Relation of 3HV fraction and thermomechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced by Azotobacter vinelandii OP.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has attracted substantial attention as a promising material for industrial applications. In this study, different PHBV films with distinct 3-hydroxyvalerate (3HV) contents produced by Azotobacter vinelandii OP were evaluated. The 3HV fraction ranged from 18.6 to 36.7 mol%, and the number-average molecular weight (Mn) was between 238 and 434 kDa. In the bioreactor, a 3HV fraction (36.7 mol%) and an Mn value of 409 kDa were obtained with an oxygen transfer rate (OTR) of 12.5 mmol L-1 h-1. Thermal analysis measurements showed decreased melting (Tm) and glass transition (Tg) temperatures, and values with relatively high 3HV fractions indicated improved thermomechanical properties. The incorporation of the 3HV fraction in the PHBV chain improved the thermal stability of the films, reduced the polymer Tm, and affected the tensile strength. PHBV film with 36.7 mol% 3HV showed an increase in its tensile strength (51.8 MPa) and a decrease in its Tm (170.61 °C) compared with PHB. Finally, scanning electron microscopy (SEM) results revealed that the PHBV film with 32.8 mol% 3HV showed a degradation upon contact with soil, water, or soil bacteria, showing more porous surfaces after degradation. The latter phenomenon indicated that thermomechanical properties played an important role in biodegradation.

Extended batch cultures for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production by Azotobacter vinelandii OP growing at different aeration rates.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a polymer produced by Azotobacter vinelandii OP. In the bioreactor, PHBV production and its molar composition are affected by aeration rate. PHBV production by A. vinelandii OP was evaluated using extended batch cultures at different aeration rates, which determined different oxygen transfer rates (OTR) in the cultures. Under the conditions evaluated, PHBV with different 3-hydroxyvalerate (3HV) fractions were obtained. In the cultures with a low OTR (6.7 mmol L-1 h-1, at 0.3 vvm), a PHBV content of 38% w w-1 with 9.1 mol % 3HV was achieved. The maximum PHBV production (72% w w-1) was obtained at a high OTR (18.2 mmol L-1 h-1, at 1.0 vvm), both at 48 h. Thus, PHBV production increased in the bioreactor with an increased aeration rate, but not the 3HV fraction in the polymer chain. An OTR of 24.9 mmol L-1 h-1 (at 2.1 vvm) was the most suitable for improving the PHBV content (61% w w-1) and a high 3HV fraction of 20.8 mol % (at 48 h); and volumetric productivity (0.15 g L-1 h-1). The findings indicate that the extended batch culture at 2.1 vvm is the most adequate mode of cultivation to produce higher biomass and PHBV with a high 3HV fraction. Overall, the results have shown that the PHBV production and 3HV fraction could be affected by the aeration rate and the proposed approach could be applied to implement cultivation strategies to optimize PHBV production for different biotechnological applications.

Increases in alginate production and transcription levels of alginate lyase (alyA1) by control of the oxygen transfer rate in Azotobacter vinelandii cultures under diazotrophic conditions

BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Azotobacter vinelandii with different 3HV fraction in shake flasks and bioreactor.

In the present study, the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Azotobacter vinelandii was evaluated in shake flasks and bioreactors, utilizing different precursors and oxygen transfer rates (OTRs). In shake flask cultures, the highest PHBV yield from sucrose (0.16 g g-1) and 3-hydroxyvalerate (3HV) fraction in the PHA chain (27.4 mol%) were obtained with valerate (1.0 g L-1). In the bioreactor, the cultures were grown under oxygen-limited conditions, and the maximum OTR (OTRmax) was varied by adjusting the agitation rate. In the cultures grown at low OTRmax (4.3 mmol L-1 h-1), the intracellular content of PHBV (73% w w-1) was improved, whereas a maximum 3HV fraction (35 mol %) was obtained when a higher OTRmax (17.2 mmol L-1 h-1, to 600 rpm) was employed. The findings obtained suggest that the PHBV production and the content of 3HV incorporated into the polymer were affected by the OTR. Based on the evidence, it is possible to produce PHBV with a different composition by varying the OTR of the culture; thus, the approach in this study could be used to scale up PHBV production.

Coenzyme Q production by metabolic engineered Escherichia coli strains in defined medium.

Coenzyme Q (CoQ) plays an important role as an electron transporter in the respiratory chain. It is formed from a benzoquinone ring and an isoprenoid chain of a specific length depending on the organism. We constructed an engineered Escherichia coli strain (menF) unable to produce demethylmenaquinone and menaquinone, compounds that compete for both chorismate, precursor of the benzoquinone ring, and the isoprenoid chain involved in CoQ biosynthesis. In addition, a mutant strain (entC) unable to produce enterobactin, high-affinity siderophore, synthesized from chorismate, and a double mutant (entC-menF) were constructed. The use of glucose or glycerol as carbon sources was also evaluated for the production of CoQ8 in these strains. The double mutant (entC-menF) showed 18% increase in CoQ8-specific content compared to the control strain; however, the single-mutant strains did not show statistically significant differences in CoQ8-specific content respect to the control, in glucose medium in bioreactor experiments. Glycerol was significantly superior compared to glucose for the production of CoQ8 in E. coli, where the CoQ8-specific content increased 126% and 53% in the control and double-mutant strain, respectively. The expression of genes related to CoQ8 biosynthesis is reported, where the entC-menF double-mutant strain showed a significant increase in the expression of CoQ8 biosynthesis-related genes when glycerol was used as sole carbon source. The control strain did not show gene expression difference between both carbon sources, indicating a possible regulation at a different level.

Poly(3-hydroxybutyrate) accumulation by Azotobacter vinelandii under different oxygen transfer strategies.

Azotobacter vinelandii OP is a bacterium that produces poly(3-hydroxybutyrate) (PHB). PHB production in a stirred bioreactor, at different oxygen transfer strategies, was evaluated. By applying different oxygen contents in the inlet gas, the oxygen transfer rate (OTR) was changed under a constant agitation rate. Batch cultures were performed without dissolved oxygen tension (DOT) control (using 9% and 21% oxygen in the inlet gas) and under DOT control (4%) using gas blending. The cultures that developed without DOT control were limited by oxygen. As result of varying the oxygen content in the inlet gas, a lower OTR (4.6 mmol L-1 h-1) and specific oxygen uptake rate (11.6 mmol g-1 h-1) were obtained using 9% oxygen in the inlet gas. The use of 9% oxygen in the inlet gas was the most suitable for improving the intracellular PHB content (56 ± 6 w w-1). For the first time, PHB accumulation in A. vinelandii OP cultures, developed with different OTRs, was compared under homogeneous mixing conditions, demonstrating that bacterial respiration affects PHB synthesis. These results can be used to design new oxygen transfer strategies to produce PHB under productive conditions.

Microbiome analysis and bacterial isolation from Lejía Lake soil in Atacama Desert.

As a consequence of the severe climatic change affecting our entire world, many lakes in the Andes Cordillera are likely to disappear within a few decades. One of these lakes is Lejía Lake, located in the central Atacama Desert. The objectives of this study were: (1) to characterize the bacterial community from Lejía Lake shore soil (LLS) using 16S rRNA sequencing and (2) to test a culture-based approach using a soil extract medium (SEM) to recover soil bacteria. This extreme ecosystem was dominated by three phyla: Bacteroidetes, Proteobacteria, and Firmicutes with 29.2, 28.2 and 28.1% of the relative abundance, respectively. Using SEM, we recovered 7.4% of the operational taxonomic units from LLS, all of which belonged to the same three dominant phyla from LLS (6.9% of Bacteroidetes, 77.6% of Proteobacteria, and 15.3% of Firmicutes). In addition, we used SEM to recover isolates from LLS and supplemented the culture medium with increasing salt concentrations to isolate microbial representatives of salt tolerance (Halomonas spp.). The results of this study complement the list of microbial taxa diversity from the Atacama Desert and assess a pipeline to isolate selective bacteria that could represent useful elements for biotechnological approaches.

Bacterial polyhydroxybutyrate for electrospun fiber production.

Nano- and microfibers obtained by electrospinning have attracted great attention due to its versatility and potential for applications in diverse technological fields. Polyhydroxyalkanoates (PHAs) are biopolymers synthesized by microorganisms such as the bacterium Burkholderia xenovorans LB400. In particular, LB400 cells are capable to synthesize poly(3-hydroxybutyrate) (PHB) from glucose. The aim of this study was to produce and characterize electrospun fibers obtained from bacterial PHBs. Bacterial strain LB400 was grown in M9 minimal medium using xylose and mannitol (10gL-1) as the sole carbon sources and NH4Cl (1gL-1) as the sole nitrogen source. Biopolymer-based films obtained were used to produce fibers by electrospinning. Diameter and morphology of the microfibers were analyzed by scanning electron microscopy (SEM) and their thermogravimetric properties were investigated. Bead-free fibers using both PHBs were obtained with diameters of less than 3µm. The surface morphology of the microfibers based on PHBs obtained from both carbon sources was different, even though their thermogravimetric properties are similar. The results indicate that the carbon source may determine the fiber structure and properties. Further studies should be performed to analyze the physicochemical and mechanical properties of these PHB-based microfibers, which may open up novel applications.

Burkholderia xenovorans LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the pha genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions.

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located at chromosome 1 of strain LB400. During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400 cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40 ± 0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.

Bacterial alginate production: an overview of its biosynthesis and potential industrial production.

Alginate is a linear polysaccharide that can be used for different applications in the food and pharmaceutical industries. These polysaccharides have a chemical structure composed of subunits of (1-4)-ß-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). The monomer composition and molecular weight of alginates are known to have effects on their properties. Currently, these polysaccharides are commercially extracted from seaweed but can also be produced by Azotobacter vinelandii and Pseudomonas spp. as an extracellular polymer. One strategy to produce alginates with different molecular weights and with reproducible physicochemical characteristics is through the manipulation of the culture conditions during fermentation. This mini-review provides a comparative analysis of the metabolic pathways and molecular mechanisms involved in alginate polymerization from A. vinelandii and Pseudomonas spp. Different fermentation strategies used to produce alginates at a bioreactor laboratory scale are described.

Bacterial production of the biodegradable plastics polyhydroxyalkanoates.

Petroleum-based plastics constitute a major environmental problem due to their low biodegradability and accumulation in various environments. Therefore, searching for novel biodegradable plastics is of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable plastics. Life cycle assessment indicates that PHB is more beneficial than petroleum-based plastics. In this report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microorganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Staining with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs produced were characterized by GC-MS. Diverse substrates for the production of PHAs in strain LB400 were analyzed.

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars.

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5 m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2 m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5 g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260 g kg(-1)) and perchlorate (41.13 µg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14 µg g(-1)) or formate (76.06 µg g(-1)) as electron donors, and sulfate (15875 µg g(-1)), nitrate (13490 µg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.