Microbial viscosifiers
This invention pertains to the production of microbial viscosifiers. 
     BACKGROUND OF THE INVENTION 
     In one of its more specific aspects, this invention pertains to the conversion of fermenter effluents into materials suitable as viscosifiers for such things as waterflood oil recovery fluids. 
     The preparation of thickening agents for use in preparing viscous waterflooding solutions from heteropolysaccharides is well known. The heteropolysaccharides are produced by bacterial action upon a variety of carbohydrate substances. Usually the fermenter liquor as recovered from the fermenter requires some treatment to produce solutions or solids useful for increasing the viscosity of fluids, such as injection water, employed in secondary oil recovery. 
     SUMMARY OF THE INVENTION 
     The present invention provides a simple and inexpensive method of increasing the viscosity of fermenter suspensions, comprising predominantly Gram-negative microbial cells themselves, over a wide range of values. 
     According to the present invention there is provided a method for treating a fermentation suspension comprising Gram-negative cells to produce microbial viscosifiers which comprises contacting the suspension with a base to increase the pH of the resulting solution. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In general, the method of this invention involves treating cultures of hydrocarbon, carbohydrate or methanol-grown cells from the fermenter with an alkali to increase the pH and the viscosity of the fermenter effluent and to produce a substance useful for increasing the viscosity of aqueous fluids employed in secondary oil recovery. 
     The fermenter effluent, to which the method of this invention is applicable is one wherein Gram-negative bacterial cells are present. 
     The method of this invention employs any base capable of raising the pH of the fermenter effluent. Preferably, an alkali metal hydroxide such as NaOH and KOH is employed, although NH 4  OH and other alkalis can be employed. The base is added to the fermentation effluent in any convenient manner and at any strength until the resulting liquor has a pH greater than 7, and preferably a pH between 9 and 12, more preferably 9 and 10. Values greater than 12 will usually not be employed. Upon the addition of the base the viscosity of the mixture increases. Intermittent dilution with water or an aqueous solution can be made, before, during, or after the addition of the base in order to facilitate mixing of the effluent and alkali. After the desired pH is attained, further dilution can be made until the mixture attains the desired viscosity. Biocides can also be added during the period of pH adjustment, either with the water or independently thereof. 
     In certain instances, the viscosity of the solution will continue to increase for a time after the desired pH is attained. Accordingly, viscosity adjustment of the mixture can be made for a period after the pH adjustment has been accomplished, during which period the mixture can be considered as aging. The aging is preferably conducted at a temperature somewhat above room temperature, although the aging can be carried out at 50° to 212° F, preferably 80° to 150° F. 
     After an aging period of about 4 weeks or less, both the viscosity and the pH of the solution will begin to decrease. Accordingly, then, there is an optimum period of time for aging to achieve maximum viscosity. Generally, aging periods longer than 4 weeks are not employed. Depending on the temperature, the time of aging will generally be at least 5 minutes to allow the alkali to produce the viscosifying properties before the solution is used. Generally a time of 1 day to 4 weeks, preferably 1-3 days will be used. In waterflood applications the material can be injected into the ground where the viscosity will continue to increase. 
     In applications using the invention, fermenter effluent can be employed, as such, or it can be evaporated to dryness or centrifuged or subjected to any conventional liquid-solid separation step to recover a solid cellular material which can be treated with a base in accordance with this invention to produce a viscosifier. By treating the total fermenter effluent, the water required, if the product is to be used as an injection fluid, is already present. Similarly when this viscosifier is used in other applications such as suspending coal fines for transmission through pipe lines, further diluents may not be necessary. 
     The alkali treated effluent is referred to herein as a solution. Although it may contain some material in the form of a suspension, probably a colloidal suspension, it has the general appearance of a solution, and hence the term solution appears to best describe this material. 
    
    
     EXAMPLE I 
     The method of this invention is illustrated by the following example. A fermenter effluent was produced as follows: A continuous fermentation was conducted at 39±1° C in a 7 liter fermenter with the organism Psuedomonas Methanica and methanol as the principal source of carbon and energy (nutrient). The medium composition was the following: 
     
         ______________________________________                                    
Recipe                                                                    
______________________________________                                    
Medium Composition                                                        
H.sub.3 PO.sub.4 (85%) ml/l                                               
                         2                                                
KCl, g/l                 1.0                                              
MgSO.sub.4 . 7H.sub.2 O, g/l                                              
                         1.5                                              
CaCl.sub.2 . 2H.sub.2 O, g/l                                              
                         0.2                                              
NaCl, g/l                0.1                                              
Trace Mineral Solution, ml/l                                              
                         5                                                
Trace Mineral Solution Recipe                                             
CuSO.sub.4 . 5H.sub.2 O, g/l                                              
                         0.06                                             
KI, g/l                  0.08                                             
MnSO.sub.4 . H.sub.2 O, g/l                                               
                         0.30                                             
H.sub.3 BO.sub.3, g/l    0.02                                             
ZnSO.sub.4 . 7H.sub.2 O, g/l                                              
                         2.0                                              
FeCl.sub.3 . 6H.sub.2 O, g/l                                              
                         4.8                                              
Na.sub.2 MoO.sub.4 . 2H.sub.2 O, g/l                                      
                         0.2                                              
H.sub.2 SO.sub.4 (to remove precipitates                                  
                         3                                                
in trace mineral                                                          
solution), ml/l                                                           
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     The pH of the medium (3.5 l) was adjusted to 4.5 with NH 3  and the fermenter and medium sterilized for 30 minutes at 121° C and 15 psig. After cooling to 40° C the sterile trace mineral solution (17.5 ml) and filtered methanol (17.5 ml) was added and pH was adjusted to 6.5 with NH 4  OH. The inoculum (500 ml) was there added to start fermentation. The fermentation run was carried out for 5 days during which time the feed rate of medium (containing 8% by wt methanol) was gradually increased from 300 to 500 ml/hr. The oxygen addition rate was also increased gradually from 6 l/min of air to a mixture of 5 l/min of air and 1 l/min of pure oxygen. Ammonium hydroxide was also added as required to maintain the pH at 6.5 and to serve as a source of nitrogen for the microorganisms. A portion of the entire fermenter effluent, including as the predominant constituent Gram-negative cells, was treated with 0.32 weight percent (based on cell content) sodium hydroxide to adjust the pH, two parts by volume of tap water being added after 24 hrs. to adjust the viscosity. Aging was conducted at 120° F. The results were as follows: 
     
                       Table I                                                     
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Time             pH         Viscosity*                                    
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Initial                                                                   
After Alkali Addn.                                                        
                 9.5        6.4                                           
24 Hours         9.0        21.7                                          
72 Hours         10.5       14.7                                          
2 Weeks          8.1        12.8                                          
3 Weeks          7.9        6.4                                           
41/2 Weeks       8.2        5.8                                           
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 *Measured at 6 rpm on Brookfield LVT with UL attachment at 120° F.
 
    
     It will be noticed that after the solution attained the desired viscosity and was aged, both the pH and the viscosity of the solution tended to decrease. Hence, aging is conducted to a desired or preselected pH or viscosity, or both, at which point the solution is ready for use as a viscosifier. 
     EXAMPLE II 
     The effect of sodium hydroxide on the viscosity of aqueous suspensions of Gram-negative cells and Gram-positive cells was investigated. The cultures were grown in an agitated medium containing 3 grams of yeast extract, 3 grams of malt extract, 5 grams of peptone, and 10 grams of glucose per liter of water, and Gram stains were made to show purity and Gram stain characteristics. The cultures grown were then centrifuged, and the packed cells were washed, recentrifuged, and then suspended in tap water so as to give the same cell density in each instance. To the test tubes containing the cell suspensions was added sufficient 1 N sodium hydroxide to raise the pH to 10. The contents of each tube were mixed and then allowed to stand for 5 minutes, after which the test tube was tipped. If the contents of the test tube had not substantially increased in viscosity, the mixture would pour, but if the viscosity increased substantially, a gel was formed which would not pour. The following table shows the effect of the sodium hydroxide on the suspensions of Gram negative (g -  ) or Gram-positive (g + ) cells. 
     
                       Table II                                                    
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                 g.sup.-  Result of                                       
Culture          or g.sup.+                                               
                          NaOH Addition                                   
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Serratia marscens                                                         
                 g.sup.-  Gel                                             
Pseudomonas species PS1                                                   
                 g.sup.-  Gel                                             
Arthobacter species H010                                                  
                 g.sup.-  Gel                                             
Pseudomonas strain BA623                                                  
                 g.sup.-  Gel                                             
Bacillus megatherium                                                      
                 g.sup.+  No change observed                              
ATCC 10778                                                                
Bacillus subtilus                                                         
                 g.sup.+  No change observed                              
Candida lipolytica                                                        
                 g.sup.+  No change observed                              
Endomyces fibriliger                                                      
                 g.sup.+  No change observed                              
ATCC 9947                                                                 
Hansenula anomala                                                         
                 g.sup.+  No change observed                              
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     Thus, after addition of the sodium hydroxide the viscosity of those suspensions containing the Gram-negative cells increased to such an extent that gel formation occurred whereas there was no observed increase in viscosity of those suspensions containing the Gram-positive cells. While the invention is applicable to any Gram-negative cells including Xanthomonas which produces extracellular gums, the invention is particularly applicable to use with Gram-negative cells which produce no extracellular material such as the four listed above. 
     EXAMPLE III 
     Xanthomonas Culture Growth 
     A lyophilized culture of Xanthomonas campestris NRRL B 1459 was received from Northern Regional Research Laboratories of the U.S. Dept. of Agriculture. The culture was grown in a nutrient broth and streaked on plates to determine purity. Gram stains showed the culture was pure. Transfers were made to YM medium in flasks. Composition of the YM medium was as follows: 
     
         ______________________________________                                    
                   g/l                                                    
______________________________________                                    
Yeast extract        3                                                    
Malt extract         3                                                    
Peptone              5                                                    
Dextrose             10                                                   
Distilled water      1000 ml                                              
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     The Xanthomonas culture was a Gram negative small rod. The Xanthomonas Campestris B 1459 was inoculated into the YM medium (500 cc medium/1 l. flask) and shaken at room temperature on a rotary shaker. After 5 days abundant growth had occurred and the culture medium had a viscous appearance. The contents of this flask were used to prepare the test solutions for viscosity measurements. The contents of the flask was used in the following scheme with viscosity measurements being made with a Brookfield Synchro-lectric Viscometer Model C at room temperature. The viscosity was measured at 0.6 RPM allowing 30 min. to reach equilibrium. Aqueous sodium hydroxide (10 N) was used to increase the pH in the runs indicated. 
     
                                           Table III                               
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                                Brookfield                                
                                Viscosity                                 
                                0.6 RPM                                   
Culture Xanthomonas Campestris B 1459                                     
                                Room Temp.                                
effluent containing cells and extracellular gum                           
                            pH  Viscosity                                 
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Sample                                                                    
1   As received from flask, 6.85                                          
                                35.5                                      
2   Sample 1 treated with sodium hydroxide,                               
                            9.1 36.5                                      
3   Supernatant liquid from Sample 1 centrifuged                          
    to remove cells         6.95                                          
                                36.0                                      
4   Sample 3 treated with sodium hydroxide                                
                            9.1 33.5                                      
5   Cells removed from sample 1 by centrifuge and                         
    washed to remove gums and then resuspended                            
    in distilled water to original volume.                                
                            6.85                                          
                                1.5                                       
6   Sample 5 cells treated with sodium hydroxide                          
                            9.1 30.0                                      
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 Supernatant liquid from the centrifuged sample (Sample 3) was treated wit
 acetone (100 cc supernatant + 200 cc acetone) which caused gum           
 precipitation. The gum was removed by twisting on a spatula and freeing  
 from acetone. The isolated gum was then resuspended in 100 cc of deionize
 water and viscosity measured.                                            
 
    
     
         Sample                      pH  Viscosity                                 
7   Isolated gum solution.  6.95                                          
                                135                                       
8   Sample 7 treated with sodium hydroxide                                
                            9.1  122                                      
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 The response of Gram positive cells was measured by taking cell effluent 
 from a fermenter Run composed of Gram positive rods. The cells were      
 recovered by centrifugation and resuspended in distilled water. The sampl
 was divided and the pH increased in one of the solutions by addition of  
 sodium hydroxide.                                                        
 
    
     
                                         Brookfield                                
                                Viscometer                                
                                6 RPM                                     
Bacillus gram positive cells                                              
                            pH  Viscosity                                 
__________________________________________________________________________
Sample                                                                    
9   Cells centrifuged and resuspended in distilled                        
    water                   6.8 2.5                                       
10  Sample 9 treated with hydroxide                                       
                            9.1 2.5                                       
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 Also tested were other Gram-positive cells (Hansenula species Y 5939).   
 These cells were treated the same way as above.                          
 
    
     
                                         Brookfield                                
                                6 RPM                                     
Hansenula gram positive cells                                             
                            pH  Viscosity                                 
__________________________________________________________________________
Sample                                                                    
11  Cells centrifuged and resuspended in                                  
    distilled water         4.0 6.                                        
12  Sample 11 treated with hydroxide                                      
                            9.1 3.5                                       
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 These data demonstrate that these gram positive cells show no increase in
 viscosity when the pH is raised and that it is the gram negative cells,  
 not the gum, which is responsible for the increase in viscosity from     
 adding a base to fermenter effluent containing gram negative cells.