Scale-dependent tipping points of bacterial colonization resistance.

Bacteria are efficient colonizers of a wide range of secluded microhabitats, such as soil pores, skin follicles, or intestinal crypts. How the structural diversity of these habitats modulates microbial self-organization remains poorly understood, in part because of the difficulty to precisely manipulate the physical structure of microbial environments. Using a microfluidic device to grow bacteria in crypt-like incubation chambers of systematically varied lengths, we show that small variations in the physical structure of the microhabitat can drastically alter bacterial colonization success and resistance against invaders. Small crypts are uncolonizable; intermediately sized crypts can stably support dilute populations, while beyond a second critical length scale, populations phase separate into a dilute region and a jammed region. The jammed state is characterized by extreme colonization resistance, even if the resident strain is suppressed by an antibiotic. Combined with a flexible biophysical model, we demonstrate that colonization resistance and associated priority effects can be explained by a crowding-induced phase transition, which results from a competition between proliferation and density-dependent cell leakage. The emerging sensitivity to scale underscores the need to control for scale in microbial ecology experiments. Systematic flow-adjustable length-scale variations may serve as a promising strategy to elucidate further scale-sensitive tipping points and to rationally modulate the stability and resilience of microbial colonizers.

Arc1 and the microbiota together modulate growth and metabolic traits in Drosophila.

Perturbations to animal-associated microbial communities (the microbiota) have deleterious effects on various aspects of host fitness, but the molecular processes underlying these impacts are poorly understood. Here, we identify a connection between the microbiota and the neuronal factor Arc1 that affects growth and metabolism in Drosophila. We find that Arc1 exhibits tissue-specific microbiota-dependent expression changes, and that germ-free flies bearing a null mutation of Arc1 exhibit delayed and stunted larval growth, along with a variety of molecular, cellular and organismal traits indicative of metabolic dysregulation. Remarkably, we show that the majority of these phenotypes can be fully suppressed by mono-association with a single Acetobacter sp. isolate, through mechanisms involving both bacterial diet modification and live bacteria. Additionally, we provide evidence that Arc1 function in key neuroendocrine cells of the larval brain modulates growth and metabolic homeostasis under germ-free conditions. Our results reveal a role for Arc1 in modulating physiological responses to the microbial environment, and highlight how host-microbe interactions can profoundly impact the phenotypic consequences of genetic mutations in an animal host.

Genome-scale metabolic modeling of Acetobacter pasteurianus 386B reveals its metabolic adaptation to cocoa fermentation conditions.

Acetobacter pasteurianus 386B has been selected as a candidate functional starter culture to better control the cocoa fermentation process. Previously, its genome has been sequenced and a genome-scale metabolic model (GEM) has been reconstructed. To understand its metabolic adaptation to cocoa fermentation conditions, different flux balance analysis (FBA) simulations were performed and compared with experimental data. In particular, metabolic flux distributions were simulated for two phases that characterize the growth of A. pasteurianus 386B under cocoa fermentation conditions, predicting a switch in respiratory chain usage in between these phases. The possible influence on the resulting energy production was shown using a reduced version of the GEM. FBA simulations revealed the importance of the compartmentalization of the ethanol oxidation reactions, namely in the periplasm or in the cytoplasm, and highlighted the potential role of ethanol as a source of carbon, energy, and NADPH. Regarding the latter, the physiological function of a proton-translocating NAD(P)+ transhydrogenase was further investigated in silico. This study revealed the potential of using a GEM to simulate the metabolism of A. pasteurianus 386B, and may provide a general framework toward a better physiological understanding of functional starter cultures in food fermentation processes.

Drosophila-associated bacteria differentially shape the nutritional requirements of their host during juvenile growth.

The interplay between nutrition and the microbial communities colonizing the gastrointestinal tract (i.e., gut microbiota) determines juvenile growth trajectory. Nutritional deficiencies trigger developmental delays, and an immature gut microbiota is a hallmark of pathologies related to childhood undernutrition. However, how host-associated bacteria modulate the impact of nutrition on juvenile growth remains elusive. Here, using gnotobiotic Drosophila melanogaster larvae independently associated with Acetobacter pomorumWJL (ApWJL) and Lactobacillus plantarumNC8 (LpNC8), 2 model Drosophila-associated bacteria, we performed a large-scale, systematic nutritional screen based on larval growth in 40 different and precisely controlled nutritional environments. We combined these results with genome-based metabolic network reconstruction to define the biosynthetic capacities of Drosophila germ-free (GF) larvae and its 2 bacterial partners. We first established that ApWJL and LpNC8 differentially fulfill the nutritional requirements of the ex-GF larvae and parsed such difference down to individual amino acids, vitamins, other micronutrients, and trace metals. We found that Drosophila-associated bacteria not only fortify the host's diet with essential nutrients but, in specific instances, functionally compensate for host auxotrophies by either providing a metabolic intermediate or nutrient derivative to the host or by uptaking, concentrating, and delivering contaminant traces of micronutrients. Our systematic work reveals that beyond the molecular dialogue engaged between the host and its bacterial partners, Drosophila and its associated bacteria establish an integrated nutritional network relying on nutrient provision and utilization.

Comparative Genomic Analysis of Closely Related Acetobacter pasteurianus Strains Provides Evidence of Horizontal Gene Transfer and Reveals Factors Necessary for Thermotolerance.

Acetobacter pasteurianus is an industrial strain used for the vinegar production. Many A. pasteurianus strains with different phenotypic characteristics have been isolated so far. To understand the genetic background underpinning these phenotypes, a comparative genomic analysis of A. pasteurianus strains was conducted. Based on bioinformatics and experimental results, we report the following. (i) The gene repertoire related to the respiratory chains showed that several horizontal gene transfer events occurred after the divergence of these strains, indicating that the respiratory chain in A. pasteurianus has the diversity to adapt to its environment. (ii) There is a clear difference in thermotolerance even between 12 closely related strains. NBRC 3279, NBRC 3284, and NBRC 3283, in particular, which have only 55 mutations in total, showed differences in thermotolerance. The Na+/H+ antiporter gene nhaK2 was mutated in the thermosensitive NBRC 3279 and NBRC 3284 strains and not in the thermotolerant NBRC 3283 strain. The Na+/H+ antiporter activity of the three strains and expression of nhaK2 gene from NBRC 3283 in the two thermosensitive strains showed that these mutations are critical for thermotolerance. These results suggested that horizontal gene transfer events and several mutations have affected the phenotypes of these closely related strains.IMPORTANCEAcetobacter pasteurianus, an industrial vinegar-producing strain, exhibits diverse phenotypic differences such as respiratory activity related to acetic acid production, acetic acid resistance, or thermotolerance. In this study, we investigated the correlations between genome sequences and phenotypes among closely related A. pasteurianus strains. The gene repertoire related to the respiratory chains showed that the respiratory components of A. pasteurianus has a diversity caused by several horizontal gene transfers and mutations. In three closely related strains with clear differences in their thermotolerances, we found that the insertion or deletion that occurred in the Na+/H+ antiporter gene nhaK2 is directly related to their thermotolerance. Our study suggests that a relatively quick mutation has occurred in the closely related A. pasteurianus due to its genetic instability and that this has largely affected its phenotype.

In vitro thermal adaptation of mesophilic Acetobacter pasteurianus NBRC 3283 generates thermotolerant strains with evolutionary trade-offs.

Thermotolerant strains are critical for low-cost high temperature fermentation. In this study, we carried out the thermal adaptation of A. pasteurianus IFO 3283-32 under acetic acid fermentation conditions using an experimental evolution approach from 37ºC to 40ºC. The adapted strain exhibited an increased growth and acetic acid fermentation ability at high temperatures, however, with the trade-off response of the opposite phenotype at low temperatures. Genome analysis followed by PCR sequencing showed that the most adapted strain had 11 mutations, a single 64-kb large deletion, and a single plasmid loss. Comparative phenotypic analysis showed that at least the large deletion (containing many ribosomal RNAs and tRNAs genes) and a mutation of DNA polymerase (one of the 11 mutations) critically contributed to this thermotolerance. The relationship between the phenotypic changes and the gene mutations are discussed, comparing with another thermally adapted A. pasteurianus strains obtained previously.

Waste valorization: Identification of an ethanol tolerant bacterium Acetobacter pasteurianus SKYAA25 for acetic acid production from apple pomace.

In present study, a potential bacterial isolate Acetobacter pasteurianus SKYAA25 was found to be very effective in the bioconversion of apple pomace to acetic acid. The isolated strain was tolerant to high ethanol concentrations of upto 14% and temperature of 42 °C. Fermentation of apple pomace alone in presence of brewing yeast produced 7.3% of bio-ethanol which was further used for acetic acid production. Apple pomace in combination with cane molasses produced 14% of bio-ethanol. The fermented bio-ethanol was used as medium for acetic acid production which yielded 52.4 g of acetic acid/100 g of DM (Dry Matter) of apple pomace. Hence, an ecofriendly process has been developed that is entirely based on microbial processing of apple pomace to produce acetic acid without involving commercial enzymes. The present bio-conversion will prove to be beneficial for utilizing food and beverage industrial waste in the production of acetic acid.

Modified semi-continuous fermentation for resuscitating nongrowing cells during high-temperature gluconic acid production by Acetobacter senegalensis.

AIMS: The formation of metabolically inactive and nongrowing cells is an inevitable by-product of intensive fermentation. This study investigated whether co-feeding can be used to resuscitate nongrowing Acetobacter senegalensis cells to enable them to produce gluconic acid in successive fermentation runs at 38°C. METHODS AND RESULTS: In the first fermentation cycle, 75 g l-1 of glucose were converted to gluconic acid. Subsequently, however, stationary-phase cells were unable to initiate a new fermentation cycle. The majority of stationary-phase cells (97%) were nonculturable on glucose at 38°C. In addition, 54 and 41% of cells contained non-active cellular dehydrogenases and a compromised cell envelope respectively. Co-feeding stationary-phase cells with a mixture of ethanol, glucose and acetic acid for 7 h enabled these cells to grow on 75 g l-1 of glucose and produce gluconic acid. Additionally, 74% of cells contained active forms of cellular dehydrogenases after 7 h of co-feeding. However, co-feeding did not improve cell envelope integrity. Quantification of cellular NAD content showed that stationary-phase cells contained moderately reduced levels of total NAD (NADt) as compared with exponential-phase cells. Interestingly, the analysis of stationary-phase cells showed that co-feeding resulted in higher levels of NADt and NADH, suggesting that the regeneration of NADH is one of the limiting factors of glucose consumption. Expression of catalase and superoxide dismutase was increased in stationary-phase cells, but analysis of protein carbonylation and lipid peroxidation did not confirm an extensive oxidative stress. CONCLUSIONS: Co-feeding with favourable nutrients may enable resuscitation of cells and utilization of less-favourable carbon sources in successive cycles. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposed a unique method for resuscitation of nongrowing cells during high-temperature fermentation. By applying this method, cells can be used for consecutive fermentation cycles.

Prevalence and Antimicrobial Properties of Lactic Acid Bacteria in Nigerian Women During the Menstrual Cycle.

The composition of vagina lactic acid bacteria (LAB) differs within the different ethnic group. This study is aimed at determining the prevalence of LAB with their antimicrobial properties in Nigerian women's vagina during different stages of the menstrual cycle. Microorganisms were isolated from vaginal swabs of ten Nigerian women during different stages of the menstrual cycle and identified by partial sequencing of the 16S rRNA gene. The antimicrobial properties of the LAB were tested against the multidrug-resistant uropathogens. The prevalence of LAB was higher during ovulation period while during menstruation period, it declined. Twenty-five LAB isolates were identified as three species, namely: Lactobacillus plantarum (15), Lactobacillus fermentum (9), Lactobacillus brevis (1) and one acetic acid bacteria - Acetobacter pasteurianus. The LAB had antimicrobial activities against the three uropathogens with zones of inhibition from 8 to 22 mm. The presence of LAB inhibits the growth of Staphylococcus sp. GF01 also in the co-culture. High LAB counts were found during ovulation period with L. plantarum as a dominant species while during menstruation, there was a decrease in the LAB counts. The isolated LAB has antimicrobial properties against the urogenital pathogens tested thus exhibiting their potential protective role against uropathogens.The composition of vagina lactic acid bacteria (LAB) differs within the different ethnic group. This study is aimed at determining the prevalence of LAB with their antimicrobial properties in Nigerian women's vagina during different stages of the menstrual cycle. Microorganisms were isolated from vaginal swabs of ten Nigerian women during different stages of the menstrual cycle and identified by partial sequencing of the 16S rRNA gene. The antimicrobial properties of the LAB were tested against the multidrug-resistant uropathogens. The prevalence of LAB was higher during ovulation period while during menstruation period, it declined. Twenty-five LAB isolates were identified as three species, namely: Lactobacillus plantarum (15), Lactobacillus fermentum (9), Lactobacillus brevis (1) and one acetic acid bacteria ­ Acetobacter pasteurianus. The LAB had antimicrobial activities against the three uropathogens with zones of inhibition from 8 to 22 mm. The presence of LAB inhibits the growth of Staphylococcus sp. GF01 also in the co-culture. High LAB counts were found during ovulation period with L. plantarum as a dominant species while during menstruation, there was a decrease in the LAB counts. The isolated LAB has antimicrobial properties against the urogenital pathogens tested thus exhibiting their potential protective role against uropathogens.

Gut microbiota affects development and olfactory behavior in Drosophila melanogaster.

It has been shown that gut microbes are very important for the behavior and development of Drosophila, as the beneficial microbes are involved in the identification of suitable feeding and egg-laying locations. However, in what way these associated gut microbes influence the fitness-related behaviors of Drosophila melanogaster remains unclear. Here, we show that D. melanogaster exhibits different behavioral preferences towards gut microbes. Both adults and larvae were attracted by the volatile compounds of Saccharomyces cerevisiae and Lactobacillus plantarum, but were repelled by Acetobacter malorum in behavioral assays, indicating that an olfactory mechanism is involved in these preference behaviors. While the attraction to yeast was governed by olfactory sensory neurons expressing the odorant co-receptor Orco, the observed behaviors towards the other microbes were retained in flies lacking this co-receptor. By experimentally manipulating the microbiota of the flies, we found that flies did not strive for a diverse microbiome by increasing their preference towards gut microbes that they had not experienced previously. Instead, in some cases, the flies even increased preference for the microbes on which they were reared. Furthermore, exposing Drosophila larvae to all three microbes promoted Drosophila development, while exposure to only S. cerevisiae and A. malorum resulted in the development of larger ovaries and in increased egg numbers in an oviposition assay. Thus, our study provides a better understanding of how gut microbes affect insect behavior and development, and offers an ecological rationale for preferences of flies for different microbes in their natural environment.

Drosophila suzukii (Diptera: Drosophilidae) Contributes to the Development of Sour Rot in Grape.

This research aimed to more clearly describe the interactions of Drosophila suzukii (Matsumura; Diptera: Drosophilidae) with microorganisms that may contribute to spoilage or quality loss of wine grapes during harvest. Experiments were conducted in controlled laboratory experiments and under field conditions to determine these effects. Laboratory trials determined the role of insect contact and oviposition to vector spoilage bacteria onto wine grapes. In the field, the roles of key organoleptic parameters in grape fruit ripening were assessed to determine their relative contribution to oviposition potential as fruit ripened. Finally, field trials determined the relationships of egg and larval infestation to sour rot levels. Non-ovipositional trials indicated elevated levels of microbiota when D. suzukii was present. D. suzukii oviposition exponentially increased the concentration of acetic acid bacteria. Both incised and sound berries showed a significant increase in concentrations of acetic acid bacteria exposed to D. suzukii. Volatile acidity was higher in treatments infested with D. suzukii. Fruit with only eggs did not develop a significant increase of volatile acidity. Larva-infested grape berries in 9.5% of samples developed higher volatile acidity after 14 d. Sound grape berries were less susceptible to the development of microbiota associated with sour rot and spoilage. D. suzukii oviposition and larval development increase risk of spoilage bacteria vectored by D. suzukii adults. Acetic acid bacteria induced fermentation and produced several volatile compounds contributing to spoilage. Spoilage bacteria may create a positive feedback loop that attracts both D. suzukii and other drosophilids, which may contribute to additional spoilage.

Commensal bacteria and essential amino acids control food choice behavior and reproduction.

Choosing the right nutrients to consume is essential to health and wellbeing across species. However, the factors that influence these decisions are poorly understood. This is particularly true for dietary proteins, which are important determinants of lifespan and reproduction. We show that in Drosophila melanogaster, essential amino acids (eAAs) and the concerted action of the commensal bacteria Acetobacter pomorum and Lactobacilli are critical modulators of food choice. Using a chemically defined diet, we show that the absence of any single eAA from the diet is sufficient to elicit specific appetites for amino acid (AA)-rich food. Furthermore, commensal bacteria buffer the animal from the lack of dietary eAAs: both increased yeast appetite and decreased reproduction induced by eAA deprivation are rescued by the presence of commensals. Surprisingly, these effects do not seem to be due to changes in AA titers, suggesting that gut bacteria act through a different mechanism to change behavior and reproduction. Thus, eAAs and commensal bacteria are potent modulators of feeding decisions and reproductive output. This demonstrates how the interaction of specific nutrients with the microbiome can shape behavioral decisions and life history traits.

Complete Genome Sequencing and Comparative Genomic Analysis of the Thermotolerant Acetic Acid Bacterium, Acetobacter pasteurianus SKU1108, Provide a New Insight into Thermotolerance.

Acetobacter pasteurianus SKU1108 is a typical thermotolerant acetic acid bacterium. In this study, the complete genome sequence of the SKU1108 strain was elucidated, and information on genomic modifications due to the thermal adaptation of SKU1108 was updated. In order to obtain a clearer understanding of the genetic background responsible for thermotolerance, the SKU1108 genome was compared with those of two closely related complete genome strains, thermotolerant A. pasteurianus 386B and mesophilic A. pasteurianus NBRC 3283. All 24 "thermotolerant genes" required for growth at higher temperatures in the thermotolerant Acetobacter tropicalis SKU1100 strain were conserved in all three strains. However, these thermotolerant genes accumulated amino acid mutations. Some biased mutations, particularly those that occurred in xanthine dehydrogenase XdhA, may be related to thermotolerance. By aligning whole genome sequences, we identified ten SKU1108 strain-specific regions, three of which were conserved in the genomes of the two thermotolerant A. pasteurianus strains. One of the regions contained a unique paralog of the thermotolerant gene xdhA, which may also be responsible for conferring thermotolerance. Thus, comparative genomics of complete genome sequences may provide novel insights into the phenotypes of these thermotolerant strains.

Assessment of the contribution of cocoa-derived strains of Acetobacter ghanensis and Acetobacter senegalensis to the cocoa bean fermentation process through a genomic approach.

Acetobacter ghanensis LMG 23848(T) and Acetobacter senegalensis 108B are acetic acid bacteria that originate from a spontaneous cocoa bean heap fermentation process and that have been characterised as strains with interesting functionalities through metabolic and kinetic studies. As there is currently little genetic information available for these species, whole-genome sequencing of A. ghanensis LMG 23848(T) and A. senegalensis 108B and subsequent data analysis was performed. This approach not only revealed characteristics such as the metabolic potential and genomic architecture, but also allowed to indicate the genetic adaptations related to the cocoa bean fermentation process. Indeed, evidence was found that both species possessed the genetic ability to be involved in citrate assimilation and displayed adaptations in their respiratory chain that might improve their competitiveness during the cocoa bean fermentation process. In contrast, other properties such as the dependence on glycerol or mannitol and lactate as energy sources or a less efficient acid stress response may explain their low competitiveness. The presence of a gene coding for a proton-translocating transhydrogenase in A. ghanensis LMG 23848(T) and the genes involved in two aromatic compound degradation pathways in A. senegalensis 108B indicate that these strains have an extended functionality compared to Acetobacter species isolated from other ecosystems.

Genomic analyses of thermotolerant microorganisms used for high-temperature fermentations.

Environmental adaptation is considered as one of the most challenging subjects in biology to understand evolutionary or ecological diversification processes and in biotechnology to obtain useful microbial strains. Temperature is one of the important environmental stresses; however, microbial adaptation to higher temperatures has not been studied extensively. For industrial purposes, the use of thermally adapted strains is important, not only to reduce the cooling expenses of the fermentation system, but also to protect fermentation production from accidental failure of thermal management. Recent progress in next-generation sequencing provides a powerful tool to track the genomic changes of the adapted strains and allows us to compare genomic DNA sequences of conventional strains with those of their closely related thermotolerant strains. In this article, we have attempted to summarize our recent approaches to produce thermotolerant strains by thermal adaptation and comparative genomic analyses of Acetobacter pasteurianus for high-temperature acetic acid fermentations, and Zymomonas mobilis and Kluyveromyces marxianus for high-temperature ethanol fermentations. Genomic analysis of the adapted strains has found a large number of mutations and/or disruptions in highly diversified genes, which could be categorized into groups related to cell surface functions, ion or amino acid transporters, and some transcriptional factors. Furthermore, several phenotypic and genetic analyses revealed that the thermal adaptation could lead to decreased ROS generation in cells that produce higher ROS levels at higher temperatures. Thus, it is suggested that the thermally adapted cells could become robust and resistant to many stressors, and thus could be useful for high-temperature fermentations.

EVALUATION OF THERMOTOLERANT ACETOBACTER PASTEURIANUS STRAINS ISOLATED FROM MOROCCAN FRUITS CATALYZING OXIDATIVE FERMENTATION AT HIGH TEMPERATURE.

Six strains of acetic acid bacteria were isolated from Moroccan local products and their potential as industrial strains was evaluated in lab-bioreactor. Three of them, namely TAV01, AF01 and CV01, isolated from traditional apple vinegar, apple and cactus fruit, respectively were selected and their responses to high temperature were assessed. Morphological and biochemical identification confirmed that these strains belong to Acetobacter species. Their growth and acetic acid production were compared with the thermoresistant reference strain, Acetobacter senegalensis and mesophilic strains of Acetobacter pasteurianus. The two strains AF01 and CV01 showed abundant growth and noticeable acetic acid production ability at high temperatures (38 to 41°C). A thermophilic character was observed for AF01 strain. Indeed, this bacterium grew better at 38 than 30°C.

Protein profile of Acetobacter pasteurianus HSZ3-21.

Acetobacter pasteurianus plays an important role in the process of traditional vinegar production and is also essential for the fermentation of Zhenjiang aromatic vinegar. In this study, we utilized the proteomic approach to analyze the proteomic profile of A. pasteurianus HSZ3-21, and 258 proteins were successfully identified by MALDI-TOF-MS and database search. The hydropathy and GO analyse combined with COG results of the identified proteins revealed the molecular biological characteristics of A. pasteurianus proteins, that is, most proteins of A. pasteurianus were related to metabolic process, binding, catalytic or cellular response. Meanwhile, our results also showed that some proteins of A. pasteurianus may be responsible for acetic acid tolerance, thermotolerance, and stress response. Therefore, the identification of 258 proteins not only deciphers protein composition and functional classification of A. pasteurianus, but also provides useful information for improving quality of Zhenjiang aromatic vinegar.

Characterization of Acetobacter pomorum KJY8 isolated from Korean traditional vinegar.

Acetobacter sp. strains were isolated from traditional vinegar collected in Daegu city and Gyeongbuk province. The strain KJY8 showing a high acetic acid productivity was isolated and characterized by phenotypic, chemotaxonomic, and phylogenetic inference based on 16S rRNA sequence analysis. The chemotaxonomic and phylogenetic analyses revealed the isolate to be a strain of Acetobacter pomorum. The isolate showed a G+C content of 60.8 mol%. It contained LL-diaminopimelic acid (LL-A2pm) as the cell wall amino acid and ubiquinone Q9 (H6) as the major quinone. The predominant cellular fatty acids were C18:1w9c, w12t, and w7c. Strain KJY8 grew rapidly on glucose-yeast extract (GYC) agar and formed pale white colonies with smooth to rough surfaces. The optimum cultivation conditions for acetic acid production by the KJY8 strain were 20°C and pH 3.0, with an initial ethanol concentration of 9% (w/v) to produce an acetic acid concentration of 8% (w/v).

Oxidation of metabolites highlights the microbial interactions and role of Acetobacter pasteurianus during cocoa bean fermentation.

Four cocoa-specific acetic acid bacterium (AAB) strains, namely, Acetobacter pasteurianus 386B, Acetobacter ghanensis LMG 23848(T), Acetobacter fabarum LMG 24244(T), and Acetobacter senegalensis 108B, were analyzed kinetically and metabolically during monoculture laboratory fermentations. A cocoa pulp simulation medium (CPSM) for AAB, containing ethanol, lactic acid, and mannitol, was used. All AAB strains differed in their ethanol and lactic acid oxidation kinetics, whereby only A. pasteurianus 386B performed a fast oxidation of ethanol and lactic acid into acetic acid and acetoin, respectively. Only A. pasteurianus 386B and A. ghanensis LMG 23848(T) oxidized mannitol into fructose. Coculture fermentations with A. pasteurianus 386B or A. ghanensis LMG 23848(T) and Lactobacillus fermentum 222 in CPSM for lactic acid bacteria (LAB) containing glucose, fructose, and citric acid revealed oxidation of lactic acid produced by the LAB strain into acetic acid and acetoin that was faster in the case of A. pasteurianus 386B. A triculture fermentation with Saccharomyces cerevisiae H5S5K23, L. fermentum 222, and A. pasteurianus 386B, using CPSM for LAB, showed oxidation of ethanol and lactic acid produced by the yeast and LAB strain, respectively, into acetic acid and acetoin. Hence, acetic acid and acetoin are the major end metabolites of cocoa bean fermentation. All data highlight that A. pasteurianus 386B displayed beneficial functional roles to be used as a starter culture, namely, a fast oxidation of ethanol and lactic acid, and that these metabolites play a key role as substrates for A. pasteurianus in its indispensable cross-feeding interactions with yeast and LAB during cocoa bean fermentation.

Complete genome sequence and comparative analysis of Acetobacter pasteurianus 386B, a strain well-adapted to the cocoa bean fermentation ecosystem.

BACKGROUND: Acetobacter pasteurianus 386B, an acetic acid bacterium originating from a spontaneous cocoa bean heap fermentation, proved to be an ideal functional starter culture for coca bean fermentations. It is able to dominate the fermentation process, thereby resisting high acetic acid concentrations and temperatures. However, the molecular mechanisms underlying its metabolic capabilities and niche adaptations are unknown. In this study, whole-genome sequencing and comparative genome analysis was used to investigate this strain's mechanisms to dominate the cocoa bean fermentation process. RESULTS: The genome sequence of A. pasteurianus 386B is composed of a 2.8-Mb chromosome and seven plasmids. The annotation of 2875 protein-coding sequences revealed important characteristics, including several metabolic pathways, the occurrence of strain-specific genes such as an endopolygalacturonase, and the presence of mechanisms involved in tolerance towards various stress conditions. Furthermore, the low number of transposases in the genome and the absence of complete phage genomes indicate that this strain might be more genetically stable compared with other A. pasteurianus strains, which is an important advantage for the use of this strain as a functional starter culture. Comparative genome analysis with other members of the Acetobacteraceae confirmed the functional properties of A. pasteurianus 386B, such as its thermotolerant nature and unique genetic composition. CONCLUSIONS: Genome analysis of A. pasteurianus 386B provided detailed insights into the underlying mechanisms of its metabolic features, niche adaptations, and tolerance towards stress conditions. Combination of these data with previous experimental knowledge enabled an integrated, global overview of the functional characteristics of this strain. This knowledge will enable improved fermentation strategies and selection of appropriate acetic acid bacteria strains as functional starter culture for cocoa bean fermentation processes.