Separating yeast and/or bacterial cells from a liquid medium
United States Patent Kanani et al.  
 [451 Apr. 15, 1975 SEPARATING YEAST AND/OR BACTERIAL CELLS FROM A LIQUID MEDIUM Inventors: Diamond Pirbhai Kanani; Peter John Sherrington; David Charles Steer, all of Stockton-on-Tees, England Imperial Chemical Industries Limited, London, England Filed: Feb. 23, 1973 Appl. No.2 335,109  
 Assignee:  
 Foreign Application Priority Data Mar. 3, l972 United Kingdom 9981/72 U.S. Cl 210/51; 210/56; 195/28 R;  
  195/104; 260/112 R; 424/204 Int. Cl. A23j 1/00 Field of Search 195/28 R, 104; 260/112 R; 424/204 [56] References Cited UNITED STATES PATENTS 3,781,264 12/1973 Akin 260/l 12 R Primary ExaminerLionel M. Shapiro Attorney, Agent, or FirmCushman. Darby &amp; C ushman [57] ABSTRACT 9 Claims, No Drawings SEPARATING YEAST AND/OR BACTERIAL CELLS FROM A LIQUID MEDIUM This invention relates to a method for separating bacterial cells from a liquid medium containing them.  
  In a process for producing a protein composition by growing bacteria upon an aqueous nutrient medium comprising an assimilable carbon source, the initial product is an aqueous suspension comprising bacterial cells.  
  Various solid-liquid separation techniques may be used to separate the bacterial cells from the suspension. For example, in some cases the suspension may be flocculated by addition of a flocculating agent, and the flocculated cells allowed to separate from the liquid in settling tanks. After settling the cells may be further concentrated by centrifugation and then dried to give, as a final product, dried bacterial cells.  
  In many cases the rate of settling of flocculated cells achievable by the separation described above is extremely slow. This is because the flocs obtained tend to be small and loose, i.e. the amount of liquid medium retained within the flocs is high and the difference between the densities of the flocs and the medium is small. Also the strength of the flocs is insufficient to permit separation by other separation techniques such as filtration or flotation and their behavior during subsequent centrifugation shows little improvement compared with the original suspension. In practice therefore the only advantage attainable by such a separation process in many cases is the removal of a proportion of the medium by settling and, in view of the low settling rates which can be achieved, it is difficult to make this stage of the separation method economical when operated upon a large scale.  
  According to the present invention we provide a method for separating bacterial cells from an aqueous medium wherein the cells are flocculated, by subjecting the medium to at least one of the steps of:  
 A. raising the pH of the medium to a value within the range 8 to 11 by treatment with an alkali, and  
 B. heating the medium to a temperature within the range 50 to 200C; followed by the step of lowering the pH to a value within the range of 2 to by treatment with an acid, and the flocculated cells are separated from the medium.  
  It is believed that steps (A) and (B) cause structural changes to occur in the walls of the bacterial cells which enable the cells to be flocculated by the method of the invention. The method is very suitable for separating bacterial cells of strains of the genera Pseudomonas, Alcaligenes, Arthrobacter, and Bacillus, for example strains of the species Pseudomonas fluorescens, Pseudomonas aeroginosa, Pseudomonas diminuta, Alcaligenes faecalis, Bacillus cereus and, particularly, Pseudomonas methylotropha (a species whose characteristics are described in our co-pending UK. Applications Nos. 58466/70 and 38938/71 corresponding to U.S. application Ser. No. 203061 now, abandoned in favor of a continuation thereof Ser. No. 416,098, and French Specification No. 2,1 17,530). Cultures of a number of strains of this last species have been deposited in the National Collection of Industrial Bacteria, Torrey Research Station, Aberdeen, Scotland, U.K. and corresponding deposits have been made in the collections of the U.S. Department of Agriculture at Peoria, Ill. and  
 the Fermentation Research Institute, Japan. The numerical designations given to these cultures are as follows:  
 NCIB NOS. 10508-15 and 10592-6 NRRL NOS. B. 5352-64 FRI NOS.- 1215-27 The method of the invention is particularly suitable for separating cells of these strains and natural or artificially derived mutants thereof from aqueous media.  
  In the method of the invention the medium may be subjected to either one of steps (A) and (B) without the other or it may be subjected to both steps. In this last case it is preferred that step (A) precedes step (B). If only one of steps (A) and (B) is to be performed it is preferably (B).  
  Whilst the method is applicable generally to separation of bacterial cells from suspension in liquid media and can be applied as a batch or continuous process, it  
 is particularly suitable for the continuous separation of bacterial cells as a step in a biomass (single cell protein) production process, e.g. in a process such as that of co-pending U.K. Application Nos. 58466/ and 3893 8/7 1. When used as a step in a continuous biomass production process, the method may conveniently be operated by passing the initial product of the process, whose pH will usually be about 6.8, through a system comprising in sequence, means for adding an alkali, a heat exchanger for raising the temperature, a cooler and means for adding acid, into a stirred tank. It is not necessary for all the elements of the system to be operational during any particular separation.  
  When alkali treatment step (A) is employed, the pH is preferably raised to a value within the range 8 to 9, for example 8.5. If heating step (B) is omitted a higher pH is preferred. The period during which the medium is maintained at an alkaline pH before treatment with acid may vary over a wide range but is preferably up to 30 mins especially 0.5 to 10 mins. The period may be very short, e.g. in the system described aboveit will be the time taken for medium to proceed from the alkali addition to the acid addition stage. For pH values within the range 8 to 9, the preferred alkali is ammonia in aqueous solution, e.g. a 1:6 NH OH:H O solution. For pH values in the range 9 to 1 1, sodium hydroxide is preferred. Other alkaline compounds which may be used include the hydroxides of potassium and calcium. During heat treatment step (B) the temperature of the medium is preferably raised to 70 to 90C, in a continuous process to C is especially suitable. The period during which the medium is maintained at a higher temperature before acid treatment may vary over a wide range but is preferably up to 30 minutes, especially 0.5 to 5 minutes. The period may be very short, the medium being subjected to flash heating, i.e. being rapidly raised to the required temperature on passing through a heat exchanger and being maintained at that temperature for a very short period, e.g. less than 0.5 minutes. In the continuous system described above a number of alternative paths may be provided for the medium after it leaves the heat exchanger. It may pass directly to the acid treatment stage or may take other more circuitous routes which result in the medium being maintained at a higher temperature for selected periods e.g. l, 2 or 3 minutes. When the medium is delayed in this way during its passage between the heat treatment and acid treatment steps the tem- 3 4 perature will fall to some extent after leaving the heat litre samples (A to F) and the pH of 5 of these (A and exchanger e.g. from 80 to 73C. C to F) was raised to 8.5 by addition of aqueous ammo- After heat treatment the&#39;medium may be cooled benia. Samples B to F were heated to selected temperafore treatment with acid. This is preferred if the floccutures at which they were maintained for selected perilated cells are eventually to be separated by settling but 5 ods, being thereafter cooled to 22C. The pH of all not if a flotation method is to be used. If a cooling step samples was reduced to 3.5 by addition of sulphuric is included the medium is preferably cooled to ambient acid causing flocculation of the cells. After acid treattemperature. However temperatures within the broad ment the samples were transferred to 1 litre graduated range 20 to 50C, particularly 20 to 30C are suitable. cylinders in such a way as to ensure thorough mixing.  
  During the acid treatment step the pH is preferably The initial rate of descent of the interface between the reduced to a value within the range 2.5 to 4.5 to cause flocculated material and the supernatant liquor was the bacterial cells to separate from the medium in large timed and the concentration of the material accumuflocs. Especially suitable pH ranges are 3 to 3.5 if the lated at the bottom of the cylinders after 16 hrs. was  
 flocculated cells are eventually to be separated by setmeasured. tling and 3.5 to 4.5 if they are eventually to be sepa- The results are shown in Table 1. rated by flotation. The acid used may suitably by Sul- In small scale tests the reproducibility of settling rates phuric, hydrochloric or phosphoric acid or an acid gas is reduced due to differences in speciflc gravities of culsuch as carbon dioxide or sulphur dioxide. When the sure media and to production f gas bubbles which method forms a p in a biomass Prod&#34;ction Process duce the rate of settling of flocs. Thus in some cases the the acid is Preferably a Phosphoric/Sulphuric acid 20 settling rate gives an inadequate indication of the exture of suitable proportions to enable the medium, after tent f fl l ti Th f in Examples 1 and 2 a Separation of the flocculated cells to be recirculated to qualitative description of the extent of flocculation has the fermentation stage of the process. After treatment also been adopted based upon visual appraisal of the wlth acld the medium ls preferably surfed whlle the size and strength of the flocs formed. In Tables 1 and flocs develop i 2 the extent of flocculation is indicated by the following The method of the invention enables large, strong Symbols flocs to be produced and a good rate of settling to be achieved. The flocs produced may vary in size over a 0 9 wide range depending upon the rate of stirring and Shght flocculatlon upon the strain of bacteria, for example the diameters Extensive Production mainly Small, light flocs may be in the range 0.1 mm to 1.0 cm. i with SatisfactPW Settling P p The acid treatment step may be followed by the steps Extensive productlon of g strong fl of settling or flotation, centrifugation, filtration or with very good settling properties. evaporation and drying to obtain dried cells as the final N.B. With sample A step (B) was omitted whilst. product of a fermentation process. Use of the method with sample B step (A) was omitted. Samples C to enables the size of the settling tanks and the number of F were subjected to both steps (A) and (B).  
 TABLE 1 Temp. after Time at lnitial Final concn. Degree of Sample heating(C) temp. (mins) Settling rate after 16 hr flocculation A 2 4 B 70 5 93 48 -ll- C 70 2 372 84 -Hl- D 70 5 392 -Hl- E 85 2 450 52 -l-H- F 85 5 354 -H+ centrifuges employed in separating the bacterial cells EXAMPLE 2 produced in a fermentation process to be reduced.  
 &#39; During the production of bacterial cellsby fermentamining approximately 2.5 g dry weight of Pseudomo tlon processes, a proportion of the protein content of &#34;as fluorescens Strain NCIB 9046 Pseudomonas cells Pmduced Passes 801mm&#34; T diminuta strain NCIB 9393, Bacillus cereus strain NClB dlum. When the cells are separated from the medium b onv mional flocculation techni ues the rotein in 55 9373 and Acalzgenes faecalzs strain NClB 8156 were y c 6 q p taken from batch cultures. In each case four 500 ml solution passes into the supernatant liquor in the set- 1 A D k d d f H tling tank and is lost. Using the method of the invention Samp es )were ta en an were treate as 0 A No treatment f th rotein in the medium is rendered insolusome 0 e p B pH adjusted to 3.5 using HCl.  
 ble by the acid treatment step and is recovered. 60  
 The invention is illustrated by the following Exam- C P adlusted 9&#34; H solutlon and after 15 minutes ad usted to 3.5 using HCl.  
  les: p D pH adjusted to 8.5 using NaOH solution, sample EXAMPLE I heated to C for l min. and then cooled to ambi- Six litres of a suspension containing 8g/l dry weight em temperature and pH adjusted to 3.5 using HC]. of Pseudomonas methylotropha str. NCIB 10593 were The initial settling rates and degree of flocculation taken from a carbon limited steady state continuous measured as described in Example 1 are given in Table culture. The suspension was divided into six separate 1 2.  
 In separate experiments samples of suspensions conv 2 Depending upon the pH and heat treatment the flocs U I I took various forms. The following types were identiinitial Degree of pH of medium fled: (i) very small, (ii) fine asbestos-fibre like, (iii) Bacterium Sample Settling flocculaon initial Rate (cm/hr) on I Sampling 5 larger fibrous, (iv) slightly granular, (v) granular, (vi) I gel balls. P d is 2 3) Under certain conditions, the flocs were formed in a 6232227 C so&#39;me 74 more gel-like state but still maintaining their shape. flotation With poor mixing in the stirred tank, gel balls and flocs E 3 were formed. Increased stirring gave full flocculation pseudomnnas B 10 whilst excessive stirring broke the flocs down. diminula C 0 7.9  
  D som ldeal flocs are large and have a high settling rate (rates in excess of 3000 ems/hr have been obtained). A o 0 Bacillus B O 0 The most ideal flocs were (-iii), (iv) and (v) above. Fine Cereus C 150 5 flocs tended to hang in the supernatant and gave rela- R g tively slow settling whilst gel balls consisted of unfloc- Alcali&#39;geries B 70 culated culture and&#39;were thus not ideal. Faemlis C 100 Examples of the results obtained under various con- D 110 -l-lditions are given in Table 3.  
 TABLE 3 Dry wt. of pH after Temp. Residence pH after Temp. in bacteria in alkali after 1 time at Cooling acid stirred medium addition heating l elevated water addition tank Comments (g/ (C) l temp. (mins) 2.4 no 0 off 3.5 25 no flocculation addition i l0.4 8.4 81.5 3 on 3.3 41 large flocs l good settling 4.75 no 50 0 on 3.5 not fine flocs addition measured good settling 4.75 no 60 l 0 on 3.5 not granular flocs. addition measured Clear supernatant 6.5 8.7 85 1 3 on 3.4 32 Flocs and gel balls quick 5 settling 6.5 8.7 85 l 0 on 3.4 32 Flocs and gel balls quick settling 6.0 8.5 80 3 on 3.5 32 Large gel flocs clear supernatant 6.0 8.5 75 O on 3.0 32.5 Large flocs quick settling EXAMPLE 3 EXAMPLE 4 A number of suspensions containing a Pseudomonas Example 2 was repeated using 4 X 200 ml samples of methylotropha strain were flocculated under differing a suspension containing approximately 8g/l dry weight conditions in a continuous flocculation apparatus. In of Pseudomonas aeroginosa strain NCIB 950 taken the apparatus medium was pumped from a feed tank from a continuous culture. The pH at the time of saminto a stirred tank, through a system comprising: pling was 7.1. The results are given in Table 4.  
 a. An alkali introduction point.  
 b. A heat exchanger wherein the tube carrying the TABLE 4 medium passed through an oil bath.  
 c. A system comprising several flow paths which en- Sample lnitial Settling Degree of abled the medium to be maintained at an elevated cm/h&#39; mcculam&#34; temperature for various periods viz. a very short A 0 0 period (zero residence), 1 min., 2 mins and 3 mins. 3 2 2 d. A cooler D 110 e. An acid introduction point.  
 Means were provided for measuring pH after alkali and acid addition and for measuring the temperature of the medium both on leaving the heat exchanger and after EXAMPLE 5 acid addition. addition was made introducing Four m] amples of a uspension containing apa NH4OHIH2O Solution into the System Acid addiproximately 5 g/l dry weight of Arthrobacter Nov. strain tion was made by introducing a 1:1 mixture of 0.5 N NRRL 33728 w r taken from batch culture. The pH 2 4 and N H3PO4 Solutions into the system at the time of sampling was 6.9. The four samples were The extent and nature of flocculation and the clarity of the supernatant liquor achieved by the method were estimated visually.  
 treated as follows:  
 A No treatment B Adjust to pH 3.5 using hydrochloric acid.  
 C Adjust to pH 10 using sodium hydroxide solution, heat to 85C for 5 minutes and adjust to pH 3.5 with hydrochloric acid.  
 The results are given in Table 5.  
 TABLE 5 Sample Initial Settling &#39;Degree of Rate cm/hr Flocculation A 0 O B 0 0 C some setting -H- We claim:  
  3. A method according to claim 1 wherein the bacterial cells belong to a strain of a species selected from the group consisting of Pseudomonas fluorescens, Pseudomonas diminuta, Alcalig enes faecalis. Pseudomonas Aeroginosa, Bacillus cereus, Pseudomonas methylotropha and Arthrobacter Nov NRRL B 3728.  
  4. A method according to claim 3 wherein the strain is selected from the group consisting of strains NRRL B. 5352 to 5364 and naturally and artifically derived mutants of these strains.  
  5. A method according to claim 2 wherein the pH is raised to a value within the range 8 to 9.  
  6. A method according to claim 1 wherein said heating is at a temperature within the range to C.  
  7. A method according to claim 1 wherein after said heating the medium is cooled to a temperature within the range 20 to 30C before being treated with the acid.  
  8. A method according to claim 1 wherein during the acid treatment step the pH is reduced to a value within the range 2.5 to 4.5.  
 9. A method according to claim 1 which forms a step in a process for the continuous production of biomass.