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PQQ (reduced)

Figure 11-2. Formation of acetic acid from ethanol by Acetobacter aceti. The oxidation of acetic acid to ethanol involves two enzymatic reactions (upper panel). In the first reaction, ethanol is oxidized to acetalde-hyde by alcohol dehydrogenase. Next, acetaldehyde dehydrogenase further oxidizes acetaldehyde to acetic acid. An intermediate hydration reaction may also occur, in which acetaldehyde hydrate is formed (not shown). Both alcohol dehydrogenase and acetaldehyde dehydrogenase enzymes require pyrroloquinoline quinone (PQQ) as a co-factor, which serves as an electron acceptor (lower panel). The reduced PQQ then supplies electrons to the electron transport chain, eventually leading to the formation of ATP via oxidative phosphorylation. Adapted from Fuchs, G., 1999.

Figure 11-2. Formation of acetic acid from ethanol by Acetobacter aceti. The oxidation of acetic acid to ethanol involves two enzymatic reactions (upper panel). In the first reaction, ethanol is oxidized to acetalde-hyde by alcohol dehydrogenase. Next, acetaldehyde dehydrogenase further oxidizes acetaldehyde to acetic acid. An intermediate hydration reaction may also occur, in which acetaldehyde hydrate is formed (not shown). Both alcohol dehydrogenase and acetaldehyde dehydrogenase enzymes require pyrroloquinoline quinone (PQQ) as a co-factor, which serves as an electron acceptor (lower panel). The reduced PQQ then supplies electrons to the electron transport chain, eventually leading to the formation of ATP via oxidative phosphorylation. Adapted from Fuchs, G., 1999.

The biochemical and physiological processes involved in the acetic acid fermentation are quite unlike those described for the lactic and ethanolic fermentations.The acetic acid fermentation is performed by obligate aerobes, and metabolism occurs not in the cytoplasm, but rather within the periplasmic space and cytoplasmic membrane. The acetic acid pathway yields energy, but not by substrate level phosphoryla-tion. Instead, the oxidation reactions are coupled to the respiratory chain which then generates ATP via electron transport and oxidative phosphorylation reactions. Finally, both the substrate and product are toxic, which no doubt contributes to the relative lack of competitors during what is essentially an open fermentation. In fact, acetic acid bacteria are rather remarkable for their ability to tolerate low pH and high acetic acid concentrations (Box 11-1).

The two main enzymes involved in the acetic acid fermentation, alcohol dehydroge-nase (AlDH) and acetaldehyde dehydrogenase (AcDH), have been characterized, and the genes coding for their expression have been cloned and sequenced (Figure 11-3). These enzymes are the "primary dehydrogenases," responsible for nearly all of the detectable oxidation activity. They are located within the cytoplasmic membrane and face into the periplasm (Figure 11-4). This means that the ethanol and acetaldehyde oxidation reactions occur in the periplasm and that ethanol can be used without having to be accumulated within the cytoplasm. Both AlDH and

Box 11β€”1. Acetic Acid Tolerance in Acetobacter

Microorganisms that produce peroxides, alcohols, acids, or other inhibitory agents must also be able to tolerate such products. In the case of acetic acid bacteria, tolerance to both ethanol and acetic acid is a necessary feature of their physiology.

During the vinegar fermentation, cells typically encounter ethanol at concentrations near 12% (more than 2.5 M). Later, as ethanol is oxidized to acetic acid, vigorous growth is maintained even when the concentration of acid reaches more than 7%. Some strains of Acetobacter can produce (and tolerate) as much as 20% acetic acid (3.3 M).To provide some perspective, Escherichia coli is inhibited by 1% or less acetic acid (Lasko et al., 2000).Thus, the means by which acetic acid bacteria deal with acetic acid stress is not only of fundamental interest, but it also has practical implications. Strains that are more acid tolerant, for example, would be expected to be more productive during commercial vinegar fermentations.

To fully appreciate the challenge faced by these cells, consider first the actual problem that exists when Acetobacter grows during the acetic acid fermentation. In a 6% (1 M) acetic acid solution at a pH of 4.5, most of the acetic acid will be in the undissociated or acid form (given that the pKa of acetate is 4.76). Since the cell membrane is permeable to the acid form, cells at that pH could accumulate, in theory, more than 0.6 M acetic acid within the cytoplasm (via simple diffusion). Accumulation of the acid will continue, in fact, until the inside and outside concentrations are equal. Of course, because the physiological pH within the cytoplasm is generally higher than the external pH (at least for most bacteria), the accumulated acid will immediately dissociate to form the anion and a free proton (in accordance to the Henderson-Hasselbach equation).This will further shift the [acidin]/[acidout] equilibrium such that even more acid will be accumulated.

At some point one of several outcomes are possible.First, unless the accumulated protons are expelled from the cytoplasm, their accumulation will eventually cause the intracellular pH to decrease to an inhibitory level. Second, if the cells relied on proton extrusion to maintain a more conducive intracellular pH, then substantial amounts of ATP (or its equivalent) would be required to drive the proton pumping apparatus, leading to a major drain on the energy resources available to the cell. Finally, if a high cytoplasmic pH is maintained, the acetate anion could, in theory, reach concentrations of 1 M or higher (under the conditions stated above).This would create severe toxicity and osmotic problems for the cell, which may be even worse than acidity problems (Russell, 1992).

Despite these apparent hurdles, researchers, primarily in Switzerland and Japan, have revealed that there may be several mechanisms that account for the ability of Acetobacter and other acetic acid bacteria to tolerate high acetic-acid and low-pH environments. These mechanisms involve both physiological and genetic responses (Table 1).As noted above, acetic acid accumulation depends on the pH gradient across the cell membrane.The higher the gradient (i.e., low pH outside and high pH inside), the greater will be the acetate that will be trapped inside. If, on the other hand, the cytoplasmic pH is allowed to drop (decreasing the gradient), then less acetate is accumulated.

Table 1. Summary of mechanisms contributing to acetic acid tolerance.

Mechanisms

Possible effect

Low pH gradient

Reduced anion accumulation

groESL operon induced

General stress response

Expression of Aar proteins

Stimulate acetate assimilation

Expression of Aap proteins

Stimulate transport systems

Expression of Asp proteins

Unknown

Aconitase expression

Stimulate acetate assimilation

Acetic acid efflux pump

Stimulate acetic acid efflux

Box 11β€”1. Acetic Acid Tolerance in Acetobacter (Continued)

In fact, this indeed seems to be the case for Acetobacter aceti, because this organism maintains only a very small pH gradient (Menzel and Gottschalk, 1985).Thus, the cell is spared the problem caused by anion (acetate) accumulation (of course, this implies that Acetobacter must still be physiologically able to tolerate a low intracellular pH).That acetate accumulation might contribute to cell inhibition is supported by the observation that resistant strains accumulate less acetate than wild-type strains that are only moderately resistant (Steiner and Sauer, 2003). The response to high acetate concentrations at a near neutral pH may, however, be quite differ-ent.These same researchers showed,for example,that A. aceti at pH 6.5 accumulated more than 3 M acetate, at an outside concentration of less than 0.4 M. However, these high-pH conditions clearly do not reflect those that this organism would likely experience.

As indicated above, intracellular acetic acid also could, in theory, be pumped out directly, provided the energy sources were available. Recently, the presence of an acetic acid efflux system was reported in Acetobacter aceti (Matsushita et al., 2005). The system was sensitive to protonophores and ionophores, agents that dissipate proton and ion gradients across the cell membrane and by a respiration inhibitor. Therefore, it appears that acetic acid efflux is energized by a proton motive force that is itself dependent on respiration activity.

Transcriptional analyses of A. aceti also have revealed the presence of genes whose expression is induced by acetic acid and that may be associated with acetic acid resistance. Examples include the groESL and the aar (acetic acid resistance) operons (Fukaya et al., 1990; Fukaya et al., 1993; Okamoto-Kainuma et al., 2002).The GroES and GroEL proteins (encoded by groESL) are part of the chaperonin family of stress-induced proteins that protect cells by stabilizing proteins and preventing their mis-folding. In A. aceti, groESL was induced by heat as well as acetic acid and ethanol. Furthermore, a groESL over-expressing strain was shown to be even more resistant to these stresses, suggesting that these gene products contribute to overall acetic acid resistance (Okamoto-Kainuma et al., 2002).

The aar operon consists of three genes, aarA, aarB, and aarC, that encode for proteins with homology to enzymes involved in the citric acid pathway. Specifically, aarA encodes for the citrate-forming enzyme citrate synthase and aarC appears to encode for coenzyme A transferase, an enzyme also involved in acetate assimilation.Thus, assimilation of acetic acid (i.e., via its oxidation) would be one way to not only de-toxify the acid, but also enable the cell to use acetate as an energy source.

Recently, another assimilation or acetate oxidation pathway was suggested to be responsible for acetate resistance (Nakano et al., 2004).These researchers used two-dimensional (2-D) gel electrophoresis to show that expression of the enzyme aconitase by A. aceti was significantly increased in response to an acetic acid shock (Nakano et al., 2004). Further, a strain harboring multiple copies of the aconitase gene (and that over-expressed aconitase) produced more acetic acid and was more acetic acid resistant compared to the parent strain.Apparently, the increased expression of aconitase, an enzyme involved in both the citric acid cycle and glyoxylate pathway, confers resistance by stimulating consumption and detoxification of intracellular acetic acid.Although effective for the cells, these strategies (the aar and aconitase) are not ones that would likely be exploited for vinegar production, since the product is consumed and the overall yield would not be enhanced.

Finally, as shown by proteome analyses (using 2-D gels), a number of proteins are synthesized by Acetobacter and Gluconobacter in response to both short-term and long-term exposure to acetic acid (Lasko et al., 1997; Steiner and Sauer, 2001).These proteins are distinct from the general stress-response proteins induced by heat shock and are thus referred to as either acetate adaptation proteins (Aaps) for adapted cells or acetate-specific stress proteins (Asps) for un-adapted cells. Although the function of most of these proteins has not yet been established, it appears (based on partial sequence analyses) that at least several of the Aaps are associated with

Box 11β€”1. Acetic Acid Tolerance in Acetobacter (Continued)

membranes and may be involved in transport processes. Indeed, acetate transport and membrane-associated respiratory proteins could both be envisioned to promote acetate resistance

(Steiner and Sauer, 2001).

References

Fukaya, M., H.Takemura, H. Okumura,Y. Kawamura, S. Horinouchi, and T. Beppu. 1990. Cloning of genes responsible for acetic acid resistance in Acetobacter aceti. J. Bacteriol. 172:2096-2104.

Fukaya M., H.Takemura, K.Tayama, H. Okumura,Y. Kawamura, S. Horinouchi, and T. Beppu. 1993 The aarC gene responsible for acetic acid assimilation confers acetic acid resistance on Acetobacter aceti. J. Ferm. Bioeng. 76:270-275.

Lasko, D.R., N. Zamboni, and U. Sauer. 2001. Bacterial response to acetate challenge: a comparison of tolerance among species.Appl.Microbiol. Biotechnol. 54:243-247.

Lasko, D.R., C. Schwerdel,J.E. Bailey, and U. Sauer. 1997.Acetate-specific stress response in acetate-resistant bacteria: an analysis of protein patterns. Biotechnol. Prog. 13:519-523.

Matsushita, K., T. Inoue, O.Adachi, and H.Toyama. 2005.Acetobacter aceti possesses a proton motive force-dependent efflux system for acetic acid. J. Bacteriol. 187:4346-4352.

Menzel, U., and G. Gottschalk. 1985.The internal pH of Acetobacterium wieringae and Acetobacter aceti during growth and production of acetic acid.Arch. Microbiol. 143:47-51.

Nakano, S., M. Fukaya, and S. Horinouchi. 2004. Enhanced expression of aconitase raises acetic acid resistance in Acetobacter aceti. FEMS Microbiol. Lett. 235:315-322.

Okamoto-Kainuma,A.,W. Yan, S. Kadono, K.Tayama,Y. Koizumi, and F. Yanagida. 2002. Cloning and characterization of groESL operon in Acetobacter aceti. J. Biosci. Bioeng. 94:140-147.

Russell,J.B. 1992.Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. J.Appl. Bacteriol. 73:363-370.

Steiner, P, and U. Sauer. 2001. Proteins induced during adaptation of Acetobacter aceti to high acetate con-centrations.Appl. Environ. Microbiol. 67:5474-5481.

Steiner, P., and U. Sauer. 2003. Long-term continuous evolution of acetate resistant Acetobacter aceti. Biotechnol. Bioeng. 84:40-44.

AcDH also contain pyrroloquinoline quinone (PQQ) as a prosthetic group (Figure 11-2). The latter serves as the primary electron acceptor during the oxidation reactions, and is responsible for the transfer of electrons to the cytochromes of the respiratory chain.

Protein analyses of purified PQQ-dependent AlDH and AcDH from several Acetobacter and Gluconobacter species have shown that these enzymes consist either of two or three subunits. These subunits contain heme-binding moieties that mediate intramolecular transport of electrons, in addition to the PQQ prosthetic groups. Acetic acid bacteria also have NAD- (or NADP)-dependent dehydrogenases present within the cytoplasm. However, the specific activities of these enzymes are up to 300 times lower than the membrane-bound, PQQ-dependent dehydro-genases. In addition, the pH optima of the latter enzymes in Acetobacter is between 4.0 and 5.0, which is much closer to the normal physiological pH than the NAD-dependent dehydrogenase enzymes, whose pH optima is above 7.0. Although the function of these enzymes has not yet been established, it has been suggested that they are involved in acetaldehyde and acetate assimilation. Finally, it should be noted that alcohols and aldehydes other than ethanol and ac-etaldehyde can serve as substrates for both of these dehydrogenases. Thus, primary alcohols such as propanol, and secondary alcohols and polyols, such as isopropanol and glycerol, all of which can be present in mashes used for vinegar production, can be oxidized and converted into acids, ketones, and other organic end products. Many of these compounds make important contributions to the aroma and flavor characteristics of vinegar (see below).

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