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

Acetic acid production by DekkerajBrettallomyces yeasts

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

Received 4 September 2001: accepted 7 January 2002

Keywords: Acetic acid. Brettanomyces. Dekkera. ethanol

Summary

271

Yeast belonging to the genera Brettanom}'ces and Dekkera are noted for spoiling cellar and bottled wine through theproduction of haze, turbidity and acetic acid. However, I was unable to find information on the use of these yeastsfor 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 aceticacid. 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 batchculture using either glucose (l00 gil) or ethanol (35 gT) as the carbon/energy source. In the initial ethanolproduction screen, 19 strains produced at least 45 g ethanol/I. In the initial acetic acid screen, 28 of the yeast strainsproduced 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 gethanol/I.

Introduction

Acetic acid is an important industrial chemical with anannual domestic production of about 2.12 x 106 metrictons in 1995. Currently, the commercial production ofglacial acetic acid is exclusively by petrochemical routes.A potential industrial use of acetic acid is the productionof environmentally friendly de-icers, including calciummagnesium acetate (CMA), as a non-corrosive road de­icer, and potassium acetate and sodium acetate, asairport runway de-icers.

More than 9.07 x 106 metric tons of rock salt arepoured onto North American highways each year toprovide safer driving conditions during the wintermonths. This heavy use of de-icing chemical destroysroadside vegetation, damages aquatic ecosystems, pol­lutes groundwater and domestic potable water supplies,and damages the highway infrastructure (Fritzsche1992). Recognizing the negative impact that using saltas 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-

* Names are necessary to report factually on available data:however. the USDA neither guarantees nor warrants the standard ofthe product. and the use of the name by USDA implies no approval ofthe product to the exclusion of others that may be suitable.

tially acceptable, non-corrosive, environmentally benignalternative to road salt (Dunn & Schenk 1980).

CMA is a mixture of calcium acetate and magne­sium acetate. It is currently manufactured by reactingglacial acetic acid with dolomitic lime (CaO-MgO) orlimestone (Ca/MgC03). In addition to its potential useas a de-icer, there are reports that CMA, when used asan additive in coal-fired combustion units. reducessulphur dioxide emissions, thus, partially mitigatingthe problem of acid rain pollution (Sharma 1991:Levendis 1991).

Two microbiological approaches have been previouslyproposed 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 ethanoleffluent is aerobically converted to acetic acid byAcetobacter. In the other approach, Clostridium ther­moaceticum is used to convert glucose to acetic acid.High concentrations of acetic acid are produced in theyeast-Acetobacter process, however, the oxygen require­ment for Acetobacter conversion makes the processenergy intensive. The thermophilic process (55-60°C)requires only a single bioreactor, but it is also energyintensive and results in low final acetic acid levels.

The genus Dekkera and its anamorph, the genusBrettanomyces, are characterized by multipolar budding(van der Walt 1984a,b: Smith et al. 1990), the presenceof a Custers effect (also called a negative Pasteur effect)(Custers 1940: Scheffers 1961, 1966, 1967: Scheffers &

272 S.N. Freer

Wiken 1969: Wijsman er al. 1984), a coenzyme Q thatcontains nine isoprene units (Yamada er al. 1980), andthe formation of acetic acid (Custers 1940). Yeastsbelonging to these genera are often noted for spoilingcellar and bottled wine through the production of haze,turbidity and acetic acid (Sponholz 1993), as well asbeing used in the secondary fermentation of Iambic beer.However. I was unable to find any information on theuse of these yeasts for the express purpose of acetic acidproduction. Sixty yeast strains belonging to these andseveral other genera were screened for their ability toproduce ethanol and/or acetic acid from either glucoseor ethanol in batch culture.

Growrh, erhanol, carbohydrare and aceric acid analysis

Growth was measured by the increase in optical densityat 600 nm. Glucose. ethanol and acetic acid werequantified by high-pressure liquid chromatography(HPLC) on a Spectra-Physics chromatograph (ThermoFerinizara, Woodstoch, GA) fitted with a BioRad HP­87H column (Bio-Red Laboratories, Hercules, CA). Themobile phase was 5 mM sulphuric acid. The compoundsof interest were detected with a Waters 410 differentialrefractometer (Millipore, Watas Chromatography Divi­sion, Marborough, MA).

Results

Materials and methods

Organisms, media and grOlvrh condirions

Producrion ot' aceric acid and erhclI/olFom eirher glucoseor erhanol

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

Table 1. Growth. acetate and ethanol production from glucose(anaerobic fermentation).

The capacity of 60 yeast strains to produce ethanol andacetic acid, when grown anaerobically, from 100 gglucose/I is presented in Table 1. In general, the yeaststested were reasonably efficient at fermenting glucose.Only three yeasts (Brerranomyces spp. NRRL YB-5241.YB-3695 and YB-3694) produced no ethanol fromglucose. Of the 60 yeasts tested, 19 produced at least45 g ethanol/L while only 10 yeasts produced less than

B. bruxellensisY-1412 25.84 1.1 45.6 0.0Y-1411 24.78 0.0 48.5 0.0B. lall/bicusY-1330 16.05 0.0 51.9 0.0B. clallsselliiY-2290 33.89 3.0 49.0 0.0Y-1414 30.44 0.0 49.4 0.0B. anoll/alusY-1415 30.40 0.4 47.1 2.8Y-12670 31.17 0.0 50.4 0.0Brellil/lOlI/)"ces sp.YB-5260 13.67 3.4 48.9 0.3D. illll!l"lI/ediaYB-4553 41.37 0.0 48.9 0.0Y-I092 38.11 1.0 48.5 0.0YB-4241 32.01 1.3 49.4 0.0D. bruxellellsisY-17534 37.65 1.0 45.0 0.0Y-12961 21.74 1.0 50.6 0.0D. anoll/alaY-17521 29.79 0.0 48.9 0.0'{-I 7520 29.60 0.0 48.7 0.0Y-17522 29.30 0.0 50.1 0.0Z. bailiiY-2227 8.09 1.0 49.6 3.4Z. bi.\]'orusY-7558 21.31 0.0 46.1 3.0P. Iel'll/ell/ansY-1619 14.19 2.0 45.6 0.0

Residual glucose(g;J)

Ethanol(gl)

.-\cetate(g I)

Growth(A600 )

Culture"(NRRL)

The following yeasts were tested in this study: Brerrano­myces bruxellensis Y-1412. Y-1411. Y-1441: B. lambicusY-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 Y­7706: Brerranolllyces spp. YB-5226, YB-5225, YB-5234,YB-5231. YB-5240. YB-5233. YB-5227. YB-5260. Y­5740. YB-5243. YB-5235. YB-5241. YB-3695. YB-3696.YB-954. YB-3694. Y-7447. YB-5230. YB-3998. YB­5212. Y-5242: Dekkera inrermedia YB-5164. YB-4553.Y-I092. YB-4241: D. bruxellensis Y-17523. Y-17535. Y­17525. Y-17524. Y-17534. Y-12961: D. naadenensis Y­17526: D. anolllaia Y-17521. Y-17520. Y-17522: Eeniellanana Y-17527, Y-17533: Zygosaccharomyces bailii Y­2227: Z. bisporus Y-7558: Pichia lIlembranaefaciens Y­2026, 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 ARSCulture Collection, Peoria, IL and are designated bytheir 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 cellsfrom a fresh slant to 20 ml of YP medium containing20 g glucose/I. The cultures were incubated aerobicallyon 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 at7000 x g for 10 min, washed once in sterile distilledwater, and suspended in 20 ml of sterile distilled water,The experimental flasks were inoculated using 0.9 ml ofcell suspension. For acetic acid production, initialscreening was performed in 125 ml baffled flasks con­taining 30 ml of basal medium prepared with either100 g glucose/lor 35 g ethanol!1. For ethanol produc­tion. 30 ml of basal medium containing 100 g glucosellin 50 ml flasks were inoculated and capped with serumstoppers and vented with No. 26 gauge sterile needles.The experimental cultures were incubated at either 24 or30°C on a rotary (New Brunswich Scientific Co., Inc.,Edison, NJ) shaker at 250 rev/min.

Acetate Fom yeasts

10 g ethanol/I. The six strains that produced the mostethanol were B. lambicus NRRL Y-1330. D. bruxellensisNRRL Y-12961. B. anomalus NRRL Y-12670. D. ano­mala NRRL Y-17533. B. c!aussenii NRRL Y-1414 andD. intermedia NRRL YB-4241. As expected, none of theyeasts produced large amounts of acetic acid whengrown fermentatively.

The capacity of the yeasts to produce acetic acid whengrown aerobically in medium initially containing 35 gethanol/l is presented in Table 2. All of the yeasts tested,except Brettanomyces sp. NRRL YB-5235, utilized, tovarying degrees. ethanol as a carbon/energy source.However, 24 of the 60 yeast strains tested produced nodetectable acetic acid when grown on ethanol. Twenty­three of the yeasts produced greater than 5 g acetic acid1 while II of the strains tested produced greater than20 g acetic acid/I. The five strains that produced themost acetic acid from ethanol were D. bruxellensisNRRL Y-17525. B. intermedius NRRL Y-2394. B. cus­tersianus NRRL Y-6653. D. intermedius NRRL Y-5164and D. anomala NRRL Y-17520. All of these yeastsproduced over 25 g acetic acid/!. however, none com­pletely utilized the initial 35 g ethanol:!.

The capacity of the yeasts to produce acetic acid andethanol when grO\vn aerobically in medium initiallycontaining 100 g glucose(1 is also presented in Table 2.Forty-seven of the yeast strains tested produced detect­able levels of ethanol under these growth conditions.

273

Twenty-six of the yeasts produced no detectable aceticacid when grO\vn aerobically on glucose. Sixteen of thesestrains produced no detectable ethanol either, eventhough many of them completely utilized the initial100 g glucoserl. No other fermentation products, in­cluding glycero!. were detected by HPLC in the culturebeers of these yeasts. Twenty-eight of the strains testedproduced at least 5 g acetic acidrl. Of these, 16 producedover 20 g acetic acid/!. The five strains that produced themost acetic acid from glucose were D. intermedia NRRLY-5164 and NRRL YB-4553. B. intermedius NRRL Y­2394 and NRRL Y-2395. and D. bruxellensis NRRL Y­17525. All of these cultures produced over 29 g aceticacid/!. however, none of these cultures completelyutilized the 100 g glucose:l initially present in themedium.

Effect ot'temperature

All of the cultures were tested for ethanol and/or aceticacid production at 30°C using glucose (100 gil) orethanol (35 g/l) as the carbon/energy source (data notshown). In genera!. the cultures made about the sameamount of product, regardless of the growth tempera­ture. However, the cultures grown at 30°C tended toproduce the product more rapidly. When grown at30 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. bruxellcnsisY-1412 48.93 2.4 , ~ 35.37 22.9 21.8 14.4B. clausseniiY-2290 12.19 10.0 26.5 34.61 24.6 15.4 41.2B. anomalusY-1415 23.05 17.6 16.7 39.51 22.4 13.8 17.1B. cuslersiallllS

Y-6653 24.81 28.0 10.1 82.63 0.0 0.0 53.4B. illlermediusY-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 31.2YB-3363 31.58 24.0 13.8 '-' ..,.., 23.7 I 1.0 43.6~.'l.;:.._"'\~'"

D. illlcrmcdiaYB-5164 35.49 27.7 6.5 33.99 31.7 6.1 48.1YB-4553 39.98 24.3 10.7 33.72 30.0 24.3 57.4Y-I092 17.92 18.3 18.0 32.09 21.2 16.7 33.4YB-4241 20.75 18.3 17.4 33.15 21.6 21.6 20D. bruxellensisY-17523 24.70 23.4 8.5 27.13 24.0 6.2 67.9Y-17535 29.52 " ~ 15.6 28.56 18.7 10.5 49.1Y-17525 31.95 33.0 10.4 34.96 31.6 15.7 26.1Y-17524 31.50 24.6 14.4 29.70 26.9 7.0 52.3Y-17534 30.21 22.0 15.2 31.26 19.0 8.5 52D. anomalaY-17521 10.50 17.8 26.4 29.00 23.0 18.6 29.81'-17520 24.20 25.3 18.5 26.66 24.4 10.2 24.4Y-17522 12.39 14.8 ...,") ...

29.19 20.4 7.7 58.8.... .;;.._1

" Only cultures that produced at least 20 g acetic acid from either ethanol (left) or glucose (right) are listed.

274

completion, as measured by the presence of a constantresidual carbon source and no further increase in aceticacid production. The only exception to this was B. bru."\'­e/lensis NRRL Y-1411. This strain produced 0.9 and19.9 g acetic acid/I when grown on ethanol at 24°C and30 0c, respectively (data not shown). When glucose wasthe carbon/energy source, NRRL Y-1411 produced 18.4and 18.8 g acetic acid/l when grown at the two temper­atures (data not shown).

Discussion

Acetic acid is believed to be produced in yeast by theoxidation of acetaldehyde, which can be formed eitherby the pyruvate dehydrogenase bypass or by theoxidation of ethanol. In both pathways, acetaldehydeis a free intermediate that is converted to acetate byaldehyde dehydrogenase and subsequently activated toacetyl-CoA by acetyl-CoA synthetase. Two aldehydedehydrogenases (one constitutive and one regulated)have been described in yeasts (Seegmiller 1955: Stein­man & Jakoby 1967; Carrascosa et al. 1981). It has beensuggested that the constitutive aldehyde dehydrogenasefunctions in the pyruvate dehydrogenase bypass andthat the regulated aldehyde dehydrogenase functions inthe oxidation of ethanol.

Apparently, acetate is excreted into the medium if it isnot completely activated to acetyl-CoA. In D. anomala(Geros et al. 2000), acetyl-CoA synthetase is repressedby glucose. Thus. when grown aerobically in mediumcontaining high concentrations of glucose, conditionsthat favor the excretion of acetic acid are invoked.Similarly, acetate is produced in Saccharomyces cerevi­siae if there is insufficient acetyl-CoA synthetase activitypresent for the complete oxidation of acetate to acetyl­CoA (Postma et al. 1989). However, in D. bruxe/lensis(syn. B. abstinens: Smith 1998) acetyl-CoA synthetaseactivity does not appear to be repressed by glucose: thus,even under culture conditions where high amounts ofacetic acid are produced, the pathway does not appearto be blocked (Carrascosa et al. 1981).

The results of the screen for acetic acid productionindicated that the majority of the Dekkera/Brettanomy­ces spp. deposited in the ARS Culture Collection werecapable of producing acetic acid when either glucose orethanol \vas used as a carbon/energy source. Overall. thefive strains that produced the most acetic acid were D.intermedia NRRL YB-4553 and YB-5164. B. intermedi­us NRRL Y-2394. D. bruxe/lensis NRRL Y-17525. andD. anomala NRRL Y-17520. All of these strains fer­mented glucose efficiently (Table I), and produced over24 g acetic acid/I from both glucose and ethanol(Table 2).

In generaL the efficiency of acetic acid production wasreasonably low. This was probably due, in part, to theinhibitory effects that acetic acid had upon the yeasts, asthe 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 acidproduced. When grown aerobically, the pH values of theculture beer after 7 days incubation ranged from 4.2 to6.8. while the anaerobic cultures ranged from 5.1 to 6.3(data not shown).

Finally, not all of the strains produced acetic acidfrom both glucose and ethanol. Three strains. NRRL Y­2395, NRRL Y-1411 and NRRL Y-1296L producedacetic acid from glucose. Little, if any, acetic acid wasproduced when ethanol was used as the carbon/energysource, although the yeasts grew well (Table 2 and datanot shown). Conversely, three strains, NRRL Y-6653,NRRL Y-5240 and NRRL Y-5235, produced aceticacid when ethanol was the carbon/energy source, butproduced no detectable acetic acid from glucose (Table 2and data not shown). Further studies on the functioningof the enzymes involved in acetaldehyde oxidation areneeded to fully explain these findings.

References

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Custers. lv!.TJ. 1940 Onderzoekingen over het gistgeslacht Brcl/allo­I11YCCS. PhD thesis. Technische Hoogeschool te Delft. Delft. TheNetherlands.

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Suppheo by U.S. DeDt of AgricuftureNational Center for Agricultural

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