Acetobacter Strains Research Articles

Isolation and Characterization of Acetic Acid Bacteria from Pineapple, Sugarcane, Apple, Grape, Pomegranate, and Papaya Fruit

The present research was carried out with the aim of isolating acetic acid bacteria (AAB) from pineapple, sugarcane, apple, grape, pomegranate, and papaya fruits. The characteristics of acetic acid bacterial isolates were identified by morphological and biochemical tests. Among the different fruits, pineapple possessed the maximum (38.46%) AAB producers, followed by sugarcane (22.90%), grape (18.29%), apple (14.63%), papaya (4.34%) and pomegranate (1.38%). Reference strain Acetobacter pasteurianus (DSM-2324) was collected from Deutsche Sammlung Von Microorganismen and Zellkulturen (DSMZ), Germany. Strains of Acetobacter pasteurianus were identified through morphological and biochemical characteristics. The isolated strains (A) were characterized and compared with the reference strain (B). Different levels of ethanol (1%, 2%, 5%, and 10%), glucose (10%, 20%, 25%, and 30%) and temperature (25 °C, 30 °C, 34 °C, and 37 °C) were used in standard medium to observe the growth characteristics of strain A and B. Both the strains revealed similar trends grown on ethanol, glucose and temperature and showed better growth at 2% ethanol, 20% glucose and 30 °C temperature. The members of the genus Acetobacter and Gluconobacter are confirmed with this research.

Occurrence and diversity pattern of CRISPR-Cas systems in Acetobacter genus provides insights on adaptive defense mechanisms against to invasive DNAs.

The Acetobacter genus is primarily known for its significance in acetic acid production and its application in various industrial processes. This study aimed to shed light on the prevalence, diversity, and functional implications of CRISPR-Cas systems in the Acetobacter genus using a genome mining approach. The investigation analyzed the CRISPR-Cas architectures and components of 34 Acetobacter species, as well as the evolutionary strategies employed by these bacteria in response to phage invasion and foreign DNA. Furthermore, phylogenetic analysis based on CAS1 protein sequences was performed to gain insights into the evolutionary relationships among Acetobacter strains, with an emphasis on the potential of this protein for genotyping purposes. The results showed that 15 species had orphan, while20 species had complete CRISPR-Cas systems, resulting in an occurrence rate of 38% for complete systems in Acetobacter strains. The predicted complete CRISPR-Cas systems were categorized into I-C, I-F, I-E, and II-C subtypes, with subtype I-E being the most prevalent in Acetobacter. Additionally, spacer homology analysis revealed against such the dynamic interaction between Acetobacter strains and foreign invasive DNAs, emphasizing the pivotal role of CRISPR-Cas systems in defending against such invasions. Furthermore, the investigation of the secondary structures of CRISPR arrays revealed the conserved patterns within subtypes despite variations in repeat sequences. The exploration of protospacer adjacent motifs (PAMs) identified distinct recognition motifs in the flanking regions of protospacers. In conclusion, this research not only contributes to the growing body of knowledge on CRISPR-Cas systems but also establishes a foundation for future studies on the adaptive defense mechanisms of Acetobacter. The findings provide valuable insights into the intricate interplay between bacteria and phages, with implications for industrial applications and potential biotechnological advancements.

A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe's vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.

Yeast strains do have an impact on the production of cured cocoa beans, as assessed with Costa Rican Trinitario cocoa fermentation processes and chocolates thereof.

The microbiological and metabolic outcomes of good cocoa fermentation practices can be standardized and influenced through the addition of starter culture mixtures composed of yeast and bacterial strains. The present study performed two spontaneous and 10 starter culture-initiated (SCI) cocoa fermentation processes (CFPs) in Costa Rica with local Trinitario cocoa. The yeast strains Saccharomyces cerevisiae IMDO 050523, Hanseniaspora opuntiae IMDO 020003, and Pichia kudriavzevii IMDO 060005 were used to compose starter culture mixtures in combination with the lactic acid bacterium strain Limosilactobacillus fermentum IMDO 0611222 and the acetic acid bacterium strain Acetobacter pasteurianus IMDO 0506386. The microbial community and metabolite dynamics of the cocoa pulp-bean mass fermentation, the metabolite dynamics of the drying cocoa beans, and the volatile organic compound (VOC) profiles of the chocolate production were assessed. An amplicon sequence variant approach based on full-length 16S rRNA gene sequencing instead of targeting the V4 region led to a highly accurate monitoring of the starter culture strains added, in particular the Liml. fermentum IMDO 0611222 strain. The latter strain always prevailed over the background lactic acid bacteria. A similar approach, based on the internal transcribed spacer (ITS1) region of the fungal rRNA transcribed unit, was used for yeast strain monitoring. The SCI CFPs evolved faster when compared to the spontaneous ones. Moreover, the yeast strains applied did have an impact. The presence of S. cerevisiae IMDO 050523 was necessary for successful fermentation of the cocoa pulp-bean mass, which was characterized by the production of higher alcohols and esters. In contrast, the inoculation of H. opuntiae IMDO 020003 as the sole yeast strain led to underfermentation and a poor VOC profile, mainly due to its low competitiveness. The P. kudriavzevii IMDO 060005 strain tested in the present study did not contribute to a richer VOC profile. Although differences in VOCs could be revealed in the cocoa liquors, no significant effect on the final chocolates could be obtained, mainly due to a great impact of cocoa liquor processing during chocolate-making. Hence, optimization of the starter culture mixture and cocoa liquor processing seem to be of pivotal importance.

Some lactobacillus, leuconostoc and acetobacter strains in traditional turkish yoghurt, cheese, kefir samples as a probiotic candidate

Lactic acid bacteria which are important for production of fermented milk products contain may strains called Lactobacillus, Streptococcus, Lactococcus and Leuconostoccus. As a result, lactic acid bacteria are called ‘milk-souring (fermenting)’ organisms. In addition to the fermentation abilities of Lactobacillus spp., it is important for aroma, texture and acid formation and comprises the most important group of lactic acid bacteria. Their critical importance comes from their metabolic capacity and probiotic features. In this research, yogurt, cheese and kefir samples were collected from cities in Turkey and used to isolate. Isolates were identified phenotypically and genotypically characterized. The probiotic features antibacterial activity against Staphylococcus aureus ATCC6538, Listeria monocytogenes DSM12464, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC51299, and Salmonella Enteritidis ATCC 130762; bile and acid salt tolerance, susceptibility to chloramphenicol, erythromycin, penicillin G, gentamicin, vancomycin, streptomycin, kanamycin, and tetracycline of isolates were determined. Isolates, were identified as Lactobacillus paracasei subspecies (subsp.) paracasei, Lactobacillus delbrueckii subsp. bulgaricus, Acetobacter ghanensis, Acetobacter fabarum, Acetobacter subsp., Leuconostoc pseudomesenteroides, and Leuconostoc mesenteroides subsp. mesenteroides. Some isolates were tolerant of acid and bile salt, some strains were resistant to antibiotics, and some could inhibit pathogens. In this study, isolates were determined to have probiotic features. As a result of the study, it was determined that some isolates showed probiotic properties and had strong antibacterial activity. Isolates can be use as natural alternative in infections.

Antibacterial Activity of Silver Functionalized Bacterial Cellulose isolated from Acetobacter Tropicalis Jcp OK356208.1 from Rotten Orange

Adequate disposal of solid waste products is one of the uncertainties of mankind. The removal of rotten fruits and vegetables has been an increasing issue in the municipal waste management. The recycling of rotten fruits and vegetables into some value added products will be a promising approach for the future waste management. Many established research works indicate the isolation of Acetobacter species from rotten fruits and vegetables. In the present study, five types (AB01, AB02, AB03, AB04, AB05) of different Acetobacter strains were isolated from a variety of rotten fruits samples. Out of that, we successfully screened out Acetobacter tropicalis jcp OK356208.1 from rotten orange samples. The isolate was identified by both phenotypic and genotypic methods. The isolates were screened for their production capacity of both vinegar and cellulose. Hence, through this present investigation, the isolation of a commercially valuable Acetobacter strain was made possible which can be successfully employed for the production of vinegar and cellulose under optimum conditions. Bacterial cellulose is a biopolymer with magnificent implementations in most of the fields such as biomedical, industrial, food and other diverse biotechnological areas. Exploitation of BC as a wound healing material is one of the keen areas of research field.

Immunomodulatory effects of six Acetobacter pasteurianus strains in RAW-Blue macrophage

In this study, we investigated the immunological properties of six strains of Acetobacter pasteurianus through nuclear factor-kappa B/activator protein-1 (NF-κB/AP-1) transcription factor activation and nitric oxide (NO) and cytokine production in macrophages. We found that the six A. pasteurianus strains had no significant inhibitory effect on the cell viability of RAW-Blue™ cells at the concentration of (25, 50, 100 CFU/macrophage). The production of NO and cytokines (TNF-α, IL-1β, and IL-6) showed different abilities of immune activation for each strain, and it was 0.7 to 0.9 times higher than that of the LPS (100 ng/mL, v/v) positive control and 7 to 8 times superior to that of the negative control group. To explore the underlying mechanism, we evaluated the mRNA expression of pro-inflammatory genes. Consequently, we found that inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression including genes expression of cytokines were elevated by the six A. pasteurianus treatment. These results suggested that the six strains of A. pasteurianus have an excellent industrial application value as a functional material for the purpose of enhancing immune function.

Comprehensive deciphering prophages in genus Acetobacter on the ecology, genomic features, toxin-antitoxin system, and linkage with CRISPR-Cas system.

Acetobacter is the predominant microbe in vinegar production, particularly in those natural fermentations that are achieved by complex microbial communities. Co-evolution of prophages with Acetobacter, including integration, release, and dissemination, heavily affects the genome stability and production performance of industrial strains. However, little has been discussed yet about prophages in Acetobacter. Here, prophage prediction analysis using 148 available genomes from 34 Acetobacter species was carried out. In addition, the type II toxin–antitoxin systems (TAs) and CRISPR-Cas systems encoded by prophages or the chromosome were analyzed. Totally, 12,000 prophage fragments were found, of which 350 putatively active prophages were identified in 86.5% of the selected genomes. Most of the active prophages (83.4%) belonged to the order Caudovirales dominated by the families Siphoviridae and Myroviridae prophages (71.4%). Notably, Acetobacter strains survived in complex environments that frequently carried multiple prophages compared with that in restricted habits. Acetobacter prophages showed high genome diversity and horizontal gene transfer across different bacterial species by genomic feature characterization, average nucleotide identity (ANI), and gene structure visualization analyses. About 31.14% of prophages carry type II TAS, suggesting its important role in addiction, bacterial defense, and growth-associated bioprocesses to prophages and hosts. Intriguingly, the genes coding for Cse1, Cse2, Cse3, Cse4, and Cas5e involved in type I-E and Csy4 involved in type I-F CRISPR arrays were firstly found in two prophages. Type II-C CRISPR-Cas system existed only in Acetobacter aceti, while the other Acetobacter species harbored the intact or eroded type I CRISPR-Cas systems. Totally, the results of this study provide fundamental clues for future studies on the role of prophages in the cell physiology and environmental behavior of Acetobacter.

Coproduction of bacterial cellulose and pear vinegar by fermentation of pear peel and pomace.

Bacterial cellulose (BC)-derived materials are given significant attention due to their porous fibrous texture, high crystallinity and extraordinary physico-mechanical properties. The main reason for the restricted use of BC is its high production cost. To reduce the production cost, the suitability of pear residue for the production of BC and pear vinegar was investigated. Komagataeibacter rhaeticus and Komagataeibacter intermedius with high fermentation ability screened from the surface of vinegar film of millet fermentation were used to produce BC and pear vinegar simultaneously. Through response surface optimization, the maximum yield of BC from pear residue medium was 10.94 ± 0.42g/L, which was higher than the synthesis medium generally used for Acetobacter strains. When pear residue medium was incubated at 30°C for 7days, the contents of total acid and soluble solids were greater than 0.3g/100mL and 3%, respectively, which met the standard requirements for fruit vinegar. The flavour components of pear vinegar were determined using gas chromatography-mass spectrometry. The pear vinegar showed similar flavour characteristics to conventional fruit vinegar. This research not only solved the utilization of agricultural resources but also avoided the discharge of waste liquid when producing BC. In addition, a more environmentally friendly and less expensive way to produce BC and pear vinegar was achieved.

현미발효식초의 발효특성 및 3T3-L1 지방세포 분화억제 효과

The brown rice vinegar was prepared by adding 10% of vinegar starter, and incubated using the strain Acetobacter pasteurianus JBA172705. The alcohol concentration of brown rice liquor was adjusted to 6%, and seed vinegar was added to 10%. Then, it was cultured by shaking for 16 days (30°C, 120 rpm). After fermentation, brown rice vinegar was extracted with 70% ethanol to evaluate the anti-obesity effect using 3T3-L1 adipocyte cells. The total acidity of brown rice vinegar increased to 6.43% after 16 days of fermentation, and acetic acid increased 15 times and succinic acid 2.5 times or more. The total phenolics and free amino acid contents decreased during the acetic acid fermentation period, and were 48.53 mg GAE/mL and 18.79 mg% (w/v) at the 16th day of fermentation. At a concentration of 100 μg/mL of brown rice vineagar extract (BRV 16d), the lipid accumulation rate was 72.2%. The leptin expression level was reduced by more than 50% according to the treatment of brown rice vinegar extract. It was confirmed that the PPARγ and SREBP-1c expression levels decreased at a concentration of 100 μg/mL. As a result, brown rice vinegar is expected to be effective in anti-obesity by suppressing the expression of transcription factors that induce obesity.

Characterization of nanocellulose production by strains of Komagataeibacter sp. isolated from organic waste and Kombucha

Bacterial nanocellulose production is gaining popularity owing to its applications in food, cosmetics and medical industry. Three Acetobacter strains isolated from organic waste and fermented tea were identified using 16S rDNA sequencing and their ability to produce nanocellulose was studied. Strain isolated from Kombucha has 99% homology with Komagataeibacter rhaeticus DSM 16663 T. This is the first report where nanocellulose productivity of this strain with different carbon sources such as glucose, glycerol, fructose and sucrose has been studied. 1% glycerol was found to be optimal concentration, with up to 69% of the utilized carbon converted to nanocellulose. Maximum productivity of 4.5 g/L of bacterial nanocellulose was obtained. Average nitrogen and phosphorus consumption rate was 45 mg/L/day each. Physical properties such as crystallinity, fibril dimensions, and glass transition temperature were studied. Bacterial cellulose was 80% crystalline when glycerol and glucose were used as carbon source and 73% for fructose and sucrose. Renewable materials such as bacterial cellulose with their unique properties are the future for applications in the field of cosmetics, composite and wound care.

Microbial Dynamics between Yeasts and Acetic Acid Bacteria in Kombucha: Impacts on the Chemical Composition of the Beverage.

Kombucha is a traditional low-alcoholic beverage made from sugared tea and transformed by a complex microbial consortium including yeasts and acetic acid bacteria (AAB). To study the microbial interactions and their impact on the chemical composition of the beverage, an experimental design with nine couples associating one yeast strain and one AAB strain isolated from original black tea kombucha was set up. Three yeast strains belonging to the genera Brettanomyces, Hanseniaspora, and Saccharomyces and three strains of Acetobacter and Komagataeibacter species were chosen. Monocultures in sugared tea were analyzed to determine their individual microbial behaviors. Then, cultivation of the original kombucha consortium and cocultures in sugared tea were compared to determine the interactive microbial effects during successive phases in open and closed incubation conditions. The results highlight the main impact of yeast metabolism on the product’s chemical composition and the secondary impact of bacterial species on the composition in organic acids. The uncovered microbial interactions can be explained by different strategies for the utilization of sucrose. Yeasts and AAB unable to perform efficient sucrose hydrolysis rely on yeasts with high invertase activity to access released monosaccharides. Moreover, the presence of AAB rerouted the metabolism of Saccharomyces cerevisiae towards higher invertase and fermentative activities.

Investigating the antibacterial effects of some Lactobacillus, Bifidobacterium and acetobacter strains killed by different methods on Streptococcus mutans and Escherichia coli.

Although there are many health advantages assigned to different live bacteria such as probiotics, some health threatening effects have also been reported. For example, live bacteria can transfer antibiotic resistance genes to other commensal and opportunistic bacteria of gastrointestinal tract. Recently, it was shown that using killed bacteria have some advantages over live ones. In this research, heat, paraformaldehyde and ozone killing methods were used to kill the bacteria. Acetobacter cerevisiae, Lactobacillus acidophilus, Bifidobacterium lactis and traditional vinegar and fermented dairy product (Kumeh) derived bacteria were killed and their antibacterial activity against Streptococcus mutans and Escherichia coli was investigated. To identify the bacteria isolated from the traditional products, 16S rDNA gene was partially sequenced. The gene analysis showed vinegar and Kumeh derived bacteria were Acetobacter pasteurianus and Lactobacillus crustorum (LcK) strains respectively. The S. mutans growth inhibition was detected in the all concentrations of all killed samples. However, generally, E. coli showed more resistant to the killed bacteria than S. mutans and the antibacterial effect of heat-killed bacteria against E. coli was not observed in the all concentrations for some killed bacteria. Among the pathogenic bacteria, S. mutans was the most sensitive one to the killed bacteria with 70% of reduction in its viability. In conclusion, this research showed that different killed bacteria had different effects on other bacteria and the killing method showed an impact on these effects. Overall, paraformaldehyde-killed L.crustorum (LcK) showed the best antibacterial activity against S. mutans; about 70% decrease in bacterial viability.

Characteristics of Biocellulose from Sago Liquid Waste with Different Ammonium Sulfate Concentration

This research aims to utilize sago liquid waste to produce biocellulose with different concentration of ammonium sulfate and to know characteristics of biocellulose. Production of biocellulose was done in sago liquid waste substrate with different concentration of ammonium sulfate, namely 0.5, 1, 1.5 and 2% (w/v) by using bacteria local strain Acetobacter xylinum LKN6. The observed parameters included thickness, yield, crude fiber and organoleptic.The analysis results showed that ammonium sulfate concentration given a different to the thickness, yield, crude fiber and organoleptic. The best characteristic of biocellulose obtained in the treatment of 1.5% ammonium sulfate concentration. The biocellulose from sago liquid waste potential as dietary fiber food.

Spontaneous fermentation tunes the physicochemical properties of sweet potato starch by modifying the structure of starch molecules

The effects of spontaneous fermentation on the molecular and physicochemical characteristics of sweet potato starch stored in tank during twelve months were investigated. From starch slurry collected during spontaneous fermentation, eight isolates showed amylolytic activity, which included two Acetobacter strains, five Bacillus strains and one Gluconacetobacter strain. By spontaneous fermentation, the amylose content and the average molecular weight of starch were significantly decreased. Besides, the native and fermented starches showed different amylopectin chain-length distribution patterns. Among them, no significant differences in granular morphology, granule size distribution, and crystalline structure. However, the thermal and pasting properties as well as the hardness of the starch gel differed significantly. Pearson's correlation analysis showed that the physicochemical properties was mainly influenced by the changes in the amylose content, amylopectin chain-length distribution as well as the average molecular weight of starch. These findings demonstrated the feasibility of spontaneous fermentation as a tool for modifying starches.

Aroma profile of pilot plant-scale produced fruit vinegar using a thermotolerant Acetobacter pasteurianus strain isolated from Moroccan cactus

In this study, new thermotolerant Acetobacter pasteurianus CV01 strain recently isolated from local product of Morocco has been investigated for its ability to perform efficient acetous fermentation at a large-scale. Firstly, the thermotolerance basis bioconversion of CV01 strain was compared to other mesophilic and thermotolerant acetic acid bacteria. Subsequently, CV01 strain was assessed for its ability to produce and tolerate high amount of acetic acid at optimal and thermal stress conditions in lab-scale bioreactor. It was found that the studied strain exhibited thermotolerant properties compared to reference strains and could withstand the increase in temperature during acetous fermentation in fermenter. Furthermore, gas chromatography-mass spectrometry (GC-MS) was used on the samples prepared with solid phase microextraction (SPME) to determine the volatile compounds of the pilot plant produced apple vinegar based on developed start-up and semi-continuous fermentation protocol. The operation strategy in the 500-L pilot plant scale acetator allowed achieving 7.3% (w/v) of final acetic acid concentration recording high yield and acetification rate. The aroma profile of experimentally produced vinegar was found different from that of the commercial reference one. According to the literature, the results obtained show that major volatile compounds found in pilot-plant produced apple vinegar are related to good aromatic note descriptors which could have a positive impact on the organoleptic quality of industrial vinegar. Consequently, it can be concluded that CV01 Acetobacter strain is well suited for large-scale production of high quality fruit vinegar.

Characterization of Bacteria in Nigerian Yogurt as Promising Alternative to Antibiotics in Gastrointestinal Infections

ABSTRACTGastrointestinal infections are endemic in Nigeria and several factors contribute to their continual survival, including bacterial resistance to commonly used antibiotics. Nigerian yogurts do not include probiotics, and limited information is available about the antimicrobial properties of the fermenters in the yogurt against gastrointestinal pathogens. Therefore, the antimicrobial potentials of bacteria in Nigeria-produced yogurts against intestinal pathogens were investigated in this study. Viable counts of lactic acid bacteria (LAB) in 15 brands of yogurt were enumerated and the bacteria identified by partial sequencing of 16S rRNA gene. Susceptibility of the gastrointestinal pathogens (Salmonella, Shigella and E. coli ) to antibiotics by disc diffusion method, to viable LAB by the agar overlay method, and to the cell-free culture supernatant (CFCS) of the LAB were investigated. Co-culture analysis of LAB and pathogens were also done. Viable counts of 1.5 × 1011 cfu/ml were observed in some yogurt samples. Two genera were identified: Lactobacillus (70.7%) and Acetobacter (29.3%). The Lactobacillus species reduced multidrug-resistant gastrointestinal pathogens by 4 to 5 log while the zones of inhibition ranged between 11 and 23. The Lactobacillus and Acetobacter strains examined displayed good activities against the multidrug-resistant tested pathogens. This is the first report of antimicrobial activities of acetic acid bacteria isolated from yogurt in Nigeria.

Effect of chitosan and SO2 on viability of Acetobacter strains in wine

Wine spoilage is an important concern for winemakers to preserve the quality of their final product and avoid contamination throughout the production process. The use of sulphur dioxide (SO2) is highly recommended to prevent wine spoilage due to its antimicrobial activity. However, SO2 has a limited effect on the viability of acetic acid bacteria (AAB). Currently, the use of SO2 alternatives is favoured in order to reduce the use of chemicals and improve stabilization in winemaking. Chitosan is a biopolymer that is approved by the European authorities and the International Organization of Vine and Wine to be used as a fining agent and antimicrobial in wines. However, its effectiveness in AAB prevention has not been studied.Two strains of Acetobacter, adapted to high ethanol environments, were analysed in this study. Both chitosan and SO2 effects were compared in artificially contaminated wines. Both molecules reduced the metabolic activity of both AAB strains. Although AAB populations were detected by culture independent techniques, their numbers were reduced with time, and their viability decreased following the application of both products, especially with chitosan.

Effect of ammonium and amino acids on the growth of selected strains of Gluconobacter and Acetobacter

Acetic acid bacteria (AAB) are a group of microorganisms highly used in the food industry. However, its use can be limited by the insufficient information known about the nutritional requirements of AAB for optimal growth. The aim of this work was to study the effects of different concentrations and sources of nitrogen on the growth of selected AAB strains and to establish which nitrogen source best encouraged their growth. Two strains of three species of AAB, Gluconobacter japonicus, Gluconobacter oxydans and Acetobacter malorum, were grown in three different media with diverse nitrogen concentrations (25, 50, 100, and 300mgN/L and 1gN/L) as a complete solution of amino acids and ammonium. With this experiment, the most favourable medium and the lowest nitrogen concentration beneficial for the growth of each strain was selected. Subsequently, under these conditions, single amino acids or ammonium were added to media individually to determine the best nitrogen sources for each AAB strain. The results showed that nitrogen requirements are highly dependent on the nitrogen source, the medium and the AAB strain. Gluconobacter strains were able to grow in the lowest nitrogen concentration tested (25mgN/L); however, one of the G. oxydans strains and both A. malorum strains required a higher concentration of nitrogen (100–300mgN/L) for optimal growth. In general, single nitrogen sources were not able to support the growth of these AAB strains as well as the complete solution of amino acids and ammonium.

Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were phylogenetically more closely related each other than with Gluconobacter.