Influence of l-amino acids on aggregation and biofilm formation in Azotobacter chroococcum and Trichoderma viride.

AIM: The effects of l-amino acids on growth and biofilm formation in Azotobacter chroococcum (Az) and Trichoderma viride (Tv) as single (Az, Tv) and staggered inoculated cultures (Az-Tv, Tv-Az) were investigated. METHODS AND RESULTS: A preliminary study using a set of 20 l-amino acids, identified 6 amino acids (l-Glu, l-Gln, l-His, l-Ser, l-Thr and l-Trp) which significantly enhanced growth and biofilm formation. Supplementation of these amino acids at different concentrations revealed that 40 mmol l-1 was most effective. l-Glu and l-Gln favoured planktonic growth in both single and in staggered inoculated cultures, while l-Trp and l-Thr, enhanced aggregation and biofilm formation. Addition of l-Glu or l-Gln increased carbohydrate content and planktonic population. Principal component analysis revealed the significant role of proteins in growth and biofilm formation, particularly with supplementation of l-Trp, l-Thr and l-Ser. Azotobacter was found to function better as biofilm under staggered inoculated culture with Trichoderma. CONCLUSIONS: The results illustrate that amino acids play crucial roles in microbial biofilm formation, by influencing growth, aggregation and carbohydrates synthesized. SIGNIFICANCE AND IMPACT OF THE STUDY: The differential and specific roles of amino acids on biofilm formation are of significance for agriculturally important micro-organisms that grow as biofilms, colonize and benefit the plants more effectively.

Encystment of Azotobacter nigricans grown diazotrophically on kerosene as sole carbon source.

Encystment of Azotobacter nigricans was induced by its diazotrophic cultivation on kerosene. Its growth and nitrogenase activity were affected by kerosene in comparison to cultures grown on sucrose. Electron microscopy of vegetative cells showed that when nitrogenase activity was higher and the poly-beta-hydroxybutyrate granules were not present to a significant extent, peripheral bodies were abundant. After 8 days of culture on kerosene, the presence of cysts with intracellular bunches of poly-beta-hydroxybutyrate granules was observed. Germination of cysts bears germinating multicelled yet unbroken capsule cysts with up to three cells inside. This is the first report of encystment induction of Azotobacter species grown on kerosene.

Studies on intracellular degradation of polyhydroxyalkanoic acid-polyethylene glycol copolymer accumulated by Azotobacter chroococcum MAL-201.

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [PHB] when grown in glucose containing nitrogen-free Stockdale medium. The same medium supplemented with valerate alone and valerate plus polyethylene glycol (PEG) leads to the accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV] and PEG containing PHBV-PEG polymers, respectively. The intracellular degradation of these polymers as studied in carbon-free Stockdale medium showed a rapid degradation of PHB followed by PHBV, while it was least in case of PHBV-PEG. The rate of such degradation was 44.16, 26.4 and 17.0 mg h(-1)l(-1) for PHB, PHBV and PHBV-PEG, respectively. During the course of such of PHBV and PHBV-PEG degradation the 3HB mol% of polymers decreased significantly with increase of 3HV mol fraction, the EG mol% in PHBV-PEG, however, remained constant. After 50h of degradation the decrease in intrinsic viscosity and molecular mass of PHBV-PEG were 37.5 and 43.6%, respectively. These values appeared low compared to PHB and PHBV. Moreover, the increasing EG content of polymer retarded their extent of degradation. Presence of PEG, particularly of low molecular weight PEG was inhibitory to intracellular PHA depolymerise (i-PHA depolymerase) activity and the relative substrate specificity of the i-PHA depolymerase of MAL-201 appeared to be PHB > PHBV > PHBV-PEG.

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum.

Food industry wastewater served as a carbon source for the synthesis of poly-beta-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5 degrees C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11 degrees C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3-7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.

Degradation of 2,4,6-trichlorophenol by Azotobacter sp. strain GP1.

A bacterium which utilizes 2,4,6-trichlorophenol (TCP) as a sole source of carbon and energy was isolated from soil. The bacterium, designated strain GP1, was identified as an Azotobacter sp. TCP was the only chlorinated phenol which supported the growth of the bacterium. Resting cells transformed monochlorophenols, 2,6-dichlorophenol, and 2,3,6-trichlorophenol. Phenol and a number of phenolic compounds, including 4-methylphenol, all of the monohydroxybenzoates, and several dihydroxybenzoates, were very good carbon sources for Azotobacter sp. strain GP1. The organism utilized up to 800 mg of TCP per liter; the lag phase and time for degradation, however, were severely prolonged at TCP concentrations above 500 mg/liter. Repeated additions of 200 mg of TCP per liter led to accelerated degradation, with an optimum value of 100 mg of TCP per liter per h. TCP degradation was significantly faster in shaken than in nonshaken cultures. The optimum temperature for degradation was 25 to 30 degrees C. Induction studies, including treatment of the cells with chloramphenicol prior to TCP or phenol addition, revealed that TCP induced TCP degradation but not phenol degradation and that phenol induced only its own utilization. Per mol of TCP, 3 mol of Cl- was released. 2,6-Dichloro-p-benzoquinone was detected in the resting-cell medium of Azotobacter sp. strain GP1. By chemical mutagenesis, mutants blocked in either TCP degradation or phenol degradation were obtained. No mutant defective in the degradation of both phenols was found, indicating separate pathways for the dissimilation of the compounds. In some of the phenol-deficient mutants, pyrocatechol was found to accumulate, and in some of the TCP-deficient mutants, 2,6-dichlorohydroquinone was found to accumulate.

Growth of Azotobacter vinelandii with correlation of Coulter cell size, flow cytometric parameters, and ultrastructure.

When Azotobacter vinelandii is grown under nitrogen-fixing conditions, the mean cell volume fluctuates from 2.7 to 6.6 microns 3 as determined using a Coulter counter. When NH4Cl is supplied as nitrogen source, the mean cell volume fluctuates from 4.6 to 7.4 microns3. Parallel experiments using flow cytometric measurements show similar characteristic fluctuations in the narrow forward angle light scattering signal and also in cellular protein content as determined using fluorescein isothiocyanate (FITC) fluorescence. Fluctuations in the perpendicular light scatter signal during batch growth are similar for both sets of growth conditions. Changes in cell morphology and ultrastructure are also similar for both sets of growth conditions, as demonstrated by electron microscopic examination. We conclude that narrow forward angle light scatter is a close correlate of cell size, whereas right angle scatter is an indicator of morphological variations other than size.

In vivo interaction between nitrogenase molybdenum-iron protein and membrane in Azotobacter vinelandii and Rhodospirillum rubrum.

Oriented whole cell multilayers of Azotobacter vinelandii and Rhodospirillum rubrum were analyzed by electron spin resonance (ESR) spectroscopy to detect possible structural associations between nitrogenase molybdenum-iron (MoFe) protein and cytoplasmic or intracytoplasmic membrane. Initially, protocols were designed to obtain strong molybdenum-iron protein ESR signals in whole cell samples of each organism. Then, two-dimensional orientation of whole cell membranes was demonstrated in whole cell multilayers using doxyl stearate spin label in A. vinelandii and the bacteriochlorophyll a dimer triplet signal, (BCHl a)T2, from the intracytoplasmic membrane-bound photosynthetic apparatus of R. rubrum. Subsequent analysis of the low-field signals, g = 4.3 and g = 3.6, of molybdenum-iron protein in whole cell multilayers of each organism showed orientation-dependent characteristics, although the properties of each were different. Specifically, as the normal to the membrane plane was rotated from perpendicular to parallel with the ESR magnetic field, the amplitude of the g = 3.6 signal decreased from maximum to about 37% of maximum in A. vinelandii and from maximum to about 88% of maximum in R. rubrum. The angular dependence of the g = 4.3 peak during rotation varied in A. vinelandii, but decreased from maximum to about 63% of maximum in R. rubrum. These data suggest that the molybdenum-iron protein of nitrogenase was oriented in response to the physical orientation of cellular membranes and that a structural association may exist between this nitrogenase component and membrane in these organisms.

Thermoresistance of azotobacter vinelandii ATCC 12837 in defined and dialysed soil media: filtrable forms.

Thermoresistance of Azotobacter vinelandii ATCC 12837 in nitrogen-free (defined) medium and dialysed soil medium was established. These microorganisms showed thermoresistance in the defined medium, but in the dialysed soil medium they were very sensitive to heat. Thermosensitivity in the dialysed soils medium was due to the production of germinal cells (filtrable forms) and not to the presence of any kind of cytoplasmic inclusions such as poly-beta-hydroxybutyric acid, metachromatic, glycogen or unstaining granules.

Lipid metabolism during encystment of Azotobacter vinelandii.

The formation of cysts by Azotobacter vinelandii involves the synthesis of lipids as major metabolic products. Cells which encyst at low levels in aging glucose cultures undergo the same pattern of lipid synthesis as cells which undergo reasonably synchronous encystment in beta-hydroxybutyrate or n-butanol. The accumulation of poly-beta-hydroxybutyrate (PHB) precedes the synthesis of 5-n-heneicosylresorcinol and 5-n-tricosylresorcinol (AR1), which is then followed in about 6 h by the synthesis of the 5-n-alkylresorcinol galactosides (AR2). In the mature cyst, PHB, AR1, and AR2 account for 8, 5.6, and 4.5%, respectively, of the dry weight. Phospholipid formation levels off 4 h postinduction, which coincides with the final cell division, but fatty acids synthesis continues at a very low level throughout encystment, suggesting some turnover of fatty acid. Distribution studies show that AR1 and AR2 are found in roughly equal amounts in the exine and central body of the cysts, with only trace amounts recovered from the intine. Studies of cysts labeled during encystment with [14C]beta-hydroxybutyrate or during vegetative growth with [14C]glucose suggest that the exine structure is synthesized during encystment, but that the intine is composed largely of vegetative cell components.

True morphology of the Azotobacteraceae-filterable bacteria.

The history of bacteriology up to the 1950s contains many references to filterable forms of bacteria but misconceptions regarding the nature of filterability eventually made the entire phenomenon an unresolvable paradox. Although the existence of filterable azotobacter has often been reported, so much doubt in these results has been expressed that now Bergey does not even mention filterability. In 1965 we reported the resistance of soil azotobacter to gamma rays and concluded that the possibilty of a very small Azotobacter form could not be dismissed and as a result of studies on survival in soil, we suggested the possible existence in soil of a still undiscovered phase of the Azotobacter cyst. We report here the discovery of these small, soil azotobacter and provide evidence to support this claim.

Studies on azotobacters prevailing in Egyptian soils.

Soil samples from different locations in Egypt, representing various types of soils, different degrees of fertility and under various standing crops, were used for isolating Azotobacter strains. Sixty Azotobacter isolates were obtained, and A. chroococcum was found to be the most predominant species (56 isolates), while A. vinclandii was sporadically found (4 isolates). Other species of the genus Azotobacter were not detected in the examined soil samples. Isolates were studied for their morphological, cultural, and physiological properties. Thirteen isolates, differing in morphological features, were selected for studying their pleomorphic character.

Relationship between calcium and uroinic acids in the encystment of Azotobacter vinelandii.

Encystment of Azotobacter vinelandii (ATCC 12837) in modified Burk nitrogen-free medium (pH 7.0) containing 0.2 percent beta-hydroxybutyrate occurs optimally in 0.37 to 0.44 mM solutions of calcium ions. Suspension of cells in media deficient in calcium results in abortive encystment characterized by the release of viscous cyst coat material. Mature cysts rupture in ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, suggesting that calcium is a structural component of the cyst coat. Maximal stimulation of encystment by calcium ions occurs prior to the completion of the cyst exine or outer coat. The uronic acid composition of cyst components is dependent on calcium levels in the medium. Uronic acids account for 31.7 percent of the intine (inner coat) and 13 percent of the exine dry weight, and only mannuronic and guluronic acids are present in these fractions. These can be extracted as homo- and heteropolymeric sequence "blocks" characteristic of alginic acids. The polyuronic acid fraction of both the cyst coats contain approximately equal amounts of heteropolymeric (mannuronic acid/guluronic acid) blocks. The exine, however, is richer in polyguluronic acid and the intine is richer in polymannuronic acid. As a result, the mannuronic acid/guluronic acid ratio of the exine is lower than that of the intine. Slimes that form in abortive encystment are rich in polymannuronic acid and have a high mannuronic acid/guluronic acid ratio. A polymannuronic acid 5-epimerase is active in the mature cyst central body and the encystment culture fluid.

Variation in the Azotobacter population from several habitats in the botanical garden in Poznan.

The abundance of Azotobacter was estimated in the rhizosphere of 13 plant species from four habitats in the Botanical Garden in Poznan. The results have shown that within the particular habitats the plant exerts a pronounced influence on the abundance of Azotobacter. The morphological differences between Azotobacter populations obtained from various habitats confirm the specific effect of the plant on this bacterium.

Critical-point drying: rapid method for the determination of bacterial extracellular polymer and surface structures.

The relative amount of extracellular polymer which remains about Azotobacter vinelandii, Zoogloea ramigera, Klebsiella pneumoniae, and Diplococcus pneumoniae after critical-point drying was studied by electron microscopy. The results obtained with this technique are compared to those obtained with methods that illustrate extracellular polymer, such as freeze-etching and ruthenium red staining. Comparative results indicate critical-point drying to be a rapid, reliable method for the determination of capsule-like polymer surrounding bacterial cells. In addition, critical-point drying can be used to observe morphogenetic changes, such as vesicle production.