Influence of carbon sources on the viability and resuscitation of Acetobacter senegalensis during high-temperature gluconic acid fermentation.

Much research has been conducted about different types of fermentation at high temperature, but only a few of them have studied cell viability changes during high-temperature fermentation. In this study, Acetobacter senegalensis, a thermo-tolerant strain, was used for gluconic acid production at 38 °C. The influences of different carbon sources and physicochemical conditions on cell viability and the resuscitation of viable but nonculturable (VBNC) cells formed during fermentation were studied. Based on the obtained results, A. senegalensis could oxidize 95 g l- 1 glucose to gluconate at 38 °C (pH 5.5, yield 83%). However, despite the availability of carbon and nitrogen sources, the specific rates of glucose consumption (qs) and gluconate production (qp) reduced progressively. Interestingly, gradual qs and qp reduction coincided with gradual decrease in cellular dehydrogenase activity, cell envelope integrity, and cell culturability as well as with the formation of VBNC cells. Entry of cells into VBNC state during stationary phase partly stemmed from high fermentation temperature and long-term oxidation of glucose, because just about 48% of VBNC cells formed during stationary phase were resuscitated by supplementing the culture medium with an alternative favorite carbon source (low concentration of ethanol) and/or reducing incubation temperature to 30 °C. This indicates that ethanol, as a favorable carbon source, supports the repair of stressed cells. Since formation of VBNC cells is often inevitable during high-temperature fermentation, using an alternative carbon source together with changing physicochemical conditions may enable the resuscitation of VBNC cells and their use for several production cycles.

Simultaneous production of acetic and gluconic acids by a thermotolerant Acetobacter strain during acetous fermentation in a bioreactor.

The activity of bacterial strains significantly influences the quality and the taste of vinegar. Previous studies of acetic acid bacteria have primarily focused on the ability of bacterial strains to produce high amounts of acetic acid. However, few studies have examined the production of gluconic acid during acetous fermentation at high temperatures. The production of vinegar at high temperatures by two strains of acetic acid bacteria isolated from apple and cactus fruits, namely AF01 and CV01, respectively, was evaluated in this study. The simultaneous production of gluconic and acetic acids was also examined in this study. Biochemical and molecular identification based on a 16s rDNA sequence analysis confirmed that these strains can be classified as Acetobacter pasteurianus. To assess the ability of the isolated strains to grow and produce acetic acid and gluconic acid at high temperatures, a semi-continuous fermentation was performed in a 20-L bioreactor. The two strains abundantly grew at a high temperature (41°C). At the end of the fermentation, the AF01 and CV01 strains yielded acetic acid concentrations of 7.64% (w/v) and 10.08% (w/v), respectively. Interestingly, CV01 was able to simultaneously produce acetic and gluconic acids during acetic fermentation, whereas AF01 mainly produced acetic acid. In addition, CV01 was less sensitive to ethanol depletion during semi-continuous fermentation. Finally, the enzymatic study showed that the two strains exhibited high ADH and ALDH enzyme activity at 38°C compared with the mesophilic reference strain LMG 1632, which was significantly susceptible to thermal inactivation.

The role of protein modifications in senescence of freeze-dried Acetobacter senegalensis during storage.

BACKGROUND: Loss of viability is one of the most important problems during starter culture production. Previous research has mostly focused on the production process of bacterial starters, but there are few studies about cellular protein deterioration causing cell defectiveness during storage. In the present study, we investigated the influence of storage temperature (-21, 4, 35°C) on the cellular protein modifications which may contribute to the senescence of freeze-dried Acetobacter senegalensis. RESULTS: Heterogeneous populations composed of culturable cells, viable but non-culturable cells (VBNC) and dead cells were generated when freeze-dried cells were kept at -21 and 4°C for 12 months whereas higher storage temperature (35°C) mainly caused death of the cells. The analysis of stored cell proteome by 2D-DiGE demonstrated a modified pattern of protein profile for cell kept at 4 and 35°C due to the formation of protein spot trains and shift of Isoelectric point (pI). Quantification of carbonylated protein by ELISA showed that the cells stored at 4 and 35°C had higher carbonylated protein contents than fresh cells. 2D-DiGE followed by Western blotting also confirmed the carbonylation of cellular proteins involved in translation process and energy generation. The auto-fluorescent feature of cells kept at 35°C increased significantly which may be an indication of protein glycation during storage. In addition, the percentage of cellular unsaturated fatty acid and the solubility of cellular proteins decreased upon storage of cells at higher temperature suggesting that peroxidation of fatty acids and possibly protein lipidation and oxidation occurred. CONCLUSIONS: High storage temperature induces some deteriorative reactions such as protein oxidation, lipidation and glycation which may cause further protein modifications like pI-shift, and protein insolubility. These modifications can partly account for the changes in cell viability. It can also be deduced that even moderate carbonylation of some critical cellular proteins (like ribosomal proteins) may lead to VBNC formation or death of freeze-dried bacteria. Moreover, it seems that other mechanisms of biomolecule deterioration preceding protein carbonylation lead to VBNC formation under very low storage temperature.