Intensification of synthesis of the exopolysaccharide ethapolan by Acinetobacter sp. 12S grown on a mixture of substrates

Enhanced synthesis of the exopolysaccharide (EPS) ethapolan by Acinetobacter sp. 12S was observed when the bacterium was grown on a mixture of two energetically nonequivalent substrates (ethanol and glucose) taken in a molar proportion of 3.1:1. The efficiency of carbon transformation into EPSs was maximum when sodium ions were absent in the medium, the concentration of nitrogen source was reduced to 0.3-0.45 g/l, and the inoculum was grown on ethanol. Such conditions provided an increase in the maximum specific growth rate and its attainment in earlier cultivation terms. Molasses as a substitution for glucose was inefficient. The activities of the key enzymes of C2-metabolism in Acinetobacter sp. 12S cells grown on the substrate mixture was 1.1 to 1.7 times lower than they were during growth on ethanol alone. The activity of isocitrate lyase in cells grown on the substrate mixture declined to an even greater extent (by 4 to 7 times), indicating that the role of the glyoxylate cycle in such cells is insignificant.     

Formation of the exopolysaccharide ethapolan by Acinetobacter sp. IMV B-7005 on a fumarate-glucose mixture

Our studies enabled us to intensify the synthesis of the microbial exopolysaccharide (EPS) ethapolan produced by Acinetobacter sp. IMV B-7005 grown on a mixture of fumarate (an energy-excessive substrate) and glucose (an energy-deficient substrate). Supplementing glucose-containing medium with sodium (potassium) fumarate at a molar ratio of 4:1 resulted in a 1.3-2.2-fold increase of the EPS amount synthesized and in a 1.3-2-fold increase of the EPS yield relative to the biomass compared to monosubstrate cultivation. The conversion of the carbon of both substrates to EPS was the highest if the carbon/nitrogen ratio in the cultivation medium was 70.5 and inoculum grown on glucose monosubstrate was used.     

Structure of an acylated exopolysaccharide synthesized by Acinetobacter sp

The complex preparation ethapolan synthesized by Acinetobacter sp. consists of neutral (minor component) and two acidic exopolysaccharides (EPS) one of which is acylated. On the basis of chemical modification of EPS, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and octasaccharide fragments, Smith degradation, 1H- and 13C NMR analysis the following structure of the acylated polysaccharide repeating unit has been established (scheme): It is suggested that in the acylated EPS at least one glucose residue and the galactose residue are O-acylated.     

Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.     

Removal of uranium by an exopolysaccharide from Pseudomonas sp.

Summary Accumulation of uranium (U) is reported for isolated exopolysaccharide produced by Pseudomonas sp. EPS-5028. A maximum uptake of 96 g U/mg polymer was observed. In contrast, the maximum accumulation of uranium by deacylated polysaccharide was 46 g/mg. This metal-complexing capacity observed suggests that the anionic reactive sites on the structure could be responsible for this activity. Metal uptake was affected by pH and was not affected by temperature. Expolysaccharide from Pseudomonas sp. EPS-5028 obeyed the Freundlich isotherm indicating single layer adsorption. Offprint requests to: F. Congregado     

Effects of growth substrate and respiratory chain composition on bioenergetics in Acinetobacter sp. strain HO1-N.

The relationship between respiratory chain composition and efficiency of coupling phosphorylation to electron transport was examined in Acinetobacter sp. strain HO1-N. Cells containing only cytochrome o as a terminal oxidase displayed the same stoichiometries of adenosine 5'-triphosphate synthesis and proton extrusion as cells which contained both cytochromes o and d as terminal oxidases. In addition, CO inhibition and photo-relief of cytochromes o or d did not alter the efficiency of energy coupling. These findings indicate that adenosine 5'-triphosphate synthesis is coupled to electron transport through both cytochromes o and d in Acinetobacter.     

Degradation of p-benzyloxyphenol by Acinetobacter sp.

Abstract Acinetobacter sp. utilized p -benzyloxyphenol as sole carbon source and degraded it to p -hydroxybenzaldehyde, p -hydroxybenzoic acid, protocatechuic acid and catechol. The intermediates were identified by paper chromatography, TLC, IR, GC and HPLC. Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase during the degradation of p -benzoloxyphenol.     

Identification and synthesis of acyl-phosphatidylglycerol in Acinetobacter sp. HO1-N

A minor phospholipid from Acinetobacter sp. HO1-N was identified as acyl-phosphatidylglycerol. Acyl-phosphatidylglycerol synthesis by outer-membrane preparations appeared to be a result of phospholipase A activity.     

Intensification of biosurfactant synthesis by <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> IMV B-7241 on a hexadecane-glycerol mixture

The possibility of enhanced biosurfactant (BS) synthesis by the cultivation of Acinetobacter calcoaceticus IMV B-7241 on a mixture of energetically nonequivalent substrates (hexadecane and glycerol) was shown. Based on theoretical calculations of the energy requirements for biomass production and the synthesis of surface-active trehalose monomycolate from the energy-deficient substrate (glycerol), the concentration of the energy-excessive substrate (hexadecane), which increased the efficiency of the substrate carbon conversion to BS, was determined. The synthesis of extracellular BS on a mixture of hexadecane and glycerol in a molar ratio of 1: 7 at C/N ratio of 30 increased 2.6–3.5-fold compared to that on single-substrate media. Increased BS synthesis by Acinetobacter calcoaceticus IMV B-7241 grown on a hexadecane-glycerol mixture was accompanied by a 1.3–2.4-fold increase in activities of the enzymes involved in their biosynthesis, as well as by simultaneous functioning of two anaplerotic pathways (the glyoxylate cycle and the phosphoenolpyruvate carboxylase reaction).     

Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp.

An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp. was found to contain D-galactopyranuronic acid (GalpA), 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (QuipNAc4NAc), 2-acetamido-2-deoxy D-mannopyranuronic acid (ManpNAcA) and one uncommon unit of D-galactopyranuronic acid with the carboxyl group amide-linked to glycine [GalpA(Gly)]. The polysaccharide was studied by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis. The following linear structure of the repeating unit was established: -->3)-alpha-D-GalpA(Gly)-(1-->4)-beta-D-ManpNAcA-(1-->4)-alp ha-D-Galp A-(1-->3)-alpha-D-QuipNAc4NAc-(1-->. A preliminary phylogenetic assignment for the bacterium is also reported.     

Induction and repression of the collagenase synthesis in Acinetobacter sp.]

The synthesis of collagenase in Acinetobacter sp. was found to be inducible by denatured collagen and by its high molecular weight fragments. The presence in the inducer of part of the tertiary structure appear to be indispensable. On the other hand, an addition of Casamino acids, meat protein hydrolysate, or a mixture of amino acids with a similar composition to gelatin does not stimulate collagenase synthesis. Enzyme production was severely repressed in the early phase of growth by glucose, arabinose, and ribose, single amino acids, proline, hydroxyproline, alanine, glutamic acid or casein acid hydrolysate. A mechanism of repression similar to catabolite repression was involved in the phenomenon caused by carbohydrates. However, the fact that cyclic adenosine 3'5-monophosphate did not overcome the repression caused by amino acids or Casamino acids, in contrast to classical catabolite repression, suggests that these two forms of repression may be distinct.     

Specific features of the synthesis of the exopolysaccharide ethapolan on a mixture of energy-deficient growth substrates

Intensification of the synthesis of the microbial exopolysaccharide ethapolan by Acinetobacter sp. B-7005 was shown to occur on a mixture of energy-deficient growth substrates (acetate + glucose). When the bacterium grew on the substrate mixture, both substrates were utilized simultaneously; acetate was taken up by means of active transport at the expense of the energy of the proton-motive force. When acetate was present in the form of a sodium salt, the activities of acetyl-CoA synthetase and phosphoenolpyruvate synthetase (the key enzyme of gluconeogenesis) were tenfold higher than in the presence of potassium acetate, and the indexes of ethapolan synthesis were two times higher. The positive effect of Na⁺ on ethapolan synthesis is supposed to consist in the creation of ion gradients on the membrane, necessary for the generation of the proton-motive force. Simultaneous functioning of the glyoxylate cycle and pyruvate carboxylase reaction, as well as an increase in the activity of isocitrate lyase, malate synthease, and phosphoenolpyruvate synthetase, provide evidence of increased gluconeogenesis in the presence of the acetate + glucose mixture (as compared to gluconeogenesis on the corresponding monosubstrates).     

Hydrolytic dehalogenation of 4-chlorobenzoic acid by an Acinetobacter sp

Acinetobacter sp. strain ST-1, isolated from garden soil, can mineralize 4-chlorobenzoic acid (4-CBA). The bacterium degrades 4-CBA, starting with dehalogenation to yield 4-hydroxybenzoic acid (4-HBA) under both aerobic and anaerobic conditions, suggesting that the dehalogenating enzyme in the strain is not an oxygenase; the enzyme may catalyze halide hydrolysis. To identify the oxygen source of the C(4)-hydroxy groups in the dehalogenation step, we used H(2)(18)O as the solvent under anaerobic conditions. When resting cells were incubated in the presence of 4-CBA and H(2)(18)O under a nitrogen gas stream, the hydroxy group on the aromatic nucleus of the 4-HBA produced was derived from water, not from molecular oxygen. This dehalogenation was hydrolytic, because analysis of the mass spectrum of the trimethylsilyl derivative of one of the metabolites, (18)O-labeled 4-HBA, showed that 80% of the C4-hydroxy groups were labeled with (18)O. Hydrolytic dehalogenation of 4-CBA in intact cells has not been reported earlier. To identify substrate specificity, we next examined the ability of the strain to dehalogenate 4-CBA analogues and dichlorobenzoic acids. The results of metabolite analysis by high-pressure liquid chromatography showed that the strain dehalogenated 4-bromobenzoic acid and 4-iodobenzoic acid, yielding 4-HBA, suggesting that these compounds could be further degraded and mineralized by the strain via the beta-ketoadipate pathway, as occurs with 4-CBA. This strain, however, did not dehalogenate 4-fluorobenzoic acid, 2- and 3-chlorobenzoic acids, or 2,4-, 3,4-, and 3,5-dichlorobenzoic acids during 4 days of incubation, implying that the dehalogenating enzyme of the strain has high substrate specificity.     

Degradation of lignin and lignin derivatives by Acinetobacter sp.

An Acinetobacter sp. utilized 2-methoxy-4-formylphenoxyacetic acid, dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin as sole carbon source. It also degraded ¹⁴C-labelled DHP lignin and teakwood lignin. Vanillic acid, protocatechuic acid and catechol were separated from 2-methoxy-4-formylphenoxyacetic acid grown cultures. Both protocatechuic acid and catechol were formed from dehydrodivanillyl alcohol, dehydrodiisoeugenol and conidendrin. On the dimeric lignin model substances this Acinetobacter sp. produced protocatechuate 3,4-dioxygenase and catechol 1,2-dioxygenase.     

Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp. Y16

A homogeneous exopolysaccharide, designated As1-1, was obtained from the culture medium of the mangrove endophytic fungus Aspergillus sp. Y16 and purified by anion-exchange and gel-permeation chromatography. Results of chemical and spectroscopic analyses, including one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopy showed that As1-1 was mainly composed of mannose with small amounts of galactose, and that its molecular weight was about 15 kDa. The backbone of As1-1 mainly consists of (1 → 2)-linked α-d-mannopyranose units, substituted at C-6 by the (1 → 6)-linked α-d-mannopyranose, (1→)-linked β-d-galactofuranose and (1→)-linked β-d-mannopyranose units. As1-1 possessed good in vitro antioxidant activity as evaluated by scavenging assays involving 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals. The investigation demonstrated that As1-1 is an exopolysaccharide different from those of other marine microorganisms, and could be a potential antioxidant and food supplement.     

Utilization of complex phenolic compounds by Acinetobacter sp.

Summary Acinetobacter sp. utilized the [ring-¹⁴C]dehydropolymer of coniferyl alcohol (DHP) (sp. act. 1.4 × 10⁴ dpm/mg), ¹⁴C-labelled teakwood lignin (sp. act. 2.5 × 10⁴ dpm/mg), guaiacolglyceryl ether, 2-methoxy-4-formylphenoxyacetic acid, p-benzyloxyphenol, dehydrodivanillyl alcohol, dehydrodiisoeugenol, veratrylglycerol--guaiacyl ether, conidendrin, black liquor lignin and indulin as sole carbon sources. The bacterium produced p-coumaric acid, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid and catechol as intermediates from lignins. Acinetobacter sp. produced catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase during the degradation of lignins. Correspondence to: A. Mahadevan