the experiments
I The recent Carnegie Institution of Washington Publica-tion 600 (1953) contains an extensive bibliography on algalculture. The material mentioned in the following references ispertinent to our work.
reported in this paper. Various algal cultures havebeen grown in flasks, carboys, and deep-tank fer-mentors in an attempt to determine whether, underthese conditions, any of the strains tested were superiorto the others in terms of mass yield.
EXPERIMENTAL RESULTSThe Growth of Various Algal Cultures in Flasks
To determine the yields of various algal cultures ina simple medium,2 the serial transfer technique wasemployed. Pure cultures were streaked on agar slants(basal medium with 1.5 per cent agar) and incubated7 days at 23 C over four fluorescent lamps having alight intensity of 175 foot candles. The growth on theslants was scraped off and washed into 25 ml of basalmedium contained in 250-ml Erlenmeyer flasks. Theflasks were incubated for 7 days under conditions asdescribed above. During the incubation period, theflasks were shaken about 5 minutes by hand twicedaily. At the end of the incubation period, 0.5 ml of thegrowth in the flasks was transferred to 25 ml of freshbasal medium in 250-ml Erlenmeyer flasks. Incubationthen proceeded for 7 days as above. Three serial trans-fers were made as described. Sterile technique wasobserved throughout the process. Turbidimetric read-ings were obtained on the growth in the flask aftereach transfer. An Evelyn turbidimeter with a 540-mufilter was employed. The instrument was set with theuninoculated medium as a blank.The data in table 1 reveal that of the 47 cultures
tested, 6 did not survive to the third transfer, andamong the survivors some appear to be declining andpossibly would not survive a fourth transfer. Thelatter observation indicates that the minimal require-ments of some of the algae were not satisfied by thebasal medium. It will be noted that most of the or-ganisms tested are in the primitive class, Chlorophyceae.To determine yields in dry weight three serial trans-
fers of various algal cultures were carried out as above,except that the volume of the basal medium was in-
2 The composition of the basal medium is as follows:NH4NO3, 100 mg; MgSO 7H20, 40 mg; KH2PO4, 40 mg; FeSO-7H20, 0.4 mg; CaCI2 anhydrous, 20 mg; sodium acetate, 1 g;dextrose, 5 g. Distilled water was added to make a liter. ThepH of the medium was unadjusted.
* Evelyn turbidimeter using a 540-m,u filter. Uninoculatedmedia = 100. NG = No growth.
creased to 100 ml contained in a 500-ml Erlenmeyerflask. Dry weights were obtained by centrifuging an
aliquot from the culture in the third serial transfer,washing twice in distilled water, and transferring thecells in a small amount of water to a tared crucible and
TABLE 2. Yields in dry weight of various algal culturesgrown in 100 ml media in 500-ml flasks*
* Yield obtained after third serial transfer in 100 ml basalmedium. Incubation 7 days at 23 C over 4 flourescent lamps.Light intensity 175 f.c.
drying to constant weight at 110 C in a Thelco oven.The yield in dry weight of various algal cultures afterthe third transfer in the basal medium is shown in table 2.Two of the cultures, Chlorella vulgaris Beyerinck (cultureno. 22) and Chlorella vulgaris var. viridis (culture no. 99)appear to be markedly superior in terms of dry weightyields.
The Growth of Various Algal Cultures in 20-Liter CarboysThe inocula for these cultures were prepared as
follows: the growth from a slant of an algal culturewas washed into 100 ml of basal medium contained ina 500-ml Erlenmeyer flask. The flask was incubated for7 days at 23 C over four fluorescent lamps having alight intensity of 175 foot candles. During the incuba-tion period the flask was shaken by hand twice dailyfor 5 minutes. The content of the flask was transferred,using sterile technique, into a 20-liter glass carboycontaining 12 liters of the N-rich B medium. The carboycontaining the medium had previously been autoclavedfor 20 minutes at 15 pounds steam pressure and cooledunder air pressure with water. The carboys were fittedwith a motor-driven agitator for stirring the medium
at the rate of 300 rpm, an aerator through which sterileair was passed at the rate of 1 liter per liter of mediumper minute, an air outlet, and a device through whichsterile samples could be obtained during the fermenta-tion. Sterile techniques were used throughout the proc-ess. After the carboy was inoculated, it was placed ina water bath maintained thermostatically at a tem-perature of 25 C ±t 2 C. The level of the circulatingwater in the bath was kept at the level of the mediumin the carboy. One 150-watt projector flood lamp wasmounted approximately 12 inches above the carboyand arranged so that the light projected directly down-ward. Samples were withdrawn during the fermenta-tion period, the volume of the medium in the carboywas measured for final dry weight calculation and thecells were then centrifuged, freeze-dried and storedunder refrigeration in a brown bottle. The final dryweight calculations on the yields of various algal cul-tures grown as described above for 5 days are containedin table 3. Chlorella vulgaris var. viridis (culture no. 99)and Chlorella pyrenoidosa (culture no. 8) appear to besuperior in terms of dry weight yield under the de-scribed conditions. Chemical analyses were made onsome of the cultures grown in 20-liter carboys. The dataare contained in table 4, and reveal a striking similaritybetween the various cultures. The nitrogen content ofmost of the cultures is approximately 6 to 7 per centwhich, on an ash-free basis, would give a protein con-tent of about 40 per cent.
TABLE 3. Yields in dry weight of various algal culturesgrown in 12 liters of media in 20-liter carboys*
99 Chlorella vulgaris var. viridis 7.099 Chlorella vulgaris var. viridis 9.999 Chlorella vulgaris var. viridis 8.699 Chlorella vulgaris var. viridis 10.299 Chlorella vulgaris var. viridis 9.399 Chlorella vulgaris var. viridis 12.299 Chlorella vulgaris var. viridis 9.699 Chlorella vulgaris var. viridis 9.8
* Grown in B medium plus 0.2 per cent yeast ext., with aera-tion and stirring for 5 days at 25 C 4i 2, and 12 inches under a150-watt projector flood lamp.
TABLE 4. Chemical analysis of algal cells grownin 20-liter carboys
CULTURENO.
7192899
77
127119
99
35
CULTURE NAME
Stichococcus subtilisChlorococcum humicolaGloecystis grevilleiChlorella vulgaris var.
viridisChlamydomonasapplanata
Oobcystis naegeliiCoelastrum probo-scideum var. gracilis
Chlorella vulgaris var.viridis
Gloecystis grevillei
PERCENTC
46.345.6639.346.74
46.78
49.0439.1
43.88
PERCENTH
7.266.896.537.92
PERCENTN
6.727.117.296.65
7.48 6.10
7.06.53
6.654.74
6.95 6.76
35.22 6.28 4.38
PERCENTPRO-TEIN*
42444541
38.1
41.529.6
42.2
27.2
* Per cent protein = per cent N X 6.23.
Growth of Various Algal Culturesin Deep-Tank Fermentors
Various algal cultures were grown in 100-, 500-, and1000-gallon glass-lined fermentation tanks. Media B3and C4 were used in these fermentations. Before in-oculation, the medium in the fermentor was sterilizedfor 30 to 45 minutes at 15 pounds steam pressure.Inocula were prepared by scraping off the agar surfacegrowth from one or more Roux bottle pure cultures ora number of test tube cultures, into sterile water,which was used to inoculate six liters of sterile nutrientmedium in a 9-liter bottle.One to three such bottles were incubated at room
temperature for about 6 to 7 days with aeration of themedium. A 150-watt light bulb in a reflector shadeplaced about a foot above the bottles supplied artificialillumination. In some cases the algal cultures grown inthe 20-liter carboys, as described above, served as theinocula. Table 5 indicates the preparation of the inoculafor the various runs. After inoculation, the medium inthe fermentor was continuously illuminated with abeam of artificial light from a 150-watt G. E. projectorflood lamp directed into the tank through the sight-glass in the manhole cover at the top of the fermentor.Agitation of the culture by means of a mechanicalstirrer insured the uniform exposure of the algal cells
3 The composition of B medium is as follows (g per L):Dextrose (Cerelose) 10, glycine 2, ammonium acetate 1, MgSO4.7H20 0.50, K2HPO4 0.25, CaCl2 0.1387, H3BO3 0.1124, thiamin(sterilized separately and added aseptically to the tank mediumat time of inoculation) 0.10, FeCl3-6H20 0.0485, ZnCl2 0.0416,MnCl2-4H20 0.0144, (NH4)2 MoO4 0.0081, CuSO4-5H20 0.0052,CoCl2-6H20 0.0020.
4 The composition of C medium is as follows (g per L):Dextrose (Cerelose) 10, Enzyme hydrolyzed casein (N-Zamine, type A) 2, sodium acetate 1, NH4NO3 0.1, KH2PO40.04, MgSO4-7H20 0.04, CaCl2 0.02, FeSO4.7H20 0.0001.
RoomRoomRoomRoomRoomRoomRoomRoomRoomRoomRoomRoomRoom23 C23 C23 C
INTER-MEDIATECULTURE,
TYPE
NoneNoneNoneNone
*
NoneNoneNoneNoneNoneNoneNoneNoneNone
tt
AERATED, ILLUMINATED BOTTLE CULTURE
No.used
2212422132211111
Age indays
771452766777737785
* Six L of medium in a 9-L bottle. Aerated and illuminated for 7 days at room temperature.t Fifty ml of medium in a 250-ml flask. Illuminated at room temperature for 4 days.t One hundred ml of medium in a 500-ml flask. Illuminated at room temperature for 6 days.
to the light. At the same time, sterile air was continu-ously forced through the medium by way of either a
single open-end pipe or a sparger at the rate of 0.4 to1.8 volumes of air per one volume of medium perminute. The mechanical stirrer also aided in distribu-ting the air uniformly throughout the medium. An airpressure of about 5 pounds per square inch was main-
RoomRoomRoomRoomRoomRoomRoomRoomRoomRoomRoomRoomRoomBath, 24-28 CBath, 24-28 CBath, 24-28 C
tained in the fermentor. An antifoam agent, such as1 per cent octadecanol in lard oil, was used to controlfoaming when needed. The temperature of the culturewas maintained at 20 to 24 C. After 142 to 280 hours,the algal cells were removed from the culture mediumin a large Sharples centrifuge. Table 6 lists the cellyields and other pertinent data of the fermentor runs.
* Weight of cells as harvested in the centrifuge bowl.Note 1. In runs 1 through 10 Chlorella vulgaris was used. In runs 11 and 12 Stichococcus subtilis was used. In runs 13, 14 and
15 Chlorella pyrenoidosa was used.Note 2. The first four runs were in 100-gal tanks; all others were in 500-gal tanks except runs 10, 12 and 14 which were in 1000-
gal tanks.Note 3. Medium C was used throughout except in runs 13, 14 and 15, where medium B was used.Note 4. The rate of stirring in runs 1 through 4 was 137 rpm. All others were stirred at 100 rpm.
* For the fermentation conditions see tables 5 and 6.
Chemical analyses were made on the cells grown intank run numbers 13 and 15. The culture was the samefor both runs, Chlorella pyrenoidosa (culture no. 8).The dry weight yields for both tanks as well as thefermentation conditions were approximately similar,as seen in table 6. However, the chemical analyses onthe cells grown in run numbers 13 and 15 show a widediscrepancy between the batches, as seen from the datain table 7. The cells from run number 13 containedapproximately 50 per cent more nitrogen, aminonitrogen, and protein as compared with the cells fromrun number 15; there is no explanation at present forthis difference.
DISCUSSION
The primary purpose of the work reported in thispaper was to gain some experience with the growth ofvarious algal cultures in different size vessels. No at-tempt was made to achieve optimum growth conditions.It was surprising that even with what would appearto be inadequate light conditions, especially in thedeep-tank fermentors, the algal yields reported in thispaper compare favorably with the yields reported inthe literature. Cook (1951) reports maximum yields of0.48 g dry weight per liter each day, while Myers(1951) achieved yields of 2.45 g per liter per day. Bothworkers used Chlorella pyrenoidosa growing auto-trophically and depending upon photosynthesis forits energy. As reported in this paper, maximum yields,with Chlorella vulgaris var. viridis, of 12.2 g dry weightper liter in 5 days (table 3) were obtained, which, ona daily basis, would be 2.4 g per liter per day. It is ofinterest that the heterotrophic growth obtained in our
that achieved by others with algae growing auto-tropically.Although it appears from the data presented in this
paper that Chlorella vulgaris var. viridis was the mostefficient algal strain tested, in terms of cell yield, otherstrains under other conditions might be superior. Thechoice of the culture used must depend upon the pur-
pose of the problem. It is a dubious point to strive forlarge-scale cell yields, which immediately involveseconomic considerations, when the utilization of thecells, once obtained, has not been the primary concern.
Spoehr et al. (1949) have shown that control of theenvironmental factors may change the protein andlipid content of the cell. Certain algal cells have beenfound to contain sterols (Klosty and Bergmann, 1952;Carter et al., 1939) and vitamins (Dam, 1944). Itappears likely, therefore, that the choice of algae strainswould influence the choice of environmental conditionsas much as the environmental conditions would influ-ence the choice of the algae. For obtaining algal prod-ucts such as sterols and vitamins, where rigorouslycontrolled techniques would have to be employed, itseems likely that the use of modified deep-tank fer-mentors offers many advantages for the large-scaleproduction of algal cells.
SUMMARY
Forty-one of forty-seven algal cultures were sub-cultured three times in a simple medium in flasks.Two of the cultures, Chlorella vulgaris Beyerinck andChlorella vulgaris var. viridis were markedly superior interms of dry weight yields.
Algal cultures were grown in a simple medium in20-liter carboys and in a medium supplemented witheither thiamin and glycine or hydrolyzed casein(N-Z-amine) in 100-, 500-, and 1000-gallon deep-tankfermentors. Dry weight yields and chemical analysesof the cells are reported.
REFERENCES
CARTER, P. W., HEILBRON, 1. M., AND LYTHGOE, B. 1939The lipochromes and sterols of the algal classes. Proc.Roy. Soc. (London). Series B., 128, 82-109.
COOK, PAUL M. 1951 Chemical engineering problems in large-scale cu'ture of algae. Ind. Eng. Chem., 43, 2385-2389.
DAM, HENRIK 1944 Vitamin K in unicellular photosynthesiz-ing organisms. Am. J. Botany, 31, 192-193.
GOTAAS, H. B., OSWALD, J., LUDWIG, H. F., AND LYNCH, V.1951 Algae symbiosis in sewage oxidation ponds. Secondprogress report. Series No. 44, Issue No. 3. U. of Cal.Institute of Engineering Research, Berkeley, Cal.
KETCHUM, B. H., LILLICK, L., AND REDFIELD, A. C. 1949The optimum growth and optimum yields of unicellularalgae in mass culture. J. Cellular and Comp. Physiol.,33, 267-280.
KLOSTY, M., AND BERGMANN, W. 1952 Sterols of Algae. III.The occurrence of ergosterol in Chlorella pyrenoidosa.J. Am. Chem. Soc., 74, 1601.
experiments with low light conditions was similar to
129
MYERS, J., PHILLIPS, JR., J. N., AND GRAHAM, Jo-RUTH. 1951
Oin the mass culture of algae. Plant Physiol., 26, 539-548.MYERS, J., AND JOHNSTON, J. A. 1949 Carbon and nitrogen
b)alance of Chlorella during growth. Plant Physiol., 24,111-119.
MYERS, J., AND CLARK, L. B. 1944 Culture conditions andthe development of the photosynthetic mechanism. II.An apparatus for the continuous culture of Chlorella. J.Gen. Physiol., 28, 103-112.
MYERS, J. 1951 Physiology of the algae. Ann. Rev. Micro-biol., 5, 157-180.
SPOEHR, H. A., SMITH, J. H. C., STRAIN, H. H., MILNER, H. W.,AND HARDIN, G. J. 1949 Fatty acid anitibacterials fromplants. Carnegie Inst. Wash. Publ. 586.
VON WITSCH, H. 1948 Beobachtungen zur physiologie deswachstums von Chlorella in massenkulturen. Biol.Zentr., 67, 95-100.
In Vitro Cellulose Digestion by Rumen Microorganismsand Its Stimulation by Fishery By-Products'
ROBERT A. MAcLEOD AND CHARLES A. BRUMWELL
Pacific Fisheries Experimental Station, Fisheries Research Board of Canada, Vancouiver, British Colutmbia, Canada
Received for publication November 27, 1953
The importance of the role played by rumen micro-organisms in the nutrition of ruminants is well recog-nized (Huffman, 1953). As a result, techniques havebeen developed to study the activities of rumen micro-organisms in vitro in an attempt to determine whatfactors may influence their activities in vivo (Pearsonand Smith, 1943; Marston, 1948; Burroughs et al.,1950).Because the possibility of including such fishery by-
products as whale solubles, herring solubles, and herringstickwater in the diet of cattle and sheep has not beenstudied extensively, it was considered of interest todetermine the effect of adding these various fisherymaterials on the breakdown of cellulose by rumen
microorganisms in vitro. In the course of the studieswhich resulted it was found that under carefully definedconditions the rate of cellulose digestion was greatlyincreased by the inclusion of the fishery supplements inthe fermentation medium. When known compoundswere tested under the same conditions, a mixture ofamino acids was discovered to have even more activitythan the fishery supplements tested. These results anda discussion of their possible implications are presentedbelow.
EXPERIMENTAL METHODS
In vitro rumen fermentations were carried out in aseries of 25 x 200 mm test tubes. Cellulose digestionwas measured by a technique developed by Block andHenderson (1953) wherein the difference in the weightof a role of vegetable parchment before and afterfermentation is determined.
I A preliminary report of this investigation appeared inFisheries Research Board of Canada Progress Reports of thePacific Coast Stations, No. 96, p. 16, 1953.
Rumen Liquid Inoculum
Rumen liquid was obtained at an abattoir from thepaunches of freshly killed cattle. The cattle had notbeen fed for a period of 12 hours prior to slaughter buthad had access to water. In the period immediatelypreceding the fast, the animals had been fed alfalfa ortimothy hay.At each collection of rumen liquid, samples from at
least four animals were pooled and the liquid wasstrained through cheesecloth to free it of gross particles.During the collection, a stream of carbon dioxide gaswas directed upon the scene of operations. In this step,as well as in all subsequent steps, every effort was madeto maintain conditions as nearly anaerobic as possible.The rumen liquid, after straining, was diluted withair-free water to give the desired inoculum concentra-tions. Each fermentation tube contained 20 ml ofinoculum in a final volume of 40 ml.
Fermentation MediumThe composition of the fermentation medium is
shown in, table 1. Cellulose was introduced as a roll ofcarefully weighed vegetable dialysis parchment stapledat one end to prevent it from unrolling. Nitrogen wasadded at a level of 24 mg in each tube and was suppliedeither as urea or as a combination of urea and supple-ment. The level of nitrogen was maintained constantin all tubes to avoid the possibility that stimulation byan active supplement could be due to the presence of ahigher level of total nitrogen in the supplemented tubes.The salt mixture used was patterned after one alreadyemployed in in vitro rumen fermentations (Burroughset al., 1950) but modified to include additional ironand phosphate as more recent findings of the sameworkers have shown that higher concentrations of these
