Bulking agent in dry anaerobic digestion as a key factor for the enhancement of biogas production.

Dry anaerobic digestion (dry-AD) is an attractive process for solid wastes such as agri-food waste. However, some limitations mainly associated to lack of effective mixing, can hinder the methane production capacity of the systems. Bulking agent (BA) has been proposed as a solution to the compaction issues in systems without mechanical agitation, such as leaching bed reactors. However, effects of BA are still not clear, and, thus, the factors to consider for its dose has not been optimized yet. This work studies the effect of BA in dry-AD. Two substrates with different characteristics were proposed as models, bean peel as a lignocellulosic substrate and a mixture of food waste as a readily biodegradable substrate. Inert plastic rings were used as BA at different BA:S ratios. Assessed BA:S ratio did not affect the performance of methane production for the lignocellulosic waste, but it did significantly affect to the easily biodegradable substrate, showing up to a 28% of methane production increase. This result could be due to the presence of lignocellulosic compounds in the bean peel, behaving like a natural BA. In assays with an increased bed height, the compaction of the system was more severe, resulting in the rapid acidification of the processes. At these conditions, the positive effect of BA addition was more marked, allowing methane production and no acidification of the system. Thus, the addition of BA is a suitable strategy for improving methane production or stability in dry-AD systems without requiring the stirring of the systems.

Establishing a green biodesulfurization process for iron ore concentrates in stirred tank and leaching column bioreactors using Acidithiobacillus thiooxidans.

The presence of sulfur impurities in complex iron ores represents a significant challenge for the iron mining and steel-making industries as their removal often necessitates the use of hazardous chemicals and energy-intensive processes. Here, we examined the microbial and mineralogical composition of both primary and secondary iron concentrates, identifying the presence of Sulfobacillus spp. and Leptospirillum spp., while sulfur-oxidizing bacteria were absent. We also observed that these concentrates displayed up to 85% exposed pyrrhotite. These observations led us to explore the capacity of Acidithiobacillus thiooxidans to remove pyrrhotite-sulfur impurities from iron concentrates. Employing stirred tank bioreactors operating at 30°C and inoculated with 5·106 (At. thiooxidans cells mL-1), we achieved 45.6% sulfur removal over 16 days. Then, we evaluated packed leaching columns operated at 30°C, where the At. thiooxidans enriched system reached 43.5% desulfurization over 60 days. Remarkably, sulfur removal increased to 80% within 21 days under potassium limitation. We then compared the At. thiooxidans-mediated desulfurization process, with and without air supply, under potassium limitation, varying the initial biomass concentration in 1-m columns. Aerated systems facilitated approximately 70% sulfur removal across the entire column with minimal iron loss. In contrast, non-aerated leaching columns achieved desulfurization levels of only 6% and 26% in the lower and middle sections of the column, respectively. Collectively, we have developed an efficient, scalable biological sulfur-removal technology for processing complex iron ores, aligning with the burgeoning demand for sustainable practices in the mining industry.

Opportunities and obstacles in microbial synthesis of metal nanoparticles.

Metallic nanoparticles (MeNPs) are widely used in many areas such as biomedicine, packaging, cosmetics, colourants, agriculture, antimicrobial agents, cleaning products, as components of electronic devices and nutritional supplements. In addition, some MeNPs exhibit quantum properties, making them suitable materials in the photonics, electronic and energy industries. Through the lens of technology, microbes can be considered nanofactories capable of producing enzymes, metabolites and capping materials involved in the synthesis, assembly and stabilization of MeNPs. This bioprocess is considered more ecofriendly and less energy intensive than the current chemical synthesis routes. However, microbial synthesis of MeNPs as an alternative method to the chemical synthesis of nanomaterials still faces some challenges that need to be solved. Some of these challenges are described in this Editorial.

Continuous bioreactors enable high-level bioremediation of diesel-contaminated seawater at low and mesophilic temperatures using Antarctic bacterial consortia: Pollutant analysis and microbial community composition.

In 2020, more than 21,000 tons of diesel oil were released accidently into the environment with most of it contaminating water bodies. There is an urgent need for sustainable technologies to clean up rivers and oceans to protect wildlife and human health. One solution is harnessing the power of bacterial consortia; however isolated microbes from different environments have shown low diesel bioremediation rates in seawater thus far. An outstanding question is whether Antarctic microorganisms that thrive in environments polluted with hydrocarbons exhibit better diesel degrading activities when propagated at higher temperatures than those encountered in their natural ecosystems. Here, we isolated bacterial consortia, LR-30 (30 °C) and LR-10 (10 °C), from the Antarctic rhizosphere soil of Deschampsia antarctica (Livingston Island), that used diesel oil as the only carbon substrate. We found that LR-30 and LR-10 batch bioreactors metabolized nearly the entire diesel content when the initial concentration was 10 (g/L) in seawater. Increasing the initial diesel concentration to 50 gDiesel/L, LR-30 and LR-10 bioconverted 33.4 and 31.2 gDiesel/L in 7 days, respectively. The 16S rRNA gene sequencing profiles revealed that the dominant bacterial genera of the inoculated LR-30 community were Achromobacter (50.6%), Pseudomonas (25%) and Rhodanobacter (14.9%), whereas for LR-10 were Pseudomonas (58%), Candidimonas (10.3%) and Renibacterium (7.8%). We also established continuous bioreactors for diesel biodegradation where LR-30 bioremediated diesel at an unprecedent rate of (34.4 g/L per day), while LR-10 achieved (24.5 g/L per day) at 10 °C for one month. The abundance of each bacterial genera present significantly fluctuated at some point during the diesel bioremediation process, yet Achromobacter and Pseudomonas were the most abundant member at the end of the batch and continuous bioreactors for LR-30 and LR-10, respectively.

Rational engineering of natural polyhydroxyalkanoates producing microorganisms for improved synthesis and recovery.

Microbial production of biopolymers derived from renewable substrates and waste streams reduces our heavy reliance on petrochemical plastics. One of the most important biodegradable polymers is the family of polyhydroxyalkanoates (PHAs), naturally occurring intracellular polyoxoesters produced for decades by bacterial fermentation of sugars and fatty acids at the industrial scale. Despite the advances, PHA production still suffers from heavy costs associated with carbon substrates and downstream processing to recover the intracellular product, thus restricting market positioning. In recent years, model-aided metabolic engineering and novel synthetic biology approaches have spurred our understanding of carbon flux partitioning through competing pathways and cellular resource allocation during PHA synthesis, enabling the rational design of superior biopolymer producers and programmable cellular lytic systems. This review describes these attempts to rationally engineering the cellular operation of several microbes to elevate PHA production on specific substrates and waste products. We also delve into genome reduction, morphology, and redox cofactor engineering to boost PHA biosynthesis. Besides, we critically evaluate engineered bacterial strains in various fermentation modes in terms of PHA productivity and the period required for product recovery.

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

Biofilm Formation Is Crucial for Efficient Copper Bioleaching From Bornite Under Mesophilic Conditions: Unveiling the Lifestyle and Catalytic Role of Sulfur-Oxidizing Bacteria.

Biofilm formation within the process of bioleaching of copper sulfides is a relevant aspect of iron- and sulfur-oxidizing acidophilic microorganisms as it represents their lifestyle in the actual heap/dump mining industry. Here, we used biofilm flow cell chambers to establish laminar regimes and compare them with turbulent conditions to evaluate biofilm formation and mineralogic dynamics through QEMSCAN and SEM-EDS during bioleaching of primary copper sulfide minerals at 30°C. We found that laminar regimes triggered the buildup of biofilm using Leptospirillum spp. and Acidithiobacillus thiooxidans (inoculation ratio 3:1) at a cell concentration of 106 cells/g mineral on bornite (Cu5FeS4) but not for chalcopyrite (CuFeS2). Conversely, biofilm did not occur on any of the tested minerals under turbulent conditions. Inoculating the bacterial community with ferric iron (Fe3+) under shaking conditions resulted in rapid copper recovery from bornite, leaching 40% of the Cu content after 10 days of cultivation. The addition of ferrous iron (Fe2+) instead promoted Cu recovery of 30% at day 48, clearly delaying the leaching process. More efficiently, the biofilm-forming laminar regime almost doubled the leached copper amount (54%) after 32 days. In-depth inspection of the microbiologic dynamics showed that bacteria developing biofilm on the surface of bornite corresponded mainly to At. Thiooxidans, while Leptospirillum spp. were detected in planktonic form, highlighting the role of biofilm buildup as a means for the bioleaching of primary sulfides. We finally propose a mechanism for bornite bioleaching during biofilm formation where sulfur regeneration to sulfuric acid by the sulfur-oxidizing microorganisms is crucial to prevent iron precipitation for efficient copper recovery.

Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering.

Seaweeds emerge as promising third-generation renewable for sustainable bioproduction. In the present work, we valorized brown seaweed to produce l-lysine, the world's leading feed amino acid, using Corynebacterium glutamicum, which was streamlined by systems metabolic engineering. The mutant C. glutamicum SEA-1 served as a starting point for development because it produced small amounts of l-lysine from mannitol, a major seaweed sugar, because of the deletion of its arabitol repressor AtlR and its engineered l-lysine pathway. Starting from SEA-1, we systematically optimized the microbe to redirect excess NADH, formed on the sugar alcohol, towards NADPH, required for l-lysine synthesis. The mannitol dehydrogenase variant MtlD D75A, inspired by 3D protein homology modelling, partly generated NADPH during the oxidation of mannitol to fructose, leading to a 70% increased l-lysine yield in strain SEA-2C. Several rounds of strain engineering further increased NADPH supply and l-lysine production. The best strain, SEA-7, overexpressed the membrane-bound transhydrogenase pntAB together with codon-optimized gapN, encoding NADPH-dependent glyceraldehyde 3-phosphate dehydrogenase, and mak, encoding fructokinase. In a fed-batch process, SEA-7 produced 76 g L-1l-lysine from mannitol at a yield of 0.26 mol mol-1 and a maximum productivity of 2.1 g L-1 h-1. Finally, SEA-7 was integrated into seaweed valorization cascades. Aqua-cultured Laminaria digitata, a major seaweed for commercial alginate, was extracted and hydrolyzed enzymatically, followed by recovery and clean-up of pure alginate gum. The residual sugar-based mixture was converted to l-lysine at a yield of 0.27 C-mol C-mol-1 using SEA-7. Second, stems of the wild-harvested seaweed Durvillaea antarctica, obtained as waste during commercial processing of the blades for human consumption, were extracted using acid treatment. Fermentation of the hydrolysate using SEA-7 provided l-lysine at a yield of 0.40 C-mol C-mol-1. Our findings enable improvement of the efficiency of seaweed biorefineries using tailor-made C. glutamicum strains.

Fed-Batch mcl- Polyhydroxyalkanoates Production in Pseudomonas putida KT2440 and ΔphaZ Mutant on Biodiesel-Derived Crude Glycerol.

Crude glycerol has emerged as a suitable feedstock for the biotechnological production of various industrial chemicals given its high surplus catalyzed by the biodiesel industry. Pseudomonas bacteria metabolize the polyol into several biopolymers, including alginate and medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs). Although P. putida is a suited platform to derive these polyoxoesters from crude glycerol, the attained concentrations in batch and fed-batch cultures are still low. In this study, we employed P. putida KT2440 and the hyper-PHA producer ΔphaZ mutant in two different fed-batch modes to synthesize mcl-PHAs from raw glycerol. Initially, the cells grew in a batch phase (µ max 0.21 h-1) for 22 h followed by a carbon-limiting exponential feeding, where the specific growth rate was set at 0.1 (h-1), resulting in a cell dry weight (CDW) of nearly 50 (g L-1) at 40 h cultivation. During the PHA production stage, we supplied the substrate at a constant rate of 50 (g h-1), where the KT2440 and the ΔphaZ produced 9.7 and 12.7 gPHA L-1, respectively, after 60 h cultivation. We next evaluated the PHA production ability of the P. putida strains using a DO-stat approach under nitrogen depletion. Citric acid was the main by-product secreted by the cells, accumulating in the culture broth up to 48 (g L-1) under nitrogen limitation. The mutant ΔphaZ amassed 38.9% of the CDW as mcl-PHA and exhibited a specific PHA volumetric productivity of 0.34 (g L-1 h-1), 48% higher than the parental KT2440 under the same growth conditions. The biosynthesized mcl-PHAs had average molecular weights ranging from 460 to 505 KDa and a polydispersity index (PDI) of 2.4-2.6. Here, we demonstrated that the DO-stat feeding approach in high cell density cultures enables the high yield production of mcl-PHA in P. putida strains using the industrial crude glycerol, where the fed-batch process selection is essential to exploit the superior biopolymer production hallmarks of engineered bacterial strains.

Photochemistry of P,N-bidentate rhenium(i) tricarbonyl complexes: reactive species generation and potential application for antibacterial photodynamic therapy.

In this work, we describe the photoisomerization of facial rhenium(i) tricarbonyl complexes bearing P,N-bidentate pyridyl/phosphine ligands with different chelating rings and anions: RePNBr, RePNTfO, and RePNNBr, which are triggered under irradiation at 365 nm in solutions. The apparent photodegradation rate constants (k app) depend on the coordinating ability of the solvent, being lowest in acetonitrile. The k app value increases as the temperature rises, suggesting a reactive IL excited state thermally populated from the MLCT excited state involved. Using the Eyring equation, positive activation enthalpies (ΔH ≠) accompanied by high negative values for the activation entropy (ΔS ≠) were obtained. These results suggest whatever the P,N-ligand or anion, the reaction proceeds through a strongly solvated or a compact transition state, which is compatible with an associative mechanism for the photoisomerization. A 100-fold decrease in the log10 CFU value is observed for E. coli and S. aureus in irradiated solutions of the compounds, which follows the same tendency as their singlet oxygen generation quantum yield: RePNBr > RePNTfO > RePNNBr, while no antibacterial activity is observed in the darkness. This result indicates that the generation of singlet oxygen plays a key role in the antibacterial capacity of these complexes.

Channelling carbon flux through the meta-cleavage route for improved poly(3-hydroxyalkanoate) production from benzoate and lignin-based aromatics in Pseudomonas putida H.

Lignin-based aromatics are attractive raw materials to derive medium-chain length poly(3-hydroxyalkanoates) (mcl-PHAs), biodegradable polymers of commercial value. So far, this conversion has exclusively used the ortho-cleavage route of Pseudomonas putida KT2440, which results in the secretion of toxic intermediates and limited performance. Pseudomonas putida H exhibits the ortho- and the meta-cleavage pathways where the latter appears promising because it stoichiometrically yields higher levels of acetyl-CoA. Here, we created a double-mutant H-ΔcatAΔA2 that utilizes the meta route exclusively and synthesized 30% more PHA on benzoate than the parental strain but suffered from catechol accumulation. The single deletion of the catA2 gene in the H strain provoked a slight attenuation on the enzymatic capacity of the ortho route (25%) and activation of the meta route by nearly 8-fold, producing twice as much mcl-PHAs compared to the wild type. Inline, the mutant H-ΔcatA2 showed a 2-fold increase in the intracellular malonyl-CoA abundance - the main precursor for mcl-PHAs synthesis. As inferred from flux simulation and enzyme activity assays, the superior performance of H-ΔcatA2 benefited from reduced flux through the TCA cycle and malic enzyme and diminished by-product formation. In a benzoate-based fed-batch, P. putida H-ΔcatA2 achieved a PHA titre of 6.1 g l-1 and a volumetric productivity of 1.8 g l-1 day-1 . Using Kraft lignin hydrolysate as feedstock, the engineered strain formed 1.4 g l- 1 PHA. The balancing of carbon flux between the parallel catechol-degrading routes emerges as an important strategy to prevent intermediate accumulation and elevate mcl-PHA production in P. putida H and, as shown here, sets the next level to derive this sustainable biopolymer from lignin hydrolysates and aromatics.

Expanding the Reach of Recombineering to Environmental Bacteria.

Broadening the application of recombineering technologies in biotechnologically important bacteria poses significant challenges. Aparicio et al. present a vital breakthrough for efficient single-stranded recombineering by utilizing a thermoinducible system in Pseudomonas putida. This offers a simple genome-editing tool towards creating superior biocatalysts for the synthesis of chemicals and for bioremediation endeavors.

Metabolic Rearrangements Causing Elevated Proline and Polyhydroxybutyrate Accumulation During the Osmotic Adaptation Response of Bacillus megaterium.

For many years now, Bacillus megaterium serves as a microbial workhorse for the high-level production of recombinant proteins in the g/L-scale. However, efficient and stable production processes require the knowledge of the molecular adaptation strategies of the host organism to establish optimal environmental conditions. Here, we interrogated the osmotic stress response of B. megaterium using transcriptome, proteome, metabolome, and fluxome analyses. An initial transient adaptation consisted of potassium import and glutamate counterion synthesis. The massive synthesis of the compatible solute proline constituted the second longterm adaptation process. Several stress response enzymes involved in iron scavenging and reactive oxygen species (ROS) fighting proteins showed higher levels under prolonged osmotic stress induced by 1.8 M NaCl. At the same time, the downregulation of the expression of genes of the upper part of glycolysis resulted in the activation of the pentose phosphate pathway (PPP), generating an oversupply of NADPH. The increased production of lactate accompanied by the reduction of acetate secretion partially compensate for the unbalanced (NADH/NAD+) ratio. Besides, the tricarboxylic acid cycle (TCA) mainly supplies the produced NADH, as indicated by the higher mRNA and protein levels of involved enzymes, and further confirmed by 13C flux analyses. As a consequence of the metabolic flux toward acetyl-CoA and the generation of an excess of NADPH, B. megaterium redirected the produced acetyl-CoA toward the polyhydroxybutyrate (PHB) biosynthetic pathway accumulating around 30% of the cell dry weight (CDW) as PHB. This direct relation between osmotic stress and intracellular PHB content has been evidenced for the first time, thus opening new avenues for synthesizing this valuable biopolymer using varying salt concentrations under non-limiting nutrient conditions.

Cascaded valorization of seaweed using microbial cell factories.

Sustainable production from seaweed has grown into an area of intense research and development. Meanwhile, more than 30 million tonnes of seaweed are produced, of which 70% are used as food and 30% have other applications such as feed, fertilizer, chemicals, and energy. Towards biorefining seaweed in an environmentally friendly and economically viable manner, we need efficient approaches that convert its biomass and residuals into added value products. Smart cell factories and fermentation strategies which can be integrated into future seaweed biorefineries are at the heart of the development and therefore receive increasing attention. Here, we review advances in the field including novel fermentation routes from seaweed to chemicals, materials, pharmaceuticals, fuels and energy, and discuss challenges and opportunities.

Engineering the Osmotic State of Pseudomonas putida KT2440 for Efficient Cell Disruption and Downstream Processing of Poly(3-Hydroxyalkanoates).

In the last decade, the development of novel programmable cell lytic systems based on different inducible genetic constructs like the holin-endolysin and lysozyme appears as a promising alternative to circumvent the use of costly enzymes and mechanical disrupters for downstream processing of intracellular microbial products. Despite the advances, upon activation of these systems the cellular disruption of the biocatalyst occurs in an extended period, thus delaying the recovery of poly(3-hydroxyalkanoate) (PHA). Herein the osmotic state of Pseudomonas putida KT2440 was engineered by inactivating the inner-membrane residing rescue valve MscL, which is responsible mainly for circumventing low-osmolarity challenges. Then the major outer membrane porin OprF and the specific porin OprE were overproduced during PHA producing conditions on decanoate-grown cells. The engineered P. putida strains carrying each porin showed no impairment on growth rate and final biomass and PHA yield after 48 h cultivation. Expression of both porins in tandem in the mutant strain KTΔmscL-oprFE led to a slight reduction of the biomass synthesis (∼10%) but higher PHA accumulation (%wt) relative to the cell dry mass. Each strain was then challenged to an osmotic upshift for 1 h and subsequently to a rapid passage to a hypotonic condition where the membrane stability of the KTΔmscL-oprFE suffered damage, resulting in a rapid reduction of cell viability. Cell disruption accounted for >95% of the cell population within 3 h as reported by colony forming units (CFU), FACS analyses, and transmission electron microscopy. PHA recovery yielded 94.2% of the biosynthesized biopolymer displaying no significant alterations on the final monomer composition. This study can serve as an efficient genetic platform for the recovery of any microbial intracellular compound allowing less unit operation steps for cellular disruption.

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid.

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2 -eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.

Biochemistry, genetics and biotechnology of glycerol utilization in Pseudomonas species.

The use of renewable waste feedstocks is an environment-friendly choice contributing to the reduction of waste treatment costs and increasing the economic value of industrial by-products. Glycerol (1,2,3-propanetriol), a simple polyol compound widely distributed in biological systems, constitutes a prime example of a relatively cheap and readily available substrate to be used in bioprocesses. Extensively exploited as an ingredient in the food and pharmaceutical industries, glycerol is also the main by-product of biodiesel production, which has resulted in a progressive drop in substrate price over the years. Consequently, glycerol has become an attractive substrate in biotechnology, and several chemical commodities currently produced from petroleum have been shown to be obtained from this polyol using whole-cell biocatalysts with both wild-type and engineered bacterial strains. Pseudomonas species, endowed with a versatile and rich metabolism, have been adopted for the conversion of glycerol into value-added products (ranging from simple molecules to structurally complex biopolymers, e.g. polyhydroxyalkanoates), and a number of metabolic engineering strategies have been deployed to increase the number of applications of glycerol as a cost-effective substrate. The unique genetic and metabolic features of glycerol-grown Pseudomonas are presented in this review, along with relevant examples of bioprocesses based on this substrate - and the synthetic biology and metabolic engineering strategies implemented in bacteria of this genus aimed at glycerol valorization.

The Transcription Factor ArcA Modulates Salmonella's Metabolism in Response to Neutrophil Hypochlorous Acid-Mediated Stress.

Salmonella Typhimurium, a bacterial pathogen with high metabolic plasticity, can adapt to different environmental conditions; these traits enhance its virulence by enabling bacterial survival. Neutrophils play important roles in the innate immune response, including the production of microbicidal reactive oxygen species (ROS). In addition, the myeloperoxidase in neutrophils catalyzes the formation of hypochlorous acid (HOCl), a highly toxic molecule that reacts with essential biomolecules, causing oxidative damage including lipid peroxidation and protein carbonylation. The bacterial response regulator ArcA regulates adaptive responses to oxygen levels and influences the survival of Salmonella inside phagocytic cells. Here, we demonstrate by whole transcriptomic analyses that ArcA regulates genes related to various metabolic pathways, enabling bacterial survival during HOCl-stress in vitro. Also, inside neutrophils, ArcA controls the transcription of several metabolic pathways by downregulating the expression of genes related to fatty acid degradation, lysine degradation, and arginine, proline, pyruvate, and propanoate metabolism. ArcA also upregulates genes encoding components of the oxidative pathway. These results underscore the importance of ArcA in ATP generation inside the neutrophil phagosome and its participation in bacterial metabolic adaptations during HOCl stress.

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential.

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.