Analysis of alcohol dehydrogenase activity and fermentation characteristics of Acetobacter pasteurianus JST-S strain

Alcohol dehydrogenase activity (ADH), acetic acid yield, and tolerance to temperature and acid-induced stress are important bacterial strain parameters for the industrial production of acetic acid or vinegar. In this study, we evaluated and compared multiple features between A. pasteurianus JST-S strain, screened in the laboratory, and A. pasteurianus CICC 20001, a commonly used industrial strain. The ADH enzyme activity peaked at 8.22 U mg−1 for JST-S compared with the 7.62 U mg−1 for CICC 20001. Further, the ADH protein level was higher in JST-S than in the CICC 20001 strain. Comparative analysis of growth and cell morphology of the two strains indicated that the acetic acid tolerance of JST-S is superior to that of CICC 20001. Further, when the two strains were used for semi-continuous fermentation in 4 batches, the total acid production in fermentation broth with the JST-S peaked at 62.96 ± 1.42 g L−1 compared with 56.83 ± 1.12 g L−1 for the CICC 20001 strain. Thus, the JST-S strain seems to have better fermentation characteristics than the commonly used industrial strain. Based on all our observations, we propose that A. pasteurianus JST-S may be applied for cost-effective industrial production to obtain a high concentration of acetic acid.

2 essential for acetic acid bacteria oxidation of ethanol into acetic acid [14] .
An increasing number of studies have been undertaken to establish the relationship between the key enzymes of the ethanol oxidizing respiratory chain and acetic acid yield [5,14,15] . However, there is very little data that elucidates the relationship between the enzyme dynamics in the ethanol oxidizing respiratory chain, the enzymatic protein expression and the tolerance of acetic acid bacteria under high temperature, acid, and alcohol stress.
In this study, the ADH activity and the tolerance of the A. pasteurianus JST-S strain during the fermentation process were compared to that of A. pasteurianus CICC 20001. The acid resistance mechanism of A. pasteurianus JST-S was evaluated by growth assay, scanning electron microscopy and by examining ADH enzyme activity and expression by SDS-PAGE gel electrophoresis. Additionally, we carried out semi-continuous fermentation to assess the suitability of each strain for application in industrial vinegar fermentation.

Bacterial strains and media
A. pasteurianus CICC 20001 (also known as Huniang 1.01) was obtained from the China Center of Industrial Culture Collection. A. pasteurianus JST-S was isolated from solid fermentation substrate of vinegar (Yantai Di Boshi brewing machine Co., Ltd., Yantai, China) stored in the laboratory at the College of Food Science and Engineering, the Key Laboratory for Agricultural Products Processing of Anhui Province, Hefei University of Technology. All Acetobacter strains were maintained on seed medium consisting of 10 g L -1 glucose, 5 g L -1 yeast extract, 1.1 g L -1 MgSO 4 , 3.3 g L -1 K 2 HPO 4 . YPGD medium (5 g L -1 yeast extract, 5 g L -1 peptone, 5 g L -1 glucose, 5 g L -1 glycerol, 17 g L -1 agar) supplemented with different concentrations of acetic acid was used in growth-monitoring experiments. Fermentation medium contained 10 g L -1 glucose, 5 g L -1 yeast extract, 1.1 g L -1 MgSO 4 ꞏ7H 2 O, 3.3 g L -1 K 2 HPO 4 , and different concentrations of ethanol. Fermentation medium were divided into 50 mL aliquots in 250 mL Erlenmeyer flasks and then sterilized at 121 °C for 20 min prior to the addition of ethanol and acetic acid. To prepare seed culture, cells were cultured on a Zhp Serias thermostat incubator with shaker (ZHP-Y2102L; Shanghai Sanfa Scientific Instruments Co., Ltd., Shanghai, China) at 30 °C for 24 h at 150 rpm. Fermentation medium was inoculated with 10% seed culture and incubated at 32 °C at 150 rpm. The chemicals used in the study were obtained from Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China.

Determination of ADH enzyme activity
To determine ADH enzyme activity in response to varying concentrations of ethanol and acetic acid, strains were inoculated and cultivated in fermentation medium containing 2%, 4%, 6%, or 8% (v/v) ethanol or 0.3%, 0.6%, 0.9%, or 1.2% (v/v) acetic acid at 32 °C for 60 h at 150 rpm. In addition, strains were also inoculated in fermentation media and cultivated at 28 °C, 32 °C, 36 °C, or 40 °C for 60 h at 150 rpm in order to evaluate ADH enzyme activity under different temperatures. Following incubation, samples were centrifuged at 8000 × g, at 4 °C for 10 min and washed 3 times using 0.01 mol L -1 PBS (pH 7.4) buffer. The crude enzyme was extracted using methods described previously [15][16] . ADH activity was determined with potassium ferricyanide as an electron acceptor coupled with catalytic dehydrogenation of substrate by the enzyme [17] . All ADH activity assays were performed at 25 °C. One unit of enzyme activity was defined as the amount of enzyme catalyzing the oxidation of 1 μmol of substrate per minute. The protein concentration was measured by the modified Lowry method and bovine serum albumin as a standard protein [18] .

Analysis of ADH protein expression by SDS-PAGE gel electrophoresis
Strains were cultivated at 32 °C for 24 h in fermentation media with 6% (V/V) ethanol and 0.3% (V/V) acetic acid. The ADH enzyme extracts were obtained from 20 mL fermentation broth and analyzed by SDS-PAGE gel electrophoresis. Briefly, the fermentation broth was centrifuged at 8000 × g, at 4 °C The suspended cells were lysed by ultrasonic cell disruption (JY92-II, Xinzhi Biotechnology Co. Ltd., Ningbo, China) using pulses (200 W) lasting 5-10 s, 60 times on ice followed by centrifugation at 10000 × g at 4 °C for 12 min and storing the supernatant containing ADH enzyme at -80 °C.
Sample aliquots (30.0 µL) were mixed with 10 µL protein sample loading buffer and boiled alongside protein ladder aliquots (20 µL) in a boiling water bath for 10 min and loaded on SDS-PAGE gels for electrophoresis, which was run initially at 60 V for 30 min followed by 80 V for 4 h. Following protein separation, the gels were Coomassie-stained for 30 min followed by overnight de-staining. The samples were then photographed on a gel-imager (Bio-Rad, USA).

Growth analysis of the strains
To observe and compare bacterial growth, strains were cultivated at 30 °C for 24 h. They were then transferred to the same Erlenmeyer flasks sterilized at 121 °C for 20 min and diluted 10 8 times with stroke-physiological saline solution (0.9 g NaCl per 100 mL distilled water). The samples (0.1 mL) were then inoculated in YPGD medium containing 1.5%, 1.8% or 2.1% (v/v) acetic acid at 32 °C for 3 days.

Morphological analysis of strains under scanning electron microscope
Morphological features of strains were analyzed using the scanning electron microscope (SEM), following previously reported procedures with some modifications [19][20][21] . Briefly, liquid culture was centrifuged at 8000 × g for 10 min at 4 °C followed by suspension into larger 50 mL fermentation medium containing 6% (v/v) ethanol and 2% (v/v) acetic acid and incubation at 32 °C for 14 h. Bacterial suspensions were pelleted by centrifugation and cell pellets were washed three times with 0.01 mol L -1 PBS (pH 7.4) buffer followed by fixation at 4 °C by 25% (v/v) glutaraldehyde for 4 h and subsequent centrifugation. The samples were dehydrated using a stepwise increment of ethanol concentration (35%, 60%, 85%, 95%, 100% (v/v)) and vacuum freeze-dried for 6 h. Finally, bacterial cells were coated with gold for 90 s and observed under a scanning electron microscope (JEM-2100F; JEOL, Japan).

Analysis of repeated batch fermentation
In order to study the fermentation characteristics of A. pasteurianus CICC 20001 and JST-S, the strains were inoculated in fermentation media containing 8% (V/V) ethanol and cultivated at 32 °C for 96 h. Subsequently, the fermented broth was collected and rapidly centrifuged at 8000 × g for 10 min at 4 °C to obtain cells. Cells were collected under aseptic conditions and again inoculated in fermentation media containing 8% (v/v) ethanol and cultivated at 32 °C for 60 h. To perform semi-continuous fermentation, the process was repeated four times. The acidity of fermentation broth was measured by 0.1 mol L -1 NaOH with phenolphthalein as an indicator of pH [12,[22][23][24][25] . Samples were collected every 12 h and total acetic acid content was determined using the above titration method. The bacterial biomass was measured as described previously [26] .

ADH enzyme activity of JST-S and CICC 20001 strains under varying initial ethanol contents
We first set out to determine the strain specific dynamics of ADH enzymatic activity under varying ethanol concentration in the culture broth. Our results are shown in Fig. 1. The ADH enzyme activity of both strains showed dynamic trends with an initial proportional increase when ethanol concentration was within a range of 2-4%. The enzyme activity of A. pasteurianus CICC 20001 and JST-S peaked with values 5.91 U mg -1 and 6.48 U mg -1 , respectively, when ethanol concentration was 4% and after 36 h of fermentation. Higher concentrations of ethanol (6-8%) resulted in reduced peak activity of ADH enzymes in both strains. At 8% ethanol concentration, the peak ADH enzyme activity, which occurred at 42 h of fermentation, was the lowest for both CICC 20001 and JST-S strains at 3.31 U mg -1 and 4.15 U mg -1 , respectively. These results implied that strain growth may be inhibited under high alcohol conditions, which led to the prolonged fermentation time for ADH enzyme activity to peak. The peak ADH enzyme activity of A. pasteurianus JST-S at alcohol concentrations 2 and 4% was higher than that of the CICC 20001 strain, which indicated that the ability of A. pasteurianus JST-S to oxidize ethanol might be better than that of the CICC 20001 strain.

ADH enzyme activity of JST-S and CICC 20001 strains at different temperatures
Temperature is an important factor affecting enzyme activity [27] . Accordingly, we observed significant differences in the enzyme activity in either of the A. pasteurianus strains with alterations in incubation temperature (Fig. 2)

ADH activity in JST-S and CICC 20001 strains under different acid stress conditions
Previous studies have shown that vinegar yield was directly proportional to the stability of ADH enzyme activity under high acidity [11] . A comparative analysis of ADH activity from both strains revealed that with initial acetic acid concentration in the range of 0.3%-0.6%, the ADH activity in either strain increased gradually and peaked after 30 h of fermentation (Fig. 3). When initial acetic acid content was 0.6%, the ADH activity peaks of both CICC 20001 and JST-S strains were at the highest, 7.05 U mg -1 and 7.89 U mg -1 , respectively. With further increase in initial acetic acid concentration (0.9%-1.2%), the activity of ADH enzyme decreased in both strains. The peak value of ADH enzyme activity was not only the most attenuated (CICC 20001: 2.41 U mg -1 and JST-S: 3.72 U mg -1 ) at 1.2% initial acetic acid concentration, but was also delayed by 6 h compared to the lower initial acetic acid concentrations (Fig. 3). Thus, an initial acetic acid concentration within the range of 0.3-0.6% resulted in higher enzyme activity, achievable after shorter fermentation durations in either strain. However, further increase in the acid concentration may result in the repression of enzyme activity or protein expression, based on the highly attenuated peak activity values attained after longer durations of fermentation. A comparative analysis of the enzyme activity suggests that the A. pasteurianus JST-S strain might present superior acid resistance than the CICC 20001 strain.

JST-S strain shows better growth at higher acetic acid concentrations
We cultivated A. pasteurianus CICC 20001 and JST-S strains in YPGD media supplemented with acetic acid at different concentrations and compared the growth of the two strains. As shown in Fig. 4, the number of A. pasteurianus CICC 20001 and JST-S colonies decreased with an increase in acetic acid concentration. While the number of colonies showed no obvious visual differences at 1.5% acetic acid concentration, at 1.8%, the number of colonies in either strain decreased significantly, indicating that 1.8% acetic acid had a slight inhibitory effect on the growth rate of the strains. At 2.1% concentration, the number of colonies decreased dramatically, with the number of A. pasteurianus JST-S strain colonies significantly higher than that of the CICC 20001 strain (Fig. 4). Thus, high concentrations of acetic acid negatively affect the growth of either bacterial strain, with A. pasteurianus JST-S strain displaying superior acetic acid tolerance, compared with the CICC 20001 strain.

Cell morphology is better conserved in the JST-S strain under acid stress
SEM is a conventional method to study the surface changes of microbiological cells [28][29][30] . We examined the cell morphology and membrane response to high acetic acid concentration (2%) in culture media for the A. pasteurianus JST-S and CICC 20001 strains (Fig. 5). Acetic acid, a metabolic byproduct of acetic acid bacteria, exhibits significant toxicity to the cells on accumulation in the cell-culture media [30] . Resistance to acetic acid has been shown to be dependent on the cell membrane characteristics [31][32] . As shown in Fig. 5, A. pasteurianus JST-S and CICC 20001 strains showed irregular rod-shaped cells having rough surface, along with the appearance of some irregularities on the cell surface of some cells displaying ruptured folds as indicated by the white arrows. These results suggest that high acetic acid concentration in the media environment can lead to detrimental changes in osmotic pressure within some A. pasteurianus JST-S and CICC 20001 cells, resulting in their rupture. We hypothesize that the surface structure of acetic acid bacteria was altered to adapt to the new acetic acid environment in order to maintain ion transport or transmembrane transport and combat acetic acid stress. Further, A. pasteurianus JST-S cells displayed higher degree of surface smoothness and less disrupted cells compared with A. pasteurianus CICC 20001, implying a lower susceptibility of the JST-S strain to the toxic effects of acetic acid.

Comparative analysis of ADH protein expression using SDS-PAGE
The molecular weight of the ADH protein as observed on SDS-PAGE appeared slightly different between the two strains (Fig. 6). The molecular weight range of subunit I and subunit II is reported to be 71~85 kDa and 44~55 kDa, respectively [9] . As seen in Fig. 6 Another clear band appeared above the 43 kDa molecular weight marker band and was estimated to be approximately 44 kDa. The two ADH bands obtained from the JST-S strain were stronger compared with those from the A. pasteurianus CICC 20001, suggesting that ADH protein expression could be higher in the A. pasteurianus JST-S than in the CICC 20001 strain.  Repeated batch fermentation is a common practice in industrial deep acetic acid fermentation owing to the higher acetic acid production rate and yield along with reduced running costs [16] . The results of repeated batch fermentation of A. pasteurianus CICC 20001 and JST-S are shown in Fig. 8 and Table  1. Both A. pasteurianus CICC 20001 and JST-S strains showed similar trends in total acetic acid production with respect to time with repeated batch fermentation (Fig. 8). In all 4 fermentation batches (A-D in Fig. 8), the JST-S strain showed a higher average acetic acid production capacity at 62.96 ± 1.42 g L -1 , while the average acetic acid content in A. pasteurianus CICC 20001 was significantly lower at 56.83 ± 1.12 g L -1 (Table 1). These results imply that A. pasteurianus JST-S has better fermentation characteristics in terms of acetic acid production (As shown in Table 1) and could be used for cost-effective industrial acetic acid production.

Conclusion
In our study, we found that ADH enzyme activity of A. pasteurianus JST-S strain was decisively superior to that of the CICC 20001 strain under varying fermentation conditions. Furthermore, higher ADH enzymatic protein expression in the JST-S strain, as analyzed by SDS-PAGE, in addition to superior growth characteristics and conservation of cell morphology under high acetic acid concentrations implied greater tolerance of the JST-S strain to extreme acidic conditions. The results of semi continuous fermentation suggested that A. pasteurianus JST-S strain could better withstand the additional stress induced by repetitive batch culturing in the industrial production setting. Future studies to elucidate the metabolic mechanisms resulting in higher tolerance of JST-S strain and adaptability to industrial production should be attempted with an aim to further improve acid yield by genetic engineering and proteomic manipulations. This research must also be undertaken to the study production of the different flavors of vinegar.