Purchased by U.S. Department of Agriculture for Official Use.

BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIV, PAGES 23-31 (1972)

Continuous to Produce Xanthan Biopolymer: Effect of Dilution Rate*

R. W. SILlVIAN and P. ROGOVIN, N ol'thern Regional Research ,t Peoria, Illinois 61604

Summary

Single-stage continuous to produce xanthan gum have been run at dilution rates (D) from 0.023 to 0.196 hel . Xanthan production rate (XPR) was a function of D. XPR increased from 0.34 g/hr/kg at D = 0.023 he1 to the maximum 0.84 g/hr/kg at D = ca. 0.15 hr-1• At D > 0.15 hr-1 XPR de­ creased and at the highest D studied (0.196 hr-1) was 0.69 g/hr/kg. Yield of xanthan from glucose consumed was 81-89%. Steady states ended between 6.5 and 8.7 turnovers when a variant strain occurred.

INTRODUCTION This Laboratory previously reported a successful single-stage con­ tinuous fermentation to produce a biopolymer, xanthan, with Xantho­ monas campestris NRRL B-1459.1 The earlier work indicated that xanthan production rate (XPR) ,vas a function of pH and dilution rate (D). D studied in that report were 0.023 to 0.0285 hr-1• This report covers the effects of increasing D up to 0.196 hr-1 under similar condi­ tions (i.e., single-stage single-feed chemostat).

* Presented at the American Chemical Society, Division of Microbial Chemistry and Technology, \Vashington, D.C., September 12-17, 1971. t This is a laboratory of the Northern Marketing and Nutrition Research Division, Agricultural Research Service, U.S. Department of Agriculture. The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned. 23 © 1972 by John Wiley & Sons, Inc. 24 SILMAN AND ROGOVIN

EXPERIMENTAL Inoculum Viable cultures of X. campestris NRRL B-1459A were maintained on TGY slants,l as before, but the inoculum buildup scheme was altered slightly (Table I).

Fermentation Fermentations ·were conducted according to the system described previously.! The fermenter was an 8-liter glass and stainless steel .~ tank of conventional configuration constructed in the Northern Laboratory's shops.l There were four, evenly spaced baffles. Agita­ tion was provided by one, si.x-bladed, disk turbine impeller mounted

TABLE I Inoculum Buildup of Xanthomonas campestris NRRL B-1459A for an 8-Liter Fermenter

Less than 4-day-old TGY' slant culture I ..I 7 ml ::'IY bin 18 X 150 mm incubated 24 hr on rotary (170 rpm, 1 in. eccentricity) at a 20° angle at 28°C

:,1 ALL ...I :35 ml ::'IY in aOO-ml Erlenmeyer incubated 24 hr on rotary shaker (245 rpm, 2 in. eccentricity) at 28°C

1 ALL 200 ml :\IY in 1000-ml Erlenmeyer incubated 24 hr on rotary shaker (245 rpm, 2 in. eccentricity) at 28°C

ca. 160 ml (5-6% inoculum)

Fermenter

n TGY: 0.5 '10 tryptoue. 0.2 % glucose, 0.5 % yeast extract, 0.1 % E:,HPO" and 2.0% agar. h :\IY: 0.:3% malt extract, 0.:3% yeast extract, 1.0% glucose, and 0.5% peptone. BlOTECH':'OLOGY A':'D BIOE':'GI':'EERING, VOL. XIV, ISSUE 1 XANTHAN BIOPOLYMER BY CONTINUOUS FERMENTATION 25 on a centered vertical shaft. Sterile air (1 vIvImin) was introduced through a single-hole sparger directly below the agitator. Fermenter temperature was controlled at 28°C ± 1°C. Medium composition is given in Table II. The medium was con­ tinously sterilized in the pilot plant;2 20-liter carboys were filled aseptically from the sterile medium storage tank, and the medium was subsequently transferred to the 8-liter feed described previously.! About 3000 g of medium were transferred from the 8-liter feed bottle to ~he fermenter, inoculated, and allowed to incu­ bate under batch conditions until the latter part of the growth phase. Medium was fed at desired rates from the 8-liter feed bottle, which rested on a weighing scale. Feed rates ·were calculated from the weight change per time interval. Product was discharged contin­ uously from the fermenter as in previous ,York.!

Analytical Analytical procedures remained the same as in previous work.! Cell mass could not be measured gravimetrically because the medium had suspended solids; however, optical density (OD) measurements at 650 m,u served as an index of relative growth. Broth viscosities were measured with a Brookfield LVT rotational at 30 rpm. Total reducing sugars, calculated as D-glucose, were determined by the Shaffer-Hartmann method.3 Xanthan concentration was deter­ mined either by direct isolation of polymer with methanol from cell­ free broth2or by estimation from broth viscosity. Viable cell counts

TABLE II­ Medium Composition (PMU)

Ingredient g/lOO g

Brown-Forman DDS· 0.8 Urea 0.04 K,HPO, 0.5 Glucose 1.G-2.5 MgSO, 0.025 GE60 Antifoam 0.03 Tap water 96-97.5 pH 7.0 before continuous sterilization at 138°C for 5 min

a DDS = distiller's dried solubles. 26 SILMAN AND ROGOVIN were determined by spreading 0.1--0.2 ml amounts of appropriate sample dilutions on :MY agar plates and counting colonies after 72 hr incubation at 28°C.

RESULTS AND DISCUSSION Continuous fermentations were run at D ranging from 0.023 to I 0.196 llf- . The course of a typical fermentation at D = 0.054 hrl is plotted in Figure 1. The fermentation ran for 17 hr as a batch fermentation and then 120 hr as a continuous fermentation, "lvhich was Q.5 turnovers (Q). One Q equals a quantity of feed equal to the fermenter holdup. Steady-state values were: pH 6.3; viscosity, 5200 cP; xanthan, 1.22%; glucose, 0.75%; and aD, 10.5. XPR "lvas 0.66 g/hr/kg with yield basis glucose consumed ca. 82%. A graphical presentation of steady-state results for other D are shown in Figure 2. XPR increased sharply from 0.34 g/hr/kg at D = 0.023 llf-I to 0.66 g/hr/kg at D = 0.054 hr-I and then increased gradually to 0.84 I g/hr/kg at D = ca. 0.15 hr- . XPR decreased to 0.69 g/hr/kg at D = 0.196 hrl . Yield of xanthan from glucose consumed was 81-89% for the entire range of D studied. The pH increased from .5.9 to 7.2 with increasing D and reflects decreasing xanthan concen­ tration. The aD ranged from 8.7 to 10.7 (Table III). Viable cell counts gave concentrations of 4-7 X 10 9 cells/ml. o o 1 6 2 6 3.9 5.2 6 5 a.o Batch Feed at 0 = 0.054 hr-1 ~ 'a.J.O "- 0"00 pH "'f'0 0 -00_0_0-0 6.0 ::;: Viscosity 6·/i.-·-l:l.·A-·-oA-·_··A-·_.-A 2.5 i 1 c-e ji ~ 4 .2.0, 0.0» ;;; ". b Cells 10~12 ..r;; l fr t -00.. _0_0 0_·0 ;';-3 -15' .I>. :: ~. \~ .' Xanthan a ~ I ... A--.....A·--....----A----A .~ 2 Q 10 \i.""1 Glucose S ~ ~ ,flJ1t;·· ..:::.y...... 4 g ;;: ~05i.,;t'.. ~'f 2~ O~O ----c2:-z0-4f:.0-S:-z0-a:-z0--=1-==00:--:1-==20:--:17.40:-' Age, Hours Fig. 1. Course of single-stage continuous xanthan fermentation at dilution rate (D) = 0.054 hr-1 with PMU medium (2.25% glucose). BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIV, ISSUE 1 XANTHAN BIOPOLYMER BY CONTINUOUS FERMENTATION 27

OD1.6~------, ~...... • ~1.4 \~. '" ~ 1.2 .0.. I «. \\ 100~ ~5 c: A\ ~ 1.0 e/e,e~& __e-i"_e,e ---e Yield 80 ~ ~ 4 ~ 0 8 '. .r..... Ii: . ... .;.( ~"'XPR ~ 3 ~ 0.6 / I. _'::: ..0. '. >: ~0.4 /\ ''--0 pH 7.2 0" • 0_-li(1"'0-0--. Xanthan g02 /' ~ 64 :;';. ~. 12>,0 A'b,

~ 0 0' ...... b, Viscosity L.....-:-.L~..l,-,---.l~.....L..~~~.:.:.....J 5.6 o 0.04 0.08 0.12 0.16 0.20 Dilution Rate, hr-1 Fig. 2. Steady-state results for single-stage continuous xanthan fermentation with D from 0.023 to 0.196 hel.

TABLE III Comparison of Steady States for Various Dilution Rates (D)

Xanthan Glucose produc- consump- Yield Vis- Xan- tion tion (glucose D, cosity, than, rate, rate, consumed), hr-l cP % pH g/hr/kg g/hr/kg % OD

0.0233 7150 1.48 5.85 0.34 0.42 81 10.5 0.041 6000 1.33 6.15 0.55 0.64 86 10.4 0.054 5200 1. 22 6.30 0.66 0.81 82 10.5 0.080 2600 0.89 6.70 0.71 0.85 83 10.6 0.106 1400 0.70 6.80 0.76 0.89 85 9.6 0.140 800 0.60 7.05 0.84 0.94 89 9.2 0.154 550 0.54 7.0 0.83 0.97 85 10.7 0.196 160 0.34 7.2 0.69 0.82 84 8.7

Steady states could not be maintained indefinitely and ended at Q's between 6.5 to 8.7. When steady state ended, the viscosity decreased drastically, pH increased slightly, cell concentration (by aD) decreased then increased again, and an increased proportion of the cells in the population measured 1 X 20-30 IJ. instead of the normal 1 X 3 IJ.. Plate counts shmved increasing presence of small 28 SILMAN AND ROGOVIN colonies (2-2.5 mm) vs. the normal large colonies (4-5 mm). The viable cell count decreased then increased as small-colony cells in­ creased. These changes preceded a new steady state, which was attained about 5 Q after the first steady state ended. The ne,v steady state at the same D had much lower viscosity, higher pH, slightly lower OD, a diminished plate count composed exclusively of small-colony cells, and only long forms (1 X 20-:30 jJ.) visible under the . 'When small-colony cells were present in the inoculum, their per­ centage remained relatively constant during batch growth. When feeding began, the small-colony cells gradually replaced the large­ colony cells because they had a selective advantage. Viscosity de­ creased as the small-colony cells took over. In a run when these small-colony cells were in the inoculum, the long cell form seen under the microscope did not appear until more than 90% of the colonies were of the small type at about 8..5 Qshowing that the short-to long­ cell morphology change and the large- to small-colony change are separate processes. A similar culture change from large-to small­ colony cells also occurs when proper culture maintenance practices are not observed as reported by Cadmus et al.4 They reported that cultures from small colonies ,vere capable of producing large colonies "'hen replated. It is significant that over a nearly tenfold range in D (0.023-0.196 hr-1) the range of Q (6 ..5-8.7) was so narrow. Evi­ dently, specific growth rate has little effect on initiation of culture changes. XPR was not constant over the nearly tenfold range of D studied (Table III). Because absolute measurement of cell mass by OD was hampered by suspended solids and because plate counts gave only viable counts, interpretations of the XPR vs. D graph (Fig. 2) based upon specific rates must be considered postulations. Since OD was uniform from D = 0.023 to 0.1.54 hr-1, the plot of specific XPR must be similar to that of XPR; i.e., increasing over that range of D. To account for this increase, we suggest that specific XPR is inversely proportional to diffusion resistance caused by viscosity. At D = 0.196 hr-1, XPR decreased because cell mass decreased. This D is ca. 75% of maximum specific growth rate as calculated by Moraine and Rogovin. 5 Lindblom and Patton6 stated that under certain single-stage condi­ tions of operation Xanthomonas underwent a dissociation change BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIV. ISSUE 1 XANTHAN BIOPOLYMER BY CONTINUOUS FERMENTATION 29

which destroyed its ability to produce polysaccharide. They re­ ported overcoming this culture change by a three-stage continuous system. They report sampling for only 95 hr, '."hich period was only 3.0 total Q for their overall D of 0.032 hr-I • Rogovin7 reported a single-stage system wherein D was about 0.02 hr-1 for 14 days (6.4 Q). In the present work, the lowest Q was 6.5. To evaluate practical aspects of the process, yearly productions were calculated for various values of D when operations were con­ ducted under the following conditions: 6.5 Q, 1 kg holdup, 300 days/ year, 1 day downtime between runs (Table IV). The variation in annual xanthan production for 6.5 Q plotted vs. D in Figure 3 is not so pronounced for D = 0.054 to 0.154 hr-1 as the XPR vs. D shown in Figure 2. This flattening of the annual production curve is due to the incorporation of downtime and the inclusion of the fermenter content at termination of the run. Since maximum annual produc­ tion is nearly constant from D = 0.054 to 0.140 hr-r, the choice of D for a process to make crude drum-dried product is simplified by cost considerations. A process run at D near 0.05 hr-I would be more economical because even though each run is 6.5 Q. plus 1 Q for the fermenter contents at end of the run, there are more runs per year at the higher D (Table IV). AD of approximately 0.05 hr-1 would require: (1) less frequent inoculum preparation; (2) less media

TABLE IV

" Annual Production a

Time of Time of Time of Runs per Xanthan Xanthan D, batch, feed, run, year, per run, per year, hr- 1 hr hr days No. g/run g/year

0.0233 18 279 1:3.4 22 110 2420 0.041 18 158 8.4 36 100 3600 0.054 17 120 6.7 45 91 4100 0.080 17 81 5.1 59 66 3900 0.106 11 61 4.0 75 53 3980 0.140 11 46 3.4 88 45 3960 0.154 11 42 3.2 94 40 3760 0.196 11 33 2.8 107 26 2780

• Basis: 6.5 turnovers, 1 day downtime between runs, 300 days per year, and 1 kg holdup. 30 SILMAN AND ROGOVIN

00 0.04 0.08 0.12 0.16 0.20 Oilution Rate, hr-1 Fig. 3. Annual xanthan production for continuous fermentations of 6.5, 13, and 26 turnovers (Q) of fermenter contents. preparation; (3) less steam for processing and product recovery; and (4) smaller size equipment. An approximate relationship of process costs for D values in the range of 0.054-0.140 hr-1 can be obtained from the reciprocals of the xanthan concentrations. For D of 0.054 and 0.140 hr-1, the cost factor is:

Dilution rate, hr-1 0.054 0.14 Xanthan, % 1.22 0.60 I/Xanthan 0.82 1.67 Cost factor 1.0 2.04 Increasing the length of time of steady state should lower cost. However, at a D of 0.054 hr-1, increasing the duration of steady state two- and fourfold increased the annual production only 8 and 12%, respectively (Fig. 3).

CONCLUSIONS This study on single-stage continuous fermentation to produce xanthan biopolymer has provided the follo,ving information for dilu­ tion rates of 0.023 to 0.196 hr-1 : (1) Xanthan production rate was a function of dilution rate. (2) Maximum xanthan production rate of 0.84 g/hr/kg was ob­ 1 tained when dilution rate was 0.15 hr- • BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIV, ISSUE 1 XANTHAN BIOPOLYMER BY CONTINUOUS FERMENTATION 31

(3) Yields of xanthan from glucose over the range of dilution rates studied were relatively constant. (4) Duration of fermentations under the conditions studied were limited to 6.5-8.7 turnovers 'when a less productive variant strain occurred.

The assistance of James H. Johnson and Thomas W. Larsen in conducting the experiments is gratefully acknowledged. References

1. R. W. Silman and P. Rogovin, Biotechnol. Bioeng., 12, 75 (1970). 2. P. Rogovin, R. F. Anderson, and I\I. C. Cadmus, J. Biochem. lVlicrobiol. Technol. Eng., 3, 51 (1961). 3. P. A. Shaffer and A. F. Hartmann, J. Bioi. Chem., 45, 365 (1921). 4. M. C. Cadmus, K. A. Burton, A. 1. Herman, and P. Rogovin, Bacteriol. Proc., 1971, A47 (1971). 5. R. A. Moraine and P. Rogovin, Biotechnol. Bioeng., 8, 511 (1966). 6. G. P. Lindblom and J. T. Patton, U.S. Patent 3,328,262 (June 27, 1967). 7. S. P. Rogovin, U.S. Patent 3,485,719 (Dec. 23, 1969). Accepted for Publication September 15, 1971