Purchased by U.S. Department of Agriculture for Official Use. BIOTECHNOLOGY AND BIOENGINEERING, VOL. XIV, PAGES 23-31 (1972) Continuous Fermentation to Produce Xanthan Biopolymer: Effect of Dilution Rate* R. W. SILlVIAN and P. ROGOVIN, N ol'thern Regional Research Laboratory,t Peoria, Illinois 61604 Summary Single-stage continuous fermentations 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 test tube incubated 24 hr on rotary shaker (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 bottle 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 viscometer 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).