Appl Microbiol Biotechnol (2006) 70: 366–373 DOI 10.1007/s00253-005-0073-z
APPLIED MICRO BIAL AND CELL PHYSIOLOGY
Janja Trcek . Hirohide Toyama . Jerzy Czuba . Anna Misiewicz . Kazunobu Matsushita
Correlation between acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria Received: 9 March 2005 / Revised: 24 June 2005 / Accepted: 24 June 2005 / Published online: 17 August 2005 # Springer-Verlag 2005
Abstract In this study, we compared the growth properties and molecular characteristics of pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) among highly acetic acid-resistant strains of acetic acid bacteria. Gluconacetobacter europaeus exhibited the highest resistance to acetic acid (10%), whereas Gluconacetobacter intermedius and Acetobacter pasteurianus resisted up to 6% of acetic acid. In media with different concentrations of acetic acid, the maximal acetic acid production rate of Ga. europaeus slowly increased, but specific growth rates decreased concomitant with increased concentration of acetic acid in medium. The lag phase of A. pasteurianus was twice and four times longer in comparison to the lag phases of Ga. europaeus and Ga. intermedius, respectively. PQQ-dependent ADH activity was twice as high in Ga. europaeus and Ga. intermedius as in A. pasteurinus. The purified enzymes showed almost the same specific activity to each other, but in the presence of acetic acid, the enzyme activity decreased faster in A. pasteurianus and Ga. intermedius than in Ga. europaeus. These results suggest that high ADH activity in the Ga. europaeus cells and high acetic acid stability of the purified enzyme represent two of the unique features that enable this species to grow and stay metabolically active at extremely high concentrations of acetic acid. J. Trcek (*) Limnos, Podlimbarskega 31, Ljubljana, 1000, Slovenia e-mail:
[email protected] Fax: +386-1-3651507 J. Trcek . H. Toyama . K. Matsushita Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan J. Czuba . A. Misiewicz Institute of Agricultural and Food Biotechnology, Rakowiecka 36, Warsaw, 02-532, Poland
Introduction The industrial production of high-acid-percentage vinegar (≥10%) is mainly performed in Frings acetators which enable production of vinegar in a few days (Ebner and Follmann 1983). Recently published data showed that bacterial population in this kind of bioreactor is essentially more heterogeneous than we thought, according to previous investigations based on the methods available at that time (Boesch et al. 1998; Kittelmann et al. 1989; Sievers and Teuber 1995; Sokollek et al. 1998; Trcek et al. 1997; Trcek and Raspor 1999). In 1992, Sievers et al. (1992) isolated a new species, Gluconacetobacter europaeus, a predominating species in the industrial bioreactors for high-acid vinegar production. In the last years, four new species were published and likewise isolated from industrial bioreactors for the production of vinegar (Boesch et al. 1998; Schüller et al. 2000; Sokollek et al. 1998). Acetic acid is detrimental already at 0.5% for the majority of microorganisms (Conner and Kotrola 1995). Although the acetic acid bacteria are naturally resistant to acetic acid, substantial differences in tolerance to acetic acid exist among species. The mechanisms of acetic acid resistance have been so far studied in Acetobacter aceti (Fukaya et al. 1990, 1993; Menzel and Gottschalk 1985; Nakano et al. 2004; Steiner and Sauer 2003). Since vinegar-producing species of the genus Gluconacetobacter generally exhibit much higher acetic acid resistance than A. aceti, Gluconacetobacter species must have developed special characteristics for surviving and remaining metabolically active at extreme growth conditions in industrial vinegar bioreactors (low pH, high concentrations of acetic acid). Therefore, they are able to overgrow and to eliminate the other acetic acid bacterial species from submerged bioreactors. The highest resistance against acetic acid was described for the following species: Ga. europaeus, Gluconacetobacter intermedius, Gluconacetobacter oboediens, and Gluconacetobacter entanii (Boesch et al. 1998; Schüller et al. 2000; Sievers and Teuber 1995; Sokollek et al. 1998).
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Since no comparative study between Acetobacter sp. and Gluconacetobacter sp. has been reported so far, the aim of this study was to compare the growth characteristics, acetic acid production rate, and acetic acid tolerance among Ga. europaeus, Ga. intermedius and Acetobacter pasteurianus. Since these species exhibited an obvious difference in acetic acid production rate and in their maximal acetic acid tolerance, we hypothesized differences in characteristics of their pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH). For testing our hypothesis, we purified the ADHs from Ga. europaeus, Ga. intermedius, and A. pasteurianus and characterized them in more detail. Besides, we also compared the partial sequences encoding the subunit I of ADH among species analyzed in this work as well as to some other representatives of acetic acid bacteria.
Materials and methods Strains and growth conditions All strains used in this study were isolated from industrial vinegar bioreactors. Vinegars used as sources of isolation are described in Table 1. Isolation of the species belonging to genus Gluconacetobacter was described previously (Trcek et al. 2000). A. pasteurianus was isolated on semiliquid medium containing 0.1% glucose, 0.045% (NH4)2HPO4, 0.013% K2SO4, 0.030% MgSO4·7H2O, 1% baker’s yeast extract (mixture of equal amounts of pressed baker’s yeast and 10% acetic acid), and 0.3% of agar by a method described previously (Czuba et al. 1991). The strains are deposited into international culture collections: the Belgian Coordinated Collections of Microorganisms (BCCM), German Collection of Microorganisms and Cell Cultures (DSMZ), and Collection of Industrial Microorganisms in Warsaw (CIM) (Table 1).
Table 1 Strains of acetic acid bacteria used in this study Strain
Source of isolation
Gluconacetobacter europaeus V3 (LMG 18494) Gluconacetobacter intermedius JK3 (DSM 13111) Acetobacter pasteurianus (KKP 584)
Nonfiltered red wine vinegar (10 vol%) produced in Frings acetators installed in Ljubljana (Slovenia) Nonfiltered cider vinegar (5.5 vol%) produced in Frings acetators installed in Ljubljana (Slovenia) Polish submerged vinegar bioreactor started with seed vinegar from generator
LMG Laboratorium voor Microbiologie, Gent; DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen; KKP Kolekcja Kultur Przemysłowych, Warsaw
The restriction analysis of the PCR-amplified 16S-23S rDNA spacer region Amplification of the 16S-23S rDNA and restriction analysis were performed as described previously (Trcek and Teuber 2002). The length of the restriction fragments was calculated relative to that of the DNA marker by linear regression of the semilogarithmic curve (mobility vs logarithm of DNA fragment length) and compared to the database described elsewhere (Trcek and Teuber 2002). Sequencing of the adh subunit I The available adhA (adh subunit I) sequences from the EMBL/Genbank/DDBJ databases were used for designing degenerate primers for PCR amplification and sequencing. The primers and procedure used for amplification and sequencing of a part of the adhA (position 1,236–1,719 with the sequence of adhA of Acetobacter europaeusT accession no. Y09480) is described elsewhere (Trcek 2005). The forward and reverse strands of partial adhA were sequenced. The sequences were aligned by a Clustal_X 1.8 program (Thompson et al. 1997) using the default settings of a multiple alignment tool. The percentage of similarity was calculated by the Jotun–Hein algorithm included in the MegAlign program (DNASTAR Inc., USA). Determination of physiological parameters Each strain was grown in an AE broth (0.3% yeast extract, 0.4% peptone, and 0.5% glucose) containing 1% (v/v) of acetic acid and 1% (w/v) of ethanol. Five milliliters of culture with a density of around 70 Klett units (corresponds to A600=0.5) was used as an inoculum in a 500-ml baffled flask containing 50 ml AE broth with a defined concentration of acetic acid and ethanol. Six milliliters of this culture from an exponential growth phase was used as an inoculum for a bioprocess performed in a 3-l baffled flask containing 600 ml AE broth with a defined concentration of acetic acid and ethanol. The flasks were incubated at 30°C and 200 rpm. Growth was monitored by measuring the optical density with a Klett–Summerson colorimeter using a red filter (640–700 nm). The exponential growth phase was identified in a plot of log(Klett Units) vs time. The specific growth rates (μ) were calculated by linear regression of ln(Klett Units) vs time, with growth rate as the regression coefficient. The acidity of the culture medium was measured with 1 N NaOH with phenolphthalein as indicator. The remaining glucose and ethanol concentration in the medium was measured spectrophotometrically at 600 nm in reaction with glucose- and alcohol-dehydrogenases. The reactions were done with phenazine methosulfate (PMS) coupled with 2,6-dichlorophenol indophenol (DCIP) as an artificial electron acceptor in KPB buffer pH 7.0 (Ameyama 1982).
368
The high acid-containing samples were neutralized with 1 N NaOH before analysis. Preparation of membrane and soluble proteins Cells were harvested by centrifugation at 6,500 rpm for 10 min and washed twice with ice-cold 50 mM KPB (pH 5.8). The washed cells were suspended at 0.1 g of wet weight per 3 ml in the same buffer and passed twice through a French pressure cell press at 16,000 psi. The intact cells were removed by centrifugation at 9,000 rpm for 10 min, and the obtained supernatant was centrifuged at 45,000 rpm for 90 min to separate the soluble fraction from the membrane fraction. The membrane fraction was homogenized with 10 mM KPB (pH 6.0). The proteins from the soluble fraction were precipitated with 10% TCA for 1 h at 4°C. The precipitates were washed twice with acetone and dissolved in water. Purification of PQQ-dependent ADH Pyrroloquinoline-quinone-dependent ADHs of A. pasteurianus, Ga. europaeus, and Ga. intermedius were purified from solubilized membrane fraction by a diethylaminoethyl (DEAE)–Toyopearl column followed by a hydroxyapatite column as described by Matsushita et al. (1992). The ferricyanide reductase activity of ADH was measured colorimetrically with potassium ferricyanide as an electron acceptor at 660 nm (Ameyama 1982). The reaction mixture containing the enzyme fraction, ethanol, and potassium ferricyanide was prepared in McIlvaine buffer pH 4.0 in the case of Ga. europaeus, Ga. intermedius, and A. pasteurianus, and in McIlvaine buffer pH 5.0 in the case of Gluconobacter oxydans. Protein contents were measured by a modified Lowry’s method (Lowry et al. 1951) with bovine serum albumin as a standard protein. Errors were calculated as standard deviations. For comparing the stability of the purified enzymes in buffer with different concentrations of acetic acid, the protein and detergent (Triton X-100) contents were adjusted to 1 mg/ml and 0.1%, respectively, in each sample of the purified enzyme. Equal volumes of enzyme suspension and 10 mM KPB (pH 6) buffer containing an appropriate concentration of acetic acid were mixed and incubated for 30 min at 4°C. The ferricyanide reductase activity of ADH was measured as described above.
(AJ635212), Gluconacetobacter xylinusT LMG 1515 (AJ6352220), Gluconacetobacter hanseniiT LMG 1527 (AJ635209), Gluconacetobacter liquefaciensT LMG 1382 (AJ635211), A. pasteurianusT DSM 3509 (AJ635216), Acetobacter pomorumT LTH 2458 (AJ635215), A. acetiT DSM 3508 (AJ635213), and Gluconacetobacter polyoxogenes (D00635) were obtained from the GenBank/EMBL/ DDBJ databases.
Results Taxonomic characterization of the isolates The taxonomic characterization of strains belonging to genus Gluconacetobacter has been described previously (Trcek et al. 2000). A strain isolated from the Polish vinegar bioreactor was identified in this work by restriction analysis of the 16S-23S rDNA as belonging to the group A. pasteurianus/A. pomorum. Additional sequencing of the PCR-amplified partial 16S rRNA gene showed 99% identity to the 16S rRNA sequences of A. pasteurianus deposited in the GenBank/EMBL/DDBJ databases under accession numbers AJ419834 and AB086016. Sequence comparison of the partial gene encoding AdhA The deduced partial AdhA sequences were aligned and compared to a homologous region of some other species of genera Acetobacter, Gluconacetobacter, and Gluconobacter (Fig. 1). The partial AdhA of Ga. europaeus V3 exhibited 100% amino acid identity to Ga. europaeusT, “Ga. polyoxogenes,” Ga. intermediusT, and Ga. intermedius JK3. The AA identity between partial AdhA of Ga. europaeus and the homologous region of Ga. xylinusT, Ga. hanseniiT, and Ga. liquefaciensT was 94.4, 90.7, and 84.0%, respectively. A comparison of the partial AdhA sequence of A. pasteurinaus KKP 584 to Ga. intermedius JK3 and Ga. europaeus V3 showed only 73% AA identity. The aligned sequences revealed in Gluconobacter strains an additional stretch of 15 nucleotides at position 229 nt (position 1,461 with the sequence of the adhA of A. europaeusT, accession no. Y09480). Consequently, both Gluconobacter species contain in the middle of deduced partial AdhA sequence an additional five amino acids (Fig. 1).
Nucleotide sequence deposition numbers
Influence of acetic acid concentration on specific growth rates and acetic acid production
The nucleotide sequence of the partial adhA of A. pasteurianus KKP 584 was deposited into the EMBL database under accession number AJ888874. The adhI sequences of Gluconobacter cerinusT DSM 9533 (AJ635218), G. oxydans DSM 2343 (CP000009), Ga. europaeusT DSM 6160 (Y09480), Ga. intermedius JK3 (AJ635222), Ga. intermediusT DSM 11804 (AJ635221), Ga. europaeus V3
Among the analyzed strains, Ga. europaeus exhibited the highest resistance to acetic acid. The strain grew in AE broth containing 7% acetic acid and 3% ethanol, both added into the medium at the beginning of cultivation (Fig. 2). A. pasteurianus grew well in medium with 3% acetic acid and 3% ethanol (3a/3e), but the length of its lag growth phase was twice (4 days) as long as of Ga. euro-
369 G. cerinusT DSM 9533 G. oxydans DSM 2343 Ga. intermedius JK3 "Ga. polyoxogenes" Ga. intermediusT DSM 11804 Ga. europaeus V3 Ga. europaeusT DSM 6160 Ga. xylinusT LMG 1515 Ga. hanseniiT LMG 1527 Ga. liquefaciensT LMG 1382 A. pasteurianusT DSM 3509 A. pasteurianus KKP 584 A. pomorumT LTH 2458 A. aceti T DSM 3508
DALWTLNGKPWYGIPGDLGGHNFAAMAYSPQTKLVYIPAQQVPFVYDPQKGGFKAHHDSWNLGLDMNKIGLLDDNDPQHKADKA DALWTLTGKPWLGIPGELGGHNFAAMAYSPKTKLVYIPAQQIPLLYDGQKGGFKAYHDAWNLGLDMNKIGLFDDNDPEHVAAKK DALYTLTGKEWYGIPGDLGGHNFAAMAFSPKTGLVYIPAQQVPFLYTNQVGGFTPHPDSWNLGLDMNKVGIPDSPE-----AKQ DALYTLTGKEWYGIPGDLGGHNFAAMAFSPKTGLVYIPAQQVPFLYTNQVGGFTPHPDSWNLGLDMNKVGIPDSPE-----AKQ DALYTLTGKEWYGIPGDLGGHNFAAMAFSPKTGLVYIPAQQVPFLYTNQVGGFTPHPDSWNLGLDMNKVGIPDSPE-----AKQ DALYTLTGKEWYGIPGDLGGHNFAAMAFSPKTGLVYIPAQQVPFLYTNQVGGFTPHPDSWNLGLDMNKVGIPDSPE-----AKQ DALYTLTGKEWYGIPGDLGGHNFAAMAFSPKTGLVYIPAQQVPFLYTNQVGGFTPHPDSWNLGLDMNKVGIPDSPE-----AKQ DALYTLTGKDWYGIPGDLGGHNFAAMAYSPKTGLVYIPAQQVPFLYTNQVGGFTPHPDSWNLGLDMNKVGIPDTPE-----AKQ DALYTLTGKDWYGIPGDLGGHNFAAMAFSPKTGLVYIPAQQVPFLYTNQKGGFLAHPDSWNLGLDMNKVGIPDSPE-----AKA EALWTLTGKDWYGIPGDLGGHNFAAMAFSPRTGLVYIPAQQVPFVYSNQVGGFKAHPDSWNLGLDMNKVGLPDKQD-----AKD DGLYTLNGKFWYGIPGPLGAHNFMAMAYSPKTHLVYIPAHQIPFGYKNQVGGFKPHADSWNVGLDMTKNGLPDTPE-----ART DGLYTLNGKFWYGIPGPLGAHNFMAMAYSPKTHLVYIPAHQIPFGYKNQVGGFKPHADSWNVGLDMTKNGLPDTPE-----ART DGLYTLNGKFWYGIPGPLGAHNFMAMAYSPKTHLVYLPAHQIPFGYKNQVGGFKPHPDSWNVGLDMTKNGLPDTPE-----ART EGLYTLTGKDWYGIPGPLGAHNFMEMAYSPRTHLIYLPAHQIPFGYKNQSGGFKPHPDSWNLGLDMTKTGLPDTPE-----ARA : :.*:**.** * **** **.*** **:**:* *:*:**:*:*: * * *** .: *:**:****.* *: * :
G. cerinusT DSM 9533 G. oxydans DSM 2343 Ga. intermedius JK3 "Ga. polyoxogenes" Ga. intermediusT DSM 11804 Ga. europaeus V3 Ga. europaeusT DSM 6160 Ga. xylinusT LMG 1515 Ga. hanseniiT LMG 1527 Ga. liquefaciensT LMG 1382 A. pasteurianusT DSM 3509 A. pasteurianus KKP 584 A. pomorumT LTH 2458 A. aceti T DSM 3508
QFLKDLKGWIVAWDPQKQQAAFTVDHKGPWNGGLLATAGGVLFQGLATGEFHAYDATTGKDLFKFPAQSAIIAPPVTYTAHGK DFLKVLKGWTVAWDPEKMAPAFTINHKGPWNGGLLATAGNVIFQGLANGEFHAYDATNGNDLYSFPAQSAIIAPPVTYTANGK AFVKDLKGWIVAWDPQKQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLFHFAADSGIIAPPVTYLANGK AFVKDLKGWIVAWDPQKQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLFHFAADSGIIAPPVTYLANGK AFVKDLKGWIVAWDPQKQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLFHFAADSGIIAPPVTYLANGK AFVKDLKGWIVAWDPQKQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLFHFAADSGIIAPPVTYLANGK AFVKDLKGWIVAWDPQKQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLFHFAADSGIIAPPVTYLANGK AFVKDLKGWIVAWDPKTQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLFHFAADSGIIAPPVSYLGSDK AFVKDLKGYILAWDPQKQAEAWRVDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGTDLFHFAADSGIIAPPVTYLAKGK AFIKDLKGWIVAWDPIKQQEAFRVDHKGPWNGGIVATGGDLLFQGLANGEFHAYDATNGNDLFSFAAQSGIIAPPVTYLANGK AYIKDLHGWLLAWDPVKMETVWKIDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLYKFDAQSGIIAPPMTYSVNGK AYIKDLHGWLLAWDPVKMETVWKIDHKGPWNGGILATGGDLLFQGLANGEFHAYDATNGSDLYKFDAQSGIIAPPMTYSVNGK AYIKDLHGWLLAWDPVKMETVWKIDHKGPWNGGVLATGGDLLFQGLANGEFHAYDATNGSDLYKFDAQSGIIAPPMTYSVNGK AYMQDLKGWLLAWDPVKLQAQWSIERAGGWDGGVLATGGDLVFQGLATGEFHAYDATNGKDLFKYDLQSGIIANPVTYSVNGK .* : ::: * *:**::**.*.::*****.*********.*.**: : :*.*** *::* ::: *:*: :**** .
84 84 79 79 79 79 79 79 79 79 79 79 79 79
167 167 162 162 162 162 162 162 162 162 162 162 162 162
Fig. 1 Alignment of the partial amino acid sequences of subunit I of the PQQ-dependent ADHs from genera Acetobacter, Gluconacetobacter and Gluconobacter. The region is positioned in the middle of AdhA (412 to 573 AA with AdhA of Ga. europaeus,
accession no. Y09480). Asterisks indicate conserved amino acid, colons indicate conservative amino acid replacements, and dots indicate the semiconservative amino acid replacements. Quotation marks (“ ”) represent the not-validly published species
paeus (2 days) (Fig. 3). At higher concentration of acetic acid (AE broth 4a/3e), A. pasteurianus grew weakly and finally stopped growing upon reaching an acidity of 5.5%. Ga. intermedius also grew well in AE broth 3a/3e without lag phase (Fig. 3b). In contrast to Ga. europaeus and Ga. intermedius, A. pasteurianus cannot overoxidize acetic acid in AE broth 3a/3e (Fig. 3c), although it overoxidized acetic acid at lower concentrations of acetic acid. In the same medium, Ga. europaeus started to metabolize glucose
just before exhausting all ethanol, and acetic acid was further overoxidized after 2 days of consuming the ethanol (Fig. 3a). Ga. intermedius started to use glucose 2 days before oxidizing all ethanol, but the overoxidation of acetic acid proceeded 8 days after consuming all the ethanol (Fig. 3b). The length of the lag phase of Ga. europaeus was 2 days in AE medium with 3, 4, and 5% acetic acid. Further increasing acetic acid concentrations to 6 and 7% pro-
Fig. 2 Comparison of acetic acid production and growth rates of Ga. europaeus V3 in 3-l baffled shake flasks containing 600 ml AE broth with 3% of ethanol (3e) and 3–7% of acetic acid (3a–7a). Growth is presented in a semilogarithmic plot
11
AE 3a/3e
10
AE 4a/3e AE 5a/3e
9
AE 6a/3e AE 7a/3e Cell density (Klett Units)
8 7 Acidity (%)
1000
6 5 4 3
100
2 1 0
10 0 2 4 6 8 10 12 14 16 18 20
0 2 4 6 8 10 12 14 16 18 20
Time (days)
Time (days)
370 6
400 350
5
300 4
250
3
200 150
2
100 1
Growth (Klett units)
Acidity (%)/Ethanol (%)/Glucose (g/L)
A
50
0
0 0
2
4
6
8
10
12
14
16
18
20
Tim e (days)
180
6
160 5 140 120
4
100 3 80 2
60
Growth (Klett Units)
Acidity (%)/Ethanol (%)/Glucose (g/L)
B
40 1 20 0
0 0
2
4
6
8
10
12
14
16
18
20
Tim e (days)
6
Purification of PQQ-dependent ADH from Ga. europaeus, Ga. intermedius, and A. pasteurianus
90 5
80 70
4
60 50
3
40 2
30 20
1
10 0
0 0
2
4
6
8
10
12
14
16
18
20
Tim e (days)
Legend:
longed the lag phase to about 3 and 5 days, respectively (Fig. 2). Ga. europaeus did not exhibit a stationary growth phase after the ethanol oxidation phase in AE broth 3a/3e, but it continuously grew further on (Fig. 2). However, the same strain exhibited a typical stationary growth phase of about 6 days in AE broth 4a/3e, and thereafter started to overoxidize acetic acid (Fig. 2). In AE medium with 5% and higher concentrations of acetic acid, Ga. europaeus did not overoxidize acetic acid. Ga. europaeus exhibited the maximal acetic acid production rate in AE broth 7a/3e. The maximal acetic acid production rate slowly increased concomitant with the amount of acetic acid added into the medium from 0.64, 0.75, 0.77, 0.94 to 1.08 g l−1 h−1 in media with 3, 4, 5, 6, and 7% acetic acid. In the bioprocesses performed in 3-l baffled flasks, the growth rate and the final biomass yields were both affected by concentration of acetic acid added to the medium (Fig. 2). An increased amount of acetic acid decreased the final biomass yield. The maximal specific growth rates of Ga. europaeus decreased with the addition of 4% acetic acid (AE broth 4a/3e) to 89% (0.58 h−1) of that obtained in AE broth 3a/3e (0.65 h−1). A further increasing of acetic acid concentration to 5 and 6% decreased the specific growth rates to about 71% (0.46 h−1) and 58% (0.38 h−1) of that obtained in AE broth 3a/3e, respectively. An additional increase in acetic acid concentration to 7% did not have a significant effect on the maximal specific growth rate.
100
acidity % (w/v)
glucose (g/L)
Klett Units
ethanol % (v/v)
Growth (Klett Units)
Acidity (%)/Ethanol (%)/Glucose (g/L)
C
3 Fig. 3 Cultivation of Ga. europaeus V3 (a), Ga. intermedius JK3 (b), and A. pasteurianus KKP 584 (c) in 3-l baffled flasks containing 600 ml AE broth with 3% of acetic acid and 3% of ethanol. Processes were started with 6 ml of inoculum taken from an exponential phase of smaller-scale processes performed in the same type of medium. The flasks were incubated at 30°C and 200 rpm
Cells were harvested at the late logarithmic growth phase from media containing 5.5–6.0% acetic acid in medium AE (3a/3e). The membrane fraction prepared from the cells exhibited specific ADH activity between 16 and 18 U/mg for Ga. europaeus and Ga. intermedius and 8 U/mg for A. pasteurianus. ADHs were further purified to almost homogeneity as judged from the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Two subunits of ca. 72 and 45 kDa were detected by SDS-PAGE in Ga. europaeus and Ga. intermedius, and three subunits ADH of ca. 74, 44, and 16 kDa in A. pasteurianus (Fig. 4; Table 2). The presence of the third subunit in ADHs of Ga. intermedius and Ga. europaeus was also checked with immunoblot analysis using antibody raised against ADH subunit III of A. aceti (data not shown). The antibody did not crossreact neither with the purified enzymes nor with the membrane proteins or proteins from the cytoplasmic fractions.
371
A
B M
M 1 2 34 kDa
kDa
97.4 66.2
97.4
42.7
C 1 2 3 4
M
66.2
kDa 97.4 66.2
42.7
42.7
31.0
31.0
1 2 3 4 ADH subunit I subunit II
31.0
21.5
14.4
subunit III
14.4
14.4
Fig. 4 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the purified PQQ-dependent ADH from Ga. europaeus V3 (a), Ga. intermedius JK3 (b), and A. pasteurianus KKP 584 (c). Lane 1 indicates membrane fraction (60 μg); lane 2, solubilized mem-
brane fraction; lane 3, pooled fraction after DEAE-Toyopearl chromatography (40 μg); and lane 4, pooled fraction after hydroxyapatite chromatography (20 μg)
Table 2 Summary of ADH purification from Ga. europaeus V3, Ga. intermedius JK3, and A. pasteurianus KKP/584 Ga. europaeus Purification step
Protein (mg)
(U) Membrane fraction Solubilized fraction DEAE–Toyopearl Hydroxylapatite
Ga. intermedius
ADH activity
Recovery (%)
Protein (mg)
(U/mg)
48.0
792
16.5
3.6
382
106
2.0 0.8
330 143
165 179
(U) 100
Recovery (%)
Protein (mg)
(U/mg)
ADH activity (U)
Recovery (%) (U/mg)
49.4
892
18.1
100.0
48.4
395
8.2
48.2
8.3
773
93.5
86.6
4.6
247
31.7
41.7 18.1
2.2 1.2
590 227
66.1 25.4
1.5 0.7
192 147
Acetic acid resistance of the purified PQQ-dependent ADH The analysis of substrate specificity did not show any specific differences for ADHs from both Gluconacetobacter species in comparison to ADHs from Acetobacter and Gluconobacter (data not shown). However, ADH from Ga. europaeus showed higher resistance to acetic acid in comparison to ADH from A. pasteurianus and Ga. interFig. 5 Comparison of ADH acetic acid-stability among different species of acetic acid bacteria representing the genera Gluconacetobacter, Acetobacter, and Gluconobacter. Each of the purified ADH (0.5 mg/ml) was incubated for 30 min in 10 mM KPB buffer (pH 6) containing different concentrations of acetic acid. Error bars show standard deviations of three replicated measurements
A. pasteurianus
ADH activity
262 192
129 205
100 62.4 48.7 37.3
medius (Fig. 5). Ga. europaeus enzyme retained 2.7% of the original activity after 30 min of incubation at 4°C in 10 mM KPB buffer containing 14% acetic acid, whereas ADHs from Ga. intermedius and A. pasteurianus lost all the activity at 12% acetic acid, and ADH from Gluconobacter frateurii lost all the activity at 6% acetic acid (Fig. 5). At 10% acetic acid, ADH of Ga. intermedius retained 15% of the original one, whereas A. pasteurianus only retained 2.3%.
372
Discussion With the aim to compare the growth characteristics among Ga. europaeus, Ga. intermedius, and A. pasteurianus, we performed a laboratory production of acetic acid in 3-l baffled shake flasks containing 600 ml of AE medium with 3% ethanol and 3% acetic acid. We observed that the lag phase of A. pasteurianus was substantially longer than the lag phases of Ga. europaeus and Ga. intermedius. This observation might explain the previous findings describing Ga. europaeus as a predominant species in submerged vinegar bioreactors, as well as the presence of a recently described species, Ga. intermedius (Boesch et al. 1998; Sievers and Teuber 1995). In submerged bioreactors, the bacterial biomass is about two times diluted in every cycle, and thus, the slow growing microorganisms are sooner or later washed out. Interestingly, Ga. intermedius exhibited a shorter lag phase than Ga. europaeus, but its maximal acetic acid resistance was 40% lower than that of Ga. europaeus, suggesting a different adaptation mechanism against acetic acid between both species. The cultivation experiments with Ga. europaeus in AE media with different concentrations of acetic acid showed higher maximal acetic acid production in medium with 7% acetic acid in comparison to those obtained in media with lower concentrations of acetic acid. This might reflect a higher turnover number or higher expression of Ga. europaeus PQQ-dependent ADH at a higher concentration of acetic acid. This observation is supported also with the industrial practice of vinegar production, which usually keeps acetic acid concentration at about 6% at the beginning of each oxidizing cycle in submerged bioreactors for production of high-acid vinegar. Two subunits of ADH had been described before in Ga. polyoxogenes, a phylogenetically closely related species to Ga. europaeus (Tayama et al. 1989; Thurner 1997). In this work, we have shown that ADH from Ga. intermedius also does not contain the third subunit of ADH as judged from the SDS-PAGE gel. We detected two or more times higher ADH activity in Ga. europaeus and Ga. intermedius than in A. pasteurianus. Since ADH activity of these purified enzymes does not differ substantially, it is possible that the expression level of ADH in these species is different. Higher ADH activity may produce a bigger energy pool available for other membrane-associated processes, such as a potential acetate/acetic acid export system (Steiner and Sauer 2003; Matsushita et al. 2005), which might also explain a capability of Ga. europaeus to resist higher concentrations of acetic acid. We observed an absolute conservation (100%) of partial AdhA sequence in both vinegar-producing Gluconacetobacter species, but there is an obvious difference in this region between Gluconacetobacter spp. and Acetobacter spp. All the other three analyzed species of the genus Gluconacetobacter (Ga. xylinus, Ga. hansenii, and Ga. liquefaciens) do not exhibit 100% AA identity to the homologous region of Ga. europaeus, which suggest different
characteristics of their ADHs in comparison to the ADH of Ga. europaeus. Different acetic acid stability of ADH between Ga. europaeus and Ga. intermedius correlates well with the acidity of vinegar which was used as a source for the isolation of each strain. Although we were not able to detect differences in their partial adhA sequences, differences might exist in the other part of adhA. Ga. intermedius was isolated from cider vinegar, where sugar content in apples usually limits the final acidity of vinegar to 6%. In contrast to Ga. intermedius, the strain of Ga. europaeus originates from 10% wine vinegar. Although A. pasteurianus also originates from industrial vinegar bioreactors with acetic acid concentration higher than 6%, we were not able to reawake its high acetic acid resistance under laboratory conditions used in this study. Thus, the acetic acid resistance in Ga. europaeus and Ga. intermedius seems to be a stable character, in contrast to a more transient one in A. pasteurianus. Strains of Ga. europaeus species are important components of starter cultures for high-acid vinegar production. Although some studies have been published on acetic acid resistance in A. aceti, there are no data for Gluconacetobacter spp. This study is a first attempt toward understanding high acetic acid resistance of Ga. europaeus in comparison to A. pasteurianus. We have shown that PQQdependent ADH from Ga. europaeus exhibits significantly higher acetic acid stability in comparison to A. pasteurianus and thus enable oxidation of ethanol to acetic acid with Ga. europaeus at higher maximal acetic acid concentrations than with A. pasteurianus. Since the understanding of acetic acid resistance in Ga. europaeus has a potential application in the vinegar industry, we are presently characterizing this species in more detail. Acknowledgements This work was supported by a grant-in-aid for postdoctoral fellows from Japan Society for the Promotion of Science (JSPS) (awarded to K.M. and J.T.) and in part by the Ministry of Education, Science and Sport of the Republic of Slovenia (research project L4-5007 to J.T.). J.T. is a recipient of JSPS postdoctoral fellowship. Dr. O. Adachi is acknowledged for a sample of purified ADH from G. frateurii IFO 3264.
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