Spray Drying of Serratia marcescens in the Jet Spray Dryer E. W. Comings,* Harry Higa, J. E. Myers, Henry Koffler, and H. A. McLain Department of Chemical Engineering, University of Petroleum and Minerals, Dhahran, Saudi Arabia
The jet spray dryer was used to dry suspensions of Serratia marcescens, a bacterium, confirming the ability of its unique control of particle time of contact with air to handle a highly heat-sensitive material in high air temperatures. Recoveries of viable cells ranged from 1.8 to 9 1.8 % . For more than half of the drying runs the viable recovery was between 40 and 60 % which compares favorably with acceptable treatments. The moisture content of the dried product was between 4 and 8 wt % . The particle size of the dried product, as measured in one experiment, had a mass mean diameter of 5.1 p with a geometric mean deviation of 1.6. Half-life values for the dried organisms stored under various conditions were a maximum of 245 days for bacteria stored under partial vacuum (pressure of 0.05 mmHg) at -30 OC.
The design and operation of the jet spray dryer are described by Comings and Coldren (1957),McLain et al. (1957), Bradford and Briggs (1963),and Comings e t al. (1960). Measurements were reported from a large number of drying runs in which the feed stock was an aqueous solution containing 5 wt % sodium sulfate. Two runs were also reported in which skim milk was dried. The drying of whole milk is described by Bradford and Briggs (1963). Other animal products have been successfully dried such as blood. The present paper describes the use of the jet-spray dryer with an aqueous suspension of Serratia marcescens as feed stock. The dryer is a device in which the stream of aqueous suspension is atomized as it issues from a nozzle into a stream of cocurrent air moving a t near-sonic velocity. The atomizing or primary air, having a temperature higher than that of the liquid droplets, serves the additional function of providing heat for the vaporization of moisture in the droplets. A secondary, slower-moving, low temperature, cocurrent stream of air is supplied in an annular space around the primary air and serves mainly to control the rate and symmetry of radial expansion of the primary jet and to prevent turbulent recirculation of the droplets from the jet. Control of the temperatures and the rates of flow of the three streams limits the time of contact and path of flow of each particle and the “wet bulb” mechanism then functions to hold the particle temperature to low values. The jet spray dryer possesses the inherent characteristic that successful drying is accomplished if the liquid is atomized into a very fine spray in the nozzle. This results in a dried product of even smaller size, depending on the concentration of solids in the original feed. For example, a spherical droplet containing 3% solids (by volume), with an initial diameter of 20 p , may be shown by calculations to have a diameter of 6.2 after complete drying. If the drop size were increased, the volume of water to be evaporated per drop would increase by a third power relation, necessitating a similar exponential increase in the length of the apparatus essential for carrying out this cocurrent process. The drying of living cells without interfering with their viability (i.e., their ability to reproduce on a suitable medium) is complicated by the fact that biological material is devitalized by heat. Drying of cells therefore must be a t relatively low temperatures and as rapid as possible. The success with which the jet spray dryer drys living cells is an index of its ability to deliver an unaltered product of heat-sensitive material. The rate of drying is dependent on three factors: (1)the coefficient of convective heat transfer, (2) the surface area through which heat is transferred, and (3) the temperature difference between the surrounding air and the entrained 12
Ind. Eng. Chem., Fundam., Vol. 16,No. 1, 1977
droplet. All three quantities are large in the jet spray dryer. High heat-transfer coefficients are obtained because of the extremely turbulent state of the atomizing primary air jet; large surface areas per unit volume of feed are produced because of the small size of the droplets formed in the nozzle; and temperature differences of several hundred degrees may be obtained by preheating the primary air. The combination of these three factors produces drying times as small as several milliseconds. A particle must a t no time have an excessive temperature. Throughout the early part of the drying process while moisture is evaporating from its surface, the particle remains a t its “wet-bulb” temperature which is low. During some of this time the temperature of the surrounding air stream may be several hundred degrees higher than the drop temperature. By the time the particle has become dry the protection of evaporative cooling is no longer available, and the particle temperature may rise to that of surrounding air. By a proper choice of the mass ratio of hot primary air to liquid feed, the air by this time will have been cooled by the evaporation from the droplets to a temperature not much higher than that of the drying particle. An additional protective factor is the diluting and cooling effect of the secondary air, supplied a t a little above room temperature, which mixes with the primary jet and suspended droplets after the initial rapid atomizing and drying period.
Material and Methods Apparatus. The spray drying apparatus is described by McLain et al. (1957). Test Organism. Freeze-dried or lyophilized cells of Serratia marcescens (The original lyophilized culture was received from Fort Detrick, Frederick, Md.), which were suspended in 2% dextrin and 1%neutralized ascorbic acid prior to jet-spray drying, were maintained as stock cultures a t -20 “C. T o prepare cultures for use, the stock material was streaked on plates of nutrient agar, and after incubation a t 30 “C for 48 h selected colonies were transferred to nutrient agar slants, and again incubated a t 30 “C for 48 hours. These slants were kept at 4 “C for a period not exceeding 3 weeks, and were used for inoculation purposes. Growth of Cells. The feed stock consisted of a suspension of S. marcescens in 2% dextrin and 1%neutralized ascorbic acid. The organisms were grown successively in two tryptose broth media termed Tryptose Broth A and Tryptose Broth B. The former medium was used to obtain the inoculum for the latter which was employed in larger quantities for the production of S. marcescens. The compositions of the media are listed in Table I.
Table I Tryptose Broth A
-
Tryptose Broth B
?6 (wt/v)
Bacto-tryptose Cerelose Tap water
%
2
Bacto-tryptose
2
(wt/v)
1
Cerelose (sterilized separately) 0.03 M NaH2PO1 0.03 M NaiHPOd Dow-Corning antifoam Tap water
1
(wt/v)
5 5 0.6
(v/v) (v/v) (v/v)
The Tryptose Broth A medium was dispensed in 500-ml Erlenmeyer flasks so that each flask contained 150 ml of broth. The flasks were then autoclaved a t 15 psig for 20 min. Tryptose Broth B medium, contained in a 19-1.carboy, was made in the following manner: 240 g of Bacto-tryptose and 600 ml each of 0.03 M solutions of NaH2P04 and Na2HP04 were mixed in the carboy and made up to 10.75 1. with tap water. In this case, steam sterilization was allowed to proceed for 1.5 h. In all other cases the sterilization time was 20 min. Subsequently, 493 ml of 24.3% cerelose solution (sterilized and cooled) and 7.0 ml of sterile Dow-Corning antifoam A emulsion were added to the buffered tryptose solution which had been cooled to room temperature. The inoculum was started by transferring the growth of a 24-h nutrient-agar-slant cultur of S. marcescens into 150 ml of sterile Tryptose Broth A. The inoculated medium was agitated on a reciprocating shaking machine (3-in. stroke, 90 cycledmin) at 25 "C for 12 h. Subsequently, aliquots of this culture were used to inoculate flasks containing Tryptose Broth A. (The inoculum was 6% of the final culture volume.) These cultures were allowed to grow for 4 h under the conditions mentioned before. The 4-h cultures were used to inoculate the prepared Tryptose Broth B medium (the inoculum was 6% of the final volume). Three 19-1. carboys giving a total culture volume of 36 1. were employed a t one time. The cultures were grown a t 30 "C for 20 h while being stirred (1725 rpm) and aerated (15 1. of air/min). Preparation of the Feed Suspension. The 20-h cultures were centrifuged in a Sharples Super Centrifuge (rotor size: 1&-in.i.d. X 10 in. The Sharples Corp., Philadelphia, Pa.) and the bacterial paste obtained from 36 1. of culture was suspended in 4 1. of medium containing 360 g of dextrin and 180 g of ascorbic acid which had been adjusted to pH 7.0 with 50% NaOH. The resulting suspension was thoroughly mixed and filtered through a pad of cheese cloth into a 19-1. carboy. The filtrate was made up to 18 1. with tap water; the concentration of dextrin and neutralized ascorbic acid thus was 2 and 1%, respectively. This final mixture constituted the feed suspension employed in spray drying. Product Analyses. The spray-dried product was analyzed for the following. ( I ) Total or direct count of the bacterial cells per gram of spray-dried product. One hundred milligrams of dried product was suspended in 10 ml of a 1%solution of peptone. The count was obtained by the use of the Petroff-Hausser counting chamber (Kolmer and Boener, 1945). (2) Viable number of bacterial cells per gram of spray-dried product. The viable counts were obtained by the pour-plate method. An unseeded hase layer (Gratia, 1936; Schmidt and Moyes, 1944; Snyder, 1947) of Wilson's peptone agar (Thomas e t al., 1954) was employed in order to prevent the occurrence of bottom-spreading colonies. At least three plates containing between 100 to 300 colonies were selected for the purpose of
counting. The results obtained were expressed as number of viable cells per milliliter of sample. One hundred milligrams of the spray dried product was reconstituted in 10 ml of an ice cold 1%solution of peptone. I t was therefore possible to express the counts obtained as the number of viable cells per gram of spray-dried product. The percent viability of the organisms was obtained by determining the ratio of viable count to total count and multiplying this value by 100. The viable recovery of the spray-dried organisms was found by dividing the percent viability of the spray-dried product by the percent viability of the feed suspension which was sampled adjacent to the rotameter in the feed line of the spray dryer. (3) Moisture content of the spray-dried product was expressed as percent. The moisture content was determined by the method of Flosdorf and Webster (1937). The difference in weights before and after desiccation of the sample was assumed to be due to the loss of moisture contained in the spray-dried product. The moisture determination of a given sample was done in duplicate and the difference in values obtained did not exceed 0.3%.
Experimental Section Spray Drying Results. The results of the spray-drying runs are reported in Table 11. The primary air rate for most experiments was approximately 500 lb/h, and the secondary air rate approximately 5000 lb/h. Primary air temperatures between 400 and 700 O F were employed, while the range of secondary air temperature was 75 to 150 O F . Feed rates were from 2.5 to 5.0 gal/h. Samples of dried cells were taken by inserting probes at two sampling points, one point (I) being a t the base of the drying chamber and the other (IT) at the inlet to the collection system. At each point the air with the entrained particles passed through a 1-in. diameter glass cyclone. The particles were collected in a test tube attached to the base of each cyclone and analyzed for moisture content and percent viability. Because of the greater exposure time of the material collected a t the downstream sampling point, it might be expected that the sample collected there would have a slightly lower moisture content and viability. However, no such relation was observed and the minor differences noticed in the analytical results were apparently due to experimental variations. Therefore, the arithmetic averages of the measured product properties for each run are reported in Table 11. The viable recovery of dried organisms reported in Table I1 ranges from 1.8 to 91.8%. Because visual and graphical inspection of these results indicated no definite trends, the data were subjected to a statistical multiple regression analysis of nonlinear form. Three measured variables were chosen initially: the feed rate (XI), the primary air temperature ( X ? ) and , the secondary air temperature (X{). The dependent variable, percent viable recovery in the Cyclone I sample, was designated X,I. A mathematical model of the form:
was hypothesized where XO was the predicted percent viable recovery and the b's were coefficients determined by the method of least squares, Le., determined so that the sum of the squares of the errors of estimate in Xi,was a minimum for this surface. By feeding the observations X,,, X I , Xz,and X : j onto a tape ahead of the regression program on an Electrodata digital computor, the equation of "best fit" for the measured and independent variables was determined as well as R 2 (the coefficient of determination) which indicated the extent to Ind. Eng. Chem., Fundam., Vol. 16, No. 1, 1977
13
which a “good fit” had been obtained or the proportion of ao2 explained by the equation. For the data given
Xo = 583.50666 + 55.73586Xi - 1.12270X2 - 6.71646X3 + 6.48815X1’ i- 0.00068X22 + 0.02892X72 - 0.05828X1X2
- 0.67298Xixj i- 0.00586X2X:j
which predicts the percent viable recovery (X,) from the three measured variables. The above equation accounts for only about 20% of the variance in the percent viable recovery [R2 = 0.19571, and a large portion of the variance is probably due to other than the three measured variables selected, as well as to random errors. This equation applied to a few experiments, including some with extremes of observed viability, gave the predicted values which are compared to observed values as listed in Table 111. The narrow range of the predicted values is an indication that the best prediction is the mean percent recovery; such a value would be the best prediction if the selected variables considered had no effect on the percent viable recovery. Such a conclusion is also indicated by the low coefficient of determination.
A similar analysis was made in which six independent viables were selected (feed rate, primary air temperature, secondary air temperature, exit air temperature, feed temperature, and product moisture content). This led to an equation accounting for only 13% of the observed variance in viable recovery. I t is significant that approximately half of the experiments gave viable recoveries lying between 40 and 60%. About 1hof the runs yielded greater viability and a fourth lesser viability. The arithmetic average viable recovery for all runs was 48%. The statistical analysis indicates incomplete understanding of the factors which control product viability. Whether these include the procedure for collecting, handling and analyzing the samples, factors within the dryer, or variables in the preparation of the feed is not clear. The statistical analysis, however, supports the conclusion that the variables controlling the operation of the dryer including a primary air temperature of 700 O F were chosen so that within the range of variation used they do not adversely effect the product viability. This is supported by inspection of the data for individual runs, which clearly shows that the jet spray dryer operated successfully and produced a product of high viability. In those
Table 11. Summary of Drying Results Primary air Run no.
Temp, “F
34 38 40 41 42 45 49 50 54 56 59 60 61 62 66 67 70 71 80 81 86 87 92 93 114 115 116 117 118 119 120
500 500 503 500 501 502 500 400 400 500 399 400 400 400 588 590 579 577 562 559 401 402 565 564 500 593 644 651 652 640 693 695 702 703 703 702 400 498 501 506 400
121
122 123 124 125 126 127 128 129 132 134 14
718
Velocity at nozzle throat ft/s
Secondary air Rate, lb/h
Temp,
1386 1383 1387 1274
537 513 503
1212
459 451 436 424 484 487 521 523 522 519 452 456 479 489 490 490 511 508 502 501 506 473 459 463 463 508 482 477 491 494 498 498 535 494 490 488 525 502
126 108 126 127 150 110 125 150 126 125 125 153 149 127 101 101
1277 1188 1166 1144 1322 124d 1245 1250 1230 1398 1363 1401 1410 1389 1390 1227 1217 1373 1375 1332 1418 1446 1443 1447 1528 1456 1455 1448 1445 1434 1431 1233 1269 1269 1264 1212 1448
Ind. Eng. Chem., Fundam., Vol. 16, No. 1, 1977
500
O F
100
101 100 86 125 153 125 125 100 86 83 107 97 88 105 107 78 77 77 76 127 129 127 125 125 79
Rate, lblh 5280 5290 5520 5770 4830 5390 5420 5180 5410 5210 5130 4930 5160 4020 4820 3640 4890 4800 5200 4860 5070 4790 5030 4860 5300 5620 5440 5050 4960 5660 5170 5190 5760 3950 5750 4250 5680 5650 5670 5010 5810 4570
Feed Total cells/ml
x 109 82.4 104.8 51.6 68.4 81.2
100.6 53.6 89.5 48.1 55.3 58.8 59.5 44.2 44.2 64.6 64.6 62.5 62.5 53.8 53.8 52.2 52.2 63.4 63.4 66.6 53.5 47.0 49.4 49.4 84.0
82.4 82.4 79.8 79.8 76.6 79.6 32.8 89.0 69.3 69.3 81.2 38.4
Viable cellslml x 10s’ 60.0 98.6 24.0 48.0 74.5 81.0 42.8 73.3 39.0 28.3 28.8 28.8 31.5 31.5 54.2 54.2 51.7 51.7 42.8 42.8 43.7 43.7 48.6 48.6 51.3 43.5 38.6 37.9 37.9 67.5 69.0 69.0 63.0 63.0 65.0 65.0 16.6 57.3 49.6 49.6 64.5 23.0
Table I1 (Continued) Feed
Run no. 34
38 40 41 42 45 49 60
54 56 59 60 61 62 66 67
70 71 80 81
86 87 92 93 114 115 116 117 118 119 120 121 122
123 124 125 126 127 128 129 132 134
Product
Total solids, wt. %
Rate, gallh
Heat added to air Btullb of water evaporated
2.43 3.10 2.37 3.04 3.30 3.20 2.75 2.94 2.77 2.90 2.94 2.94 2.89 2.89 3.16 3.16 2.37 2.37 3.41 3.41 3.74 3.74 3.61 3.61 3.96 6.25 3.10 4.32 4.32 6.02 3.93 3.93 3.02 3.02 3.43 3.43 2.93 3.24 3.12 3.12 3.23 3.10
3.00 3.00 3.00 3.00 5.00 4.00 5.00 3.00 3.00 2.50 2.50 2.50 4.00 4.00 3.00 4.00 2.50 5.00 3.00 3.00 5.00 4.57 5.00 4.00 2.50 2.50 2.50 2.50 5.00 5.00 3.00 4.00 3.00 4.00 3.00 4.00 2.50 3.00 3.00 3.00 2.50 5.00
4840 3810 4630 4730 3300 2670 2540 4290 3680 5480 4420 5520 3510 2270 2610 2020 2980 1490 3040 2320 2010 2930 2520 3180 3290 3400 3320 4740 2090 2550 4230 3230 3570 2650 3530 2550 3650 5040 3940 4060 5020 2220
Outlet air temp, O F
141
122 125 139 140 103 117 139 123 142 134 155 142 126 123 118 129 105 124 109 104 144 137 141 122
107 109 128
103 100 138 131 119 115 123 124 144 151 144 148 144 112
Table 111 ~~
__
Obsd 90 viable recovery: X,I
Predicted % viable recovery: X ;
38 66
42.5
67
15.4 83.5 58.9
40.7 41.7 41.1 43.5 49.3
Run no.
116 124
6.0
runs where product viability was a t the low end of its range the statistical analysis implies that the cause more than likely was an undetermined factor rather than jet-spray dryer operating variables. Table I1 lists the moisture content of the dried samples. The arithmetic average of results obtained with samples collected a t Cyclone I (at the end of the drying chamber) was 6.1 wt %. The arithmetic average of results obtained with Cyclone I1 samples, taken approximately 10 ft farther down stream a t
Moisture, wt % 5.0 6.5 6.0
5.8 7.4 5.3 5.7 2.6 5.6 6.2 5.5 4.7 8.1 7.8 4.4 7.3 5.7 6.8 5.9 4.1 14.7 6.2 4.7 4.2 5.6 7.9 6.4 5.5 6.5 6.9
... ...
3.1 6.1 6.0 6.8 6.1
5.8 5.6 5.7 4.1 7.4
Viable recovery, %
85.8 46.6 54.6 91.2 55.7 60.4
51.0 31.8 61.1 50.5 55.7 65.5 52.2 54.6 7.2 22.4 26.8 35.3 43.3 17.8 1.83 30.5 52.0 50.8 17.6 44.1 80.2 48.3 53.6 84.3 56.7 55.4 50.4 58.2 58.2 50.5 48.0 69.5 85.5 81.5 44.3 29.8
Viable cells/g of dry product X 10" 1184 1350 440 853 980 845 " I
1
