Sei sulla pagina 1di 5

ll/orld Journal or Microbiology & Biotechnology 18: 271-275. 2002. © 2002 Khl1Fer Academic Publishers. Prill/ed in the Netherlal/(I.s-.

Academic Publishers. Prill/ed in the Netherlal/(I.s-. 271 Acetic acid production by Dekkeraj Brettallomyces

271

Acetic acid production by Dekkeraj Brettallomyces yeasts

S.N. Freer Fermentation Biochemistr,v Research Unit, National Center for Agricultural Utilization Research, USDA *, Agricultural Research Service, 1815 N. Universi(v Street, Peoria, lL 61604, USA Tel.: + 1-309-6816472, Fax: + 1-309-6816427, E-mail: ,li-eersn@ncaur.usda.gov

Received 4 September 2001: accepted 7 January 2002

Keywords:

Summary

Acetic acid. Brettanomyces. Dekkera. ethanol

Yeast belonging to the genera Brettanom}'ces and Dekkera are noted for spoiling cellar and bottled wine through the production of haze, turbidity and acetic acid. However, I was unable to find information on the use of these yeasts for the expressed purpose of acetic acid production. Sixty yeast strains belonging to these, and several other genera, from the ARS Culture Collection, Peoria, IL, were screened for their ability to produce both ethanol and/or acetic acid. For ethanol production, the strains were grown anaerobically at 24 and 30°C in batch culture using glucose (l00 gil) as the carbon/energy source. For acetic acid production, the strains were grown aerobically in batch culture using either glucose (l00 gil) or ethanol (35 gT) as the carbon/energy source. In the initial ethanol production screen, 19 strains produced at least 45 g ethanol/I. In the initial acetic acid screen, 28 of the yeast strains produced at least 5 g acetic acid/l from 100 g glucose/I. while 23 strains produced at least 5 g acetic acid/l from 35 g ethanol/I.

Introduction

Acetic acid is an important industrial chemical with an annual domestic production of about 2.12 x 10 6 metric tons in 1995. Currently, the commercial production of

glacial acetic acid is exclusively by petrochemical routes.

A potential industrial use of acetic acid is the production

of environmentally friendly de-icers, including calcium magnesium acetate (CMA), as a non-corrosive road de- icer, and potassium acetate and sodium acetate, as airport runway de-icers. More than 9.07 x 10 6 metric tons of rock salt are poured onto North American highways each year to provide safer driving conditions during the winter months. This heavy use of de-icing chemical destroys roadside vegetation, damages aquatic ecosystems, pol- lutes groundwater and domestic potable water supplies, and damages the highway infrastructure (Fritzsche 1992). Recognizing the negative impact that using salt

as a de-icer has upon the environment, in the mid-1970s,

the US Department of Transportation's Federal High- vay Administration funded research to develop substi- tutes for the chloride salts currently used as de-icers. Calcium magnesium acetate was identified as a poten-

report

however. the USDA neither guarantees nor warrants the standard of the product. and the use of the name by USDA implies no approval of the product to the exclusion of others that may be suitable.

* Names

are

necessary

to

factually

on

available

data:

tially acceptable, non-corrosive, environmentally benign alternative to road salt (Dunn & Schenk 1980). CMA is a mixture of calcium acetate and magne- sium acetate. It is currently manufactured by reacting glacial acetic acid with dolomitic lime (CaO-MgO) or

limestone (Ca/MgC0 3 ). In addition to

as a de-icer, there are reports that CMA, when used as an additive in coal-fired combustion units. reduces sulphur dioxide emissions, thus, partially mitigating the problem of acid rain pollution (Sharma 1991:

Levendis 1991). Two microbiological approaches have been previously proposed for the fermentative production of acetic acid; CMA from corn (Yang et al. 1997). In one approach, glucose is fermented by yeast and the resultant ethanol effluent is aerobically converted to acetic acid by Acetobacter. In the other approach, Clostridium ther- moaceticum is used to convert glucose to acetic acid. High concentrations of acetic acid are produced in the yeast-Acetobacter process, however, the oxygen require- ment for Acetobacter conversion makes the process energy intensive. The thermophilic process (55-60°C) requires only a single bioreactor, but it is also energy intensive and results in low final acetic acid levels. The genus Dekkera and its anamorph, the genus Brettanomyces, are characterized by multipolar budding (van der Walt 1984a,b: Smith et al. 1990), the presence of a Custers effect (also called a negative Pasteur effect) (Custers 1940: Scheffers 1961, 1966, 1967: Scheffers &

its potential use

272

Wiken 1969: Wijsman er al. 1984), a coenzyme Q that contains nine isoprene units (Yamada er al. 1980), and the formation of acetic acid (Custers 1940). Yeasts belonging to these genera are often noted for spoiling cellar and bottled wine through the production of haze, turbidity and acetic acid (Sponholz 1993), as well as being used in the secondary fermentation of Iambic beer. However. I was unable to find any information on the use of these yeasts for the express purpose of acetic acid production. Sixty yeast strains belonging to these and several other genera were screened for their ability to produce ethanol and/or acetic acid from either glucose or ethanol in batch culture.

Materials and methods

Organisms, media and grOlvrh condirions

The following yeasts were tested in this study: Brerrano- myces bruxellensis Y-1412. Y-1411. Y-1441: B. lambicus Y-1413. Y-1330: B. c1ausseniiY-2290. Y-1414. YB-4087:

B. anolllalus Y-1415. Y-12670: B. cusrersianus Y-6653. B. inrermedius Y-2394. Y-2395. YB-3363: B. naadenensis Y- 7706: Brerranolllyces spp. YB-5226, YB-5225, YB-5234, YB-5231. YB-5240. YB-5233. YB-5227. YB-5260. Y-

5740. YB-5243. YB-5235. YB-5241. YB-3695. YB-3696.

YB-954. YB-3694. Y-7447. YB-5230. YB-3998. YB-

5212. Y-5242: Dekkera inrermedia YB-5164. YB-4553.

Y-I092. YB-4241: D. bruxellensis Y-17523. Y-17535. Y- 17525. Y-17524. Y-17534. Y-12961: D. naadenensis Y- 17526: D. anolllaia Y-17521. Y-17520. Y-17522: Eeniella nana Y-17527, Y-17533: Zygosaccharomyces bailii Y- 2227: Z. bisporus Y-7558: Pichia lIlembranaefaciens Y- 2026, Y-2089: P.ferlllenrans Y-1619; P. anomala Y-366:

Candida krusei Y-7179: Issarchenkia orienralis Y-5396. All of the organisms were obtained from the ARS Culture Collection, Peoria, IL and are designated by their NRRL enumerations. The basal medium (YP) consisted of 20 g peptone!1 and 109 yeast extract /1. Inocula were prepared by transferring a loop of cells from a fresh slant to 20 ml of YP medium containing 20 g glucose/I. The cultures were incubated aerobically on a rotary shaker at 200 rev/min and 28°C for 48 h. and 0.2 ml was used to inoculate 20 ml of fresh medium. After 24 h. the cells were harvested by centrifugation at 7000 x g for 10 min, washed once in sterile distilled water, and suspended in 20 ml of sterile distilled water, The experimental flasks were inoculated using 0.9 ml of cell suspension. For acetic acid production, initial screening was performed in 125 ml baffled flasks con- taining 30 ml of basal medium prepared with either 100 g glucose/lor 35 g ethanol!1. For ethanol produc- tion. 30 ml of basal medium containing 100 g glucosell in 50 ml flasks were inoculated and capped with serum stoppers and vented with No. 26 gauge sterile needles. The experimental cultures were incubated at either 24 or 30°C on a rotary (New Brunswich Scientific Co., Inc., Edison, NJ) shaker at 250 rev/min.

S.N. Freer

Growrh, erhanol, carbohydrare and aceric acid analysis

Growth was measured by the increase in optical density at 600 nm. Glucose. ethanol and acetic acid were quantified by high-pressure liquid chromatography (HPLC) on a Spectra-Physics chromatograph (Thermo Ferinizara, Woodstoch, GA) fitted with a BioRad HP- 87H column (Bio-Red Laboratories, Hercules, CA). The mobile phase was 5 mM sulphuric acid. The compounds of interest were detected with a Waters 410 differential refractometer (Millipore, Watas Chromatography Divi- sion, Marborough, MA).

Results

Producrion ot' aceric acid and erhclI/olFom eirher glucose or erhanol

The capacity of 60 yeast strains to produce ethanol and acetic acid, when grown anaerobically, from 100 g glucose/I is presented in Table 1. In general, the yeasts tested were reasonably efficient at fermenting glucose. Only three yeasts (Brerranomyces spp. NRRL YB-5241. YB-3695 and YB-3694) produced no ethanol from glucose. Of the 60 yeasts tested, 19 produced at least 45 g ethanol/L while only 10 yeasts produced less than

Table

1. Growth.

acetate

and

ethanol

(anaerobic fermentation).

production

from

glucose

Culture"

Growth

.-\cetate

Ethanol

Residual glucose

(NRRL)

(A 600 )

(g

I)

(gl)

(g;J)

B.

bruxellensis

Y-1412

25.84

1.1

45.6

0.0

Y-1411

24.78

0.0

48.5

0.0

B.

lall/bicus

Y-1330

16.05

0.0

51.9

0.0

B.

clallssellii

Y-2290

33.89

3.0

49.0

0.0

Y-1414

30.44

0.0

49.4

0.0

B.

anoll/alus

Y-1415

30.40

0.4

47.1

2.8

Y-12670

31.17

0.0

50.4

0.0

Brellil/lOlI/)"ces sp.

 

YB-5260

13.67

3.4

48.9

0.3

D.

illll!l"lI/edia

YB-4553

41.37

0.0

48.9

0.0

Y-I092

38.11

1.0

48.5

0.0

YB-4241

32.01

1.3

49.4

0.0

D.

bruxellellsis

Y-17534

37.65

1.0

45.0

0.0

Y-12961

21.74

1.0

50.6

0.0

D.

anoll/ala

Y-17521

29.79

0.0

48.9

0.0

'{-I 7520

29.60

0.0

48.7

0.0

Y-17522

29.30

0.0

50.1

0.0

Z.

bailii

Y-2227

8.09

1.0

49.6

3.4

Z.

bi.\]'orus

Y-7558

21.31

0.0

46.1

3.0

P.

Iel'll/ell/ans

Y-1619

14.19

2.0

45.6

0.0

" Only cultures that produced at least 45 g ethanoL I are listed.

Acetate Fom yeasts

10 g ethanol/I. The six strains that produced the most ethanol were B. lambicus NRRL Y-1330. D. bruxellensis NRRL Y-12961. B. anomalus NRRL Y-12670. D. ano- mala NRRL Y-17533. B. c!aussenii NRRL Y-1414 and D. intermedia NRRL YB-4241. As expected, none of the yeasts produced large amounts of acetic acid when grown fermentatively. The capacity of the yeasts to produce acetic acid when grown aerobically in medium initially containing 35 g ethanol/l is presented in Table 2. All of the yeasts tested, except Brettanomyces sp. NRRL YB-5235, utilized, to varying degrees. ethanol as a carbon/energy source. However, 24 of the 60 yeast strains tested produced no detectable acetic acid when grown on ethanol. Twenty- three of the yeasts produced greater than 5 g acetic acid 1 while II of the strains tested produced greater than 20 g acetic acid/I. The five strains that produced the most acetic acid from ethanol were D. bruxellensis NRRL Y-17525. B. intermedius NRRL Y-2394. B. cus- tersianus NRRL Y-6653. D. intermedius NRRL Y-5164 and D. anomala NRRL Y-17520. All of these yeasts produced over 25 g acetic acid/!. however, none com- pletely utilized the initial 35 g ethanol:!. The capacity of the yeasts to produce acetic acid and ethanol when grO\vn aerobically in medium initially containing 100 g glucose(1 is also presented in Table 2. Forty-seven of the yeast strains tested produced detect- able levels of ethanol under these growth conditions.

273

Twenty-six of the yeasts produced no detectable acetic acid when grO\vn aerobically on glucose. Sixteen of these strains produced no detectable ethanol either, even though many of them completely utilized the initial 100 g glucoserl. No other fermentation products, in- cluding glycero!. were detected by HPLC in the culture beers of these yeasts. Twenty-eight of the strains tested produced at least 5 g acetic acidrl. Of these, 16 produced over 20 g acetic acid/!. The five strains that produced the most acetic acid from glucose were D. intermedia NRRL Y-5164 and NRRL YB-4553. B. intermedius NRRL Y- 2394 and NRRL Y-2395. and D. bruxellensis NRRL Y- 17525. All of these cultures produced over 29 g acetic acid/!. however, none of these cultures completely utilized the 100 g glucose:l initially present in the medium.

Effect ot'temperature

All of the cultures were tested for ethanol and/or acetic acid production at 30°C using glucose (100 gil) or ethanol (35 g/l) as the carbon/energy source (data not shown). In genera!. the cultures made about the same amount of product, regardless of the growth tempera- ture. However, the cultures grown at 30°C tended to produce the product more rapidly. When grown at 30 0c, the fermentations were completed in 3-5 days, while the cultures grown at 24°C required 5-7 days for

Table 2.

Growth. acetate and/or ethanol production from either glucose or ethanol (aerobic metabolism).

Culture"

Growth

Acetate

Residual EtOH

Growth

Acetate

Ethanol

Residual glucose

(NRRLI

(/1.6(10)

(g/l)

(g/l)

(A 600 )

(gl)

(gI)

(g/l)

B.

bruxellcnsis

Y-1412

48.93

2.4

,

35.37

22.9

21.8

14.4

B.

claussenii

Y-2290

12.19

10.0

26.5

 

34.61

24.6

15.4

41.2

B.

anomalus

Y-1415

23.05

17.6

16.7

39.51

22.4

13.8

17.1

B.

cuslersiallllS

Y-6653

24.81

28.0

10.1

 

82.63

0.0

0.0

53.4

B.

illlermedius

Y-2394

29.08

29.2

9.5

 

19.45

29.6

10.5

40.2

'{-2395

6l.l6

0.0

0.8

32.25

29.1

13.5

31.2

YB-3363

31.58

24.0

13.8

'-'

23.7

 

,

,

I 1.0

43.6

 

D.

illlcrmcdia

YB-5164

35.49

27.7

6.5

 

33.99

31.7

6.1

48.1

YB-4553

39.98

24.3

10.7

33.72

30.0

24.3

57.4

Y-I092

17.92

18.3

18.0

32.09

21.2

16.7

33.4

YB-4241

20.75

18.3

17.4

33.15

21.6

21.6

20

D.

bruxellensis

Y-17523

24.70

23.4

8.5

 

27.13

24.0

6.2

67.9

Y-17535

29.52

"

15.6

28.56

18.7

10.5

49.1

Y-17525

31.95

33.0

10.4

34.96

31.6

15.7

26.1

Y-17524

31.50

24.6

14.4

29.70

26.9

7.0

52.3

Y-17534

30.21

22.0

15.2

31.26

19.0

8.5

52

D.

anomala

Y-17521

10.50

17.8

26.4

29.00

23.0

18.6

29.8

1'-17520

24.20

25.3

18.5

26.66

24.4

10.2

24.4

Y-17522

12.39

14.8

 

,")

29.19

 

20.4

7.7

58.8

.;;

1

274

completion, as measured by the presence of a constant residual carbon source and no further increase in acetic acid production. The only exception to this was B. bru."\'- e/lensis NRRL Y-1411. This strain produced 0.9 and 19.9 g acetic acid/I when grown on ethanol at 24°C and 30 0c, respectively (data not shown). When glucose was the carbon/energy source, NRRL Y-1411 produced 18.4 and 18.8 g acetic acid/l when grown at the two temper- atures (data not shown).

Discussion

Acetic acid is believed to be produced in yeast by the oxidation of acetaldehyde, which can be formed either by the pyruvate dehydrogenase bypass or by the oxidation of ethanol. In both pathways, acetaldehyde is a free intermediate that is converted to acetate by aldehyde dehydrogenase and subsequently activated to acetyl-CoA by acetyl-CoA synthetase. Two aldehyde dehydrogenases (one constitutive and one regulated) have been described in yeasts (Seegmiller 1955: Stein- man & Jakoby 1967; Carrascosa et al. 1981). It has been suggested that the constitutive aldehyde dehydrogenase functions in the pyruvate dehydrogenase bypass and that the regulated aldehyde dehydrogenase functions in the oxidation of ethanol. Apparently, acetate is excreted into the medium if it is not completely activated to acetyl-CoA. In D. anomala (Geros et al. 2000), acetyl-CoA synthetase is repressed by glucose. Thus. when grown aerobically in medium containing high concentrations of glucose, conditions that favor the excretion of acetic acid are invoked. Similarly, acetate is produced in Saccharomyces cerevi- siae if there is insufficient acetyl-CoA synthetase activity present for the complete oxidation of acetate to acetyl- CoA (Postma et al. 1989). However, in D. bruxe/lensis (syn. B. abstinens: Smith 1998) acetyl-CoA synthetase activity does not appear to be repressed by glucose: thus, even under culture conditions where high amounts of acetic acid are produced, the pathway does not appear to be blocked (Carrascosa et al. 1981). The results of the screen for acetic acid production indicated that the majority of the Dekkera/ Brettanomy- ces spp. deposited in the ARS Culture Collection were capable of producing acetic acid when either glucose or ethanol \vas used as a carbon/energy source. Overall. the five strains that produced the most acetic acid were D. intermedia NRRL YB-4553 and YB-5164. B. intermedi- us NRRL Y-2394. D. bruxe/lensis NRRL Y-17525. and D. anomala NRRL Y-17520. All of these strains fer- mented glucose efficiently (Table I), and produced over 24 g acetic acid/I from both glucose and ethanol (Table 2). In generaL the efficiency of acetic acid production was reasonably low. This was probably due, in part, to the inhibitory effects that acetic acid had upon the yeasts, as the pH values of the batch cultures were not controlled. The final pH values of the various cultures varied

S.N. Freer

greatly, depending upon the amount of acetic acid produced. When grown aerobically, the pH values of the culture beer after 7 days incubation ranged from 4.2 to 6.8. while the anaerobic cultures ranged from 5.1 to 6.3 (data not shown). Finally, not all of the strains produced acetic acid from both glucose and ethanol. Three strains. NRRL Y- 2395, NRRL Y-1411 and NRRL Y-1296L produced acetic acid from glucose. Little, if any, acetic acid was produced when ethanol was used as the carbon/energy source, although the yeasts grew well (Table 2 and data not shown). Conversely, three strains, NRRL Y-6653, NRRL Y-5240 and NRRL Y-5235, produced acetic acid when ethanol was the carbon/energy source, but produced no detectable acetic acid from glucose (Table 2 and data not shown). Further studies on the functioning of the enzymes involved in acetaldehyde oxidation are needed to fully explain these findings.

References

Carrascosa. J.M

Nunez de Castro. I. & SchetTers.

W.A. 1981 lvletabolism of acetaldehyde and Custers efl"ect in the

yeast Brel/([/IOI11)"Ci'S abslillells. AllIollie rail LccuH'ellllOek 47. 209-

215.

Viguera.

M.D

Custers. lv!.TJ. 1940 Onderzoekingen over het gistgeslacht Brcl/allo-

I11YCCS. PhD thesis. Technische Hoogeschool te Delft. Delft. The Netherlands. Dunn. S.A. & Schenk. R. U. 1980 Alternate highway deicing chemicals.

Federal Higll\I"{[Y Adl11illislralioll Rcporl FHWA-RD-79-106.

Fritzsche. CJ. 1992 Nonpoint source pollution. calcium magnesium acetate deicer. an efl"ective alternative for salt-sensitive areas.

rValer EII\"irolll11cllI alld Techllology 4, 44-51.

Geros. H

Azevedo. M.-M. & Cassio. F. 2000 Biochemical studies on

the production of acetic acid by the yeast Dckkera allol11ala. Food

Tcchllology alld Biolechllology 38. 59-62.

Levendis. Y·.A. 1991 Catalysis of the combustion of carbonaceous

particles (synthetic chars and coal) by addition of calcium acetate.

In

Calciul11

Maglicsiul11

ACelale.

All

El11ergilig

Bulk

Chel11ical

.iIJl·

EII\"irolll11elllal

Applicaliolls.

eds.

Wise.

0

Levendis.

Y.

& Metghalchi. M. pp. 221-246. Amsterdam: Elsevier. ISBN 044-

4885110.

Postma.

E

Verduyn. C. Schefl"ers. \iV.A. & van Dijken. J.P. 1989

glucose-limited

Enzymatic

analysis

of

the

Crabtree

effect

in

chemostat cultures of SaccharOlJl'\'ces cercl·islae. Applied alld EIIl"irolll11ellral Microbiology 55. 468-477.

Schefl"ers. W.A.

1961 On the inhibition of alcoholic fermentation in

B/"{.'llallol11yces yeasts under anaerobic conditions. E.ypcricmia 17.

40-46.

Scheffel'S. W.A. 1966 Stimulation of fermentation in yeasts by acetoin and oxygen. Na/urc ! LOlldoll) 210. 533-534.

1967 Effects of oxygen and acetoin on fermentation

and growth in Brettanomyces and some other yeast genera. In AI/i

XIV COllgrcsso dclla Socicla llalialla di Microbiologia lvlessina-

Taormina. Italy. 14-16 October 1967. pp. 91-107. Napoli. Italy:

Societa Italiana di lVlicrobiologia. Tipograria di Blasica. Scheffel'S. W.A. & Wiken. T.O. 1969 The Custers efl"ect (negative Pasteur effect) as a diagnostic criterion for the genus Brel/anolllY-

Scheffel'S. W.A.

ces. AllIollie rail Leeull'cllhock 35. A3 I-A32.

Seegmiller. J .E. 1955 TPN-1inked aldehyde dehydrogenase from yeast.

Alelhocl.\· ill En::Yl11ology. 1. 511-514.

Sharma.

Calcium impregnation of coals as a means for

In Calciul11 Magnesiul11

Acclalc. An El11erging Bulk Chcl11ical for Enl"ironl11clllal Applica-

sulfur emissions control in combustion.

P.K.

1991

Acetate Fom yeasts

Levendis. Y. & Metghalchi. M. pp. 273-296.

Amsterdam: Elsevier. ISBN 0444885110. Smith. M.Th. 1998 Dekkera van der Walt. In The Yeasls. A Taxonomic Sl1Idy. 4th edn. eds. Kurtzman. c.P. & Fell. J.W. pp. 174-177. Amsterdam: Elsevier. ISBN 0444813128.

Smith. ivLT

myces and Eenie!la: electrophoretic comparison of enzymes and

Yamazake. M. & Poot. G.A. 1990 Dekkera. Brellano-

lions. eds. Wise. D

DNA-DNA homology. YeaSI 6.299-310.

Sponholz. W.-R. 1993 Wine spoilage by microorganisms. In rVine

Fleet. G.H. pp. 395-420.

Switzerland: Harwood Academic Publishers. ISBN 3718651327. Steinman. C.R. & Jakoby. W.B. 1967 Yeast aldehyde dehydrogenase.

Microbiology

and Biolechnology.

ed.

Journal of Biological Chemislry 242. 5019-5023.

van der Walt. J.P. 1984a Brellanomyces KufTerath et van Lair. In The Yeasl. A Taxonomic Sl1Idy. 3rd edn. ed. Kreger-van Rij. NJ.W. pp. 562-567. Amsterdam: Elsevier Science Publishers. ISBN

0444804218.

275

van der Walt JP. 1984b Dekkera van der Walt. In The Yeasl,

A Taxonomic Sllnly. 3rd edn. ed. Kreger-van Rij. NJ.W. pp. 146- 150, Amsterdam: Elsevier Science Publishers. ISBN 0444804218,

van Kleeff. B.H.A. & Scheffers.

W.A. 1984 Inhibition of fermentation and growth in batch cultures of the yeast Brell{//lOmyces il1lermedius upon a shift from aerobic to

anaerobic conditions (Custers effect). Al1Ionie ran Leellll'enhock 50.

Wijsman. M,R

van Dijken. J.P

183-192.

Yamada. Y

Tahara. Y. & Smith. NIT

the classification of the ascosporo-

genous genus Dekkera and the asporogenolls yeast genus Brella-

nomyces. AllIonie ran Leellll'enhoek 46. 595-599,

Jin. Z. & ChoHar. B.H. 1997 Production of low-cost

acetate deicers from biomass and industrial wastes. In S/l())1'

Remoral and Ice COl1lrol Technology, pp. 60-69, Washington. DC:

Transportation

ISBN 0309062160.

Council.

Takinami-Nakamura. H

1980 The coenzyme Q system in

Yang. S.-T

Research

Board.

National

Research

Suppheo by U.S. DeDt of Agricufture

National Center for Agricultural

h

~ /+;1:., .' ·=l"nn p .,C-l~p?rr;.!, I e!ma. IHHV'!;'?

~.

0