Biofilm Formation of Two Different Marine Bacteria on Modified PDMS Surfaces is Affected by Surface Roughness and Topography.

Different strategies were tested to reduce biofilm formation of the model marine bacteria Cobetia marina and Marinobacter hydrocarbonoclasticus on cross-linked polydimethylsiloxane (PDMS) coated aluminum and cellulose acetate surfaces modified by addition of multi-walled carbon nanotubes (MWCNT) or exposure of the surfaces to bromine vapors in the presence and absence of UV irradiation. The three surface modifications explored led to important reductions in biofilm formation for the two marine bacteria, up to 30% in the case of exposure to Br2(g). Biofouling reduction could be correlated to surface properties in all cases through the introduction of a quantitative theoretical model based on an effective roughness parameter, Raeff, that accounted for the different morphological changes observed. The model considers the possibility of bacterial inclusion into large surface wells, as observed by AFM in the case of Br2(g) + UV light treatment. In addition, a linear relationship was observed between biofouling reduction and the Raeff effective roughness parameter.

Polyhydroxyalkanoates: Much More than Biodegradable Plastics.

Bacterial polyhydroxyalkanoates (PHAs) are isotactic polymers that play a critical role in central metabolism, as they act as dynamic reservoirs of carbon and reducing equivalents. These polymers have a number of technical applications since they exhibit thermoplastic and elastomeric properties, making them attractive as a replacement of oil-derived materials. PHAs are accumulated under conditions of nutritional imbalance (usually an excess of carbon source with respect to a limiting nutrient, such as nitrogen or phosphorus). The cycle of PHA synthesis and degradation has been recognized as an important physiological feature when these biochemical pathways were originally described, yet its role in bacterial processes as diverse as global regulation and cell survival is just starting to be appreciated in full. In the present revision, the complex regulation of PHA synthesis and degradation at the transcriptional, translational, and metabolic levels are explored by analyzing examples in natural producer bacteria, such as Pseudomonas species, as well as in recombinant Escherichia coli strains. The ecological role of PHAs, together with the interrelations with other polymers and extracellular substances, is also discussed, along with their importance in cell survival, resistance to several types of environmental stress, and planktonic-versus-biofilm lifestyle. Finally, bioremediation and plant growth promotion are presented as examples of environmental applications in which PHA accumulation has successfully been exploited.

Unexpected stress-reducing effect of PhaP, a poly(3-hydroxybutyrate) granule-associated protein, in Escherichia coli.

Phasins (PhaP) are proteins normally associated with granules of poly(3-hydroxybutyrate) (PHB), a biodegradable polymer accumulated by many bacteria as a reserve molecule. These proteins enhance growth and polymer production in natural and recombinant PHB producers. It has been shown that the production of PHB causes stress in recombinant Escherichia coli, revealed by an increase in the concentrations of several heat stress proteins. In this work, quantitative reverse transcription (qRT)-PCR analysis was used to study the effect of PHB accumulation, and that of PhaP from Azotobacter sp. strain FA8, on the expression of stress-related genes in PHB-producing E. coli. While PHB accumulation was found to increase the transcription of dnaK and ibpA, the expression of these genes and of groES, groEL, rpoH, dps, and yfiD was reduced, when PhaP was coexpressed, to levels even lower than those detected in the non-PHB-accumulating control. These results demonstrated the protective role of PhaP in PHB-synthesizing E. coli and linked the effects of the protein to the expression of stress-related genes, especially ibpA. The effect of PhaP was also analyzed in non-PHB-synthesizing strains, showing that expression of this heterologous protein has an unexpected protective effect in E. coli, under both normal and stress conditions, resulting in increased growth and higher resistance to both heat shock and superoxide stress by paraquat. In addition, PhaP expression was shown to reduce RpoH protein levels during heat shock, probably by reducing or titrating the levels of misfolded proteins.

Effects of aeration on the synthesis of poly(3-hydroxybutyrate) from glycerol and glucose in recombinant Escherichia coli.

Bioreactor cultures of Escherichia coli recombinants carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol under low-agitation conditions accumulated more poly(3-hydroxybutyrate) (PHB) and ethanol than at high agitation, while in glucose cultures, low agitation led to a decrease in PHB formation. Cells produced smaller amounts of acids from glycerol than from glucose. Glycerol batch cultures stirred at 125 rpm accumulated, in 24 h, 30.1% (wt/wt) PHB with a relative molecular mass of 1.9 MDa, close to that of PHB obtained using glucose.

Elimination of D-lactate synthesis increases poly(3-hydroxybutyrate) and ethanol synthesis from glycerol and affects cofactor distribution in recombinant Escherichia coli.

The effect of eliminating D-lactate synthesis in poly(3-hydroxybutyrate) (PHB)-accumulating recombinant Escherichia coli (K24K) was analyzed using glycerol as a substrate. K24KL, an ldhA derivative, produced more biomass and had altered carbon partitioning among the metabolic products, probably due to the increased availability of carbon precursors and reducing power. This resulted in a significant increase of PHB and ethanol synthesis and a decrease in acetate production. Cofactor measurements revealed that cultures of K24K and K24KL had a high intracellular NADPH content and that the NADPH/NADP(+) ratio was higher than the NADH/NAD(+) ratio. The ldhA mutation affected cofactor distribution, resulting in a more reduced intracellular state, mainly due to a further increase in NADPH/NADP(+). In 60-h fed-batch cultures, K24KL reached 41.9 g·liter⁻¹ biomass and accumulated PHB up to 63% ± 1% (wt/wt), with a PHB yield on glycerol of 0.41 ± 0.03 g·g⁻¹, the highest reported using this substrate.

Pseudomonas extremaustralis sp. nov., a Poly(3-hydroxybutyrate) producer isolated from an antarctic environment.

A Gram-negative, mobile, rod-shaped, non-spore-forming bacterium (strain 14-3(T)) was isolated from a temporary pond in Antarctica. On the basis of 16S rRNA gene sequence similarity, strain 14-3(T) was shown to belong to the genus Pseudomonas sensu stricto. Physiological and biochemical tests supported the phylogenetic affiliation. Strain 14-3(T) is closely related to Pseudomonas veronii DSM 11331(T), sharing 99.7% sequence similarity. DNA-DNA hybridization experiments between the two strains showed only moderate reassociation similarity (35.1%). Tests for arginine dihydrolase and nitrate reduction were positive, while those for denitrification, indol production, glucose acidification, urease, ss-galactosidase, esculin, caseine and gelatin hydrolysis were negative. Growth of this bacterium occurred in a range from 4 to 37 degrees C but not at 42 degrees C. It accumulated poly(3-hydroxybutyrate) when grown on sodium octanoate medium. Strain 14-3(T) therefore represents the type strain of a new species, for which the name Pseudomonas extremaustralis sp. nov. is proposed. The type strain 14-3(T) has been deposited as DSM 17835(T) and as CIP 109839(T).

Effects of granule-associated protein PhaP on glycerol-dependent growth and polymer production in poly(3-hydroxybutyrate)-producing Escherichia coli.

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular granules by many bacteria under unfavorable conditions, enhancing their fitness and stress resistance. Poly(3-hydroxybutyrate) (PHB) is the most widespread and best-known PHA. Apart from the genes that catalyze polymer biosynthesis, natural PHA producers have several genes for proteins involved in granule formation and/or with regulatory functions, such as phasins, that have been shown to affect polymer synthesis. This study evaluates the effect of PhaP, a phasin, on bacterial growth and PHB accumulation from glycerol in bioreactor cultures of recombinant Escherichia coli carrying phaBAC from Azotobacter sp. strain FA8. Cells expressing phaP grew more, and accumulated more PHB, both using glucose and using glycerol as carbon sources. When cultures were grown in a bioreactor using glycerol, PhaP-bearing cells produced more polymer (2.6 times) and more biomass (1.9 times) than did those without the phasin. The effect of this protein on growth promotion and polymer accumulation is expected to be even greater in high-density cultures, such as those used in the industrial production of the polymer. The recombinant strain presented in this work has been successfully used for the production of PHB from glycerol in bioreactor studies, allowing the production of 7.9 g/liter of the polymer in a semisynthetic medium in 48-h batch cultures. The development of bacterial strains that can efficiently use this substrate can help to make the industrial production of PHAs economically feasible.

dye (arc) Mutants: insights into an unexplained phenotype and its suppression by the synthesis of poly (3-hydroxybutyrate) in Escherichia coli recombinants.

arcA codes for a central regulator in Escherichia coli that responds to redox conditions of growth. Mutations in this gene, originally named dye, confer sensitivity to toluidine blue and other redox dyes. However, the molecular basis for the dye-sensitive phenotype has not been elucidated. In this work, we show that toluidine blue redirects electrons to O2 and causes an increase in the generation of reactive O2 species (ROS). We also demonstrate that synthesis of poly (3-hydroxybutyrate) suppresses the Dye phenotype in E. coli recombinants, as the capacity to synthesize the polymer reduces sensitivity to toluidine blue, O2 consumption and ROS production levels.

Redox driven metabolic tuning: carbon source and aeration affect synthesis of poly(3-hydroxybutyrate) in Escherichia coli.

Growth and polymer synthesis were studied in a recombinant E. coli strain carrying phaBAC and phaP of Azotobacter sp. strain FA8 using different carbon sources and oxygen availability conditions. The results obtained with glucose or glycerol were completely different, demonstrating that the metabolic routes leading to the synthesis of the polymer when using glycerol do not respond to environmental conditions such as oxygen availability in the same way as they do when other substrates, such as glucose, are used. When cells were grown in a bioreactor using glucose the amount of polymer accumulated at low aeration was reduced by half when compared to high aeration, while glycerol cultures produced at low aeration almost twice the amount of polymer synthesized at the higher aeration condition. The synthesis of other metabolic products, such as ethanol, lactate, formate and acetate, were also affected by both the carbon source used and aeration conditions. In glucose cultures, lactate and formate production increased in low agitation compared to high agitation, while poly(3-hydroxybutyrate) synthesis decreased. In glycerol cultures, the amount of acids produced also increased when agitation was lowered, but carbon flow was mostly redirected towards ethanol and poly(3-hydroxybutyrate). These results indicated that carbon partitioning differed depending on both carbon source and oxygen availability, and that aeration conditions had different effects on the synthesis of the polymer and other metabolic products when glucose or glycerol were used.

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures.

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the 'one-factor-at-a-time' technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett-Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box-Wilson design. Under such optimized conditions (22.02 g l(-1) glycerol, 1.78 g l(-1) CAS, and 1.83 g l(-1) inoculum) microaerobic batch cultures gave rise to 8.37 g l(-1) CDW and 3.52 g l(-1) PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l(-1). After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l(-1), respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.

Poly(3-hydroxybutyrate) synthesis by recombinant Escherichia coli arcA mutants in microaerobiosis.

We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% +/- 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% +/- 3%. This strain grew in a simple medium at a specific growth rate of 0.69 +/- 0.07 h(-1), whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 +/- 0.06 h(-1). The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.

New recombinant Escherichia coli strain tailored for the production of poly(3-hydroxybutyrate) from agroindustrial by-products.

A recombinant E. coli strain (K24K) was constructed and evaluated for poly(3-hydroxybutyrate) (PHB) production from whey and corn steep liquor as main carbon and nitrogen sources. This strain bears the pha biosynthetic genes from Azotobacter sp. strain FA8 expressed from a T5 promoter under the control of the lactose operator. K24K does not produce the lactose repressor, ensuring constitutive expression of genes involved in lactose transport and utilization. PHB was efficiently produced by the recombinant strain grown aerobically in fed-batch cultures in a laboratory scale bioreactor on a semisynthetic medium supplemented with the agroindustrial by-products. After 24 h, cells accumulated PHB to 72.9% of their cell dry weight, reaching a volumetric productivity of 2.13 g PHB per liter per hour. Physical analysis of PHB recovered from the recombinants showed that its molecular weight was similar to that of PHB produced by Azotobacter sp. strain FA8 and higher than that of the polymer from Cupriavidus necator and that its glass transition temperature was approximately 20 degrees C higher than those of PHBs from the natural producer strains.

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in Bacillus megaterium.

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act(Bm), a sigma(54) regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.

A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance.

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.