Continuous Ethanol Production From Jerusalem Artichoke Tubers.

I. Use of Free Cells of Kluyveromyces mamianus

ARGYRIOS MARGARITIS and PRATIMA BAJPAI, Department of Chemical and Biochemical Engineering, Faculty of Engineering Science, The University of Western Ontario, London, Ontario, Canada N6A 5B9

s- The continuous fermentation of Jerusalem artichoke juice to ethanol by free cells of Kluyvero-

myces marxianus UCD (FST) 55-82 has been studied in a continuous-stirred-tank bioreactor at 35'C and pH 4.6. A maximum ethanol yield of 90% of the theoretical was obtained at a dilution rate of 0.05 h-l. About 95% of the sugars were utilized at dilution rates lower than 0.15 h-I. Volumetric ethanol productivity and volumetric biomass productivity reached maximum values of 7 g ETOH/L/h and 0.6 g dry wt/L/h, respectively, at a dilution rate of 0.2 h-'. The mainte- nance energy coefficient for K. marxianus culture was found to be 0.46 g sugar/g biomass/h. Oscillatory behavior was observed following a change in dilution rate from a previous steady state and from batch to continuous culture. Values of specific ethanol production rate and spe- cific sugar uptake were found to increase almost linearly with the increase of the dilution rate. The maximum specific ethanol production rate and maximum specific sugar uptake rate were found to be 2.6 g ethanol/g ceWh and 7.9 sugars/g cell/h, respectively. Washout occurred at a dilution rate of 0.41 h-I.

INTRODUCTION

The world population is expected to reach the six-billion mark by the end of this century, which will undoubtedly create severe strains on existing energy and food resources. It is expected that new developments in biotechnology will play an important role in resolving part of the energy and food problems that lie ahead. One important development which has stimulated worldwide interest is the utilization of renewable carbohydrate resources for the produc- tion of ethanol as a liquid and concomitant production of single-cell protein (SCP). A wide variety of carbohydrate materials such as starchy ma- terials including corn, potatoes, grains, and various cellulosic materials, and materials containing sugars such as sugarcane, molasses, sugar beets, cheese whey, and other waste carbohydrate materials4-' have been utilized for the production of ethanol.

One carbohydrate material which has received very little attention is the Jerusalem artichoke. With the exception of the early work of Underkofler,

Biotechnology and Bioengineering, Vol. XXIV, Pp. 1473-1482 (1982) 0 1982 John Wiley & Sons, Inc. CCC 0006-3592/82/071473-10$02.00

1474 MARGARITIS AND BAJPAI

McPherson, and Fulmer8 and B ~ i n o t , ~ there is no reported information in the literature on the fermentation of Jerusalem artichokes to ethanol. The Je- rusalem artichoke has been reported to have one of the highest known carbo- hydrate yields for agricultural crops, ranging from 2400-5900 kg of sugar per acre per year, and has many advantages over traditional crops which include minimal fertilizer requirements, good growth in poor soil, and high tolerance to frost and various plant diseases.lOJ1 As far as we know, there are no re- ported literature data available on the continuous fermentation of Jerusalem artichokes to produce both ethanol and SCP. Optimization growth experi- ments on Jerusalem artichoke juice using eight different yeast and microbial cultures have been reported by Margaritis, Bajpai, and Cannell.12 Batch growth kinetic studies on Jerusalem artichoke juice using two strains of Kluy- veromyces marxianus and Saccharomyces rosei are reported by Margaritis and Bajpai. l3 These studies have shown that Kluyveromyces marxianus UCD (FST) 55-82 is the best producer of ethanol, and it has been selected for fur- ther work on the continuous production of ethanol and SCP reported in this article. The data reported in this article, using free cells of K. marxianus, along with those presented in Part 11, using immobilized cells of K. marx- ianus, l4 form the basis for comparison of the free and immobilized cell sys- tems for the continuous production of ethanol from Jerusalem artichoke tubers.

MATERIALS AND METHODS

Extraction of Juice from Jerusalem Artichoke Tubers

The tubers were washed thoroughly with water to remove any remaining soil and then cut into small slices. The sliced tubers were then placed in a dish for 1 h with an equal amount of fresh water to extract the sugars at 75°C. The slurry was then filtered through a cloth to collect the first extract containing about 10% (w/v) total sugars. The extraction was repeated four to five times using an equal amount of fresh water each time until no detect- able sugar was found. Usually two extractions were sufficient to remove about 90% of the total soluble sugars present in the tubers. The extract was then sterilized at 120°C for 20 min and stored at 0°C for further use.

Microorgankms and Culture Conditions

A strain of Kluyveromyces marxianus UCD (FST) 55-82 was used in this study. It was provided by Dr. A. Lachance of the Plant Sciences Department at The University of Western Ontario. The culture was maintained on yeast extract glucose agar slants and was subcultured at weekly intervals. The fer- mentation medium consisted of artichoke extract which was supplemented with 0.05% Tween 80, 0.01% oleic acid, and 0.01% cornsteep liquor. The artichoke extracts contained carbohydrates equivalent to 10% total sugars. The pH of the medium was adjusted to 4.5 with lN HCI. For the preparation of inoculum, the yeast was first inoculated into fresh stock medium which

ETHANOL PRODUCTION FROM JERUSALEM ARTICHOKE. I 1475

consisted of artichoke extract. The measured amount of the active yeast cul- ture in stock media was transferred to 50 mL of the medium contained in 250-mL stoppered flasks. After incubation on a New Brunswick rotary shaker at 35OC and 140 rpm for ca. 24 h, this inoculum was transferred asep- tically to 400 mL of the fermentation medium, contained in a 1-L New Bruns- wick fermentor.

Continuous Fermentation

A 1-L New Brunswick Bioflo fermentor model C-30 with a working volume of 0.4 L was used as a continuous-stirred-tank bioreactor. The reactor contents were stirred at 200 rpm with a magnetically coupled four-bladed agitator. A variable-speed peristaltic pump was used to deliver a controlled volumetric flow of media into the reactor. The bioreactor was steam sterilized and a 24- h-old inoculum of K. manrianus was added. The culture was allowed to grow for 12 h batchwise before the continuous operation was started. The tempera- ture of the reactor was maintained at 35OC. The following effluent concentra- tion measurements were made at steady-state conditions: ethanol, total and reducing sugars, free cell, and pH. It was found that steady state was reached after three to four bioreactor volume turnovers at a given dilution rate. A series of samples were taken at a given dilution rate to confirm steady state.

Analytical Methods

For the determination of cell dry weight concentration in the broth, 5-mL samples were used. The cells were centrifuged at 4000 rpm for 10 min and then washed twice with distilled water and dried to a constant weight at 105°C. Ethanol concentration was determined using gas chromatography. The instrument used was a Varian model 3700 gas chromatograph with a 12-ft-long, 0.025-in. -diam stainless-steel column packed with chromosorb 107. A 5-pL sample was injected using a 10-pL syringe at an oven tempera- ture of 175OC and inlet and flame ionization detector temperature of 200°C using helium as a carrier gas. The combustion gases were hydrogen and air. The gas chromatograph output signal was connected to a strip-chart recorder operating at a chart speed of 2 cm/min. Ethanol concentration in the sam- ples was determined from a calibration curve of known standard solutions of ethanol. Total sugars were determined by the anthrone reagent method,15 and reducing sugars were determined by the dinitrosalicylic acid method.I6

RESULTS AND DISCUSSION

The results of a single-stage continuous fermentation with artichoke juice containing approximately 10% total sugars are shown in Figures 1 and 2. Ethanol, biomass, and total sugars concentrations are plotted as a function of the dilution rate in Figure 1, and Figure 2 shows the reducing and total sugars concentrations, pH, and the mass ratio of reducing-to-total sugars as a

1476 MARGARITIS AND BAJPAI

l2O1

Fig. 1. Total sugars, ethanol, and biomass effluent concentrations as a function of dilution rate at 35OC: (m) total sugars, ( 0 ) ethanol, and ( A ) biomass.

function of the dilution rate. These data indicate that ethanol and biomass concentration decreased with the increase of the dilution rate and dropped to zero at a washout rate of 0.4 h-l. Ethanol concentration dropped very slowly at low dilution rates, i.e., D = 0.1-0.15 h-l, but above a dilution rate of 0.15 h-l, ethanol concentration declined more rapidly. The maximum ethanol concentration of 46 g/L was obtained at a dilution rate of 0.2 h-l. The concentration of total and reducing sugars in the effluent remained almost constant, at about 9 and 3.8 g/L, respectively, at dilution rates lower than 0.15 h-l, but beyond this dilution rate, the concentration of total and reducing sugars increased. The mass ratio of reducing-to-total sugars varied slightly with the increase of the dilution rate, while the pH of the effluent did not change with increasing dilution rates and remained stable at about 4.6. From these data, volumetric ethanol productivity, volumetric biomass pro- ductivity, and percent substrate utilization have been calculated and are plotted as a function of dilution rate, shown in Figure 3. Figure 3 shows that as the dilution rate increased, volumetric ethanol and biomass productivities also increased, but beyond a dilution rate of 0.2 h-l, volumetric ethanol and biomass productivities began to decline. Thus the optimum dilution rate for

ETHANOL PRODUCTION FROM JERUSALEM ARTICHOKE. I 1477

0.1 0.2 v a4 DILUTION RATE,D. (h- )

Fig. 2. Total and reducing sugars concentrations and mass ratio of reducing to total sugars and pH as a function of dilution rate: ( w ) total sugars, ( A ) reducing sugars, (0) mass ratio. and ( 0 ) pH.

the maximum volumetric ethanol and biomass productivities was found to be 0.2 h-l. The maximum volumetric ethanol and biomass productivities have been calculated to be 7 g ETOH/L/h and 0.61 g dry wt/L/h, respectively. Percent substrate utilization remained almost constant at dilution rates lower than 0.15 h-l. At these dilution rates, ca. 95% of the sugars were utilized. When the dilution rate was increased to 0.2 h-l, percent substrate utilization dropped to 8270, and at a dilution rate of 0.3 h-l, only 52% of the sugars were utilized.

Figure 4 shows a plot of ethanol yield (g ethanollg sugar utilized) and biomass yield (g biomasslg sugar utilized) as a function of the dilution rate. Ethanol yield dropped continuously with the increase of the dilution rate. The maximum ethanol yield of 90% of the theoretical was obtained at a dilu- tion rate of 0.05 hW1. Biomass yield increased slightly from 0.03 to 0.035 with the increase of the dilution rate from 0.05 to 0.15 h-l, but above a dilution rate of 0.15 h-l, it started to decline. The maintenance energy coefficient of m = 0.466 g sugar/g biomasdh was determined as the slope of the line b y plotting l/Yx/s as a function of l/D as shown in Figure 5, where YxIs is the

1478 MARGARITIS AND BAJPAI

DILUTION RATTE,D.(h” 1 Fig. 3. Percent of total sugars utilized, ethanol productivity and biomass productivity as a

function of dilution rate: (6) percent sugars utilized, ( 0 ) ethanol productivity, and (A) biomass productivity.

overall cell yield factor and D is the dilution rate. l7 Lee, Tribe, and RogerslS have observed a high maintenance energy requirement for anaerobically grown Zymomonm mobilis. They obtained values of m = 1.6 and m = 3.6 g glucose/g biomasdh for 10 and 15% feed glucose solutions, respectively. Thus, it appears that as compared to Z. mobilis, K. mamianus uses a relatively low proportion of sugar for maintenance energy.

In Figure 6, specific ethanol production rate (g ethanol/g cell/h) and spe- cific sugar uptake rate (g sugar/g cell/h) have been plotted as a function of dilution rate. These data show that specific ethanol production rate and spe- cific sugar uptake rate increased almost linearly with the increase of the dilu- tion rate. The maximum specific ethanol production rate and the maximum specific sugar uptake rate have been calculated to be 2.6 g ethanollg cell/h and 7.9 g sugars/g cell/h, respectively. Grote, Lee,and Rogers,19 using free cells of Z. mobilis ZM4 growing batchwise on 10% glucose, have reported a maximum specific ethanol productivity of 4.2 g ETOH/g cell/h and max- imum specific glucose uptake rate of 8.9 g glucose/g cell/h.

Oscillations in biomass, sugar, and ethanol concentrations were observed following a change in dilution rate from a previous steady state and from

ETHANOL PRODUCTION FROM ERUSALEM ARTICHOKE. I 1479

.05

.04

/ - = -03

.02

.01

0.1 0.2 03 0.4 DILUTION RATE, D, (h-' )

Fig. 4. Ethanol and biomass yield as a function of dilution rate: ( 0 ) ethanol and ( A ) biomass.

I

Fig. 5. Endogenous metabolism plot for K. murxiunus, reciprocal of overall cell yield versus reciprocal of dilution rate. The slope gives the maintenance energy coefficient, m .

1480 MARGARITIS AND BAJPAI

0.1 62 02 oh DILUTION RATE,D./r, ( h ' )

Fig. 6. Specific ethanol production rate and specific sugar uptake rate for K. marxianus as a function of dilution rate: ( 0 ) ethanol production and (m) sugars consumption.

batch to continuous culture. In Figure 7, typical oscillations in K. mumianus culture are shown following a change from batch to continuous culture. The data in Figure 7 indicate that the culture took about three bioreactor volume turnovers to reach the steady state.

The maximum steady-state values of the various kinetic parameters ob- tained in this continuous run are summarized in Table I.

Margaritis,20 using free cells of S. cerevisiue ATCC 4126 in a continuous- stirred-tank bioreactor with 10% glucose feed concentration at 28°C and pH 4.0, obtained a maximum ethanol productivity of 3.5 ETOH/L/h at a dilu- tion rate D = 0.15 h-I and a maintenance energy coefficient m = 1.4 g glu- c o d g biomasslh. Cysewski,21 also using free cells of S. cerevisiue ATCC 4126 with 10% glucose feed concentration supplemented with the addition of 10 mg ergosterol/L, obtained a maximum ethanol productivity of 5.95 g ETOH/L/h at a dilution rate of D = 0.2 h-' and a temperature of 35°C and pH 4.0.

On the basis of ethanol yield obtained in the present study, and carbohy- drate yields of Jerusalem artichokes reported by Chubey and Dorrelllo and Fleming and Groot-Wassink," it seems feasible to produce ethanol in the

ETHANOL PRODUCTION FROM JERUSALEM ARTICHOKE. I

BATCH CONTINUOUS 1

Fig. 7. Typical oscillations in concentrations of total sugars, reducing sugars, ethanol, and biomass when switching from batch to continuous fermentation at D = 0.05 h-I: (m) total sugars, (A) reducing sugars, (0 ) ethanol, and (0) biomass.

TABLE I Maximum Steady-State Kinetic Parameters for K. marxianus

Parameter Maximum value

Specific growth rate, pmU 0.40 0 - l )

(g ethanol/g sugars utilized) (percent of theoretical)

(g dry wt/g sugars utilized)

( 7 0 )

(g ethanol/L)

(g dry wt/L)

Ethanol yield, Y,,, 0.46 (90%)

Biomass yield, Y,,, 0.035

Initial sugar utilized 95.0

Maximum ethanol concentration, P,,, 46.0

Maximum biomass concentration, X,,, 4.35

Maximum specgic ethanol production rate 2.55

7.9

Maximum volumetric ethanol productivity 7.0

Maximum volumetric biomass productivity 0.61

Maintenance energy coefficient, m 0.466

[g ethanol (g cell)-' h-'1

[g sugars (g cell)-' h-'1

(g ethanol L-' h-')

(g dry wt L-' h-')

[g sugar (g biomass)-' h-'1

Maximum specific total sugars uptake rate

1482 MARGARITIS AND BAJPAI

range of 1400-2700 kg per acre of land per year. The yield appears to be quite attractive and shows that the Jerusalem artichoke offers excellent possi- bilities as a raw material for the production of industrial ethanol and SCP.

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Accepted for Publication December 15, 1981