JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 90, No. 3, 289-293. 2000

Effective Onion Vinegar Production Fermentation System

by a Two-Step

JUN-ICHI HORIUCHI, * TOHRU KANNO, AND MASAYOSHI KOBAYASHI Department of Chemical System Engineering, Kitami Institute of Technology, 165 Koen-cho, Kitami,

Hokkaido 090-8507, Japan

Received 10 April 2OOO/Accepted 9 June 2000

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/Z total sugar, was smoothly converted to onion alcohol containing 30.6 g/Z ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain fR-2. Stable operation was possibie and the maximum productivity was about 8.0 g/Z/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/Z/h and 37.9 g/Z, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.

[Key words: vinegar, charcoal pellet bioreactor, onion, repeated batch process, flocculating yeast]

Vinegar is widely produced from rice, malt, apples, wine, and various other starting materials (1). In a re- cent study, we reported the production of a new type of vinegar rich in amino acids, organic acids and certain minerals from commercially worthless onions that had failed to meet the quality standards required for market- ing to customers (2).

Since conventional vinegar production is usually time- consuming, many studies has been carried out with the aim of improving the production procedures (3). Vinegar fermentation is essentially a two-step process: the anaero- bic conversion of sugars to ethanol and the aerobic oxidation of ethanol to acetic acid. Hence, to improve the overall performance during vinegar production from carbohydrate resources such as agricultural products, both the ethanol fermentation and the subsequent acetic acid fermentation process need to be optimized.

Saeki (4) reported effective vinegar production from saccharified rice in a two-stage bioreactor system using microorganism cells immobilized in calcium alginate, which realized high productivity and yield levels in con- tinuous operation. However, the high cost and the fact that the immobilized cells lacked long-term stability were critical drawbacks in terms of the practical application of the system. Saeki (5) also examined a two-stage biore- actor system that used free cells. The system was rather simple and continuous operation was realized with a high yield. However, since the cell concentration was relatively low, the operation would be unstable during long-term operation. Thus, further improvements are required to establish an effective continuous vinegar pro- duction process.

With regard to ethanol fermentation, a repeated batch process using a flocculating yeast is widely recognized as a promising method for effective alcohol production due to its simple configuration, ease of cell recycling, and stable operation (6-8). The repeated batch process is considered to be more suitable than a continuous proc-

* Corresponding author.

ess for alcohol production from agricultural resources like commercially worthless onions since they are sea- sonal products and the quantity available is limited.

With regard to effective acetic acid production, we have developed a packed bed bioreactor employing char- coal pellets derived from waste mushroom medium (9). These pellets, which are characterized by their high spe- cific surface area and bacterial alfinity, have been suc- cessfully applied to continuous acetic acid production using a synthetic medium.

In this study, we report on a new type of two-step vinegar production process that combines repeated batch fermentation with the charcoal pellet bioreactor and its application in effective onion vinegar production.

MATERIALS AND METHODS

Strains and media Saccharomyces cerevisiae strain IR-2, which was originally isolated from an Indonesian fermented food and has been well-characterized (8), was used for the alcohol fermentation. Strain IR-2 was main- tained on a YM medium slant at 4°C and subcultured at about two-month intervals. The YM medium contained the following (per liter): glucose, 10 g; yeast extract, 3 g; malt extract, 3 g; peptone, 5 g. One loop of cells from the slant was inoculated into 150ml onion juice, incu- bated for 16 h at 30°C on a reciprocating shaker (150 strokes/min), and then employed as the preculture.

Acetobacter pasteurianus no. 1, which was kindly provided by Hokkaido Industrial Technology Center, was used for the acetic acid fermentation (10). The strain was maintained on an agar plate containing GYP medium at 4°C and subcultured at about two-month intervals. The GYP medium had the following com- position (per liter): glucose, log; yeast extract, log; peptone, 5 g; CH3COONa.3Hz0, 2 g; meat broth, 5 g; Tween 80 (5% water solution) 10ml; mineral solution 5 ml. The mineral solution contained the following (per liter): MgS04. 7Hz0, 40 g; MnS04. 5Hz0, 2 g; FeS04. 7Hz0, 2 g; NaCl, 2 g. One loop of cells from the slant

289

290 HORIUCHI ET AL.

TABLE 1. Composition of onion juice produced from red onion R-3

Glucose (g/l) 20.6 Fructose (g//J 20.6 Sucrose (g/f) 26.3 Ca (w/O 80.8 K (mdl) 1965.0 Na (m/l) 9.0 Mg (md0 82.3 P (mg/l) 442.0 Fe bdl) 0.78 Mn (m/l) 1.65 Zn (w/l) 1.45

PH 5.4

was inoculated into 100 ml fresh GYP medium and in- cubated on a reciprocating shaker (100 strokesimin) for 48 h at 3O”C, and then employed as a preculture. The ODem of the preculture was about 0.2.

The red onion R-3 was used as the raw material for the experiments. Onion juice for alcohol fermentation was obtained as follows. Onions were cut and pressed in a mechanical juicer. The extract was packed in a heat- sealed pouch (2.5 r) and the packed juice was promptly cooked for 30min in 90°C water. The cooked juice was then cooled and stored at -20°C. Prior to the experi- ments, defrosted juice was filtered using no. 1 filter paper and then autoclaved at 12O’C for 20min. Table 1 summarizes the main components of the onion juice. Sucrose, glucose, and fructose were the chief sugars and the average total sugar concentration was 67.3 g/l. The juice was also used as the inoculum medium for the al- cohol fermentation. In terms of sugar content and nu- trients, there is no problem in using onions that failed to meet commercial standards as a source for the medium because they are rejected mainly on account of their shape or size.

Analytical Samples from the fermentor were cen- trifuged at 4”C, 7000 Xg, for 6 min. The supernatant was used to determine the sugars, ethanol, and acetic acid by HPLC (Tosoh, Tokyo; 8020 series). Several minerals were determined by inductively coupled plas- ma atomic emission spectroscopy (ICP-AES: Hitachi, Tokyo, 3.6 system). The total sugar concentration was calculated as the sum of the sucrose, fructose, and glu- cose concentrations. The cell concentration was mea-

J. BIOSCI. BIOENG.,

sured by the dry cell method for yeast and the ODm using a spectrophotometer (Shimadzu, Kyoto; UV- 1600PC) for acetic acid bacteria. One unit of ODMo cor- responded to about 0.4 g-dry cells/l in this culture.

Fermentations Figure 1 shows a schematic represen- tation of the experimental apparatus. Basically, a jar fer- mentor for ethanol fermentation [3] and a vertical char- coal pellet bioreactor for acetic acid fermentation [5] are serially linked via a reservoir [4]. Onion alcohol pro- duced in the jar fermentor is initially stored in the onion alcohol broth reservoir, from where it is continuously fed to the charcoal pellet bioreactor by a peristaltic pump. In order to harmonize the alcohol production rate and the alcohol consumption rate (acetic acid production rate), repeated batch fermentation was inter- mittently carried out. Suspended yeast cells that inevita- bly remained in the supernatant from the jar fermentor were not removed.

The jar fermentor used for the ethanol fermentation (Biott Corp., Tokyo) had a working volume of 2.01. The culture temperature was automatically maintained at 30°C. Air was not supplied in order to minimize ethanol evaporation loss and the culture pH was not controlled. Agitation was maintained at 200rpm during the fermen- tation. Repeated batch operation was carried out as fol- lows. By seeding 1350ml autoclaved onion juice with 150ml of the preculture, the operation was commenced with a total volume of 1500ml. At the completion of the fermentation, agitation was stopped and the culture was allowed to stand for 30min to let the yeast in the broth sediment. Then, 1125ml of the clarified broth, i.e. 75% of the total, was withdrawn and stored in the onion alco- hol broth reservoir. New autoclaved onion juice was ad- ded to fill the fermentor and the agitation was restarted to begin the next fermentation. In this way, repeated batch fermentation was carried out.

For the acetic acid fermentation, a charcoal pellet bioreactor 400 mm in height and 50 mm in diameter (Tokyo Rika Kikai, Tokyo) was used. The reactor was filled with 0.5 1 charcoal pellets and the working vol- ume was estimated to be 0.5 1. The void fraction of the packed pellets was 0.43. The pellets were produced from waste mushroom medium at the Okhotsk Carbonization Center pilot plant (Ikutahara, Hokkaido). The pellets are characterized by their high specific surface area (200 m2/g) with numerous micropores (2-10pm) and their

FIG. 1. Schematic diagram of the experimental apparatus. 1, Medium reservoir; 2, peristaltic pump; 3, jar-fermentor; 4, onion alcohol broth reservoir; 5, charcoal pellet bioreactor; 6, vinegar broth reservoir.

VOL. 90, 2000

bacterial affinity. Details of the pellets and bioreactor characteristics were previously reported (9). The biore- actor filled with pellets was autoclaved for 30 min at 120°C and then employed for the experiments. The cul- ture temperature was maintained at 30°C and the alco- hol broth produced from onion juice was continuously fed from the bottom at various feed rates. Aseptic air was also supplied from the bottom, the supply rate be- ing modified in proportion to the medium feed rate so as to maintain an aerobic condition.

The ethanol and acetic acid productivity (PD, and PD,, g/[/h) was calculated as the volumetric ethanol or acetic acid production over the total fermentation time or retention time. The ethanol and acetic acid yields (Y, and Y,) were defined as the ratio of the total alcohol or acetic acid produced to the theoretical ethanol or acetic acid production, which was stoichiometricahy calculated from the consumed sugars or ethanol. The cell yield, (YXJ was calculated as the ratio of the increase in the dry cell weight to the sugars or ethanol consumption.

RESULTS AND DISCUSSION

Onion ethanol production by repeated batch culture Prior to the repeated batch operation, the characteristics of alcohol production from onion juice by strain IR-2 were investigated in a batch culture using the jar fermen- tor. Figure 2 shows the results. About 3Og/f of ethanol was successfully produced from the sugars contained in the onion juice and the cell growth was smooth. Both the sucrose and glucose concentrations steadily declined. The fructose concentration showed an initial temporari- ly increase before decreasing steadily, because sucrose is first decomposed to glucose and fructose by cellular invertase and then utilized by the yeast. The apparent increase in the fructose concentration during the initial stage of the batch cultivation indicates that the specific sucrose decomposition rate is higher than the specific fructose consumption rate in IR-2 until specific fructose consumption activity is sufficiently induced (11). The floc- culent activity of IR-2 was satisfactory for successful cell recycling. The progress and completion of the fermenta-

80

ONION VINEGAR PRODUCTION BY TWO-STEP SYSTEM 291

40 15

0 0 0 5 10 15

Time(h)

FIG. 2. Time courses of batch culture for onion alcohol produc- tion by S. cerevisiue IR-2. Symbols: 0, fructose; A, sucrose; n , glu- cose; A, ethanol; 0, dry cell weight.

tion could be monitored by observing the CO1 evolution rate.

Figure 3 shows typical time courses of the total sugar concentration, ethanol concentration, and COZ evolution rate during repeated batch fermentation for onion alco- hol production. Six batches were successfully completed in this case. Most of the total sugar was smoothly con- verted to about 3Og/Z ethanol and the residual total su- gar concentration was around 3.5 g/l. From the second batch onwards, the initial sucrose concentrations ob- tained by analyzing samples taken immediately after the replacement of the culture broth ranged from 2.0-3.0 g/l. Values within this range are pretty lower than the theoretical value, and would have been caused by rapid adsorption of sucrose on the yeast cell surface. As a result, in Fig. 3, the initial total sugar concentrations from the second batch onwards were about 45 g/l com- pared with a theoretical value of about 53 g/f. The fer- mentation time was reduced from 11 h in the first batch to 3.5 h in the last by repeating the operation; the PD,

20 30

Fermentation time (h) FIG. 3. Typical time courses of repeated batch fermentation for onion alcohol production. Symbols: m, total sugar concentration; A,

ethanol; 0, CO2 evolution rate.

292 HORIUCHI ET AL. J. BIOSCI. BIOENG.,

070

8 -

” ” ” ” ” ” 1 2 3 4 5 6 7 8 9 10 1112 13

Batch number FIG. 4. Productivity, ethanol yield, and final cell concentration

for each batch during repeated batch operation. Symbols: A, ethanol yield; n , productivity; 0, dry cell weight.

accordingly increased to about 6.5 g/l/h in the sixth batch. From the third batch, settling velocity of the yeast floes in the fermentor became almost constant (about 0.4cm/s), which indicates the high flocculating activity of the cells was maintained. As a result, no diffi- culty was encountered in repeating the batch process throughout the experiments.

Figure 4 shows the PD,, Y,, and Y,,, for each batch during the repeated batch operation. After the 13th batch, the final cell concentration and the PD, had in- creased to 3Og/f and 8.0g/Nh, while the Y, and YXjs were about 0.90-0.95 and 0.11, respectively. Cell recov- ery by sedimentation during the operation was about 80%. The Y, and PD, obtained here are sufficiently com- petitive. The juice of onion juice had no inhibitory effect on the repeated batch alcohol fermentation by strain IR- 2.

Onion vinegar production in the charcoal pellet bio- reactor Continuous cultivation in the charcoal pellet bioreactor was commenced by inoculating 300 ml of a preculture of free cells at a low dilution using a syn- thetic medium. After confirming the successful growth of the acetic acid bacteria on the surface of the pellets, onion alcohol was fed to the reactor. Figure 5 shows the time courses of onion vinegar production under various dilution rates. The operation of the reactor was con- tinued for about 50 d. Onion vinegar was successfully produced from onion alcohol. After confirming the establishment of a steady state, the dilution rate was changed stepwisely as shown in Fig. 5. With each in- crease in the dilution rate, the acetic acid concentration fell while the ethanol concentration rose. Throughout the experimental run, the operation remained stable and no breakthrough or process malfunction was observed. The Y, was in the range of 0.88-0.92 during the opera- tion, which is slightly higher than the yield previously

0 10 20 30 40 50 Time (d)

FIG. 5. Time courses of dilution rate and effluent concentrations in the charcoal pellet bioreactor during onion vinegar production. Symbols: A, ethanol; 0, acetic acid (0).

using a synthetic medium (9). This indicates that ethanol was not utilized by yeast cells under the aerobic con- dition in the charcoal pellet bioreactor because the yeast activity was inhibited by a high acetic acid concentration and low pH (3.6-3.8).

Table 2 summarizes the steady-state analysis of the effluent from the charcoal pellet bioreactor at various dilution rates. At a dilution rate of 0.058 h-l, ethanol was completely converted to acetic acid. As the dilution rate was raised, the acetic acid concentration decreased while the ethanol concentration increased. The maxi- mum productivity reached about 3.3 g/l/h at a dilution rate of 0.124 h-r. The characteristics of acetic acid fer- mentation in the charcoal pellet bioreactor were very similar to those previously obtained using a synthetic medium (9).

Table 3 summarizes the main composition of the onion juice, onion alcohol, and onion vinegar produced by the two-step fermentation system. Totally, 37.9g/l acetic acid was obtained from 67.3 g/l total sugar. Since only 29.4g/l acetic acid was produced from onion juice containing 64.Og/l total sugar by a batch culture in our previous study (2), the total acetic acid production yield was greatly improved.

TABLE 2. Steady-state analysis of continuous onion vinegar prodution in the charcoal pellet bioreactor under

various dilution rates

D RT PO2 Aeration Outlet

(h-9 0) (atm) &$$ Edm& Ac&icid pH t-1

(g%h)

0.058 17.1 0.21 1.1 0.4 37.9 3.67 2.2 0.089 11.2 0.21 1.4 5.0 32.5 3.70 2.9 0.124 8.1 0.21 1.4 9.1 26.4 3.14 3.3 0.144 7.0 0.21 1.5 13.3 20.7 3.89 3.0

D, Dilution rate; RT, retention time; POz, oxygen content; PD,, acetic acid productivity.

TABLE 3. Composition of onion juice, onion alcohol, and onion vinegar produced by the two-step fermentation system

Total sugar Ethanol Acetic acid

Concentrations (g/[) Onion juice Onion alcohol Onion vinegar

67.3 3.5 2.5 0.0 30.6 0.4 0.0 0.0 37.9

VOL. 90, 2000 ONION VINEGAR PRODUCTION BY TWO-STEP SYSTEM 293

TABLE 4. Comparison of two-step vinegar production systems

Medium Ethanol (g%h)

Aectic acid (g/l) (g/l) (gF$h)

Acetic acid yield Operating period (%) (4 Ref.

Immobilized cell system Saccharified rice 38.0 9.7 36-42 4.5-6.4 68-78 70 4 Free cell system Saccharified rice bran 46-48 1.6-3.0 51-55 3.5-5.0 77-84 55 5 This work Onion juice 30.6 8.0 37.9 3.3 86 50 (180) -

PD,, Ethanol productivity; PD,, acetic acid productivity.

Performance of two-step vinegar production system In Table 4, the performances of three two-step systems used for vinegar production are compared. Though the experimental conditions differed, the alcohol and acetic acid productivities obtained in this study are sufficiently competitive with those of the other systems.

In our experiments, the theoretical acetic acid produc- tion based on the theoretical ethanol production calcu- lated from the consumed sugar was 44.2g/l, while the actual acetic acid production was 37.9g/l. Therefore, the total acetic acid yield over the consumed sugar was 0.86, which compares well with the yields of 0.77-0.84 reported by Saeki (4, 5).

The immobilized cell system required a high cost for cell immobilization and its stability during long-term operation was insufficient, though the productivity was rather high. Our system is characterized by low operat- ing costs and low initial investment on account of its simple configuration, the ease of cell recycling in re- peated batch operation, and the inexpensiveness and long-term stability of the charcoal pellets used in the packed bed bioreactor.

Compared with the free cell system, our system is ad- vantageous in terms of its total productivity and opera- tional stability. The free cell system required the addi- tion of acetic acid to the alcohol fermentor medium to prevent its contamination, which decreased the ethanol fermentation productivity. Since high cell concentrations could be maintained both in the repeated batch fermen- tor and the packed bed bioreactor during the operation of our system, there was no contamination problem. The use of repeated batch operation for the alcohol fer- mentation also improved the operational flexibility of total system because the alcohol production rate could be easily controlled in response to changes in the opera- tional conditions of the subsequent charcoal pellet bio- reactor.

The total operation period in this study was limited to 50 d due to a difficulty in preparing sufficient onion juice for use as feed stock. However, since the charcoal pellet bioreactor has been successfully operated for more than 180d using a synthetic medium (9), operation of the system for a longer period would easily be possible.

We have previously reported that onion vinegar con- tains various amino acids, organic acids and minerals (2). The analysis in Table 1 shows that the potassium content of onion juice is extremely high, while the sodi- um content is rather low. This suggests that onion vine- gar could be used as a nutraceutical food to help prevent hypertension.

In conclusion, a two-step system combining repeated batch process with a charcoal pellet bioreactor was found to be effective for onion vinegar production. Repeated batch operation using the flocculating yeast strain IR-2 resulted in the smooth conversion of onion

juice to onion alcohol with a high productivity. A packed bed bioreactor containing charcoal pellets pro- duced from waste mushroom medium was then success- fully employed for continuous vinegar production from the onion alcohol. The two-step system was operated for 50d and proved to be competitive in terms of its high productivity, high acetic acid yield, operational stability, and low investment costs.

ACKNOWLEDGMENTS

The authors wish to thank Dr. H. Kuriyama of the National Institute of Bioscience and Human-Technology for providing S. cerevisiae IR-2, Mr. M. Kuramoto of Kuramoto Industry Corp. and Mr. S. Osumi of the Okhotsk Carbonization Center for sup- plying charcoal pellets, and Mr. K. Tabata, Ms. N. Yamauchi, Ms. M. Osugi and Mr. K. Ando for their laboratory assistance.

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