Potential of methyltransferase containing Pseudomonas oleovorans for abatement of arsenic toxicity in rice.

Arsenic (As) has become natural health hazard for millions of people across the world due to its distribution in the food chain. Naturally, it is present in different oxidative states of inorganic [As(V) and As(III)] and organic (DMA, MMA and TMA) forms. Among different mitigation approaches, microbe mediated mitigation of As toxicity is an effective and eco-friendly approach. The present study involves the characterization of bacterial strains containing arsenite methyltransferase (Pseudomonas oleovorans, B4.10); arsenate reductase (Sphingobacterium puteale, B4.22) and arsenite oxidase (Citrobacter sp., B5.12) activity with plant growth promoting (PGP) traits. Efficient reduction of grain As content by 61 % was observed due to inoculation of methyltransferase containing B4.10 as compared to B4.22 (47 %) and B5.12 (49 %). Reduced bioaccumulation of As in root (0.339) and shoot (0.166) in presence of B4.10 was found to be inversely related with translocation factor for Mn (3.28), Fe (0.073), and Se (1.82). Bioaccumulation of these micro elements was found to be associated with the modulated expression of different mineral transporters (OsIRT2, OsFRO2, OsTOM1, OsSultr4;1, and OsZIP2) in rice shoot. Improved dehydrogenase (407 %), and ß-glucosidase (97 %) activity in presence of P. oleovorans (B4.10) as compared to arsenate reductase (198 and 50 %), and arsenite oxidase (134 and 69 %) containing bacteria was also observed. Our finding confers the potential of methyltransferase positive P. oleovorans (B4.10) for As stress amelioration. Reduced grain As uptake was found to be mediated by improved plant growth and nutrient uptake associated with enhanced soil microbial activity.

Removal of gaseous tetrahydrofuran via a three-phase airlift bioreactor loaded with immobilized cells of GFP-tagged Pseudomonas oleovorans GDT4.

Tetrahydrofuran (THF) is a common highly toxic cyclic aliphatic ether that frequently exists in waste gases. Removal of gaseous THF is a serious issue with important environmental ramifications. A novel three-phase airlift bioreactor (TPAB) loaded with immobilized cells was developed for efficient THF removal from gas streams. An effective THF-degrading transformant, Pseudomonas oleovorans GDT4, which contains the pTn-Mod-OTc-gfp plasmid and was tagged with a green fluorescent protein (GFP), was constructed. Continuous treatment of THF-containing waste gases was succeeded by the GFP-labelled cells immobilized with calcium alginate and activated carbon fiber in the TPAB for 60 days with >90% removal efficiency. The number of fluorescent cells in the beads reached 1.7 × 1011 cells·g-1 of bead on day 10, accounting for 83.3% of the total number of cells. The amount further increased to 3.0 × 1011 cells·g-1 of bead on day 40. However, it decreased to 2.5 × 1011 cells·g-1 of bead with a substantial increase in biomass in the liquid because of cell leakage and hydraulic shock. PCR-DGGE revealed that P. oleovorans was the dominant microorganism throughout the entire operation. The maximum elimination capacity was affected by empty bed residence time (EBRT). The capacity was only 25.9 g m-3·h-1 at EBRT of 80 s, whereas it reached 37.8 g m-3·h-1 at EBRT of 140 s. This work provides an alternative method for full-scale removal of gaseous THF and presents a useful tool for determining the biomass of a specific degrader in immobilized beads.

Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines.

Amination of bulky ketones, particularly in (R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S) selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R) amines for the production of high-value products and to screen for those converting putrescine.IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Microbial transformation of hederagenin by Cunninghamella echinulate, Mucor subtilissimus, and Pseudomonas oleovorans.

The pentacyclic triterpenoid hederagenin (1) was subjected to biotransformation by Cunninghamella echinulate CGMCC 3.2000, Mucor subtilissimus CGMCC 3.2454 and Pseudomonas oleovorans CGMCC 1.1641. Three metabolites were obtained. On the basis of nuclear magnetic resonance and high-resolution mass spectral analyses, their structures were characterized as 3ß, 23-dihydroxyolean-12-en-28-oic acid 28-O-ß-D-glucopyranosyl ester (2), 3ß, 15α, 23-trihydroxyolean-12-en-28-oic acid (3), 1ß, 3ß, 23-trihydroxyolean-12-en-28-oic acid (4), and metabolite (3) was a new compound. This was the first report on the biotransformation of hederagenin.

Isolation of bacterial strains able to degrade biphenyl, diphenyl ether and the heat transfer fluid used in thermo-solar plants.

Thermo-solar plants use eutectic mixtures of diphenyl ether (DE) and biphenyl (BP) as heat transfer fluid (HTF). Potential losses of HTF may contaminate soils and bioremediation is an attractive tool for its treatment. DE- or BP-degrading bacteria are known, but up to now bacteria able to degrade HTF mixture have not been described. Here, five bacterial strains which are able to grow with HTF or its separate components DE and BP as sole carbon sources have been isolated, either from soils exposed to HTF or from rhizospheric soils of plants growing near a thermo-solar plant. The organisms were identified by 16S rRNA gene sequencing as Achromobacter piechaudii strain BioC1, Pseudomonas plecoglossicida strain 6.1, Pseudomonas aeruginosa strains HBD1 and HBD3, and Pseudomonas oleovorans strain HBD2. Activity of 2,3-dihydroxybiphenyl dioxygenase (BphC), a key enzyme of the biphenyl upper degradation pathway, was detected in all isolates. Pseudomonas strains almost completely degraded 2000ppm HTF after 5-day culture, and even tolerated and grew in the presence of 150,000ppm HTF, being suitable candidates for in situ soil bioremediation. Degradation of both components of HTF is of particular interest since in the DE-degrader Sphingomonas sp. SS3, growth on DE or benzoate was strongly inhibited by addition of BP.

Biotransformation of 6:2 polyfluoroalkyl phosphates (6:2 PAPs): Effects of degradative bacteria and co-substrates.

Polyfluoroalkyl phosphates (PAPs), a group of fluorotelomer alcohol (FTOH)-based surfactants commonly used in water- and grease-proof food contact paper, have been suggested as a direct source of human exposure to health-concerned perfluoroalkyl carboxylic acids (PFCAs). This study investigated factors affecting biotranformation of 6:2 polyfluoroalkyl phosphates (6:2 PAPs) by three known FTOH-degrading Pseudomonas strains (Pseudomonas butanovora, P. oleovorans, and P. fluorescens DSM 8341) under different co-substrate conditions and compared to that by activated sludge samples. The three pure strains transformed 6:2 PAPs into eight different per- and poly-fluoroalkyl carboxylic acids (PFCAs) and/or PFCA precursors. P. fluorescens DSM 8341 produced 5:2 sFTOH [CF3(CF2)4CH(OH)CH3] and P. oleovorans produced 5:2 ketone [CF3(CF2)4C(O)CH3] as the primary transformation product, respectively, with citrate having a minimal impact on the transformation. P. butanovora with lactate produced more diverse transformation products than those by any two strains. Activated sludge was more efficient at transforming 6:2 PAPs and produced more transformation products including PFHpA [CF3(CF2)5COOH] and PFPeA [CF3(CF2)3COOH], with 5:2 sFTOH as the most abundant product on day 30. The abundance of the alkane hydroxylase (alkB) gene related to alkane oxidation, the changes of total microbial population as well as their community structure in activated sludge during 6:2 PAPs biotransformation were also investigated.

Biodegradation kinetics of tetrahydrofuran, benzene, toluene, and ethylbenzene as multi-substrate by Pseudomonas oleovorans DT4.

The biodegradation kinetics of tetrahydrofuran, benzene (B), toluene (T), and ethylbenzene (E) were systematically investigated individually and as mixtures by a series of aerobic batch degradation experiments initiated by Pseudomonas oleovorans DT4. The Andrews model parameters, e.g., maximum specific growth rates (µmax), half saturation, and substrate inhibition constant, were obtained from single-substrate experiments. The interaction parameters in the sum kinetics model (SKIP) were obtained from the dual substrates. The µmax value of 1.01 for tetrahydrofuran indicated that cell growth using tetrahydrofuran as carbon source was faster than the growth on B (µmax, B = 0.39) or T (µmax, T = 0.39). The interactions in the dual-substrate experiments, including genhancement, inhibition, and co-metabolism, in the mixtures of tetrahydrofuran with B or T or E were identified. The degradation of the four compounds existing simultaneously could be predicted by the combination of SKIP and co-metabolism models. This study is the first to quantify the interactions between tetrahydrofuran and BTE.

[Biodegradation of tetrahydrofuran by combined immobilized of Pseudomonas oleovorans DT4].

A new composite matrix, calcium alginate (CA) coupled with activated carbon fiber (ACF) was designed to immobilize the cells of Pseudomonas oleovorans DT4 for tetrahydrofuran (THF) degradation. The average removal rate of the CA-ACF immobilized cells reached 24.0 mg x (L x h)(-1) with an initial THF concentration of 360 mg x L(-1) when the concentration of CA and ACF was 3% and 1.5% respectively. The mechanical strength of the mobilized cells was also significantly improved with the addition of ACF. Compared to the free suspended cells, higher stable removal efficiency (more than 80%) of CA-ACF cells was detected under different conditions of temperature and pH. The feasibility of the newly designed matrix was also reflected by the repeated batch degradation which showed that the removal activity decreased insignificantly after 80 cycles with the modified reaction system (PNS).

Biodegradation of tetrahydrofuran by Pseudomonas oleovorans DT4 immobilized in calcium alginate beads impregnated with activated carbon fiber: mass transfer effect and continuous treatment.

A novel entrapment matrix, calcium alginate (CA) coupled with activated carbon fiber (ACF), was prepared to immobilize Pseudomonas oleovorans DT4 for degrading tetrahydrofuran (THF). The addition of 1.5% ACF increased the adsorption capacity of the immobilized bead, thus resulting in an enhanced average removal rate of 30.3mg/(Lh). The synergism between adsorption and biodegradation was observed in the hybrid CA-ACF beads instead of in the system comprising CA beads and freely suspended ACF. The effective diffusion coefficient of the CA-ACF bead was not significantly affected by bead size, but the bead's value of 1.14×10(-6)cm(2)/s (for the bead diameter of 0.4 cm) was larger than that of the CA bead by almost one order of magnitude based on the intraparticle diffusion-reaction kinetics analysis. Continuous treatment of the THF-containing wastewater was succeeded by CA-ACF immobilized cells in a packed-bed reactor for 54 d with a >90% removal efficiency.

Substrate interactions during the biodegradation of BTEX and THF mixtures by Pseudomonas oleovorans DT4.

The efficient tetrahydrofuran (THF)-degrading bacterium, Pseudomonas oleovorans DT4 was used to investigate the substrate interactions during the aerobic biotransformation of THF and BTEX mixtures. Benzene and toluene could be utilized as growth substrates by DT4, whereas cometabolism of m-xylene, p-xylene and ethylbenzene occurred with THF. In binary mixtures, THF degradation was delayed by xylene, ethylbenzene, toluene and benzene in descending order of inhibitory effects. Conversely, benzene (or toluene) degradation was greatly enhanced by THF leading to a higher degradation rate of 39.68 mg/(h g dry weight) and a shorter complete degradation time about 21 h, possibly because THF acted as an "energy generator". Additionally, the induction experiments suggested that BTEX and THF degradation was initiated by independent and inducible enzymes. The transient intermediate hydroquinone was detected in benzene biodegradation with THF while catechol in the process without THF, suggesting that P. oleovorans DT4 possessed two distinguished benzene pathways.

[Characteristics of tetrahydrofuran degradation by Pseudomonas oleovorans DT4].

A tetrahydrofuran (THF)-degrading strain Pseudomonas oleovorans DT4 was isolated from the activated sludge of a pharmaceutical plant. P. oleovorans DT4 was able to utilize THF as the sole carbon and energy source under aerobic condition. 5 mmol/L of THF could be completely degraded by 3.2 mg/L inoculums of P. oleovorans DT4 in 14 h at pH 7.2 and 30 degrees C, with the cells concentration increasing to 188.6 mg/L. After the complete consumption of THF, no TOC could be detected but IC reached the stable value of about 46 mg/L, with pH decreasing to 6.54, which indicated that the substance was totally mineralized by P. oleovorans DT4. The optimum conditions for THF biodegradation in shaking flasks were pH 7.5 and temperature 37 degrees C, respectively. Results from the oxygen control experiments revealed that the oxygen supply by shaking was the satisfactory growth condition. Additionally, as the important elements for DT4, Mg2+ and Ca2+ at concentrations of 0.80 mmol/L and 0.20 mmol/L, respectively, were suitable for THF degradation. All the results contribute to the efficient bioremediation for the THF contaminated.

Biosynthesis and characterization of copolymer poly(3HB-co-3HV) from saponified Jatropha curcas oil by Pseudomonas oleovorans.

Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polymers with promising application in the formulation of plastic materials. PHAs are produced by numerous bacteria as energy/carbon storage materials from various substrates, including sugars and plant oils. Since these substrates compete as food sources, their use as raw material for industrial-scale production of PHA is limited. Therefore, efforts have been focused on seeking alternative sources for bacterial production of PHA. One substrate that seems to have great potential is the seed oil of Jatropha curcas plant. Among other favorable properties, J. curcas seed oil is non-edible, widely available, and can be cheaply produced. In this study, Pseudomonas oleovorans (ATCC 29347) was grown in a mineral salt medium supplemented with saponified J. curcas seed oil as the only carbon source under batch fermentation. Optimum PHA yield of 26.06% cell dry weight was achieved after 72 h. The PHA had a melting point (T(m)) between 150 and 160 degrees C. Results of polymer analyses by gas chromatography/mass spectrometry (GC/MS) identified only the methyl 3-hydroxybutanoate monomeric unit. However, electrospray ionization-time of flight mass spectroscopy (ESI-TOF MS) confirmed that the PHA was a copolymer with the characteristic HB/HV peaks at m/z 1155.49 (HB) and 1,169, 1,184-1,194 (HV). The data were further supported by 1H and 13C NMR analysis. Polymer analysis by gel permeation chromatography (GPC) indicated a peak molecular weight (MP) of 179,797, molecular weight (M(W)) of 166,838, weight number average mass (M(n)) of 131,847, and polydispersity (M(w)/M (n)) of 1.3. The data from this study indicate that J. curcas seed oil can be used as a substrate to produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV).

A newly isolated strain capable of effectively degrading tetrahydrofuran and its performance in a continuous flow system.

A Gram-negative strain DT4, capable of growing aerobically on tetrahydrofuran (THF) as the sole carbon and energy source was isolated from a pharmaceutical wastewater treatment plant. It was identified as Pseudomonas oleovorans by morphological and physiological characteristics as well as Biolog profiling and 16S rDNA sequence. Cells of P. oleovorans DT4 pre-cultured in THF could degrade 5 mM THF completely without lag phase. The generation time of 2.7 h and the maximum degradation rate of 203.9 mg THF/(h g dry weight) were observed, demonstrating that DT4 bears the highest THF-degrading activity in ever described strains. Furthermore, THF concentration as high as 100 mM was tolerated by the culture. Several important compounds including gamma-butyrrolactone and benzene could be directly metabolized, whereas other pollutants (e.g., tetrahydropyrane) could be cometabolized by DT4. THF removal was achieved in a continuous flow system with the maximum specific growth rate 0.113 h(-1) and half-saturation constant 1.224 mg/L, indicating the great potential of THF bioremediation in future full-scale application.

Synthesis of microbial elastomers based on soybean oily acids. Biocompatibility studies.

Biocompatibility studies of the autoxidized and unoxidized unsaturated medium-long chain length (m-lcl) co-poly-3-hydroxyalkanoates (m-lclPHAs) derived from soya oily acids have been reported. Pseudomonas oleovorans was grown on a series of mixtures of octanoic acid (OA) and soya oily acids (Sy) with weight ratios of 20:80, 28:72 and 50:50 in order to obtain unsaturated m-lcl copolyesters coded PHO-Sy-2080, PHO-Sy-2872 and PHO-Sy-5050, respectively. The PHA films were obtained by solvent cast from CHCl(3). They were all originally sticky and waxy except PHO-Sy-5050. Autoxidation of the unsaturated copolyester films was carried out on exposure to air at room temperature in order to obtain crosslinked polymers. They became a highly flexible elastomer after being autoxidized (about 40 days of autoxidation). The in vivo tissue reactions of the autoxidized PHAs were evaluated by subcutaneous implantation in rats. The rats appeared to be healthy throughout the implantation period. No symptom such as necrosis, abscess or tumorigenesis was observed in the vicinity of the implants. Retrieved materials varied in their physical appearance after 6 weeks of implantation. In vivo biocompatibility studies of the medical applications indicated that the microbial copolyesters obtained were all biocompatible and especially the PHOSy series of copolyesters had the highest biocompatibility among them.

Characterization of an extracellular polysaccharide produced by a Pseudomonas strain grown on glycerol.

A new extracellular charged polysaccharide composed mainly by galactose, with lower amounts of mannose, glucose and rhamnose, was produced by the cultivation of Pseudomonas oleovorans NRRL B-14682 using glycerol as the sole carbon source. Thermal and solid-state NMR analysis showed that this polymer is essentially amorphous, with a glass transition temperature of 155.7 degrees C. The exopolysaccharide aqueous solutions have viscoelastic properties similar to that of Guar gum, but with affinity to salts as a result of its polyelectrolyte character. In addition, the exopolysaccharide has demonstrated good flocculating and emulsifying properties and film-forming capacity. These properties make this polymer a good alternative to more expensive natural polysaccharides, such as Guar gum, in several applications in the food, pharmaceutical, cosmetic, textile, paper and petroleum industries.

In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate.

The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions.

Production and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions.

This work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems.

Structure and polymer form of poly-3-hydroxyalkanoates produced by Pseudomonas oleovorans grown with mixture of sodium octanoate/undecylenic acid and sodium octanoate/5-phenylvaleric acid.

PHAs (poly-3-hydroxyalkanoates) obtained by Pseudomonas oleovorans grown with mixed carbon sources were investigated. Mixed carbon sources were sodium octanoate/undecylenic acid and sodium octanoate/5-phenylvaleric acid. Effect of carbon source in pre-culture on PHAs structure was investigated. Main fermentation was conducted with mixture of sodium octanoate/undecylenic acid, and PHA contained both saturated and unsaturated units. When more undecylenic acid was used in the medium, the ratio of unsaturated unit increased and the T(g) of the products also changed. The PHA grown with mixture of sodium octanoate and undecylenic acid was a random copolymer, which was determined by DSC analysis. Using mixed carbon sources of sodium octanoate and 5-phenylvaleric acid, highest dry cell weight and PHA concentration were obtained when 0.02g or 0.04g of 5-phenylvaleric acid were added in 50mL medium. Cultured with sodium octanoate and 5-phenylvaleric acid, PHA containing HO (3-hydroxyoctanoate) unit and HPV (3-hydroxy-5-phenylvalerate) unit was produced. T(g) of the products fell between those of pure PHO and pure PHPV. By means of DSC analysis and fractionation method, the PHA obtained was regarded as a random copolymer.

Rapid spectrofluorometric screening of poly-hydroxyalkanoate-producing bacteria from microbial mats.

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72 h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications.

Rapid spectrofluorometric screening of poly-hydroxyalkanoate- producing bacteria from microbial mats / Cribado espectrofluorométrico rápido de bacterias productoras de polihidroxialcanoatos en tapetes microbianos

Microbial mat ecosystems are characterized by both seasonal and diel fluctuations in several physicochemical variables, so that resident microorganisms must frequently adapt to the changing conditions of their environment. It has been pointed out that, under stress conditions, bacterial cells with higher contents of poly-hydroxyalkanoates (PHA) survive longer than those with lower PHA content. In the present study, PHA-producing strains from Ebro Delta microbial mats were selected using the Nile red dying technique and the relative accumulation of PHA was monitored during further laboratory cultivation. The number of heterotrophic isolates in trypticase soy agar (TSA) was ca. 107 colony-forming units/g microbial mat. Of these, 100 randomly chosen colonies were replicated on mineral salt agar limited in nitrogen, and Nile red was added to the medium to detect PHA. Orange fluorescence, produced upon binding of the dye to polymer granules in the cell, was detected in approximately 10% of the replicated heterotrophic isolates. The kinetics of PHA accumulation in Pseudomonas putida, and P. oleovorans were compared with those of several of the environmental isolates spectrofluorometry. PHA accumulation, measured as relative fluorescence intensity, resulted in a steady-state concentration after 48 h of incubation in all strains assayed. At 72h, the maximum fluorescence intensity of each strain incubated with glucose and fructose was usually similar. MAT-28 strain accumulated more PHA than the other isolates. The results show that data obtained from environmental isolates can highly improve studies based on modeling-simulation programs, and that microbial mats constitute an excellent source for the isolation of PHA-producing strains with industrial applications (AU)

El ecosistema de los tapetes microbianos se caracteriza por fluctuaciones diarias y estacionales en diversas variables fisicoquímicas, de tal manera que los microorganismos residentes deben adaptarse frecuentemente a las condiciones cambiantes de su ambiente. Se ha destacado que, en condiciones de estrés, las células bacterianas con elevado contenido en polihidroxialcanoatos (PHA) pueden sobrevivir más tiempo que las de bajo contenido. En este estudio, se utilizó la técnica del colorante rojo Nilo para seleccionar las cepas productoras de PHA de los tapetes microbianos del Delta del Ebro, y para monitorizar la acumulación relativa de PHA durante el cultivo en el laboratorio. El número de aislados heterotrofos en TSA fue de aproximadamente 107 unidades formadoras de colonias/g tapete microbiano. De éstas, se replicaron 100 colonias elegidas al azar cultivadas en agar mineral salino limitado en nitrógeno, al que se le añadió el colorante rojo Nilo para la detección de PHA. La fluorescencia naranja, que se produce al unirse el rojo Nilo a los gránulos de polímero en la célula, se detectó en aproximadamente el 10% de los aislados heterotrofos replicados. La cinética de acumulación de PHA en Pseudomonas putida, P. oleovorans y Escherichia coli se comparó con la de los aislados ambientales por espectrofluorometría. En todas las cepas estudiadas, la acumulación de PHA, medida como la intensidad relativa de fluorescencia, alcanzó una concentración estable a las 48h de incubación. Alas 72h, la máxima intensidad de fluorescencia de las cepas incubadas con glucosa o fructosa fue casi siempre similar. La cepa MAT-28 acumuló más PHA que los otros aislados. Estos resultados ponen de manifiesto que los datos obtenidos a partir de aislados ambientales pueden ser mucho mejores que los que se basan en programas de simulación, y que los tapetes microbianos constituyen una excelente fuente de cepas productoras de PHA con aplicaciones industriales (AU)