UMYU Journal of Microbiology Research

E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668

ORIGINAL RESEARCH ARTICLE

Isolation of Bacteria with Acetic Acid Production Potential from Selected Fruits and Soil

Dienye, B. N^1*, Agwa, O. K.², Akponiya, P. E. ², Peter, C. P. ² and Princewill, C. D. ¹

¹Department of Microbiology, Rivers State University, P.M.B. 5080, Port Harcourt, Nigeria

²Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria

*Corresponding author E-mail: Dienye, B. N blessing.dienye@ust.edu.ng

ABSTRACT

Acetic acid bacteria (AAB) are widely recognized for their role in the production of various food products, particularly vinegar and wine, due to their ability to oxidize ethanol and sugars to acetic acid. This study aimed to isolate and characterize acetic acid-producing bacteria from selected fruit and soil samples collected in Port Harcourt. Unripe and over-ripe samples of banana, plantain, avocado, pineapple, and apple were homogenized and enriched in a medium containing 3% acetic acid and 4% ethanol, followed by incubation at 30^oC for 72 hours. Bacteria were isolated using the serial dilution technique and qualitatively screened for acetic acid production on glucose yeast calcium carbonate (GYC) and Carr agar media. Morphological and biochemical identification of the isolates was performed, while tolerance to varying ethanol and glucose concentrations was evaluated to assess growth performance. Acetic acid production was determined by submerged fermentation, and the acetic acid concentration was estimated titrimetrically. Of 58 bacterial isolates, 35 were confirmed as acetic acid producers, belonging to the genera Acetobacter (31%) and Gluconobacter (69%). The isolates exhibited variable tolerance to different concentrations of ethanol and glucose, with optimal growth at 5% ethanol and 20% glucose. Acetic acid production ranged from 0.24 g/100mL to 16 g/100mL, with soil-derived Gluconobacter sp. demonstrating the highest yield. The results revealed that soil and selected fruit may be potential sources of AAB, especially Gluconobacter strains, which might offer applications in acetic acid production.

Key words: Acetic acid bacteria, Gluconobacter, Acetobacter, Submerge fermentation, ethanol tolerance

INTRODUCTION

Acetic acid (AA) is a commercially important organic acid that is extensively used as food additive and preservative (Phong et al., 2017; Temel and Kösesakal, 2024). Chemical synthesis of acetic acid is possible, but approximately 10% of commercial acetic acid production is obtained by biological routes (Awad et al., 2012; Merli et al., 2021). Microbial fermentation for AA production is considered environmentally friendly and offers opportunities to exploit biological waste and farm surpluses as carbon sources (El-Askri et al., 2022). Acetic acid produced via the fermentation route gives vinegar its characteristic flavour and aroma (Onuorah et al. 2016). This organic acid serves as the primary flavouring and acidity component in vinegar (Divyashree et al., 2022).

Acetic acid is the primary metabolite of acetic acid bacteria (AAB) (Gullo et al., 2014). Louis Pasteur discovered Acetic Acid Bacteria as a microorganism responsible for the production of vinegar (Shafiei and Delvigne, 2019; Alisigwe et al., 2022). They are strict aerobic bacteria belonging to the class Alphaproteobacteria and the family Acetobacteraceae (Qiu et al., 2021). Acetic Acid Bacteria are Gram-negative, catalase-positive, rod-shaped cells with a size ranging from 0.6–1.0 × 1.0–4.0 μm, occurring singly in pairs or chains (Kubizniaková et al, 2021). AAB does not sporulate and is either motile or non-motile (Ouattara et al., 2021). They utilize substrates such as glucose and ethanol as energy sources (Es-sbata et al., 2021; Dereje et al., 2024). Acetic Acid Bacteria can partially oxidize alcohol and sugar, leading to the accumulation of several metabolites, including acetic acid, through aerobic fermentation (He et al. 2022). Acid tolerance is a unique trait of AAB microorganisms (De Roos & De Vuyst, 2018).

Acetic Acid Bacteria are widespread in nature, and their presence has been detected in a variety of natural and industrial environments (Hata et al. 2023; Garcia-Garcia et al., 2023). These heterogeneous assemblage organisms have nineteen genera currently recognized based on genetic analysis and phenotypic characteristics (Gomes et al., 2018; Qiu et al., 2021). Although a variety of bacteria can produce acetic acid, at present day most acetic acid is produced using Acetobacter and Gluconobacter (Arifuzzaman et al, 2014). Gluconobacter and Acetobacter play important roles in the production of metabolic products, including vitamin C, and some polysaccharides, such as cellulose, dextran, and levan. Their contribution extends beyond the production of fermented foods and beverages (Kadere et al., 2008; Shafiei et al, 2019; Lynch et al., 2019). Due to the usefulness of AAB across various sectors and its growing demand, there is a need to explore new, promising strains of bacteria from untapped niches. In this context, the isolation of a potential acetic acid-producing bacterial strain from selected fruits and soil was attempted in the present investigation, considering that novel and potential acetic acid-producing strains can be isolated from biodiversity niches.

MATERIALS AND METHODS

Sample Collection

Fruit samples of unripe and overripe Bananas, Plantains, Avocados, Pineapples, and Apples were procured from fruit markets in Choba and Rumuokoro and selected at random. Soil samples were collected from the farmland of the University of Port Harcourt, Choba, Obio/Akpor Local Government Area. The soil samples were obtained at a depth of 15-20cm using a soil auger, placed in a zipped polythene bag, sealed, labelled, and transferred immediately to the laboratory for analysis.

Isolation and screening of acetic acid bacteria

Isolation and screening of acetic acid bacteria were conducted according to the protocols of Bellankimath et al. (2017) and Diba et al. (2015). The collected fruit samples were washed in sterile distilled water, and each fruit sample was crushed aseptically. Five grams (5g) of each fruit sample was homogenized and immersed in a solution containing 3% acetic acid and 4% ethanol to enrich the fruit samples. The mixture was incubated at 30 °C for 3 days. Enriched fruit samples (1ml) and soil samples (1g) were separately inoculated into 9ml sterile saline (10^-1) to make appropriate dilution up to 10^-6. Aliquot (0.1ml) from the appropriate dilution was spread on already prepared GYC agar plate (10 % glucose, 1.0% yeast extract, 2.0% calcium carbonate, 1.5% agar) and Carr medium containing yeast extract 30, ethanol 2% (v/v), bromocresol green 0.02; agar 20 in g/L^-1 and at pH 6.8 supplemented with 100mg/l of pimaricin to inhibit the growth of yeast and molds and incubated at 30⁰C for 48hrs. After incubation, plates were examined for colonies with a clear halo and a yellow colour on the agar. The selected colonies were picked and streaked separately onto fresh GYC agar and Carr agar. Carr agar medium was used to selectively grow bacteria that produce acetic acid by the overoxidation of ethanol.

Maintenance of pure culture

Single colonies selected were subcultured on fresh GYC and Carr agar media using the streak plate technique and incubated at 30 °C for 24 hours until pure cultures were obtained. Purified isolates were maintained on GYC slants and then stored at 4 °C.

Identification of acetic acid bacteria

Colony morphology of acetic acid bacterial isolates

The selected acetic acid-producing isolates were macroscopically characterized on GYC plates after 24hours and the size, elevation, texture, shape, and colour were recorded. Gram reaction and motility tests were also performed (Ouattara et al., 2018).

Biochemical characteristics of acetic acid bacterial isolates

Different biochemical tests were performed following the protocol of Ouattara et al. (2021). Citrate utilization test, catalase, oxidase, indole, Methyl Red (MR), urease, Voges-Proskauer (VP), and carbohydrate (glucose, sucrose, Lactose) fermentation test, and overoxidation of ethanol using Carr medium were carried out.

Inoculum preparation

Each selected bacterial isolate from its respective slant was streaked aseptically onto a GYC agar plate and incubated at 30°C for 24 hours. A loopful of fresh colony was inoculated into 10 ml of nutrient broth and incubated for 24hours at 30 °C. Cells for all experiments were used when the optical density at 600nm was approximately 1.2. An aliquot of a 24-hour-old actively growing culture of the isolates was used at a constant inoculum size for each medium.

Screening for tolerance of selected bacterial isolates

Tolerance of the selected AAB isolates to ethanol and glucose was assessed using the methods of Rahman et al. (2024) and Josue Jr et al. (2025), with some modifications.

Ethanol tolerance assay: 100 ml of each GYC broth was prepared in a 250 ml Erlenmeyer flask and autoclaved at 121 °C for 15 minutes. After sterilization, varying ethanol concentrations (1%, 2%, 5%, and 10%) were added aseptically to the medium, which was then inoculated with an actively growing culture at a constant inoculum size and incubated at 30 °C for 72 hours. After incubation, their growth was assessed by measuring turbidity.

Glucose tolerance assay: For the determination of glucose tolerance of isolated AAB, Erlenmeyer flasks containing GYC broth were adjusted during preparation to different glucose concentrations (10%, 20%, 25%, and 30%). After sterilization, each flask was inoculated with an actively growing culture at a constant inoculum size and incubated at 30 °C for 96 hours. After incubation, the growth of isolated bacteria was assessed by measuring turbidity.

Production of Acetic Acid

All selected bacterial isolates were grown in GYC broth under submerged fermentation to determine the amount of acetic acid produced. Fermentation was carried out in 250 mL Erlenmeyer flasks containing 100 mL each of sterile GYC broth with 5% ethanol added after sterilization, and inoculated with an actively growing culture at a constant inoculum size. The flasks were incubated at 150 rpm for 96 hours at 30 °C. After fermentation, the concentration of acetic acid in the fermented broth was estimated.

Estimation of Acetic Acid production

The concentration of Acetic acid produced in the fermentation broth was estimated by titration, as described by Sahoo et al. (2020). 0.5N NaOH containing 20g of NaOH and 1000 mL of distilled water, and phenolphthalein reagents containing phenolphthalein 0.1g, ethanol 60g, and distilled water 1000 mL were prepared. 5 mL of the fermentation broth was mixed with 20 mL of distilled water, and then 3-5 drops of the phenolphthalein indicator were added. The solution was titrated against 0.5 N sodium hydroxide (NaOH). The amount of NaOH utilized was recorded, and the concentration of acetic acid produced in 100 ml of medium was calculated using the formula:

Acetic acid (g/100 ml) = Volume of NaOH (ml) used in titration × 0.03 × 20

RESULTS

Isolation and screening of acetic acid-producing bacteria

A total of 58 distinct colonies were isolated from GYC agar medium from these 58 colonies, 2 were from unripe and 5 from overripe apple, 8 were from unripe and 5 from overripe Avocado, 6 were from unripe and 6 from overripe Banana, 1 was from unripe and 1 from overripe pineapple, 8 were from unripe and 6 from overripe plantain and 10 were from farm soil sample (Figure 1). Primary screening of AAB in GYC and Carr medium was evaluated based on the ability to form a distinct, clear zone and green to yellow colonies on the surface of the medium (Plate I). Of the 58 isolates screened for acetic acid production, only 35 (60%) produced a clear zone and a colour change, while about 23 (40%) showed no acetic acid potential.

Figure 1: Frequency and Proportion of acetic acid producing bacteria isolated

Plate I: (A) Colonial morphology of acetic acid-producing bacteria on Carr agar medium containing bromocresol green. (B) Pure culture of acetic acid bacteria

Identification of selected bacterial isolates

Table 1 shows the morphological and biochemical characteristics of the selected bacterial isolates. All the bacterial isolates were nonmotile and formed creamy colonies on nutrient agar plates. Gram staining revealed that all selected bacterial isolates belong to the Gram-negative group and are rod-shaped. catalase-positive and reacted positively to the citrate test. The isolated bacteria were tentatively identified as belonging to the genera Gluconobacter and Acetobacter. The percentage of occurrence, as shown in Figure 2, reveals that Gluconobacter has the highest abundance of 69% occurrence, while Acetobacter has an abundance of 31 %

Screening for tolerance capability by selected bacterial isolates

The result showing the ethanol and glucose tolerance of the selected AAB is presented in Table 2. Results for bacterial tolerance to ethanol revealed that all isolates were tolerant (able to grow) at 1, 2, and 5% ethanol, with optimal tolerance (appreciable growth) at 5%. At 10% ethanol concentration, the growth declined gradually except for twelve (12) isolates (UAV-2, UB-2, SS-1, SS-2, SS-3, SS-4, SS-5, SS-6, SS-7, SS-8, SS-9, SS-10), which were not able to grow in 10% concentration.

The glucose tolerance test revealed that all selected AAB isolates showed good growth at 10% and 20% glucose concentrations, but appreciable growth (highest glucose tolerance) was observed at 20%. At 25% glucose concentration, all strains showed moderate growth, and at 30%, minimal growth was observed.

Evaluation of Acetic acid production

The result of the quantity of acetic acid produced by the selected AAB isolates in liquid medium is shown in Figure 3. The yield of acetic acid produced varied among the isolates, ranging from 0.24g/100ml to 16 g/100ml. Isolate SS-7 demonstrated highest acetic acid yield (16g/100ml) (Gluconobacter sp), followed by isolate SS-3 (Gluconobacter sp), SS-4 (Acetobacter sp), and SS-6 (Acetobacter sp), with a yield of 12g/100ml while the lowest acetic acid yield of 0.24g/100ml was produced by isolate OPi-1.

Table 1: Morphological and biochemical characteristics of acetic acid producing bacteria isolated from selected fruits and soil

Figure 2: Frequency of occurrence of AAB (in percentage) isolated from the selected fruit samples

Table 2: AAB growth at different ethanol and glucose concentrations

Key -= no growth, +1=low growth, +2= Moderate growth, +3= Strong positive growth.

Figure 3: Acetic acid-producing ability of isolates

DISCUSSION

In this study, the potential of bacterial strains isolated from selected fruit and soil samples to produce acetic acid was assessed. Acetic acid bacteria strains were successfully isolated from the samples using the serial dilution and spread plate method on GYC agar medium. The highest number of bacteria was recorded in soil, followed by plantain, with pineapple having the least. The observed differences in occurrence might be attributed to soil organic matter content and the availability of substrates these bacteria can utilize for growth. Comparable techniques for the isolation of AAB using GYC agar plates have been used earlier by Varshini et al. (2023) and Sahoo et al. (2020). Patel and Jariwala (2025) isolated AAB from diverse rhizospheric soil and three isolates exhibited notable acidogenic activity. Bellakinmath et al. (2017) isolated AAB from a Western Ghats soil sample and obtained 17 isolates, of which only 2 showed distinct clear zones. The occurrence of Acetic acid-producing bacteria in various natural sources, such as sugar cane juice processing water, palm wine, coconut wine, and deteriorated fruit, has been reported by many researchers (Konate et al., 2014; Kowser et al., 2015; Patil et al., 2025).

The primary selection of Acetic acid-producing bacteria was based on halo zones and yellow-coloured colonies on GYC and Carr media (Diba et al., 2015). The halo formed by the isolates was due to the hydrolysis of calcium carbonate in the GYC medium and to acetic acid produced by the AAB colonies (Dereje et al., 2024; Josue et al., 2025). Various researchers also isolated AAB with a clear halo around colonies (Ouattara et al., 2021; Rahman et al., 2024; Mamiouk and Gullo, 2013; and Bellakinmath et al., 2017). The observed colour change in Carr medium from green (bromocresol green) to yellow after incubation of the isolate could be attributed to the presence of a pH indicator and to the conversion of the ethanol present in the medium to acetic acid (Mukadam et al., 2016; Yanti et al., 2017). This work is similar to Mounir et al. (2016), who also reported that isolates obtained from apple, date cactus, and vinegar were capable of converting the ethanol present in the Carr medium into acetic acid, resulting in a colour change.

According to an earlier study, the criteria for identifying major acetic acid producers, following the characterisation pattern in the ninth edition of Bergey’s manual of systematic bacteriology, are catalase, oxidase, microscopic examination, and Gram reaction (Arifizzaman et al., 2014). The Acetic acid-producing bacteria isolated were tentatively identified as Acetobacter sp. and Gluconobacter sp. based on morphological and biochemical characteristics, with Gluconobacter sp. found to be dominant. The phenotypic characteristics indicated that the bacteria are Gram-negative, motile and non-motile bacilli with ablity to ferment carbohydrates such as glucose. The result supported the report of Bellankimath et al. (2017), who isolated Acetobacter species from deteriorated fruits. Previous studies by Kowser et al. (2015), Mounir et al. (2016), Baheshti-Maal and Shafiee (2019), Lee et al. (2024), and El-Askri et al. (2022) have revealed the presence of various Acetobacter sp. and Gluconobacter sp. from various environmental sources. Paul et al. (2024) also confirmed that species of Acetobacter and Gluconobacter are Gram-negative rods, negative for oxidase and positive for catalase and citrate.

Ethanol concentration plays a critical role in the growth and acetic acid production potential of AAB (Ouattara et al. 2018). Moreover, Islam et al. (2017) and El‐Askri et al. (2022) proposed that ethanol concentration enhances microbial growth and affects the membrane permeability and fluidity of AAB. The AAB strains isolated grew well at 5% ethanol concentration, with diminished growth at higher concentrations. The reduced growth at 10% ethanol concentration implied that high ethanol concentration slows down acetic acid yield. This is consistent with the research of Lee et al. (2015), who documented that a 5% concentration showed strong positive growth. Soumahoro et al. (2015), also reported an optimal acetic acid production at ethanol concentration between 4% and 8%, while Klawpiyapamornkun et al. (2015) reported that 4 to 6 % ethanol content accelerated the growth of AAB, but only a few grew above 10 % ethanol. El-Askri et al. (2022) reported that high concentrations of ethanol reduced growth rate and acetic acid productivity. Additionally, Lee et al. (2024) reported that ethanol concentrations of 10 % reduced growth rates. Islam et al. (2017) suggested that the decrease in growth rate might be due to excess acetic acid formation from sugar consumption in the fermentation medium, further inhibiting acetic acid production and controlling growth at relatively high ethanol concentrations. Increased growth rate at ethanol concentrations between 9% and 20%, indicating a high alcohol tolerance, has been previously reported by Chen et al. (2016) and Beheshti-Maal and Shafiee (2019), contradicting the present study.

Sugar tolerance is also essential for acetic acid fermentation (Song et al. 2022). The isolated AAB grew optimally at 20 % glucose concentration, with some strains doubling their population compared to higher concentrations. Similar findings have been reported in the studies by Kim et al. (2023) and Rahman et al. (2024). However, growth declined at concentrations above 20%, consistent with the findings of Gullo et al. (2006), who reported similar trends in AAB growth.

Almost all selected bacterial isolates produced acetic acid, with varying levels. The variation in acid produced by acetic acid-producing bacteria may be attributed to differences in bacterial strains (Es-sbata et al., 2021). Sahoo et al. (2020) documented a range of 1.1 g/L to 15.2 g/L for 13 acetic acid-producing bacteria isolated from waste fruits. Onyenegecha et al. (2020) also observed acetic acid production of 3.6g/100 mL and 1.8g/100 mL for two AAB strains in the presence of 2% ethanol. Moreover, Alisigwe et al. (2022) reported acetic acid values ranging from 24.34 g/100ml to 23.46g/100ml for strains isolated from selected waste protein sources.

CONCLUSION

Acetic acid-producing microorganisms were isolated from soil and selected fruits. Selected bacterial isolates were screened for acetic acid production potentials on GYC and Carr agar media. Thirty-five (35) of the isolates obtained produced acetic acid and belong to the Gluconobacter and Acetobacter genera. All screened isolates exhibited tolerance to 5% and 20% ethanol and glucose, respectively. The Gluconobacter strain isolated from soil had the highest acetic acid production ability

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