Abstract:
This specification discloses a process and a culture composition for growth of an alga and synthesis of biopolymer by the alga. The process involves growth of the alga and concomitant synthesis of the biopolymer in an aqueous culture containing as a source of phosphate for said alga dibasic sodium or dibasic potassium phosphate. The nitrogen source can be sodium nitrate or urea. Control of the pH of the culture is effected by injecting a mixture of carbon dioxide and air into the culture during growth of the alga and synthesis of the biopolymer. The carbon dioxide also serves as a source of carbon for growth of the alga and synthesis of the biopolymer and provides agitation/mixing within the culture chamber. Preferably, the mixture of carbon dioxide and air is injected continuously into the culture during the entire growth period of the alga.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates to the growth of an alga and synthesis thereby of biopolymer. 
     2. Description of the Prior Art 
     Processes for the growth of alga are well known. For example, U.S. Pat. No. 3,195,271 discloses a process for the growth of the alga Porphyridium cruentum and synthesis of the alga constituent carrageenin. Other processes for the growth of alga are disclosed in &#34;Algal Culture: From Laboratory to Pilot Plant&#34;, J. S. Burlew, Ed., Carnegie Inst. of Washington, Publication No. 600, Washington, D.C. (1964), and &#34;Properties and Products of Algae&#34;, J. E. Zajic, Ed., Plenum Press, N.Y. (1970). A culture for the growth of the alga Porphyridium aerugineum, known as the MCYII medium, is disclosed by Ramus, J., in the Jnl. Phycol., 8 [1], 97 (1972) and by Gantt, E. et al., in the Jnl. Phycol., 4, 65 (1968). 
     SUMMARY OF THE INVENTION 
     The invention comprises a process and culture composition for growth of an alga and concomitantly therewith the synthesis of biopolymer. The alga is grown and the biopolymer synthesized in a culture containing, as a source of phosphate for the alga, dibasic sodium or dibasic potassium phosphate. The nitrogen source can be sodium nitrate or urea. Control of the pH of the culture is effected by injecting into the culture a mixture of carbon dioxide and air during growth of the alga and synthesis of the biopolymer. The carbon dioxide also serves as a source of carbon for growth of the alga and synthesis of the biopolymer. The injected gas also provides a mechanism for culture agitation and cell turnover required to insure exposure of the alga cells to the illumination source. Preferably, injection of the mixture of carbon dioxide and air is continued throughout the entire growth period of the alga. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a plot of the apparent viscosities attained by cultures of the alga Porphyridium aerugineum at various ages of the cultures versus the initial dibasic potassium phosphate concentration of the cultures. 
     FIG. 2 is a plot of the apparent viscosities attained by cultures of the alga Porphyridium aerugineum at various initial concentrations of dibasic potassium phosphate in the cultures versus age of the cultures. 
     FIG. 3 is a plot of the apparent viscosities attained by cultures of the alga Porphyridium aerugineum containing dibasic sodium and dibasic potassium phosphate. 
     FIG. 4 is a plot of the apparent viscosities attained by cultures of the alga Porphyridium aerugineum containing urea as a nitrogen source. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     There is disclosed in the copending application of one of us, Joseph George Savins, Ser. No. 680,819, filed concurrently with this application, a process for the production of petroleum from a subterranean formation. In this process, an aqueous driving fluid is injected into the subterranean formation through an input well and passed through the formation in the direction of an output well to drive the petroleum in the formation to the output well. The aqueous driving fluid contains a thickening agent and the thickening agent is a biopolymer, a heteropolysaccharide, synthesized by an alga. The alga, in its growth cycle, in a culture thereof, concomitantly synthesizes the biopolymer as an extracellular product and the biopolymer enters into solution in the culture. The aqueous driving fluid may contain the biopolymer as an in-vivo solution or in a reconstituted form. The disclosure of the mentioned copending application is included by reference thereto in the present application. 
     The present invention is directed to a process and culture composition for the growth of alga and synthesis of biopolymer. The biopolymer may be employed as a thickening agent in aqueous driving fluids for the recovery of petroleum from a subterranean formation. However, it is to be understood that the biopolymer has other uses than as a thickening agent in the recovery of petroleum, such other uses for alga biopolymer being known to the art. 
     Whereas the process of the invention may be employed for the growth of, and synthesis of biopolymer by, any alga, it is particularly applicable to the growth of, and synthesis of biopolymer by, the alga Porphyridium aerugineum. The alga Porphyridium aerugineum, in common with other algae, requires for its growth illumination, an inorganic carbon source and certain other nutrients and nutrient-related materials. Since Porphyridium aerugineum is an obligate photoautotroph, it is customary to provide the source of inorganic carbon in the form of gaseous carbon dioxide. A standard culture for the growth of Porphyridium aerugineum is the MCYII medium previously mentioned. This culture contains the following distribution of macro and micro levels of inorganic ions, chelating agents, buffering agent, vitamins, etc. 
     
                       TABLE I______________________________________MCYII MEDIUMNaNO.sub.3          442        mgKCl                 30         mgCaCl.sub.2 . 2H.sub.2 O               36.6       mgFeCl.sub.3 . 6H.sub.2 O               1.9        mgMgSO.sub.4 . 7H.sub.2 O               100        mgNa.sub.2 . glycerophosphate . 5H.sub.2 O               90         mgTricine buffer      986        mgPII trace metal mix 10         mlVitamin B.sub.12    3.5        μgDistilled water to  1000       mlAdjust pH to 7.6 with NaOHPII Metal Mix:H.sub.3 BO.sub.3    114.0      mgMnCl.sub.2 . 4H.sub.2 O               14.4       mgZnSO.sub.4          2.2        mgCoCl.sub.2 . 6H.sub.2 O               0.4        mgFeCl.sub.3 . 6H.sub.2 O               4.8        mgNa.sub.2 EDTA       100        mgDistilled water to  100        ml______________________________________ 
    
     In accordance with one aspect of the present invention, an alga, such as Porphyridium aerugineum, is grown and biopolymer synthesized in an aqueous culture containing dissolved therein dibasic sodium or dibasic potassium phosphate as a source of phosphate for the alga. The culture also contains dissolved therein hydrated magnesium sulfate, sodium nitrate or urea, calcium chloride, ferric chloride or ethylene dinitrilo tetraacetic acid Ferric-Sodium salt (C 10  H 12  FeN 2  NaO 8 ), boric acid, hydrated ferrous sulfate, hydrated zinc sulfate, potassium chloride, manganous chloride, vitamin B 12 , hydrated cobaltous chloride, and disodium salt of ethylene diamine tetraacetic acid. The amount of dibasic potassium or dibasic sodium phosphate employed is such that the initial phosphate ion concentration in the aqueous culture is at least 14 weight parts per million. 
     In this culture, the phosphate requirements of the alga, as indicated, are provided by the dibasic sodium or dibasic potassium phosphate. The latter two compounds are relatively inexpensive as compared to sodium glycerophosphate, the phosphate source employed in the prior art culture set forth in Table I. Further, in this aspect of the present invention, pH control, or buffering, is effected by injection of a mixture of carbon dioxide and air into the culture during growth of the alga. Control of the pH of the culture by injection of a mixture of carbon dioxide and air is relatively inexpensive as compared to the tricine buffer employed in the prior art culture set forth in Table I. Accordingly, growth of the alga and synthesis of the biopolymer by the process of the invention is effected employing low cost components in the culture, thereby enabling the synthesis of biopolymer at low cost. Thus, the process of the invention is particularly applicable to the synthesis of biopolymer for such uses as a thickening agent in an aqueous driving fluid for the recovery of petroleum from a subterranean process where economics prohibits high cost materials and methods. 
     In a specific embodiment, the culture contains the components, and in the concentrations indicated, set forth in Table II following. 
     
                       TABLE II______________________________________Component            Concentration - WPPM______________________________________MgSO.sub.4 . 7H.sub.2 O                100NaNO.sub.3 or        442Urea                 155CaCl.sub.2           28FeCl.sub.3 or        1.43C.sub.10 H.sub.12 FeN.sub.2 NaO.sub.8                5.36K.sub.2 HPO.sub.4 or At least 26Na.sub.2 HPO.sub.4   At least 21H.sub.3 BO.sub.3     11.4FeSO.sub.4 . 7H.sub.2 O                2.2ZnSO.sub.4 . 7H.sub.2 O                1.44KCl                  30B.sub.12             3.5 × 10.sup.-6CoCl.sub.2 . 6H.sub.2 O                0.044(Na).sub.2 EDTA (disodium salt of                10ethylene diaminetetraacetic acid)______________________________________ 
    
     Injection of the mixture of carbon dioxide and air into the aqueous culture during growth of the alga and synthesis of the biopolymer is preferably carried out continuously during the entire growth period of the alga. Growth of the alga and synthesis of the biopolymer can be carried out under artificial illumination in which case the illumination and the injection of the mixture of carbon dioxide and air can be provided during the entire growth period of the alga. On the other hand, growth of the alga and synthesis of the biopolymer can be carried out with natural illumination in which case the growth and synthesis occur in a diurnal cycle. CO 2  /air is always continuously injected, with or without tricine buffer, diurnal or continuous illumination, as the carbon is required for nutrition. We have ascertained that by continuous injection of the mixture of carbon dioxide and air during both the daylight and dark portions of the diurnal cycle, fluctuations in pH of the culture are eliminated and both the production of alga and kinetics and levels of biopolymer synthesis are improved. 
     The relative proportions of the carbon dioxide and air in the mixture thereof injected into the aqueous culture may be as desired. However, it is preferred that the mixture contain 5% of carbon dioxide and 95% of air. 
     The following examples will be illustrative of the invention. 
     EXAMPLE 1 
     This example will illustrate the effect of the concentration of dibasic potassium phosphate in the aqueous culture on the extent of synthesis of biopolymer with time. 
     Porphyridium aerugineum was grown and biopolymer synthesized in a series of cultures containing different initial concentrations of dibasic potassium phosphate. These concentrations of dibasic potassium phosphate, in terms of phosphate ion (PO 4 ).sup..tbd. in weight parts per million, were 28, 22.5, 14.0, 8.4, and 2.8. The cultures contained 442 weight parts per million of sodium nitrate and also contained the remaining components, and in the concentrations, set forth in Table II above. The particular Porphyridium aerugineum that was employed is cataloged as isolate No. 755 in the alga culture collection maintained at Indiana State University, Bloomington, Indiana, e.g. see Starr, R. C., Amer. Jnl. Bot., 51 [9], 1013 (1964). The alga was grown and the biopolymer synthesized at an average temperature of 77° F. (25° C.). Agitation was provided by the standard shake flask culture method. The carbon source was 5% carbon dioxide entrained with 95% air and was injected continuously into the cultures. This also provided a measure of agitation. Undefined but continuous (24-hour) illumination was provided via ceiling mounted and auxiliary fluorescent lights. Levels of illumination and radiant energy incident to the culture flasks were on the order of 800-foot candles (8061.12 lux) and 10 4  ergs/cm 2  sec. (10 3  microjoules/cm 2  sec.). Samples of the cultures were removed at 4, 7, 9, 11, and 14-day intervals and the apparent viscosity at a shear rate of 1.8 sec -1  measured employing a Brookfield viscometer fitted with a U.L. adapter. The apparent viscosity of the culture is a measure of the amount of biopolymer in the culture. 
     The results are given in FIGS. 1 and 2. In FIG. 1, the concentration of the dibasic potassium phosphate is expressed as concentration of PO 4   57  ion. It will be observed from FIGS. 1 and 2 that an initial PO 4   57  level on the order of 14 weight parts per million, i.e., 26 weight parts per million of dibasic potassium phosphate leads to an acceptable level of viscosity production, i.e., concentration of biopolymer in the culture. 
     EXAMPLE 2 
     In this example, Porphyridium aerugineum was grown in a series of three cultures. One of the cultures contained 41 weight parts per million of dibasic potassium phosphate (22.5 weight parts per million of PO 4   57  ). Another culture contained 51 weight parts per million of dibasic potassium phosphate (28.0 weight parts per million of PO 4   57  ). The third culture contained 42 weight parts per million of dibasic sodium phosphate (28.0 weight parts per million of PO 4   57  ). Each of the cultures contained 442 weight parts per million of sodium nitrate and the remaining components set forth in Table II and in the concentrations given. The particular Porphyridium aerugineum that was employed was the same isolate that was employed in Example 1. Conditions of temperature, illumination, agitation and injection of a mixture of carbon dioxide and air were the same as in Example 1. Samples of the cultures were removed at various intervals and the apparent viscosities measured as in Example 1. 
     The results are given in FIG. 3. In FIG. 3, the data points for the 41 weight parts per million of dibasic potassium phosphate are signified by o, for the 51 weight parts per million of dibasic potassium phosphate by +, and for the dibasic sodium phosphate by X. It will be observed from the Figure that the viscosity kinetics and production levels are practically the same for the three sources of the PO 4   57  ion. 
     EXAMPLE 3 
     This example will illustrate the effect of employing urea as the source of nitrogen in the aqueous culture on the extent of synthesis of biopolymer with time. 
     Porphyridium aerugineum was grown and biopolymer synthesized in two aqueous cultures. Each of the cultures contained 155 weight parts per million of urea. One of the cultures contained 51 weight parts per million of dibasic potassium phosphate and the other contained 42 weight parts per million of dibasic sodium phosphate. Each also contained the remaining components set forth in Table II and in the concentrations given. The particular Porphyridium aerugineum and the reaction conditions and procedures employed were the same as in the previous examples. 
     The results are given in FIG. 4. In this Figure, the data points for the culture containing the dibasic potassium phosphate are signified by X and for the dibasic sodium phosphate by ∇. It will be observed from the Figure that the viscosity kinetics and production levels are practically the same when urea is used as the nitrogen source.