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Background Enantiomerically pure alcohols are important building blocks for production of chiral pharmaceuticals, flavors, agrochemicals and functional materials and appropriate whole-cell biocatalysts offer a highly enantioselective, minimally polluting route to these valuable compounds. At present, most of these biocatalysts follow Prelog’s rule, and thus the (S)-alcohols are usually obtained when the smaller substituent of the ketone has the lower CIP priority. Only a few anti-Prelog (R)-specific whole cell biocatalysts have been reported. In this paper, the biocatalytic anti-Prelog reduction of 2-octanone to (R)-2-octanol was successfully conducted with high enantioselectivity using whole cells of Acetobacter pasteurianus GIM1.158. Results Compared with other microorganisms investigated, Acetobacter pasteurianus GIM1.158 was shown to be more effective for the reduction reaction, affording much higher yield, product enantiomeric excess (e.e.) and initial reaction rate. The optimal temperature, buffer pH, co-substrate and its concentration, substrate concentration, cell concentration and shaking rate were 35°C, 5.0, 500 mmol/L isopropanol, 40 mmol/L, 25 mg/mL and 120 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 89.5% and >99.9%, respectively, in 70 minutes. Compared with the best available data in aqueous system (yield of 55%), the yield of (R)-2-octanol was greatly increased. Additionally, the efficient whole-cell biocatalytic process was feasible on a 200-mL preparative scale and the chemical yield increased to 95.0% with the product e.e. being >99.9%. Moreover, Acetobacter pasteurianus GIM1.158 cells were proved to be capable of catalyzing the anti-Prelog bioreduction of other prochiral carbonyl compounds with high efficiency. Conclusions Via an effective increase in the maximum yield and the product e.e. with Acetobacter pasteurianus GIM1.158 cells, these results open the way to use of whole cells of this microorganism for challenging enantioselective reduction reactions on laboratory and commercial scales.

Bacterial cellulose (BC) is a promising natural polymer that is produced by bacteria and that has unique and desirable structural, physical, and chemical properties. From the time when the remarkable properties of BC were found 15 years ago compared with plant cellulose, interest has grown in BC and it has become an article of trade in diverse applications. Following this trend, this paper reviews the progress of relevant studies, including general information about cellulose, production by microorganisms as well as BC cultivation, and its properties. The applications reviewed in the present article comprise biological and nonbiological fields. The latest use of BC in the biomedical, environmental, agricultural, electronic, food, and industrial fields is discussed with its applications in composite form. The present article attempts to amass the assorted uses of BC under one umbrella. Thus, recent advances in BC applications in different fields are thoroughly reviewed. This article concludes with the need for future research of BC to make it commercialized as vital biomaterial.

Many advances in nanomaterials synthesis have been recorded during the last 30 years. Bacterial cellulose (BC) produced by bacteria belonging to the genera Acetobacter, Rhizobium, Agrobacterium, and Sarcina is acquiring major importance as one of many eco-friendly materials with great potential in the biomedical field. The shape of BC bulk is sensitive to the container shape and incubation conditions such as agitation, carbon source, rate of oxygenation, electromagnetic radiation, temperature, and pH. The challenge is to control the dimension and the final shape of biosynthesized cellulose, by the optimization of culture conditions. The production of 3D structures based on BC is important for many industrial and biomedical applications such as paper and textile industries, biological implants, burn dressing material, and scaffolds for tissue regeneration. In our work, wild strains of Acetobacter spp. were isolated from homemade vinegar then purified and used for cellulose production. Four media of different initial viscosity were used. Cultures were performed under static conditions at 29°C, in darkness. The dimensions and texture of obtained bacterial cellulose nanofibers were studied using scanning electron microscopy (SEM). X-ray diffraction (XRD) showed that the biosynthesized material has a cellulose I crystalline phase characterized by three crystal planes. fourrier transform infrared spectroscopy (FTIR) data confirmed the chemical nature of the fibers. Thermo-gravimetric analysis (TGA) showed that BC preserves a relatively superior non-degradable fraction compared to microcrystalline cellulose.

The paper deals with the question whether toxicity could be a reason for the incomplete microbial degradation of mineral oils in the laboratory and the environment, especially in soils contaminated over a long period. The luminescence test showed inhibition by the nondegradable residues as well as by the water phase from degradation experiments. This most frequently applied test reaches its limits in the case of substances with very low solubility in water. Conclusions relating to the impact on the microbial community of a contaminated ecosystem are not admissible. Neither nondegradable residues from raffinates nor extracts from long-term contaminated soils showed inhibiting effects on the dehydrogenase activity of Acetobacter methanolicus and a mineral oil-adapted consortium Inhibition of the respiration of the pure strain A. methanolicus and of the consortium growing on glucose and spindle oil respectively was not observed either. The residues evidently contain mainly highly stable compounds which withstand microbial attack. The toxicity of such compounds per se is unlikely. Moreover, toxicity investigations with aqueous phases from degradation experiments gave no indication of the formation of metabolites, which could be responsible for the stagnation of the process.

Protein acidostability is a common problem in biopharmaceutical and other industries. However, it remains a great challenge to engineer proteins for enhanced acidostability because our knowledge of protein acidostabilization is still very limited. In this paper, we present a comparative study of proteins from bacteria with acidic (AP) and neutral cytoplasms (NP) using an integrated statistical and machine learning approach. We construct a set of 393 non-redundant AP-NP ortholog pairs and calculate a total of 889 sequence based features for these proteins. The pairwise alignments of these ortholog pairs are used to build a residue substitution propensity matrix between APs and NPs. We use Gini importance provided by the Random Forest algorithm to rank the relative importance of these features. A scoring function using the 10 most significant features is developed and optimized using a hill climbing algorithm. The accuracy of the score function is 86.01% in predicting AP-NP ortholog pairs and is 76.65% in predicting non-ortholog AP-NP pairs, suggesting that there are significant differences between APs and NPs which can be used to predict relative acidostability of proteins. The overall trends uncovered in the study can be used as general guidelines for designing acidostable proteins. To best of our knowledge, this work represents the first systematic comparative study of the acidostable proteins and their non-acidostable orthologs.

Antimicrobial packaging protects the product from the external environment and microbial contamination, conferring numerous advantages on human health. Interest in biopolymers as packaging materials has considerably increased recently. Bacterial cellulose is an interesting biomaterial produced as nanofibrils by Acetobacter xylinium and is a promising candidate due to its remarkable properties. New composite materials with antimicrobial properties were developed in this work, containing poly(vinyl alcohol) (PVA) as polymer matrix and ground bacterial cellulose (BC) as reinforcing fibres. Sorbic acid was used as an antimicrobial agent because it is a preservative recognised in the food industry. The materials obtained were studied using Fourier-transformed infrared spectroscopy (FTIR). The swelling rate of the composites was also measured. Release experiments of sorbic acid from the composite films into water were performed and the mass transfer phenomena were investigated using Fick’s law of diffusion. The antimicrobial effect was tested against Escherichia coli K12-MG1655. The results obtained indicated that the new biocomposite films could be promising antimicrobial food packaging materials.

The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains.

Cellulose is the most abundant natural polymer on Earth. It is the major constituent of higher plants, and cellulose fibers have important industrial applications including the clothing that we wear and even the paper upon which this article is written. Cellulose forms the cell wall of eukaryotic plants, algae and fungi, and is synthesized by a variety of bacteria, including Acetobacter xylinum. A. xylinum, a Gramnegative bacterium found in the soil, can frequently be isolated from decaying fruit such as apples that have fallen to the ground. In addition to serving as a model organism for the study of cellulose synthesis, A. xylinum can serve as an interesting and instructive organism in the classroom to help students understand the process of science. The major focus of this article will be upon how students can experiment with A. xylinum using an inquiry approach to their learning. Acetobacter xylinum is an aerobic soil bacterium in the family of bacteria that ferments carbohydrates to vinegar (A. aceti). A. xylinum is an unusual member of this family because it synthesizes and extrudes fibrils of cellulose as part of the metabolism of glucose. The glucose subunits that form the cellulose microfibril are extruded through pores in the cell wall of the bacteria. In standing laboratory culture of the bacteria, the cellulose fibrils bundle together to form a mat or pellicle within which the bacteria are held. The pellicle floats on the surface of the medium allowing the bacteria to obtain plenty of oxygen, which they require for growth, multiplication, and more cellulose synthesis. Cellulose biosynthesis by A. xylinum may serve the bacterium in a number of ways in their microenvironment. These may include holding the bacteria at the air interface to ensure adequate oxygen, protection from ultraviolet radiation, protection from desiccation by holding water, acting as a biofilm to hold the organisms on substrates that they may be decomposing, and/or possibly providing protection from predators or competitors. Cellulose synthesis occurs as a multi-step series of chemical reactions beginning with glucose that is catalyzed by enzymes. The pathway is believed to be as shown in Figure 1. Cellulose is considered to be a secondary metabolite, which the organism synthesizes when major nutritional needs are met. There is genetic and molecular evidence that cellulose synthesis in A. xylinum is under operon control. Some practical applications of bacterial cellulose include: sensitive diaphragms for stereo headphones, thickeners for foods, paint, ink and adhesives. It has also been used as short term protection for burned skin prior to skin grafting. Most commercial applications of cellulose produced by Acetobacter have employed static batch culture to produce sheets of cellulose that can be sterilized to kill the bacteria and washed to remove microbial waste product contaminants. One large scale commercial application is the production of bacterial cellulose in a nonaseptic medium of sugar-enriched coconut milk in the Philippines, yielding an edible product called "nata de coco." This food product has become popular in Japan as a high-fiber, low-calorie food. Recently, Japanese investigators have developed a technique to harvest Acetobacter cellulose from shallow culture pans with a windup roller device to pull the cellulose slowly from the surface of the culture medium. High yield commercial production of bacterial cellulose will require agitated (shaking) culture techniques. There has been some limited success in the laboratory studies producing cellulose in this way, but, at present, there are no commercial applications of this process.

Microbial cellulose has proven to be a remarkably versatile biomaterial and can be used in wide variety of applied scientific endeavors, such as paper products, electronics, acoustics, and biomedical devices. In fact, biomedical devices recently have gained a significant amount of attention because of an increased interest in tissue-engineered products for both wound care and the regeneration of damaged or diseased organs. Due to its unique nanostructure and properties, microbial cellulose is a natural candidate for numerous medical and tissue-engineered applications. For example, a microbial cellulose membrane has been successfully used as a wound-healing device for severely damaged skin and as a small-diameter blood vessel replacement. The nonwoven ribbons of microbial cellulose microfibrils closely resemble the structure of native extracellular matrices, suggesting that it could function as a scaffold for the production of many tissue-engineered constructs. In addition, microbial cellulose membranes, having a unique nanostructure, could have many other uses in wound healing and regenerative medicine, such as guided tissue regeneration (GTR), periodontal treatments, or as a replacement for dura mater (a membrane that surrounds brain tissue). In effect, microbial cellulose could function as a scaffold material for the regeneration of a wide variety of tissues, showing that it could eventually become an excellent platform technology for medicine. If microbial cellulose can be successfully mass produced, it will eventually become a vital biomaterial and will be used in the creation of a wide variety of medical devices and consumer products.

Vinegar or sour wine is a product of alcoholic and subsequent acetous fermentation of sugary precursors. Among acetic acid producing bacteria, only few genera (Acetobacter and Gluconobacter) are used in vinegar industry. In this paper, we intended to produce vinegar at 37 °C using two Acetobacter pasteurianus strains (S3 and S32). These species were isolated from palm (Elaeis guineensis) wine and presented potentialities for industrial vinegar production at 37 °C. Successive fermentations were carried up and semi-continuous acetous fermentation was performed to increase acid production. Concentrated bananas (Musa ssp.) juice (11°Brix) was fermented using Saccharomyces cerevisae within 7 days, yielding 6.4% alcohol. After fermentation, 60 and 58 g/L acetic acid were produced in vinegars obtained using S3 and S32 stains respectively in 34 days and 5 flow cycles. Malic and acetic acids were the most substantial acids produced in alcoholic juice with 5 631.473 and 2 833.055 mg/L respectively. Among the eight organic acids responsible for vinegars total acidity, acetic acid was major compound with 23 459.416 and 21 268.407 mg/L for S3 and S32 strains respectively. Alcohol and acetic acid fermentation efficiency were 90.9% and 85.39 - 87.63% respectively. All the results above showed that S3 and S32 strains revealed great potentialities for successful industrial vinegar production from overripe banana.

Nitrogen is an essential plant nutrient, widely applied as N-fertilizer to improve yield of agriculturally important crops. An interesting alternative to avoid or reduce the use of N-fertilizers could be the exploitation of plant growth-promoting bacteria (PGPB), capable of enhancing growth and yield of many plant species, several of agronomic and ecological significance. PGPB belong to diverse genera, including Azospirillum, Azotobacter, Herbaspirillum, Bacillus, Burkholderia, Pseudomonas, Rhizobium, and Gluconacetobacter, among others. They are capable of promoting plant growth through different mechanisms including (in some cases), the biological nitrogen fixation (BNF), the enzymatic reduction of the atmospheric dinitrogen (N(2)) to ammonia, catalyzed by nitrogenase. Aerobic bacteria able to oxidize ethanol to acetic acid in neutral or acid media are candidates of belonging to the family Acetobacteraceae. At present, this family has been divided into ten genera: Acetobacter, Gluconacetobacter, Gluconobacter, Acidomonas, Asaia, Kozakia, Saccharibacter, Swaminathania, Neoasaia, and Granulibacter. Among them, only three genera include N(2)-fixing species: Gluconacetobacter, Swaminathania and Acetobacter. The first N(2)-fixing acetic acid bacterium (AAB) was described in Brazil. It was found inside tissues of the sugarcane plant, and first named as Acetobacter diazotrophicus, but then renamed as Gluconacetobacter diazotrophicus. Later, two new species within the genus Gluconacetobacter, associated to coffee plants, were described in Mexico: G. johannae and G. azotocaptans. A salt-tolerant bacterium named Swaminathania salitolerans was found associated to wild rice plants. Recently, N(2)-fixing Acetobacter peroxydans and Acetobacter nitrogenifigens, associated with rice plants and Kombucha tea, respectively, were described in India. In this paper, recent advances involving nitrogen-fixing AAB are presented. Their natural habitats, physiological and genetic aspects, as well as their association with different plants and contribution through BNF are described as an overview.

In this paper, we present a novel DNA separation medium using bacterial cellulose fibrils. Bacterial cellulose has an intrinsic three-dimensional micrometer- to nanometer-scale network structure. Addition of this material to a low-concentration polymer solution (5 cP) enables high-resolution electrophoretic separation of DNA, even for fragments of 10-100-bp or single-nucleotide polymorphism. The newly designed medium consists of a double mesh: a 10-nm flexible mesh derived from a conventional polymer medium containing 10-nm to 1-microm rigid pores made up of 10-microm bacterial cellulose fragments.

The category, characteristic of diazotrophs isolated from inside and/or rhizosphere of graminaceous plants in recent year and the mechanism of the promoting effects on their host plant were reviewed in this paper. The current status of application of associative nitrogen-fixation inoculants and the problems in inoculation were discussed. It was indicated that the main factors influencing the effects of inoculants include the competition of indigenous micro-organism with inoculants for nutritions and energy, difference of host plant genotypes in associative relationship, and variance of environmental conditions such as the concentration of ammonium in soil solution and the oxygen partial pressure in soil air. The trends of future research in this field were prospected, for example, to isolate and identify the high nitrogen fixing efficiency strains with wider environmental adaptability, to create associative nitrogen fixing bacteria strain which is able to bear or endure higher concentration of ammonium by gene engineering technique, to induce graminaceous plant forming root nodule for nitrogen fixation and to exert the predominance of endophytic diazotrophs.

This paper investigates the anaerobic biotransformation of three isomers of phthalic acid and benzoic acid in sediment slurries under four different atmospheres [N 2 , N 2 /H 2 (19:1, v/v), CO 2 , and CO 2 /H 2 (4:1, v/v)]. Significant differences were observed in lag periods and biotransformation rates among the phthalic acid isomers and under the different atmospheres. In most cases, the relative biotransformation rates of the three isomers of phthalic acid were ortho -phthalic acid > isophthalic acid > terephthalic acid. Benzoate was transformed faster than any isomer of phthalic acid. Since biotransformation of phthalic acid isomers in sediment slurries was enhanced by high initial levels of H 2 and CO 2 in the headspace, we propose a pathway for phthalic acid biodegradation in which the initial transformation to benzoate is CO 2 –H 2 dependent. Acetogenic bacteria were investigated for their possible involvement in this transformation reaction, but when MPN counts were used to compare the growth dynamics of acetogenic bacteria with the time course of the terephthalic acid transformation under N 2 /H 2 (19:1, v/v) and CO 2 /H 2 (4:1, v/v) atmospheres, the results were inconclusive.

This paper describes the bioconversion of 2-methyl-1,3-propanediol to (R)-beta-hydoxyisobutyric acid (HIBA) by Acetobacter ALEI in a hollow fiber membrane bioreaction system arrangement that allows the integration of three liquid phases: the aqueous bioconversion phase, the organic phase consisting of a solution of trioctyl phosphine oxide (TOPO) in isooctane, and the third phase consisting of a basic stripping solution that allows reextraction of HIBA from the organic phase. A comparison of HIBA mass transfer experiments was carried out in the membrane reactor with two and three phases for different pH and TOPO concentrations. The use of the three-phase arrangement allows the extraction of high quantities of HIBA from the aqueous medium (higher than 85%) independently of the pH, whereas in the two-phase system the percentage of HIBA extracted from the aqueous medium was lower, 42% in the best case, and strongly influenced by the pH. The percentage of the extractive agent TOPO in the organic phase influenced on the mass transfer rate in both bi- and triphasic arrangements. By simply integrating the re-extraction phase in the system it was possible to increase the extraction yield by 2-fold, reduce the amount of TOPO by 4-fold, and operate at the more favorable pH 4. A bioconversion experiment was done in these conditions (pH = 4, TOPO = 5%) to confirm the advantages of including the third stripping solution. Fed-batch operation of the triphasic membrane reactor was maintained for more than 20 h, reaching an HIBA concentration in the stripping solution of 29 g L(-)(1).

Orientador: Silvia Yuko Eguchi Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: Bactérias acéticas do gênero Acetobacter foram identificadas em Usina de Açúcar e Álcool. Do total de 32 linhagens classificadas como Acetobacter sp, 11 (34,37 %) foram isoladas da peneira do "Cush-Cush", 3 (9,38 %) foram isoladas da parede do tanque de coleta do caldo de cana recém peneirado no "CushCush", 15 (46,87 %) foram isoladas do caldo de cana recém peneirado e 3 (9,38 %) foram isoladas da parede do Separador. As linhagens identificadas como bactérias acéticas foram testadas quanto a capacidade fermentativa em processo descontínuo. A taxa específica de produção de ácidos e a produtividade foram analisadas a partir das curvas de crescimento celular e de produção de ácidos. As linhagens Acetobacter sp CCT 2910, CCT 2023 e CCT 2924 apresentaram altas taxas de produção de ácidos, após 48 horas de fermentação, e as melhores taxas específicas de produção de ácidos e produtividades entre as cepas estudadas neste trabalho Abstract: Acetic acid bacteria of the Acetobacter genus have been identified at the sugarcane mill, in ethanol plants. Out of a total of 32 strains, classified as Acetobactersp., 11 (34,37 %) have been isolated in the Cush-Cush sieve, 3 (9,38 %) have been isolated from the collect tank walls of the newly filtered sugar juice in the Cush-Cush, 15 (46,87 %) have been isolated from newly filtered sugar cane and 3 (9,38 %) have been isolated from the Separator. The strains identified as acetic acid bacteria have been tested as for their fermenting capacity in a discontinued processo The specific rate of acid production and productivity have been analysed based on the cell growth chart and acid production. The CCT 2910, CCT 2023 and CCT 2924 Acetobacter sp. strains have showed high acid production rates after 48 hours of fermenting and the best specific rates of acid production and productivity among of the strains studied in this paper Mestrado Mestre em Ciência de Alimentos

This paper evaluates the use of denaturing high performance liquid chromatography (DHPLC) technology for the discrimination of genetic differences in the 16S rRNA and alcohol dehydrogenase (AdhA) genes among bacterial species based on its efficiency and sensitivity to enable the detection and discrimination of different genetic sequences. In order to optimize DHPLC protocols for the analysis of 16S rRNA gene fragments amplified from bacteria, DNA isolated from 22 different strains representing main bacterial groups of interest in food microbiology was analyzed. While the use of 16S rRNA gene did not allow to difference two wild strains of Acetobacter malorum, this region revealed as useful to differentiate them from some pathogenic bacteria as Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Listeria innocua, Clostridium perfringens or Sthapylococcus aureus, from spoilage microorganisms as Xantomonas vesicatoria and Alicyclobacillus spp., and also from lactic acid bacteria as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus sakei, Lactobacillus acidophilus, Streptococcus thermophilus and Lactococcus lactis that may suppose technological risk during vinegar production. The results demonstrate that 16S rRNA gene region is not adequate for the discrimination of the acetic acid bacteria (AAB) strains, so AdhA gene was selected to identify the two wild strains of Acetobacter malorum. Also 6 different reference strains of AAB were separated based on differences in AdhA gene region. DHPLC technology is able to discriminate between these two wild strains of A. malorum based on differences existing in the AdhA gene region. The data obtained indicate that the technique is capable of identifying most bacteria at species level and even at strain level with optimization of the protocols. This is of particular relevance in the case of AAB due to their poor recovery on culture media and difficulties in detection of viable but non cultivable cells.

Cellulose, an aggregate of unbranched polymers of β-1,4-linked glucose residues, is the major component of wood and thus paper, and is synthesized by plants, most algae, some bacteria and fungi, and even some animals. The genes that synthesize cellulose in higher plants differ greatly from the well-characterized genes found in Acetobacter and Agrobacterium sp. More correctly designated as 'cellulose synthase catalytic subunits', plant cellulose synthase (CesA) proteins are integral membrane proteins, approximately 1,000 amino acids in length. The sequences for more than 20 full-length CesA genes are available, and they show high similarity to one another across the entire length of the encoded protein, except for two small regions of variability. There are a number of highly conserved residues, including several motifs shown to be necessary for processive glycosyltransferase activity. No crystal structure is known for cellulose synthase proteins, and the exact enzymatic mechanism is unknown. There are a number of mutations in cellulose synthase genes in the model organism Arabidopsis thaliana. Some of these mutants show altered morphology due to the lack of a properly developed primary or secondary cell wall. Others show resistance to well-characterized cellulose biosynthesis inhibitors.

Lysogeny could until now not be shown for methylotrophic bacteria or for Acetobacter. In this paper we demonstrate the lysogenic state in a methylotrophic strain of the genus Acetobacter. The responsible temperate phage MO 1 belongs to group A of BRADLEY's classification system and contains double stranded DNA. Phage MO 1 was characterized by the following properties: Host range, sensitivity against environmental factors, serological behaviour, burst size, and latent period in one-step growth experiments, and especially by its response to inducing factors.

Kerwar, Suresh S. (Oregon State University, Corvallis), Vernon, H. Cheldelin, and L. W. Parks . Valine-isoleucine metabolism in Acetobacter suboxydans and the inhibition of growth by valine. J. Bacteriol. 88: 179–186. 1964.—Extracts of Acetobacter suboxydans can synthesize valine and isoleucine via acetolactate and acetohydroxybutyrate, respectively. The amounts of these amino acids synthesized from different intermediates were determined. The pathways appear to be identical to those described for yeast, Neurospora , and Escherichia coli . When exogenous valine was added to a synthetic growth medium inoculated with A. suboxydans , no growth of the culture was observed. The inhibitory effect of valine was reversed by the addition of isoleucine. The site and mechanism of valine inhibition were investigated. Threonine deaminase was inhibited by valine and isoleucine but not by leucine. Repression of the deaminase by isoleucine but not by valine was indicated. The data reported in this paper suggest that valine prevented growth of the organism through false feedback inhibition of threonine deaminase, thereby limiting isoleucine biosynthesis.

1. The unknown substance reported in the previous paper was identified as glucosone. The formation of glucosone from fructose by Gluconoacetobacter roseus is a similar reaction with the formation of reductone (enol form of glycerosone) from dihydroxyacetone by the same bacterium. A presumable pathway of ketose oxidation would be shown as follows:2. The fructose dehydrogenase of G. roseus was investigated by Thunberg method. The activity of this dehydrogenase was the same order as that of gluconic acid dehydrogenase and was inhibited by the addition of 1:5.000mol. of 2, 4-dinitrophenol, when other dehydrogenases such as glucose, gluconic acid, and mannitol dehydrogenase were not affected. The fructose dehydrogenase of this bacterium seemed to be of the same type as the dihydroxyacetone dehydrogenase of Acetobacter suboxydans.3. The author isolated a new substance from the fermented broth of fructose. The chemical structure of this compound is now under further investigation. This compound developes purple color with ferric chloride solution and gives different m.p. and Rf value from those of kojic acid, comenic acid, and rubiginol.4. Although the biological induction of kojic acid from glucosone is not yet attained, the author assumes that glucosone would be the most possible intermediate of kojic acid formation, because Bond, Knight and Walker proved in 1937 the existance of the same substance in the brothh fermented by Aspergillus cryzae.

1. The unknown substance reported in the previous paper was identified ad glucosone. The formation of glucosone from fructose by Gluoonoacetobacter roseus is a similar reaction with the formation of reductone (enol form of glycerosone) from dihydroxyacetone by the same bacteria. A presumable pathway of ketose oxidation would be shown as follows: CH2OH CO CHOH R_??_CHOH COH CHOH R -2H→CHO CO CHOH R_??_CHO COH COH R This hypothesis extremely agreed with the experimental data of MAGSANIK and CHARGAFF who had reported the formation of α, β-diketo-inositol from inosose by Acetobacter suboxydans. 2. The fructose dehydrogenase of G. roseus was investigated by ZUNEERG method. The activity of this dehydrogenase was the same order as that of gluconic acid dehydrogenase and was inhibited by the addition of 1:5000 mol. of 2, 4-dinitrophenol, when the other dehydroge-nases such as glucose, gluconic acid, mannitol dehydrogenase did not be affected. The fruc-tose dehydrogenase of this bacteria seemed to be the same type as the dihydroxyacetone dehy-drogenase of Acetobacter suboxydans. 3. The author isolated a new substance from the fermented broth of fructose. The chemi-cal structure of this compound is now under the further investigation. This compound deve-loped purple colour with, ferric chloride solution and gives different m. p. and RF value from those of kojic acid, comenic acid and rubiginol. 4. Although the biological induction of kojic acid from glucosone is not yet attained, the author thinks glucosone would be the most possible intermediate of kojic acid formation, be-cause, in 1937, BOND, KNIGHT and WALKER have proved the existance of same substance in the broth fermented by Aspergillus oryzae.

In extension of the studies on the formation of cellulose by Acetobacter acetigenum (Z-4), the action of this organism on glycerol has now been investigated; this paper records the examination of the products formed in a defined glycerol medium buffered with lactate. Preliminary observations on the action of this organism on ethylene glycol and on inositol are also reported.

2,5-Diketogluconic acid decarboxylase has been purified from extracts of Acetobacter melanogenum. This paper reports the method of purification, the properties of the purified enzyme, and some properties of the reaction product. A crystalline o-nitrophenylhydrazone derivative of the decarboxylation product has been obtained.

When Acetobacter aceti, Acetobacter rancens, Acetobacter acidum-mucosum and Acetobacter acetosum var. nairobiense were grown in defined media containing glucose as sole source of carbon, gentiobiose (O-β- d -glucopyranosyl-(1 → 6)- d -glucopyranose) and sophorose (O-β- d -glucopyranosyl-(1 → 2)- d -glucopyranose) were the principal oligosaccharide products in all cases. These were accompanied by other disaccharides and by higher saccharides. Paper chromatograms showed that each Acetobacter species synthesized the same series of sugars.

Methylotrophic bacteria which use hexulose-6-phosphate synthase for fixing formaldehyde are able to oxidize it cyclically via 6-phosphogluconate. This also holds true for Acetobacter methanolicus MB58. Generally methylotrophic bacteria oxidize formaldehyde via formate to CO2. Up to now only the presence of a membrane-bound, dye-mediated formate dehydrogenase has been demonstrated in Acetobacter methanolicus MB58. In this paper evidence for the presence of an aldehyde dehydrogenase is provided which catalyzes the oxidation of formaldehyde by means of toluylene blue as an electron acceptor. Hence Acetobacter methanolicus MB58 possesses at least two routes for the dissimilation of formaldehyde to CO2.

GUDDING, ROAR: The suitability of some media and peptones for sulfonamide testing. Acta vet. scand. 1974, 15, 366–380. — The content of p-aminobenzoic acid and folic acid has been determined in some media and peptones, and the sensitivity of Bacillus megaterium, cultivated on these substrates, towards sulfonamides has been examined. The concentrations of p-aminobenzoic acid and folic acid should be as low as possible in suitable substrates, but other factors are also of importance. The presence of antagonists towards folic acid has been demonstrated in some media, and the consequences of these inhibitors are discussed. Three of the media (Paper Disc Method-Antibiotic Sensitivity Medium, AB Biodisk; Sulphonamide Antagonist Free Medium, Mast Laboratories; and Mueller Hinton, Difco Laboratories) seem to be satisfactory for the determination of the resistance of bacteria towards sulfonamides. The application of Acetobacter medium is suggested for the detection of rest concentrations of sulfonamides in foodstuffs. detection of sulfonamide residues; p-aminobenzoic acid; folic acid.

Publisher Summary This chapter focuses on 5-keto-D-fructose and its phosphate esters. 5-ketofructose (5KF) is produced by oxidation of D-fructose through all phases of bacterial growth. It is consumed by the growing cells until the pH of the medium drops to 3.5–3.8. When growth ceases, D-fructose is converted stoichiometrically to 5KF, which then accumulates in the medium. A product isolated from Acetobacter suboxydans cultures grown on D-fructose, and whose structure was initially proposed to be 6-aldo-D-fructose, is identified to be 5KF. Method of isolation of 5KF is described. Partial reduction of 5KF, such as boron hydrides, yields a mixture of L-sorbose and D-fructose on complete reduction, a mixture of D-mannitol, n-sorbitol, and L-iditol is obtained.On paper or thin-layer chromatography, 5KF moves slower than D-fructose and L-sorbose, and also yields different colored products with the various spray reagents used to detect reducing hexoses. 5KF does not react with Disehe's eysteine-carbazole reagent for ketohexoses, but it can react specifically with o-aminobiphenyl. 5KF can be determined speetrophotometrically using specific NADPH-dependent 5KF reductases. In the presence of ATP, 5KF is effectively phosphorylated by yeast hexokinase or by liver fructokinase to yield 5KF-1-P. For preparative purposes, the most convenient procedure for the preparation of 5KF-1-P is to couple the 5KF-hexokinase system with catalytic amounts of ATP and a phosphoenolpyruvate-pyruvate kinase coupling system. The reaction is allowed to proceed to completion as determined by the appearance of pyruvate. 5KF-1-P is then recovered as the only phosphate ester in solution either by chromatography, or if desired, as the barium salt. In the presence of ATP, yeast, and rabbit-muscle, phosphofructokinase can effectively phosphorylate 5KF-1-P to 5KF-1,6-P2.

Cellulose is the most abundant renewable carbon resource on earth and is an indispensable raw material for the wood, paper, and textile industries. A model system to study the mechanism of cellulose biogenesis is the bacterium Acetobacter xylinum which produces pure cellulose as an extracellular product. It was from this organism that in vitro preparations which possessed high levels of cellulose synthase activity were first obtained in both membranous and soluble forms. We recently demonstrated that this activity is subject to a complex multi-component regulatory system, in which the synthase is directly affected by an unusual cyclic nucleotide activator enzymatically formed from GTP, and indirectly by a Ca (2+) -sensitive phosphodiesterase which degrades the activator. The cellulose synthase activator (CSA) has now been identified as bis-(3' 5')-cyclic diguanylic acid (5'G3'p5'G3'p) on the basis of mass spectroscopic data, nuclear magnetic resonance analysis and comparison with chemically synthesized material. We also report here on intermediary steps in the synthesis and degradation of this novel circular dinucleotide, which have been integrated into a model for the regulation of cellulose synthesis.

The paper presents a method for assaying activity of glycerol dehydrogenase activity in the submerged culture of Acetobacter suboxydans. The effect of substrate concentration, content of the submerged culture in the reaction mixture and pH value on the rate of enzymic oxidation of glycerol to dihygrooxyacetone by A. suboxydans has been studied.

SUMMARY: A new type of Aeetobacter, provisionally named Acetobacter acidophilum, has been isolated in pure culture from an experimental vinegar generator originally inoculated with active industrial beechwood shavings. In contrast to other bacteria, this organism needs an acidic medium for growth and has a very specific oxidizing capacity. It does not grow above pH 4·3 and oxidizes only primary alcohols. Other substrates, including glucose and acetic acid, are not oxidized at a rate higher than 1 % of the oxidation rate for ethanol. The new isolation procedure, which has been applied for the strain described in this paper, allows the isolation of the bacteria present on active shavings, suggesting that this type of bacteria belongs probably to the group of organisms responsible for the acetification in the ‘quick vinegar process’.

1. The bacterium Acetobacter xylinum produces extracellular cellulose microfibrils that form a pellicle in the medium enmeshing the bacterial cells. These microfibrils may show some localized alignment, which can be seen as birefringence when the culture is viewed between crossed Polaroid sheets. 2. An increase in birefringence can be induced by the addition of small amounts of certain classes of lipids, particularly sterols, to the cultures. 3. A crude lipid extract from Acetobacter cells induced greatly increased birefringence when added to fresh cultures of this organism. 4. When the bacterial lipids were fractionated, most of the activity was recovered in a complex, polar lipid. The lipid is secreted into the medium during growth and is unstable. The non-saponifiable portion of this lipid is shown to be a 1:1 mixture of a saturated and a monounsaturated C35 tetrahydroxy terpene with a hopane ring system in the accompanying paper by Förster et al. (1973). The saturated molecule is referred to as tetrahydroxybacteriohopane. 5. Tetrahydroxybacteriohopane is itself capable of inducing birefringence in cultures as is 22-hydroxyhopane, which was also isolated from the non-saponifiable fraction of the total lipids. 6. The mechanism of induction of birefringence (orientation of microfibrils) is not known. This is unlikely to be a specific effect, since all the above compounds are active (intact lipid, tetrahydroxybacteriohopane, 22-hydroxyhopane), as are other classes of lipid. It is suggested, however, that a common mechanism may be involved and that similar compounds may be concerned with control of microfibril alignment in the cells of higher plants.

Bacterial cellulose, produced by Acetobacter species, displays unique properties, including high mechanical strength, high water absorption capacity, high crystallinity, and an ultra-fine and highly pure fibre network structure. It is expected to be a new commodity biochemical with diverse applications, if its mass production process could be improved, especially via submerged fermentation technology. It has already found application as a food matrix (nata de coco) and as dietary fibre, as a temporary dressing to heal skin burns, as an acoustic or filter membrane, as ultra-strength paper and as a reticulated fine fibre network with coating, binding, thickening and suspending characteristics. A wet spinning process for producing textile fibres from bacterial cellulose has also been developed, and applications as a superconducting and optical fibre matrix are under study. We have been able to improve bacterial cellulose production in surface culture (up to 28 g/l), as well as in submerged culture (up to 9 g/l) via strain selection, mutation, medium composition optimization and physico-chemical fermentation parameter control. Glucose and fructose as the carbon source and acetic acid as the energy source, combined with a precise control of pH and dissolved oxygen levels, results in highly improved cellulose yields. An internal pH control in stationary surface cultures was achieved by an appropriate choice of the ratio of fructose/glucose/acetic acid. It was also demonstrated that cellulose formation could be enhanced by adding insoluble microparticles such as diatomaceous earth, silica, small glass beads and loam particles to submerged, agitated/aerated Acetobacter cultures. This microcarrier-enhanced cellulose synthesis could be the result of the formation of microenvironments with locally lowered dissolved oxygen levels because of the attachment of Acetobacter cells as a biofilm on the particles. As such, less glucose is lost as gluconate, saving it for cellulose formation and maintaining the pH profile within the desirable range. We have also developed a UV-mutation and proton enrichment strategy, which allows the selection of A. xylinum mutants, which are highly restricted in (keto)gluconate synthesis and produce cellulose more efficiently, even under oxidative culture conditions. Combining these nutritional, genetic and bioprocess-technological improvements, very high levels of bacterial cellulose have been attained. Further improvements are needed to arrive at an economical fermentation process for mass production of bacterial cellulose.

The change in the fine structure of the cellulose gel biosynthesized by Acetobacter zylinum with time was studied by means of electron microscopy. It was found that in the process of time, the inhomogeneous distribution cellulose chain arised in the vertical direction against the long axis of the amorphous fibril initially and finally many microscopic fibrils with the width of about 40∼50 A appeared simultaneously in the amorphous fibril. This microscopic fibril corresponds to the elementary fibril reported in the previous paper. It was also found that the bacterial cellulose microfibril is composed of the microscopic fibril.

Bacterial cellulose (BC) is produced by some strains of Acetobacter. The BC has unique network structure of fine fiber. The BC can be obtained with two typical methods, namely static and agitated culture. We have found some structural differences between these two types of BC. We, therefore, investigated the properties of the BCs for papermaking.When these two types of BCs were added at wet-end, both tensile strength and filler-retention of the handsheets were improved. Especially, the BC from agitated culture had higher effects on filler-retention than that from static one. Thus, the BC may be useful as a wet-end additive for papermaking.