Polyhydroxybutyrate

Effects of osmolytes on salt resistance of Halomonas socia CKY01 and identification of osmolytes-related genes by genome sequencing.[Pubmed:32653639]

J Biotechnol. 2020 Jul 9. pii: S0168-1656(20)30181-4.

Bacteria from the genus Halomonas hold promise in biotechnology as sources of salt-tolerant enzymes, biosurfactants, biopolymers, osmolytes, and as actors in bioremediation processes. In a previous work, we have identified Halomonas socia strain CKY01 having various hydrolase activities. Here, we aimed to study the survival strategies of marine bacteria. A deep genome sequencing study of H. socia CKY01 has revealed 4627 genes reaching 4,753,299 bp with 64% of GC content. This strain produced Polyhydroxybutyrate (PHB) having one gene clusters having phaC and phasin, and it has several genes responsible for the uptake, synthesis, and transport of the osmolytes such as betaine, choline, ectoine, carnitine, and proline in the bacterial genome. The addition of 60 mM glutamate, 60 mM proline and 60 mM ectoine enhanced growth 300.8%, 294.2% and 235.0%, respectively, under 10% saline conditions. In particular, ectoine and proline increased salt resistance and allowed cells to survive in more than 15% NaCl. By combining experimental and genome sequencing data, we have investigated the importance of osmolytes on the survival of this Halomonas strain.

Polyhydroxybutyrate bioresins with high thermal stability by crosslinking with resorcinol diglycidyl ether.[Pubmed:32633490]

Biomacromolecules. 2020 Jul 7.

The development of sustainable materials employing natural and non-toxic resources has been attracting much attention in the last years. In this work, we discuss in premiere the chemical combination between resorcinol diglycidyl ether (RDGE), an aromatic biobased thermosetting monomer, and Polyhydroxybutyrate (PHB), a bio-derived and biodegradable thermoplastic polyester. By this combination we searched to associate the high thermal stability of RDGE with a toughening effect by aliphatic chains of PHB. The investigations on mechanism of cross-linking reaction and on structural connectivity between the two components were realised by FTIR and NMR spectroscopies. We found that the epoxide polymerization catalysed by tertiary amines triggers the formation of crotonyl species by Polyhydroxybutyrate cleavage. Two-dimensional NMR experiments show that Polyhydroxybutyrate fragments covalently bind as side chains to the rigid aromatic network of the epoxide frame. The crosslinking between the two systems entails the formation of new ester and ether bonds. The obtained structures show a network homogeneity confirmed by a single Tg, from 85 to 47 degrees C, in function of formulation, and tan values from 87 to 53 degrees C. The combination between the two comonomers proved a positive effect. The PHB increased the toughness of RDGE based thermosets, improving the materials elasticity by increasing the chains length between the crosslinks. An important result of this study is the high thermal stability of RDGE/PHB bioresins, with the T5% varying between 330 and 310 degrees C in function of PHB ratio.

In situ based surface-enhanced Raman spectroscopy (SERS) for the fast and reproducible identification of PHB producers in cyanobacterial cultures.[Pubmed:32614341]

Analyst. 2020 Jul 2.

The production of Polyhydroxybutyrate (PHB) by autotrophic fermentation of cyanobacteria has received increasing interest in the light of carbon emission reducing process strategies. Biotechnological approaches are in development to optimize the yield of PHB, including adapted cultivation media, characterized by a limitation of key nutrients: cyanobacteria accumulate PHB as energy storage molecules under limited growth conditions. Since there is an increasing demand for fast, simple and reliable analytics, we report the establishment of surface enhanced Raman spectroscopy (SERS) as a suitable monitoring tool for up scaled PHB production processes. Both, pure Ag-colloids mixed with bacterial culture, and in situ prepared colloids (Ag-Synechocystis), generated on the cell surface directly, were successfully applied and evaluated for this purpose. SERS measurements with in situ prepared Ag-colloids improved the reproducibility of Raman signals from 54.8% to 93.9%. The measurement time could be reduced significantly, completing our secondary goal. The quality of classically and in situ prepared Ag-colloids was monitored by zeta potential measurements and scanning electron microscopy (SEM) respectively. For data interpretation and statistical model-building an in house written code in the open source software RStudio was implemented. It was applied for the differentiation of PHB producers at the cellular level, revealing heterogeneities within sample groups regarding the PHB amount accumulated. The results obtained using the statistical model were validated as well and were complementary to the reference HPLC analysis. Therefore, a fast and reliable identification in situ SERS tool for the selection of the most promising cyanobacterial PHB production was established.

Flexible and Stretchable Temperature Sensors Fabricated Using Solution-Processable Conductive Polymer Composites.[Pubmed:32602670]

Adv Healthc Mater. 2020 Jun 30:e2000380.

Accurate monitoring of physiological temperatures is important for the diagnosis and tracking of various medical conditions. This work presents the design, fabrication, and characterization of temperature sensors using conductive polymer composites (CPCs) patterned on both flexible and stretchable substrates through both drop coating and direct ink writing (DIW). These composites were formed using a high melting point biopolymer Polyhydroxybutyrate (PHB) as the matrix and the graphenic nanomaterial reduced graphene oxide (rGO) as the nanofiller (from 3 to 12 wt%), resulting in a material that exhibits a temperature-dependent resistivity. At room temperature the composites exhibited electrical percolation behavior. Around the percolation threshold, both the carrier concentration and mobility were found to increase sharply. Sensors were fabricated by drop-coating PHB-rGO composites onto ink-jet printed silver electrodes. The temperature coefficient of resistance was determined to be 0.018 / degrees C for pressed rGO powders and 0.008 / degrees C for the 3 wt% samples (the highest responsivity of all composites). Composites were found to have good selectivity to temperature with respect to pressure and moisture. Thermal mapping was demonstrated using 6 x 7 arrays of sensing elements. Stretchable devices with a meandering pattern were fabricated using DIW, demonstrating the potential for these materials in healthcare monitoring devices.

Biogas valorization via continuous polyhydroxybutyrate production by Methylocystis hirsuta in a bubble column bioreactor.[Pubmed:32585559]

Waste Manag. 2020 Jul 15;113:395-403.

Creating additional value out of biogas during waste treatment has become a priority in past years. Biogas bioconversion into valuable bioproducts such as biopolymers has emerged as a promising strategy. This work assessed the operational feasibility of a bubble column bioreactor (BCB) implemented with gas recirculation and inoculated with a Polyhydroxybutyrate (PHB)-producing strain using biogas as substrate. The BCB was initially operated at empty bed residence times (EBRTs) ranging from 30 to 120 min and gas recirculation ratios (R) from 0 to 30 to assess the gas-to-liquid mass transfer and bioconversion of CH4. Subsequently, the BCB was continuously operated at a R of 30 and an EBRT of 60 min under excess of nitrogen and nitrogen feast-famine cycles of 24 h:24 h to trigger PHB synthesis. Gas recirculation played a major role in CH4 gas-liquid transfer, providing almost fourfold higher CH4 elimination capacities (~41 g CH4 m(-3) h(-1)) at the highest R and EBRT of 60 min. The long-term operation under N excess conditions entailed nitrite accumulation (induced by O2 limiting conditions) and concurrent methanotrophic activity inhibition above ~60 mg N-NO2(-) L(-1). Adjusting the N-NO3(-) supply to match microbial N demand successfully prevented nitrite accumulation. Finally, the N feast-famine 24 h:24 h strategy supported a stable CH4 conversion with a removal efficiency of 70% along with a continuous PHB production, which yielded PHB accumulations of 14.5 +/- 2.9% (mg PHB mg(-1) total suspended solids x 100). These outcomes represent the first step towards the integration of biogas biorefineries into conventional anaerobic digestion plants.

Author Correction: The transcriptional regulator NtrC controls glucose-6-phosphate dehydrogenase expression and polyhydroxybutyrate synthesis through NADPH availability in Herbaspirillum seropedicae.[Pubmed:32572134]

Sci Rep. 2020 Jun 22;10(1):10358.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced isobutanol production by co-production of polyhydroxybutyrate and cofactor engineering.[Pubmed:32569791]

J Biotechnol. 2020 Jun 20;320:66-73.

Once cells have been used to produce biochemicals, there are only a few effective ways to utilize the residual cell mass, even though the utilization of leftover cells would aid in decreasing production costs. Here, a Polyhydroxybutyrate (PHB) and isobutanol co-production system was designed to address this challenge. The addition of the PHB operon into Escherichia coli conferred a metabolic advantage for alcohol production, generating 1.14-fold more isobutanol. Furthermore, following nitrogen source optimization and cofactor engineering, the engineered E. coli strain produced 2-fold more isobutanol and 0.25g/L PHB. Moreover, E. coli cells showed higher tolerance to isobutanol with the overexpression of PHB biosynthesis genes. This co-production system resulted in an increased biomass, higher glucose utilization, and lower acetate maintenance, leading to higher productivity regarding PHB and isobutanol yield. Thus, this novel system is applicable to future fermentation studies for the co-production of PHB and isobutanol.

Modeling of nitrogen fixation and polymer production in the heterotrophic diazotroph Azotobacter vinelandii DJ.[Pubmed:32551229]

Metab Eng Commun. 2020 May 30;11:e00132.

Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH3). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. Azotobacter vinelandii is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and Polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in A. vinelandii remain unknown to date. Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium A. vinelandii DJ (iDT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. iDT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.

Expanding the current knowledge and biotechnological applications of the oxygen-independent ortho-phthalate degradation pathway.[Pubmed:32510798]

Environ Microbiol. 2020 Jun 8.

ortho-Phthalate derives from industrially produced phthalate esters, which are massively used as plasticizers and constitute major emerging environmental pollutants. The pht pathway for the anaerobic bacterial biodegradation of o-phthalate involves its activation to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we have explored further the pht peripheral pathway in denitrifying bacteria and shown that it requires also an active transport system for o-phthalate uptake that belongs to the poorly characterized class of TAXI-TRAP transporters. The construction of a fully functional pht cassette combining both catabolic and transport genes allowed to expand the o-phthalate degradation ecological trait to heterologous hosts. Unexpectedly, the pht cassette also allowed the aerobic conversion of o-phthalate to benzoyl-CoA when coupled to a functional box central pathway. Hence, the pht pathway may constitute an evolutionary acquisition for o-phthalate degradation by bacteria that thrive either in anoxic environments or in environments that face oxygen limitations and that rely on benzoyl-CoA, rather than on catecholic central intermediates, for the aerobic catabolism of aromatic compounds. Finally, the recombinant pht cassette was used both to screen for functional aerobic box pathways in bacteria and to engineer recombinant biocatalysts for o-phthalate bioconversion into sustainable bioplastics, e.g., Polyhydroxybutyrate, in plastic recycling industrial processes.

Recent advances in production and extraction of bacterial lipids for biofuel production.[Pubmed:32464391]

Sci Total Environ. 2020 Sep 10;734:139420.

Lipid-based biofuel is a clean and renewable energy that has been recognized as a promising replacement for petroleum-based fuels. Lipid-based biofuel can be made from three different types of intracellular biolipids; triacylglycerols (TAGs), wax esters (WEs), and Polyhydroxybutyrate (PHB). Among many lipid-producing prokaryotes and eukaryotes, biolipids from prokaryotes have been recently highlighted due to simple cultivation of lipid-producing prokaryotes and their ability to accumulate high biolipid contents. However, the cost of lipid-based biofuel production remains high, in part, because of high cost of lipid extraction processes. This review summarizes the production mechanisms of these different types of biolipids from prokaryotes and extraction methods for these biolipids. Traditional and improved physical/chemical approaches for biolipid extraction remain costly, and these methods are summarized and compared in this review. Recent advances in biological lipid extraction including phage-based cell lysis or secretion of biolipids are also discussed. These new techniques are promising for bacterial biolipids extraction. Challenges and future research needs for cost-effective lipid extraction are identified in this review.

A Brief Report on an Implantation of Small-Caliber Biodegradable Vascular Grafts in a Carotid Artery of the Sheep.[Pubmed:32455730]

Pharmaceuticals (Basel). 2020 May 21;13(5). pii: ph13050101.

The development of novel biodegradable vascular grafts of a small diameter (<6 mm) is an unmet clinical need for patients requiring arterial replacement. Here we performed a pre-clinical study of new small-caliber biodegradable vascular grafts using a sheep model of carotid artery implantation. The 4 mm diameter vascular grafts were manufactured using a mix of Polyhydroxybutyrate/valerate and polycaprolactone supplemented with growth factors VEGF, bFGF and SDF-1alpha (PHBV/PCL-GFmix) and additionally modified by a polymer hydrogel coating with incorporation of drugs heparin and iloprost (PHBV/PCL-GFmix(Hep/Ilo)). Animals with carotid artery autograft implantation and those implanted with clinically used GORE-TEX(R) grafts were used as control groups. We observed that 24 h following surgery, animals with carotid artery autograft implantation showed 87.5% patency, while all the PHBV/PCL-GFmix and GORE-TEX(R) grafts displayed thrombosis. PHBV/PCL-GFmix(Hep/Ilo) grafts demonstrated 62.5% patency 24 h following surgery and it had remained at 50% 1 year post-operation. All the PHBV/PCL grafts completely degraded less than 1 year following surgery and were replaced by de novo vasculature without evidence of calcification. On the other hand, GORE-TEX(R) grafts displayed substantial amounts of calcium deposits throughout graft tissues. Thus, here we report a potential clinical usefulness of PHBV/PCL grafts upon their additional modification by growth factors and drugs to promote endothelialization and reduce thrombogenicity.

Critical overview of biomass feedstocks as sustainable substrates for the production of polyhydroxybutyrate (PHB).[Pubmed:32448640]

Bioresour Technol. 2020 Sep;311:123536.

Polyhydroxybutyrates (PHBs) are a class of biopolymers produced by different microbial species and are biodegradable and biocompatible in nature as opposed to petrochemically derived plastics. PHBs have advanced applications in medical sector, packaging industries, nanotechnology and agriculture, among others. PHB is produced using various feedstocks such as glycerol, dairy wastes, agro-industrial wastes, food industry waste and sugars. Current focus on PHB research has been primarily on reducing the cost of production and, on downstream processing to isolate PHB from cells. Recent advancements to improve the productivity and quality of PHB include genetic modification of producer strain and modification of PHB by blending to develop desirable properties suited to diversified applications. Selection of feedstock plays a critical role in determining the economic feasibility and sustainability of the process. This review provides a bird's eye view of the suitability of different waste resources for producing Polyhydroxybutyrate; providing state-of the art information and analysis.

New perspectives on butyrate assimilation in Rhodospirillum rubrum S1H under photoheterotrophic conditions.[Pubmed:32434546]

BMC Microbiol. 2020 May 20;20(1):126.

BACKGROUND: The great metabolic versatility of the purple non-sulfur bacteria is of particular interest in green technology. Rhodospirillum rubrum S1H is an alpha-proteobacterium that is capable of photoheterotrophic assimilation of volatile fatty acids (VFAs). Butyrate is one of the most abundant VFAs produced during fermentative biodegradation of crude organic wastes in various applications. While there is a growing understanding of the photoassimilation of acetate, another abundantly produced VFA, the mechanisms involved in the photoheterotrophic metabolism of butyrate remain poorly studied. RESULTS: In this work, we used proteomic and functional genomic analyses to determine potential metabolic pathways involved in the photoassimilation of butyrate. We propose that a fraction of butyrate is converted to acetyl-CoA, a reaction shared with Polyhydroxybutyrate metabolism, while the other fraction supplies the ethylmalonyl-CoA (EMC) pathway used as an anaplerotic pathway to replenish the TCA cycle. Surprisingly, we also highlighted a potential assimilation pathway, through isoleucine synthesis and degradation, allowing the conversion of acetyl-CoA to propionyl-CoA. We tentatively named this pathway the methylbutanoyl-CoA pathway (MBC). An increase in isoleucine abundance was observed during the early growth phase under butyrate condition. Nevertheless, while the EMC and MBC pathways appeared to be concomitantly used, a genome-wide mutant fitness assay highlighted the EMC pathway as the only pathway strictly required for the assimilation of butyrate. CONCLUSION: Photoheterotrophic growth of Rs. rubrum with butyrate as sole carbon source requires a functional EMC pathway. In addition, a new assimilation pathway involving isoleucine synthesis and degradation, named the methylbutanoyl-CoA (MBC) pathway, could also be involved in the assimilation of this volatile fatty acid by Rs. rubrum.