Phytohemagglutinin from Phaseolus vulgaris enhances the lung cancer cell chemotherapy sensitivity by changing cell membrane permeability.

Chemotherapy is still a prevalent strategy for clinical lung cancer treatment. However, the inevitable emerged drug resistance has become a great hurdle to therapeutic effect. Studies have demonstrated that the primary cause of drug resistance is a decrease in the chemotherapeutic medicine concentration. Several lectins have been confirmed to be effective as chemotherapy adjuvants, enhancing the anti-tumor effects of chemotherapy drugs. Here, we combined phytohemagglutinin (PHA), which has been reported possess anti-tumor effects, with chemotherapy drugs Cisplatin (DDP) and Adriamycin (ADM) on lung cancer cells to detect the sensitivities of PHA as a chemotherapy adjuvant. Our results demonstrated that the PHA significantly enhanced the sensitivity of lung cancer cells to DDP and ADM, and Western blot showed that PHA combined with DDP or ADM enhance cytotoxic effects by inhibiting autophagy and promoting apoptosis. More importantly, we found PHA enhanced the chemotherapeutic drugs cytotoxicity by changing the cell membrane to increase the intracellular chemotherapeutic drugs concentration. Besides, the combination of PHA and ADM increased the ADM concentration in the multidrug-resistant strain A549-R cells and achieved the drug sensitization effect. Our results suggest that PHA combined with chemotherapy can be applied in the treatment of lung cancer cells and lung cancer multidrug-resistant strains, and provide a novel strategy for clinical tumor chemotherapy and a new idea to solve the problem of drug resistance in clinical lung cancer.

Polyhydroxybutyrate synthesis in Camelina: Towards coproduction of renewable feedstocks for bioplastics and fuels.

Plant-based co-production of polyhydroxyalkanoates (PHAs) and seed oil has the potential to create a viable domestic source of feedstocks for renewable fuels and plastics. PHAs, a class of biodegradable polyesters, can replace conventional plastics in many applications while providing full degradation in all biologically active environments. Here we report the production of the PHA poly[(R)-3-hydroxybutyrate] (PHB) in the seed cytosol of the emerging bioenergy crop Camelina sativa engineered with a bacterial PHB biosynthetic pathway. Two approaches were used: cytosolic localization of all three enzymes of the PHB pathway in the seed, or localization of the first two enzymes of the pathway in the cytosol and anchoring of the third enzyme required for polymerization to the cytosolic face of the endoplasmic reticulum (ER). The ER-targeted approach was found to provide more stable polymer production with PHB levels up to 10.2% of the mature seed weight achieved in seeds with good viability. These results mark a significant step forward towards engineering lines for commercial use. Plant-based PHA production would enable a direct link between low-cost large-scale agricultural production of biodegradable polymers and seed oil with the global plastics and renewable fuels markets.

Valorization of agro-industrial wastes into polyhydroxyalkanoates-rich single-cell proteins to enable a circular waste-to-feed economy.

Recovering and converting carbon and nutrients from waste streams into healthy single-cell proteins (SCPs) can be an effective strategy to address costly waste management and support the increasing animal feed demand for the global food supply. Recently, SCPs rich in polyhydroxybutyrate (PHB) have been identified as an effective biocontrol healthy feed to replace conventional antibiotics-supplemented aquaculture feed. PHB, an intercellular polymer of short-chain-length (SCL) hydroxy-fatty acids, is a common type of polyhydroxyalkanoates (PHA) that can be microbially produced from various organics, including agro-industrial wastes. The complex chemical properties of agro-industrial wastes might produce SCPs containing PHA with SCL and/or medium chain-length (MCL) hydroxy-fatty acids. However, the effects of MCL-PHA-containing SCPs on aqua species' health and disease-fighting ability remains poorly understood. This study investigated the feasibility of producing various PHA-containing SCPs from renewable agro-industrial wastes/wastewaters, the effectiveness of SCL- and MCL-PHA as biocontrol agents, and the effects of these PHA-rich SCPs on the growth and disease resistance of an aquaculture animal model, brine shrimp Artemia. Zobellella denitrificans ZD1 and Pseudomonas oleovorans were able to grow on different pure substrates and agro-industrial wastes/wastewaters to produce various SCL- and/or MCL-PHA-rich SCPs. Low doses of MCL-fatty acids (i.e., PHA intermediates) efficiently suppressed the growth of aquaculture pathogens. Moreover, MCL-PHA-rich SCPs served as great food/energy sources for Artemia and improved Artemia's ability to fight pathogens. This study offers a win-win approach to address the challenges of wastes/wastewater management and feed supply faced by the aquaculture industry.

Exploitation of wasted bread as substrate for polyhydroxyalkanoates production through the use of Haloferax mediterranei and seawater.

The use of the halophile microorganism Haloferax mediterranei, able to synthesize poly(hydroxybutyrate-hydroxyvalerate) (PHBV), is considered as a promising tool for the industrial production of bioplastic through bioprocessing. A consistent supplementation of the growth substrate in carbohydrates and minerals is overall necessary to allow its PHBV production. In this work, wasted bread was used as substrate for bioplastic production by microbial fermentation. Instead of the consistent and expensive minerals supplement required for Hfx. mediterranei DSM1411 growth, microfiltered seawater was added to the wasted bread-derived substrate. The suitable ratio of wasted bread homogenate and seawater, corresponding to 40:60, was selected. The addition of proteases and amylase to the bread homogenate promoted the microbial growth but it did not correspond to the increase of bioplastic production by the microorganism, that reach, under the experimental conditions, 1.53 g/L. An extraction procedure of the PHBV from cells, based on repeated washing with water, followed or not by a purification through ethanol precipitation, was applied instead of the conventional extraction with chloroform. Yield of PHBV obtained using the different extraction methods were 21.6 ± 3.6 (standard extraction/purification procedure with CHCl3:H2O mixture), 24.8 ± 3.0 (water-based extraction), and 19.8 ± 3.3 mg PHAs/g of wasted bread (water-based extraction followed by ethanol purification). Slightly higher hydroxyvalerate content (12.95 vs 10.78%, w/w) was found in PHBV obtained through the water-based extraction compared to the conventional one, moreover, the former was characterized by purity of 100% (w/w). Results demonstrated the suitability of wasted bread, supplemented with seawater, to be used as substrate for bioplastic production through fermentation. Results moreover demonstrated that a solvent-free extraction, exclusively based on osmotic shock, could be used to recover the bioplastic from cells.

A randomized double blinded placebo controlled clinical trial for the evaluation of green coffee extract on immune health in healthy adults.

Background: The immune system functions to protect the host from a broad array of infectious diseases. Here, we evaluated the in vitro immunomodulatory effects of green coffee extract (GCE), and conducted a double-blinded, randomized and placebo-controlled trial among apparently healthy individuals. Methods: We determined the levels and functions of inflammatory and immune markers viz., phospho-NF-κB p65 ser536, chemotaxis, phagocytosis, TH1/TH2 cytokines and IgG production. We also evaluated several immunological markers such as total leukocyte counts, differential leukocyte counts, NK cell activity, CD4/CD8 ratio, serum immunoglobulin, C-reactive protein (CRP) and pro-inflammatory cytokines (IL-6 and TNF-α). Results and conclusion: GCE significantly inhibited LPS-induced NF-κB p65 ser536 phosphorylation, MCP-1-induced chemotaxis and significantly enhanced phagocytosis and IgG production. In addition, GCE modulated PMA/PHA-induced TH1/TH2 cytokine production. Clinical investigations suggested that the expression of CD56 and CD16 was markedly augmented on NK cells following GCE treatment. GCE significantly enhanced IgA production before and after influenza vaccination. Similarly, IL-6, TNF-α and CRP levels were significantly inhibited by GCE. Together, GCE confers several salubrious immunomodulatory effects at different levels attributing to optimal functioning of immune responses in the host. Taxonomy: Cell biology, Clinical study, Clinical Trial.

In silico identification of bacterial seaweed-degrading bioplastic producers.

There is an urgent need to replace petroleum-based plastic with bio-based and biodegradable alternatives. Polyhydroxyalkanoates (PHAs) are attractive prospective replacements that exhibit desirable mechanical properties and are recyclable and biodegradable in terrestrial and marine environments. However, the production costs today still limit the economic sustainability of the PHA industry. Seaweed cultivation represents an opportunity for carbon capture, while also supplying a sustainable photosynthetic feedstock for PHA production. We mined existing gene and protein databases to identify bacteria able to grow and produce PHAs using seaweed-derived carbohydrates as substrates. There were no significant relationships between the genes involved in the deconstruction of algae polysaccharides and PHA production, with poor to negative correlations and diffused clustering suggesting evolutionary compartmentalism. We identified 2 987 bacterial candidates spanning 40 taxonomic families predominantly within Alphaproteobacteria, Gammaproteobacteria and Burkholderiales with enriched seaweed-degrading capacity that also harbour PHA synthesis potential. These included highly promising candidates with specialist and generalist specificities, including Alteromonas, Aquisphaera, Azotobacter, Bacillus, Caulobacter, Cellvibrionaceae, Duganella, Janthinobacterium, Massilia, Oxalobacteraceae, Parvularcula, Pirellulaceae, Pseudomonas, Rhizobacter, Rhodanobacter, Simiduia, Sphingobium, Sphingomonadaceae, Sphingomonas, Stieleria, Vibrio and Xanthomonas. In this enriched subset, the family-level densities of genes targeting green macroalgae polysaccharides were considerably higher (n=231.6±68.5) than enzymes targeting brown (n=65.34±13.12) and red (n=30.5±10.72) polysaccharides. Within these organisms, an abundance of FabG genes was observed, suggesting that the fatty acid de novo synthesis pathway supplies (R)-3-hydroxyacyl-CoA or 3-hydroxybutyryl-CoA from core metabolic processes and is the predominant mechanism of PHA production in these organisms. Our results facilitate extending seaweed biomass valorization in the context of consolidated biorefining for the production of bioplastics.

Sustainable applications of polyhydroxyalkanoates in various fields: A critical review.

PHA is one of the most promising candidates in bio-polymer family which is biodegradable and environment-friendly in nature. In recent years, it has been applied as a biodegradable alternative for petroleum-based plastic across different domains. In literature, several research groups have scrutinised the biocompatibility and biodegradability of PHA in both in vivo settings as well as in in vitro conditions. Microbial yield polyhydroxyalkanoates (PHAs) are promoted at present as biodegradable plastics. On the other hand, only a limited number of products is being commercially manufactured out of PHAs (e.g., bottles). A succession of microbes (prokaryotes in addition to eukaryotes) has been identified as potential candidates that can disintegrate PHAs. These materials have been successfully employed in packaging industry, medical devices and implants, moulded goods, paper coatings, adhesives, performance additives, mulch films, non-woven fabrics, etc. The present paper reviews and focuses on the potential applications of PHA and its derivatives in different industries.

Conversion of Short and Medium Chain Fatty Acids into Novel Polyhydroxyalkanoates Copolymers by Aeromonas sp. AC_01.

Polyhydroxyalkanoates (PHAs) production by Aeromonas sp. AC_01 was investigated using synthetic and waste derived short and medium chain fatty acids (SMCFAs). The obtained results revealed that the analyzed bacterial strain was able to grow and synthesize PHAs using SMCFAs. The highest PHA productivity was observed in the cultivation supplemented with a mixture of acetic acid and butyric acid (3.89 mg/L·h). Furthermore, SMCFAs-rich stream, derived from acidogenic mixed culture fermentation of acid whey, was found to be less beneficial for PHA productivity than its synthetic mixture, however the PHA production was favored by the nitrogen-limited condition. Importantly, Aeromonas sp. AC_01 was capable of synthesizing novel scl-mcl copolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxytridecanoate (3HtriD) and/or 3-hydroxytetradecaonate (3HTD) with high 3HB and 3HV fractions. They were identified with alterable monomers composition depending on the culture conditions used. Moreover, in-depth thermal analyses proved that they are highly resistant to thermal degradation regardless of their monomeric composition. The obtained results confirm that Aeromonas sp. AC_01 is a promising candidate for the biotechnological production of PHAs from SMCFAs with thermal properties that can be tuned together with their chemical composition by the corresponding adjustment of the cultivation process.

Advanced application of stimuli-responsive drug delivery system for inflammatory arthritis treatment.

Inflammatory arthritis is a major cause of disability in the elderly. This condition causes joint pain, loss of function, and deterioration of quality of life, mainly due to osteoarthritis (OA) and rheumatoid arthritis (RA). Currently, available treatment options for inflammatory arthritis include anti-inflammatory medications administered via oral, topical, or intra-articular routes, surgery, and physical rehabilitation. Novel alternative approaches to managing inflammatory arthritis, so far, remain the grand challenge owing to catastrophic financial burden and insignificant therapeutic benefit. In the view of non-targeted systemic cytotoxicity and limited bioavailability of drug therapies, a major concern is to establish stimuli-responsive drug delivery systems using nanomaterials with on-off switching potential for biomedical applications. This review summarizes the advanced applications of triggerable nanomaterials dependent on various internal stimuli (including reduction-oxidation (redox), pH, and enzymes) and external stimuli (including temperature, ultrasound (US), magnetic, photo, voltage, and mechanical friction). The review also explores the progress and challenges with the use of stimuli-responsive nanomaterials to manage inflammatory arthritis based on pathological changes, including cartilage degeneration, synovitis, and subchondral bone destruction. Exposure to appropriate stimuli induced by such histopathological alterations can trigger the release of therapeutic medications, imperative in the joint-targeted treatment of inflammatory arthritis.

Phosphorus recovery in the alternating aerobic/anaerobic biofilm system: Performance and mechanism.

To balance the high phosphorus concentration in recirculated solution and the stability of biofilm system, this study explored the performance and mechanism of phosphorus uptake/release for recovering phosphorus from sewage when the phosphorus content in biofilm (Pbiofilm) changed. The results showed that the maximum phosphorus concentration in the concentrated solution reached 171.2 ± 2.5 mg·L-1 in harvest 1st-5th stages. Polyphosphate accumulating organisms (PAOs) performed a metabolic shift from glycogen accumulation metabolism (GAM) to polyphosphate accumulation metabolism (PAM) when Pbiofilm increased at each phosphorus enrichment stage, and more phosphorus was absorbed/released by PAOs. Nevertheless, the release of poly-phosphate from PAOs was inhibited after phosphorus concentration stabilized, and PAOs were unable to absorb phosphorus from wastewater as it reached the phosphorus saturation stage. To maintain the stability of the system, phosphorus had to be harvested so that the saturated phosphorus in PAOs was easily released in a new recirculated solution, resulting in adequate storage space for PAOs to absorb phosphorus. Meanwhile, the 31P NMR analysis demonstrated that phosphorus was stored in EPS and cell of PAOs, whereas EPS played a significant role than cell at the anaerobic phase. Particularly, ortho-phosphate was the major component of phosphorus release by EPS and poly-phosphate was the major part of phosphorus release by cell. Furthermore, the change of Pbiofilm had no impact on biofilm characteristics and microbial communities, whereas some PAOs would be enriched, and others that were not suitable for this process would be inhibited with repeated cycles of alternating aerobic/anaerobic operation.

PHA-680626 Is an Effective Inhibitor of the Interaction between Aurora-A and N-Myc.

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

Properties of Degradable Polyhydroxyalkanoates (PHAs) Synthesized by a New Strain, Cupriavidus necator IBP/SFU-1, from Various Carbon Sources.

The bacterial strain isolated from soil was identified as Cupriavidus necator IBP/SFU-1 and investigated as a PHA producer. The strain was found to be able to grow and synthesize PHAs under autotrophic conditions and showed a broad organotrophic potential towards different carbon sources: sugars, glycerol, fatty acids, and plant oils. The highest cell concentrations (7-8 g/L) and PHA contents were produced from oleic acid (78%), fructose, glucose, and palm oil (over 80%). The type of the carbon source influenced the PHA chemical composition and properties: when grown on oleic acid, the strain synthesized the P(3HB-co-3HV) copolymer; on plant oils, the P(3HB-co-3HV-co-3HHx) terpolymer, and on the other substrates, the P(3HB) homopolymer. The type of the carbon source influenced molecular-weight properties of PHAs: P(3HB) synthesized under autotrophic growth conditions, from CO2, had the highest number-average (290 ± 15 kDa) and weight-average (850 ± 25 kDa) molecular weights and the lowest polydispersity (2.9 ± 0.2); polymers synthesized from organic carbon sources showed increased polydispersity and reduced molecular weight. The carbon source was not found to affect the degree of crystallinity and thermal properties of the PHAs. The type of the carbon source determined not only PHA composition and molecular weight but also surface microstructure and porosity of the polymer films. The new strain can be recommended as a promising P(3HB) producer from palm oil, oleic acid, and sugars (fructose and glucose) and as a producer of P(3HB-co-3HV) from oleic acid and P(3HB-co-3HV-co-3HHx) from palm oil.

Ameliorating effect of nitrate on nitrite inhibition for denitrifying P-accumulating organisms.

Lowered air supply and organic carbon need are the key factors to reduce wastewater treatment costs and thereby, avoid eutrophication. Denitrifying PO43-- removal (DPR) process using nitrate instead of oxygen for PO43- uptake was started up in the sequencing batch reactor (SBR) at a nitrate dosing rate of 20-25 mg N L-1 d-1. Operation with a real municipal wastewater supplied with CH3COONa, K2HPO4 and KNO3 succeeded in the cultivation of biomass containing denitrifying polyphosphate accumulating organisms (DPAOs). The durations of SBR process anaerobic/anoxic/oxic cycles were 1.5 h, 3.5 h and 1 h, respectively. SBR operation resulted in a maximum PO43--P uptake of 17 mg PO43--P g-1 MLSS. The highest TN and PO43- removal efficiencies were observed during the first half of reactor operation at 77 (±10) % and 71 (±5) %, respectively. An average COD removal rate of 172 (±98) mg g-1 MLSS and a high average removal efficiency of 89 (±4) % were achieved. Nitrite effect with/without nitrate as DPR electron acceptor was investigated in batch-scale to show possibilities to use high nitrite and nitrate contents simultaneously as electron acceptors for the anoxic phosphate uptake. Nitrate attenuation against nitrite toxicity can be economically justified in full-scale treatment applications in which wastewater has a high nitrogen content. Nitrate attenuated nitrite toxicity (caused by nitrite content at 5-100 mg NO2--N L-1) when using supplemental additions of nitrate (at concentrations of 45-200 mg NO3--N L-1) in batch tests. Illumina sequencing emphasized that during biomass adaption microbial community changed by lowered aerobic cycle length and by lowered nitrate dosing towards representation of key DPAO/PAO- organisms, such as Candidatus Accumulibacter, Xanthomonadaceae, Comomonadaceae, Saprospiraceae and Rhodocyclaceae. This study showed that DPAO biomass adaption to nitrate maintained an efficient COD, nitrogen and phosphorus removal and the biomass can be applied for treatment of wastewater containing high nitrite and nitrate content.

The influence of medium composition on the microbial secretory production of hydroxyalkanoate oligomers.

With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) in Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.

Uremic Toxins in the Progression of Chronic Kidney Disease and Cardiovascular Disease: Mechanisms and Therapeutic Targets.

Chronic kidney disease (CKD) is a progressive loss of renal function. The gradual decline in kidney function leads to an accumulation of toxins normally cleared by the kidneys, resulting in uremia. Uremic toxins are classified into three categories: free water-soluble low-molecular-weight solutes, protein-bound solutes, and middle molecules. CKD patients have increased risk of developing cardiovascular disease (CVD), due to an assortment of CKD-specific risk factors. The accumulation of uremic toxins in the circulation and in tissues is associated with the progression of CKD and its co-morbidities, including CVD. Although numerous uremic toxins have been identified to date and many of them are believed to play a role in the progression of CKD and CVD, very few toxins have been extensively studied. The pathophysiological mechanisms of uremic toxins must be investigated further for a better understanding of their roles in disease progression and to develop therapeutic interventions against uremic toxicity. This review discusses the renal and cardiovascular toxicity of uremic toxins indoxyl sulfate, p-cresyl sulfate, hippuric acid, TMAO, ADMA, TNF-α, and IL-6. A focus is also placed on potential therapeutic targets against uremic toxicity.

Resource recovery in aerobic granular sludge systems: is it feasible or still a long way to go?

Lately, wastewater treatment plants are much often being designed as wastewater-resource factories inserted in circular cities. Among biological treatment technologies, aerobic granular sludge (AGS), considered an evolution of activated sludge (AS), has received great attention regarding its resource recovery potential. This review presents the state-of-the-art concerning the influence of operational parameters on the recovery of alginate-like exopolysaccharides (ALE), tryptophan, phosphorus, and polyhydroxyalkanoates (PHA) from AGS systems. The carbon to nitrogen ratio was identified as a parameter that plays an important role for the optimal production of ALE, tryptophan, and PHA. The sludge retention time effect is more pronounced for the production of ALE and tryptophan. Additionally, salinity levels in the bioreactors can potentially be manipulated to increase ALE and phosphorus yields simultaneously. Some existing knowledge gaps in the scientific literature concerning the recovery of these resources from AGS were also identified. Regarding industrial applications, tryptophan has the longest way to go. On the other hand, ALE production/recovery could be considered the most mature process if we take into account that existing alternatives for phosphorus and PHA production/recovery are optimized for activated sludge rather than granular sludge. Consequently, to maintain the same effectiveness, these processes likely could not be applied to AGS without undergoing some modification. Therefore, investigating to what extent these adaptations are necessary and designing alternatives is essential.

Seemingly trivial secondary factors may determine microbial competition: a cautionary tale on the impact of iron supplementation through corrosion.

Microbial community engineering aims for enrichment of a specific microbial trait by imposing specific cultivation conditions. This work demonstrates that things may be more complicated than typically presumed and that microbial competition can be affected by seemingly insignificant variables, like in this case the type of acid used for pH control. Aerobic bioreactors pulse fed with acetate operated with hydrochloric acid resulted in the enrichment of Plasticicumulans acidivorans, and changing the pH controlling agent to sulfuric acid shifted the community towards Zoogloea sp. Further research demonstrated that the change in community structure was not directly caused by the change in acid used for pH control, but resulted from the difference in corrosive strength of both acids and the related iron leaching from the bioreactor piping. Neither system was iron deficient, suggesting that the biological availability of iron is affected by the leaching process. Our results demonstrate that microbial competition and process development can be affected dramatically by secondary factors related to nutrient supply and bioavailability, and is way more complex than generally assumed in a single carbon substrate limited process.

Production and Properties of Microbial Polyhydroxyalkanoates Synthesized from Hydrolysates of Jerusalem Artichoke Tubers and Vegetative Biomass.

One of the major challenges in PHA biotechnology is optimization of biotechnological processes of the entire synthesis, mainly by using new inexpensive carbon substrates. A promising substrate for PHA synthesis may be the sugars extracted from the Jerusalem artichoke. In the present study, hydrolysates of Jerusalem artichoke (JA) tubers and vegetative biomass were produced and used as carbon substrate for PHA synthesis. The hydrolysis procedure (the combination of aqueous extraction and acid hydrolysis, process temperature and duration) influenced the content of reducing substances (RS), monosaccharide contents, and the fructose/glucose ratio. All types of hydrolysates tested as substrates for cultivation of three strains-C. necator B-10646 and R. eutropha B 5786 and B 8562-were suitable for PHA synthesis, producing different biomass concentrations and polymer contents. The most productive process, conducted in 12-L fermenters, was achieved on hydrolysates of JA tubers (X = 66.9 g/L, 82% PHA) and vegetative biomass (55.1 g/L and 62% PHA) produced by aqueous extraction of sugars at 80 °C followed by acid hydrolysis at 60 °C, using the most productive strain, C. necator B-10646. The effects of JA hydrolysates on physicochemical properties of PHAs were studied for the first time. P(3HB) specimens synthesized from the JA hydrolysates, regardless of the source (tubers or vegetative biomass), hydrolysis conditions, and PHA producing strain employed, exhibited the 100-120 °C difference between the Tmelt and Tdegr, prevailing of the crystalline phase over the amorphous one (Cx between 69 and 75%), and variations in weight average molecular weight (409-480) kDa. Supplementation of the culture medium of C. necator B-10646 grown on JA hydrolysates with potassium valerate and ε-caprolactone resulted in the synthesis of P(3HB-co-3HV) and P(3HB-co-4HB) copolymers that had decreased degrees of crystallinity and molecular weights, which influenced the porosity and surface roughness of polymer films prepared from them. The study shows that JA hydrolysates used as carbon source enabled productive synthesis of PHAs, comparable to synthesis from pure sugars. The next step is to scale up PHA synthesis from JA hydrolysates and conduct the feasibility study. The present study contributes to the solution of the critical problem of PHA biotechnology-finding widely available and inexpensive substrates.

Biosynthesis of polyhydroxyalkanoates from vegetable oil under the co-expression of fadE and phaJ genes in Cupriavidus necator.

The acyl-CoA dehydrogenase (FadE) and (R)-specific enoyl-CoA hydratase (PhaJ) are functionally related to the degradation of fatty acids and the synthesis of polyhydroxyalkanoates (PHAs). To verify this, a recombinant Cupriavidus necator H16 harboring the plasmid -pMPJAS03- with fadE from Escherichia coli strain K12 and phaJ1 from Pseudomonas putida strain KT2440 under the arabinose promoter (araC-PBAD) was constructed. The impact of co-expressing fadE and phaJ genes on C. necator H16/pMPJAS03 maintaining the wild-type synthase on short-chain-length/medium-chain-length PHA formation from canola or avocado oil at different arabinose concentrations was investigated. The functional activity of fadEE.c led to obtaining higher biomass and PHA concentrations compared to the cultures without expressing the gene. While high transcriptional levels of phaJ1P.p, at 0.1% of arabinose, aid the wild-type synthase to polymerize larger-side chain monomers, such as 3-Hydroxyoctanoate (3HO) and 3-Hydroxydecanoate (3HD). The presence of even small amounts of 3HO and 3HD in the co-polymers significantly depresses the melting temperature of the polymers, compared to those composed of pure 3-hydroxybutyrate (3HB). Our data presents supporting evidence that the synthesis of larger-side chain monomers by the recombinant strain relies not only upon the affinity of the wild-type synthase but also on the functionality of the intermediate supplying enzymes.

The relevance of enzyme specificity for coenzymes and the presence of 6-phosphogluconate dehydrogenase for polyhydroxyalkanoates production in the metabolism of Pseudomonas sp. LFM046.

Reconstruction of genome-based metabolic model is a useful approach for the assessment of metabolic pathways, genes and proteins involved in the environmental fitness capabilities or pathogenic potential as well as for biotechnological processes development. Pseudomonas sp. LFM046 was selected as a good polyhydroxyalkanoates (PHA) producer from carbohydrates and plant oils. Its complete genome sequence and metabolic model were obtained. Analysis revealed that the gnd gene, encoding 6-phosphogluconate dehydrogenase, is absent in Pseudomonas sp. LFM046 genome. In order to improve the knowledge about LFM046 metabolism, the coenzyme specificities of different enzymes was evaluated. Furthermore, the heterologous expression of gnd genes from Pseudomonas putida KT2440 (NAD+ dependent) and Escherichia coli MG1655 (NADP+ dependent) in LFM046 was carried out and provoke a delay on cell growth and a reduction in PHA yield, respectively. The results indicate that the adjustment in cyclic Entner-Doudoroff pathway may be an interesting strategy for it and other bacteria to simultaneously meet divergent cell needs during cultivation phases of growth and PHA production.