Effective Onion Vinegar Production by a Two-step Fermentation System

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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, Ki tami,

Hokkaido 090-8507, Japan

Received 10 Apr il 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 cerevis iaestrain fR-2. Stable operation was possibie and

the maximum productiv ity 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 produc tivity and acetic acid concentration

of about 3.3 g/Z/h and 37.9

g/Z,

respec tively. The total acetic acid yield calculated from the amount of sugar

consumed was 0.86. The two-step sys tem was operated for 50 d and proved to be competitive with other systems

in terms of its high productivity , high acetic acid yield, operational stabili ty and low production costs.

[Key words:

vinegar, charcoal pellet bioreactor, onion, repeated batch process, locculating yeast]

Vinegar is widely produced from rice, malt, apples,

wine, and various other start ing 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 cel ls. The system was rather

simple and continuous operation was realized with a

high yield. However, since the cell concentration was

relat ively 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-

cifi c 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 HORIUC HI ET AL.

TABLE 1. Co mposit ion 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 cel l 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. Basical ly, a jar fer-

mentor for ethanol fermentation [3] and a vert ical 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 init ially 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 cel ls 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 ju ice 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 clarif ied 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 fil l 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. Schem atic diagram of the exper imental apparatus. 1, Medium reservoir; 2, per istal tic pum p; 3, jar- fermentor; 4, onion alcohol broth

reservoir; 5, charcoa l pellet bioreactor; 6, vinegar b roth rese rvoir.



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. Asept ic 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 successfu lly produced from the sugars contained in

the onion juice and the cell growth was smooth. Both

the sucrose and glucose concentrations steadi ly 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 initia l

stage of the batch cultivation indicates that the specif ic

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

recycl ing. The progress and completion of the fermenta-

80

ONION VINEGAR PROD UCTION BY TWO-STEP SYSTEM 291

40

15

0

0

0

5

10 15

Time(h)

FIG. 2. Time courses of batch cul ture for onion alcohol produc-

t ion by S. cerevisiue IR-2. Sym bols: 0, fructose; A, sucrose; n , glu-

cose; A, ethanol; 0, dry cel l weight.

tion could be monitored by observing the CO1 evolution

rate.

Figure 3 shows typica l 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 successful ly 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 t ime courses of repeated batch fermentation for onion alcohol production. Symbo ls: m, total sugar concentration; A,

ethanol; 0, CO2 evolution rate.



292 HORIU CHI ET AL.

J . BIOSCI. BIOENG.,

070

8 -

” ” ” ” ” ”

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

Batch number

FIG. 4. Productivi ty, ethanol yield, and f inal cel l concentration

for each batch dur ing repeated batch operation. Symbo ls: A, ethanol

yield;

n

, productivi ty; 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 ce lls 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 successful ly

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 acet ic 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 di lut ion rate and eff luent concentrations

in the charcoa l pellet bioreactor during o nion vinegar production.

Symb ols: 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

activ ity 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 character istics 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 charcoa l pellet bioreactor under

various dilution rates

D R T

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, Dilut ion rate; RT, retention t ime; POz, oxygen content; PD ,,

acetic acid productivi ty.

TABLE 3. Compo sit ion of onion juice, onion alcohol, and onion

vinegar produced by the two-step fermentation syste m

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 PRODU CTION BY TWO-STEP SYSTEM

293

TABLE 4. Comparison of two-step vinegar production syste ms

Medium

Ethanol

(g%h)

Aectic acid

(g/l) (g/l)

(gF h)

Ace tic acid yield Operating period

(%)

(4

Ref .

Immobi l ized cel l system Sacchar i f ied r ice 38.0

9.7 36-42 4.5-6.4 68-78 70 4

Free cel l system Sacchar i f ied r ice 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 p roductivi ty; PD,, ace tic acid productivi ty.

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 stabil ity. The free cel l 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 flex ibili ty of

total system because the alcohol production rate could

be easi ly 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 difficu lty in preparing sufficient onion juice

for use as feed stock. However, since the charcoal pellet

bioreactor has been successful ly operated for more than

180d using a synthetic medium (9), operation of the

system for a longer period would easi ly 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 i s rather low. This suggests that onion vine-

gar could be used as a nutraceut ical 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.

AC KN OWLED GMEN T S

The authors wish to thank Dr. H . Kur iyama of the National

Insti tute of Bioscience and Human-Technology for providing S.

cerevisiae IR-2, Mr. M. Kuramoto of Kuramoto Industry Co rp.

and Mr. S. Osumi of the Okhotsk Carbonization Center for sup-

plying charcoal pel lets, and Mr. K. Tabata, M s. N. Yamauchi, Ms .

M. O sugi and Mr. K. Ando for their laboratory assistance.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

REFERENCES

Itoh, H .: Shokusu (Vinegar) . Jozo Kyokai-shi , 73, 200-208

(1978) (in Japanese).

Ho&&i, J., Kanno, T., and Kobayashi, M.: New vinegar

production from onions. J. Biosci . Bioeng., 88, 107-109

(1999).

Mor i , A.: Vinegar production by bioreactor. New Food In-

dustry, 34, l-11 (1992). (in Japanese)

Saeki , A.: Continuous production of vinegar with immobi l ized

Saccharomyco des ludwigii cel ls and immobi l ized Acetobacier

aceti cel ls entrapped in calcium alginate gel beads. Nippon

Shokuhin Kogyo G akkaishi , 37, 722-725 (1990). ( in Japanese)

Saeki , A.: Continuous vinegar production using twin bioreac-

tors made from ethanol fermentor and acetic acid fermentor.

Nippon Shokuhin Kogyo G akkaishi , 38, 891-896 (1991). ( in

Japanese)

Comberbach, D. M. and Bu’ lock, J. D.: Continuous ethanol

fermentation in the gas- l i f t tower fermentor. Biotechnol. Lett.,

6, 129-134

(1984).

Kida, K., Mor imura, S., Kume , K., Suruga, K., and Sonoda,

Y.: Repeated-batch ethanol fermentation by a f locculating

yeast, Saccharomyces cerevisiae IR-2. J. Ferment. Bioeng., 71,

340-344 (1991).

Kuriyama, H., Seiko, Y., Mnrakam i, T., Kobayashi, H., and

Sonoda, Y.: Continuous ethanol fermentation with cel l recycl-

ing using f locculating yeast. J. Fermen t. Tech nol., 63, 159-165

(1985).

Hor iuchi , J., Tabata, K., Kanoo, T., and Kobayashi, M.:

Con-

t inuous acetic acid production by a packed bed bioreactor em-

ploying charcoal p el lets der ived from wa ste mushroom medium

J. Biosci . Bioeng., 89, 126-130 (2000).

Miyazak i , S. , Otsubo, M. , Aok i , H . , and Sawaya, T . : Acetic

acid fermentation with quince, asparagus and all that

using so-

lated acetic acid bacter ia. Nihon Shokuhin Kagaku Kougaku

Kaishi, 43, 858-865 (1996). (in Japanese)

Fontana, A., Ghommidh, C., Guiraud, J. P., aud Navarro,

J. M.: Continuous alcohol fermen tation of sucrose using f loc-

culating yeast. The l imits of invertase activi ty. Biotechnol.

Lett., 14, 505-510 (1994).

Effective Onion Vinegar Production by a Two-step Fermentation System

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