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The SO2 resistance of South African acetic acid bacteria and their effect on fermentation
Acetic acid bacteria are well known to be able to produce high concentrations of volatile acidity (VA) from ethanol. VA, which consists predominantly of acetic acid, can contribute to the complexity of red wine. It can, however, spoil the wine if the concentration becomes too high and the legal limit of VA in S.A. bottled wines is 1.2 g/L. A study in which more than 7 000 Australian wines were tested showed that 33% of these wines contained high VA levels, which should be disturbing to the winemakers (7). Acetic acid is also known to inhibit yeast during alcoholic fermentation and may thus contribute to sluggish or even stuck fermentation. The effective control of acetic acid bacteria is therefore very important to the winemaker. The aim of this study was thus to determine the SO2 resistance of five acetic acid bacteria strains in white grape juice and the effect of their metabolites on yeast numbers and alcoholic fermentation.
Materials and methods
The SO2 resistance of five acetic acid bacteria strains was determined in white grape juice. These five strains, which included Acetobacter aceti, A. liquefaciens, A. hansenii, A. pasteurianus and Gluconobacter oxydans, were isolated from South African musts and wines. Sterile Colombard juice (18.5oB, acid 6.4 g/L, pH 3.22), of which the pH was changed to 3.4, 3.6 and 3.8 with sterile KOH, were used. Of these juices, 180 mL was inoculated in a sterile 250 mL Erlenmeyerflask. The acetic acid bacteria were grown overnight in a preculture consisting of glucose and yeast extract to OD 1 at 600 nm and 180 microlitres were inoculated in the juice which gave an initial cell count of 104- 105 cfu/mL. After this the SO2 was added at a total of 20, 40 and 60 mg/L and the molecular SO2 concentration determined. By plating a sample from these juices every day, the growth of the acetic acid bacteria. were monitored After this the acetic acid bacteria were killed by Velcorin addition and the sugar and VA levels determined. Certain metabolites produced by the bacteria in the juice were determined by GC and GC-MS. In the musts, where the acetic acid bacteria utilised some of the sugar, the sugar concentration was increased to 18.5oB. The juices were then inoculated with the yeast Vin 13 and sterile diammoniumphosphate added at 0.75 g/L. The yeast numbers were also determined in each juice by plating and the sugar concentration determined after 6 days, and 10 days (after 6 days, the control fermentations in which no acetic acid bacteria grew prior to yeast inoculation, were fermented dry).
Results
In figure 1 the growth of the five strains of acetic acid bacteria in sterile Colombard juice at pH 3.8 can be seen. It is clear that the different bacteria strains do not grow at the same rate at the same pH value. However, the growth of each strain was the same at the different pH values (pH 3.4, 3.6 and 3.8). In figures 2, 3 and 4 the growth of A. hansenii, A. aceti and A. liquefaciens can be seen at different molecular SO2 concentrations. It is clear that the A. hansenii strain was the most SO2 resistant, with 0.65 mg/L molecular SO2 killing it effectively over 4 days. A. aceti in figure 2 was more SO2 sensitive, with 0.1 mg/L killing it effectively. In figure 3 can be seen that A. liquefaciens was also more sensitive to this molecular SO2 concentration, but at lower concentrations its numbers started to increase later. A. pasteurianus was more SO2 resistant than A. aceti and A. liquefaciens, but less than A. hansenii, with 0.59 mg/L molecular SO2 killing it after one day. In figure 5 the SO2 resistance of the five strains at 0.05 mg/L SO2 can be seen. It is clear that the resistance differs from strain to strain. Figure 6 shows the growth of the yeast Vin 13 in juice in which the acetic acid bacteria had grown with no SO2 additions.
Table 1 shows the different molecular SO2 concentrations and the VA production by the bacteria at different pH values. It also indicates the sugar values after six days' fermentation and ten days' fermentation. It is clear that where the bacteria were not inhibited to a large degree by the SO2, high concentrations of VA were formed, which subsequently inhibited the yeast if one looks at the residual sugar level after fermentation.
(See also Table 2)
Discussion
Acetic acid bacteria occur naturally on grapes and thus also in the must during fermentation (4, 8). It has also been shown that acetic acid bacteria can inhibit yeast (3, 9). It is clear that the different pH levels did not influence the growth of individual acetic acid bacteria strains. The difference of growth of different strains at the same pH can however be attributed to strain differences. The weak growth of the G. oxydans strain is surprising, due to the fact that this species normally dominates in fresh must and prefers glucose over ethanol as a carbon source. This strain also exhibited the weakest SO2 resistance. There was a difference in SO2 resistance in the Acetobacter strains. Boulton et al (1995) stated that 0.8 mg/L molecular SO2 was enough to control A. aceti. This correlates with 0.64 mg/L being necessary to control the A. hansenii. The use of SO2 is thus one of the most important weapons in the arsenal of the winemaker to control the unwanted growth of these micro-organisms. It is, however, only the free, molecular form of SO2 in juice or wine which acts in an antimicrobial manner. The proportion of molecular SO2 increases with a degrease in pH. The inhibition of acetic acid bacteria should, however, be done at an early stage of the winemaking process, due to the inhibition of yeast by acetic acid bacteria. In the juice to which little or no SO2 was added, the acetic acid bacteria exhibited good growth, yeast counts stayed low after subsequent yeast inoculation and these fermentations were sluggish or stuck after the control fermentation had been fermented dry. This is especially true for the juices in which the strains grew which produced the most VA. It has been shown that acetic acid inhibits yeast exponentially with an increase in concentration (10). This is especially true at higher alcohol concentrations. These juices had a low initial sugar concentration and the inhibitory effect of the acetic acid would probably have been higher, if the must had a higher sugar and subsequent higher ethanol concentration.
Some acetic acid bacteria are also able to produce SO2 binding substances, like gluconic, 2 ketogluconic and 2,5 diketogluconic acids from glucose. The binding of SO2 to these substances, robs it of its antimicrobial activity. This could have happened in the must in which the A. liquefaciens strain grew (1, 11).
The lactic acid bacteria Lactobacillus kunkeei, have also been shown to inhibited yeast. These bacteria produce high concentrations of acetic acid in juice, which could contribute to stuck or sluggish fermentations. Fermentations to which acetic acid was added at the same rate at which the bacteria produced it, did not inhibit the yeast to the same degree as in fermentations in which the bacteria grew with the yeast (5, 6). The bacteria can thus accumulate nutrients before the yeast, thus preventing the yeast from absorbing it. It can also inhibit yeast in some other unknown way. This could also be the case with acetic acid bacteria.
The metabolism of acetic acid bacteria can also influence the organic acidity of the wine. These bacteria can metabolise malic and citric acid. Joyeux et al (1984) found that some acetic acid bacteria decreased the malic acid concentration in must from 4.7 g/L to 1.8 g/L. It also seems as if they are able to break down tartaric acid (3). This could influence the organoleptic qualities of the wine. The difference in the organic acid concentration in this study gives an indication that the bacteria's Krebs cycle was active during growth.
Acetic acid bacteria can also produce other compounds like acetoin from lactic acid. This compound with its buttery aroma is considered unwanted in wine and it can also bind SO2. Acetic acid bacteria are also able to metabolise glycerol, even from 6.4 g/L to 2.3 g/L, according to Drysdale and Fleet (1989) Glycerol is considered to be a wanted compound in wine.
Conclusion
It is clear that acetic acid bacteria can be controlled by the effective addition of SO2. It seems as if a molecular SO2 concentration of 0.8 to 1 mg/L can inhibit the growth of these bacteria in juice. This was 60 mg/L total SO2 at pH 3.4. This is however, not a given, due to strain differences that may occur. This can be achieved by settling the must and the addition of SO2 at a low pH.
The South African wine industry must nowadays compete more globally and the production of consistent quality wine at a lower production cost is thus important. This can only be achieved when all the negative factors that can influence wine quality are identified and eliminated. Acetic aid bacteria are one of these and more research is needed on this type of micro-organism.
References
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This is the second of two articles that focus on the M. Sc. research (Oenology) of Wessel du Toit at the Department of Viticulture and Oenology and the IWBT, University of Stellenbosch.
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