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Rice straw is an important low-cost feedstock for bio-based economy. This report presents a study in which rice straw was used both as a source for isolation of bacteria producing the biodegradable polyester polyhydroxyalkanoate (PHA), as well as the carbon source for the production of the polymer by the isolated bacteria. Of the 100 bacterial isolates, seven were found to be positive for PHA production by Nile blue staining and were identified as Bacillus species by 16S rRNA gene sequence analysis. Three isolates showed 100% sequence identity to B. cereus, one to B. paranthracis, two with 99 and 100% identity to B. anthracis, while one was closely similar to B. thuringiensis. For use in PHA production, rice straw was subjected to mild alkaline pretreatment followed by enzymatic hydrolysis. Comparison of pretreatment by 2% sodium hydroxide, 2% calcium hydroxide and 20% aqueous ammonia, respectively, at different temperatures showed maximum weight loss with NaOH at 80 °C for 5 h, but ammonia for 15 h at 80 °C led to highest lignin removal of 63%. The ammonia-pretreated rice straw also led to highest release of total reducing sugar up to 92% on hydrolysis by a cocktail of cellulases and hemicellulases at 50 °C. Cultivation of the Bacillus isolates on the pretreated rice straw revealed highest PHA content of 59.3 and 46.4%, and PHA concentration of 2.96 and 2.51 g/L by Bacillus cereus VK92 and VK98, respectively.

Polyhydroxyalkanoates (PHAs) are versatile biodegradable polymers produced by bacteria and are suitable for many downstream applications. They can be produced inexpensively from mixed microbial cultures under feast and famine conditions in the presence of biobased volatile fatty acids (VFAs). Here, we investigated the effect of changing the sludge retention time (SRT) and the addition of fermented cellulosic primary sludge (CPS) as a carbon source on the selection of PHA-storing biomass when applying the feast and famine strategy under aerobic and anoxic conditions, respectively. Increasing the SRT from 5 to 7–10 days enhanced PHA yields under feast conditions from 0.18 gCODPHA/gCODVFA (period 1) to 0.40 gCODPHA/gCODVFA (period 2). The use of fermented CPS as a carbon source (period 3) increased PHA yields to 0.62 gCODPHA/gCODVFA despite the presence of biodegradable non-VFA fractions. Microbial characterization by denaturing gradient gel electrophoresis and fluorescence in situ hybridization revealed high microbial speciation during the three experimental periods. In period 3, the dominant genera were Thauera, Paracoccus, and Azoarcus, which accounted for ∼95% of the total microbial biomass., The effect of sludge retention time and bio-based volatile fatty acids on the selection of polyhydroxyalkanoate-storing biomass.

Plasticizers are the most used polymer additives world-wide. Nowadays, conventional plasticizers (e.g. phthalates) do not meet the requirements in terms of renewability, biodegradability and cytotoxicity that have become necessary, especially if they are compounded with biopolymers. In this study, novel bioplasticizers are synthesized from levulinic acid via a protecting-group-free three-step process. After FT-IR and NMR characterization of the synthesized molecules, their plasticization effect has been tested with poly(3-hydroxybutyrate) (PHB) as a model semicrystalline biopolyester characterized by a narrow processing window, slow re-crystallization and high brittleness, which limit its processability and diffusion. The proposed bioplasticizers show remarkable miscibility with PHB and low leaching. The bioplasticizers also show a remarkable plasticization effect in terms of reducing the glass transition and melting temperatures (17 °C and 8 °C, respectively), which are comparable with the performance of the best commercially available green plasticizers. Furthermore, flexibility and crystallinity are positively affected, leading to an overall reduction in the typical brittleness of PHB. The observed effects result in an expansion of the temperature range in which PHB can be processed without thermal degradation. Moreover, the incorporation of the levulinic acid-based additives does not significantly affect the typical biodegradability and biocompatibility of PHB, showing their promising features as bioplasticizers for both environmental and biomedical applications.

The accumulation of petrochemical plastic waste is detrimental to the environment. Polyhydroxyalkanoates (PHAs) are bacterial-derived polymers utilized for the production of bioplastics. PHA-plastics exhibit mechanical and thermal properties similar to conventional plastics. However, high production cost and obtaining high PHA yield and productivity impedes the widespread use of bioplastics. This study demonstrates the concept of cyclic fed-batch fermentation (CFBF) for enhanced PHA productivity by Bacillus thuringiensis using a glucose-rich hydrolyzate as the sole carbon source. The statistically optimized fermentation conditions used to obtain high cell density biomass (OD600 of 2.4175) were: 8.77 g L−1 yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised three cycles of 29 h, 52 h, and 65 h, respectively. After the third cyclic event, cell biomass of 20.99 g L−1, PHA concentration of 14.28 g L−1, PHA yield of 68.03%, and PHA productivity of 0.219 g L−1 h−1 was achieved. This cyclic strategy yielded an almost threefold increase in biomass concentration and a fourfold increase in PHA concentration compared with batch fermentation. FTIR spectra of the extracted PHAs display prominent peaks at the wavelengths unique to PHAs. A copolymer was elucidated after the first cyclic event, whereas, after cycles CFBF 2–4, a terpolymer was noted. The PHAs obtained after CFBF cycle 3 have a slightly higher thermal stability compared with commercial PHB. The cyclic events decreased the melting temperature and degree of crystallinity of the PHAs. The approach used in this study demonstrates the possibility of coupling fermentation strategies with hydrolyzate derived from lignocellulosic waste as an alternative feedstock to obtain high cell density biomass and enhanced PHA productivity.

Liquid cultures of Vibrio sp. SI9, isolated from the outer tissue of the sea anemone Radianthus crispus, was found to produce three new O-isocrotonyl-3-hydroxybutyric acid derivatives, O-isocrotonyl-3-hydroxypentanoic acid (1), O-isocrotonyl-3-hydroxyhexanoic acid (2), and O-(Z)-2-hexenoyl-3-hydroxybutyric acid (3), together with the known O-isocrotonyl-3-hydroxybutyric acid (4). The structures of 1–3 were established by NMR spectroscopy and mass spectrometry, coupled with anisotropy-based chiral analysis, revealing the same R-configuration for all congeners 1–4. The compounds 1–4 were weakly growth-inhibitory against a marine fish ulcer pathogenic bacterium, Tenacibaculum maritimum NBRC16015. Structural similarities among 1–4, the O-isocrotonylated 3-hydroxybutyrate oligomers 5, and microbial biopolymer polyhydroxyalkanoates (PHA) suggest the presence of a common biosynthetic machinery, and hence a possible dehydrative modification at the hydroxy terminus of PHA.

Одержано нові продукти біосинтезу штаму Pseudomonas sp. PS-17: біогенні поверхнево-активні речовини (рамноліпідний біокомплекс, дирамноліпід та екзополісахарид) і біополімер полігідроксіалканоат. Вивчено процес термічного перетворення цих продуктів у повітряному середовищі в інтервалі температур 293–1273 К. За результатами термолізу та ІЧ-спектроскопії виявлено утворення рамноліпідного біокомплексу між рамноліпідами та екзополісахаридом при підкисленні супернатанту культуральної рідини до рН=3. Методом UV-Vis спектроскопії (за максимумом поглинання при 239 нм) ідентифіковано клітинний полімер – полігідроксіалканоат. За даними термічного аналізу визначено температуру плавлення полігідроксіалканоату (319 К) та температуру початку його деструкції (499 К). З використанням методу Віка визначено теплостійкість полімеру, що склала 321 К. Biogenic surfactants (rhamnolipid biocomplex, dirhamnolipid and exopolysaccharide) and polyhydroxyalkanoate biopolymer which are the new biosynthesis products of Pseudomonas sp. PS-17 strain have been obtained. The thermal transformation of these products has been studied in the air in the temperature range of 293–1273 K. Thermolysis and IR spectroscopy revealed a formation of rhamnolipid biocomplex between rhamnolipids and exopolysaccharide upon acidification of the culture fluid supernatant to pH = 3. Cellular polymer – polyhydroxyalkanoate – was identified by UV-Vis spectroscopy. According to the thermal analysis, the melting point and initial degradation temperature of polyhydroxyalkanoate were determined. Using the Vicat method, the heat resistance of the polymer was determined.

Producción Científica, Methylocystis hirsuta, a type II methanotroph, has been experimentally demonstrated to be able to efficiently synthesize polyhydroxyalkanoates (PHA) from biogas under nutrient-limited conditions. A mechanistic model capable of describing the relevant processes of M. hirsuta, which is currently not available, would therefore lay a solid foundation for future practical demonstration and optimization of the PHA synthesis technology using biogas. To this end, dedicated batch tests were designed and conducted to obtain experimental data for different mechanistic processes of M. hirsuta. Through utilizing the experimental data of well-designed batch tests and following a step-wise model calibration/validation protocol, the stoichiometrics and kinetics of M. hirsuta are reported for the first time, including the yields of growth and PHA synthesis on CH4 (0.14 ± 0.01 g COD g–1 COD and 0.25 ± 0.02 g COD g–1 COD), the CH4 and O2 affinity constants (5.1 ± 2.1 g COD m–3 and 4.1 ± 1.7 g O2 m–3), the maximum PHA consumption rate (0.019 ± 0.001 g COD g–1 COD d–1), and the maximum PHA synthesis rate on CH4 (0.39 ± 0.05 g COD g–1 COD d–1). Through applying the developed model, an optimal O2:CH4 molar ratio of 1.6 mol O2 mol–1 CH4 was found to maximize the PHA synthesis by M. hirsuta. Practically, the model and parameters obtained would not only benefit the design and operation of bioreactors performing PHA synthesis from biogas, but also enable specific research on selection for type II methanotrophs in diverse environments., Australian Research Council (ARC) through Future Fellowship (FT160100195), Junta de Castilla y León y EU-FEDER (CLU 2017-09) y (UIC 71), European Union’s Horizon 2020 research and innovation program. grant agreement no. 790231.

In this study, adsorption treatment of phenol pollutant using electrospun poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is highlighted for the first time. Both P(3HB) and P(3HB-co-3HHx) were extracted from bacterial cells and nanofibres were fabricated through electrospinning technique for the adsorption of phenol. The electrospun nanofibres were optimised after preliminary study of the electrospinning parameters (i.e. applying different voltages, polymer concentrations and mixed solvent ratios). The effects of adsorption under different variables such as contact of time, pH, initial concentration and temperature were studied. The raw polymers were characterised with gas chromatography (GC), gas permeation chromatography (GPC) while the electrospun fibres were investigated using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and viewed under field emission scanning electron microscope (FESEM). P(3HB) had higher molecular weight as well as higher crystallinity and thermal stability than P(3HB-co-3HHx). The maximum phenol adsorption capacity of optimised nanofibre with weight of 0.14 g was 35.7 mg/g. The adsorption trend of the isotherm followed the Freundlich (R2 = 0.99) and adsorption kinetics of pseudo-second-order. The equilibrium contact time was 10 min with 99.8% of the phenol removed and attained 99.9% removal after optimal contact time of 270 min for all samples. This innocuous product has strong affinity towards organic pollutants and zero by-products were produced after the adsorption.

The chlorophyll ethanol-extracted silkworm excrement was hardly biologically reused or fermented by most microorganisms. However, partial extremely environmental halophiles were reported to be able to utilize a variety of inexpensive carbon sources to accumulate polyhydroxyalkanoates. In this study, by using the nile red staining and gas chromatography assays, two endogenous haloarchaea strains: Haloarcula hispanica A85 and Natrinema altunense A112 of silkworm excrement were shown to accumulate poly(3-hydroxybutyrate) up to 0.23 g/L and 0.08 g/L, respectively, when using the silkworm excrement as the sole carbon source. The PHA production of two haloarchaea showed no significant decreases in the silkworm excrement medium without being sterilized compared to that of the sterilized medium. Meanwhile, the CFU experiments revealed that there were more than 60% target PHAs producing haloarchaea cells at the time of the highest PHAs production, and the addition of 0.5% glucose into the open fermentation medium can largely increase both the ratio of target haloarchaea cells (to nearly 100%) and the production of PHAs. In conclusion, our study demonstrated the feasibility of using endogenous haloarchaea to utilize waste silkworm excrement, effectively. The introduce of halophiles could provide a potential way for open fermentation to further lower the cost of the production of PHAs.

The synthesis of polyhydroxybutyrate (PHB) by photosynthetic non-sulfur bacteria is a potential approach for producing biodegradable plastics. In this work, acetate was used as a single carbon source to study the effect on PHB formation in Rhodopseudomonas sp. cultured in a cylindrical four-liter photobioreactor under semi-continuous mode. The cultivation process is divided into a symmetrical growth phase and a PHB accumulation phase separated temporally. The symmetrical growth phase (nutrient sufficient conditions) was followed by a sulfur-limited phase to promote PHB accumulation. The main novelty is the progressive lowering of the sulfur concentration into Rhodopseudomonas culture, which was obtained by two concomitant conditions: (1) sulfur consumption during the bacterial growth and (2) semi-continuous growth strategy. This caused a progressive lowering of the sulfur concentration into Rhodopseudomonas culturedue to the sulfur-free medium used to replace 2 L of culture (50% of the total) that was withdrawn from the photobioreactor at each dilution. The PHB content ranged from 9.26% to 15.24% of cell dry weight. At the steady state phase, the average cumulative PHB was &gt, 210 mg/L. Sulfur deficiency proved to be one of the most suitable conditions to obtain high cumulative PHB in Rhodopseudomonas culture.

Polyhydroxyalkanoate (PHA) is a microbial storage polymer that is naturally produced by certain bacteria. This is the first study on the ability of this particular species Massilia haematophila to synthesize a PHA copolymer containing 3-hydroxyvalerate (3HV) monomer. Using the statistical design on Massilia haematophila UMTKB-2, this study highlights the optimization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HB-co-3HV), copolymer production for shaken-flask cultivation. Moreover, the mechanical and thermal features of the polymers were determined. The production of P(3HB-co-3HV) by Massilia haematophila UMTKB-2 using optimal conditions provided by response surface methodology (RSM) yielded 5.0 g/l of P(3HB-co-7 mol% 3HV), which was higher than the value obtained from unoptimized conditions such as 4.40 g/l of P(3HB-co-4mol% 3HV). This result showed a 14% increase in copolymer concentration and a two-fold increase in 3HV composition. In this study, the P(3HB-co-3HV) synthesized was determined as a block copolymer and its thermal properties were better than P(3HB). Using RSM, the optimization conditions were successfully obtained for this bacterium, and this result is a starting platform for additional studies of a larger scaled PHA production from Massilia haematophila UMTKB-2 using bioreactors.

Aiming to address the issue of poor bioavailability of most anti-tumor medicines against colorectal cancer, we developed a targeted anticancer nanocarrier using biocarriers able to both bind and easily release their load in a controlled manner. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) carriers were obtained via the emulsification-diffusion method, loaded with 5-fluorouracil and then characterized in terms of particle morphology and size (SEM, DLS), drug uptake and release. The cytotoxic potential of the 5-fluorouracil-loaded polymer nanocarriers on human adenocarcinoma cells (HT-29 cell line) was investigated. The in vitro studies clearly demonstrated that while the nanocarriers themselves slightly alter HT-29 cell viability, when loaded with 5-fluorouracil they significantly decrease cell viability, suggesting that the polymer itself exhibits low cytotoxicity and the drug-loaded carrier acts in an efficient manner to kill HT-29 human adenocarcinoma cells.

Biopolymers are a promising alternative to petroleum-based plastic raw materials. They are bio-based, non-toxic and degradable under environmental conditions. In addition to the homopolymer poly(3-hydroxybutyrate) (PHB), there are a number of co-polymers that have a broad range of applications and are easier to process in comparison to PHB. The most prominent representative from this group of bio-copolymers is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). In this article, we show a new kinetic model that describes the PHBV production from fructose and propionic acid in Cupriavidus necator (C. necator). The developed model is used to analyze the effects of process parameter variations such as the CO2 amount in the exhaust gas and the feed rate. The presented model is a valuable tool to improve the microbial PHBV production process. Due to the coupling of CO2 online measurements in the exhaust gas to the biomass production, the model has the potential to predict the composition and the current yield of PHBV in the ongoing process.

Pseudomonas citronellolis SJTE-3 was isolated as a highly efficient microorganism for biodegradation and valorization of drilling fluids (DF) wastewater. The strain metabolised DF and oily mud exhibiting up to 93%, 86%, 85% and 88% of chemical oxygen demand (COD), n-dodecane, n-tetradecane and naphthalene removal efficiency respectively. Enhanced bioconversion was enabled through production of biosurfactants that reduced the surface tension of water by 53% and resulted in 43.3% emulsification index (E24), while synthesizing 24% of dry cell weight (DCW) as medium-chain-length polyhydroxyalkanoates (PHA). Expression from the main pathways for alkanes and naphthalene biodegradation as well as biosurfactants and PHA biosynthesis revealed that although the alkanes and naphthalene biodegradation routes were actively expressed even at stationary phase, PHA production was stimulated at late stationary phase and putisolvin could comprise the biosurfactant synthesized. The bioconversion of toxic petrochemical residues to added-value thermoelastomers and biosurfactants indicate the high industrial significance of P. citronellolis SJTE-3.

Escherichia coli contains 12 chaperone-usher operons for biosynthesis and assembly of various fimbriae. In this study, each of the 12 operons was deleted in E. coli MG1655, and the resulting 12 deletion mutants all grew better than the wild type, especially in the nutrient-deficient M9 medium. When the plasmid pBHR68 containing the key genes for polyhydroxyalkanoate production was introduced into these 12 mutants, each mutant synthesized more polyhydroxyalkanoate than the wild-type control. These results indicate that the fimbria removal in E. coli benefits cell growth and polyhydroxyalkanoate production. Therefore, all 12 chaperone-usher operons, including 64 genes, were deleted in MG1655, resulting in the fimbria-lacking strain WQM026. WQM026 grew better than MG1655, and no fimbria structures were observed on the surface of WQM026 cells. Transcriptomic analysis showed that in WQM026 cells, the genes related to glucose consumption, glycolysis, flagellar synthesis, and biosynthetic pathways of some key amino acids were upregulated, while the tricarboxylic acid cycle-related genes were downregulated. When pBHR68 was introduced into WQM026, huge amounts of poly-3-hydroxybutyrate were produced; when the plasmid pFW01-thrA*BC-rhtC, containing the key genes for l-threonine biosynthesis and transport, was transferred into WQM026, more l-threonine was synthesized than with the control. These results suggest that this fimbria-lacking E. coli WQM026 is a good host for efficient production of polyhydroxyalkanoate and l-threonine and has the potential to be developed into a valuable chassis microorganism. IMPORTANCE In this study, we investigated the interaction between the biosynthesis and assembly of fimbriae and intracellular metabolic networks in E. coli. We found that eliminating fimbriae could effectively improve the production of polyhydroxyalkanoate and l-threonine in E. coli MG1655. These results contribute to understanding the necessity of fimbriae and the advantages of fimbria removal for industrial microorganisms. The knowledge gathered from this study may be applied to the development of superior chassis microorganisms.

Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.

Polyhydroxyalkanoate (PHA) synthase is an enzyme that polymerizes the acyl group of hydroxyacyl-coenzyme A (CoA) substrates. Aeromonas caviae PHA synthase (PhaCAc) is an important biocatalyst for the synthesis of a useful PHA copolymer, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)]. Previously, a PhaCAc mutant with double mutations in asparagine 149 (replaced by serine [N149S]) and aspartate 171 (replaced by glycine [D171G]) was generated to synthesize a 3HHx-rich P(3HB-co-3HHx) and was named PhaCAc NSDG. In this study, to further increase the 3HHx fraction in biosynthesized PHA, PhaCAc was engineered based on the three-dimensional structural information of PHA synthases. First, a homology model of PhaCAc was built to target the residues for site-directed mutagenesis. Three residues, namely tyrosine 318 (Y318), serine 389 (S389), and leucine 436 (L436), were predicted to be involved in substrate recognition by PhaCAc. These PhaCAc NSDG residues were replaced with other amino acids, and the resulting triple mutants were expressed in the engineered strain of Ralstonia eutropha for application in PHA biosynthesis from palm kernel oil. The S389T mutation allowed the synthesis of P(3HB-co-3HHx) with an increased 3HHx fraction without a significant reduction in PHA yield. Thus, a new workhorse enzyme was successfully engineered for the biosynthesis of a higher 3HHx-fraction polymer.

An overview of the main polyhydroxyalkanoates (PHA) recovery methods is here reported, by considering the kind of PHA-producing bacteria (single bacterial strains or mixed microbial cultures) and the chemico-physical characteristics of the extracted polymer (molecular weight and polydispersity index). Several recovery approaches are presented and categorized in two main strategies: PHA recovery with solvents (halogenated solvents, alkanes, alcohols, esters, carbonates and ketones) and PHA recovery by cellular lysis (with oxidants, acid and alkaline compounds, surfactants and enzymes). Comparative evaluations based on the recovery, purity and molecular weight of the recovered polymers as well as on the potential sustainability of the different approaches are here presented.

A novel generation of gels based on medium chain length poly(3-hydroxyalkanoate)s, mcl-PHAs, were developed by using ionic interactions. First, water soluble mcl-PHAs containing sulfonate groups were obtained by thiol-ene reaction in the presence of sodium-3-mercapto-1-ethanesulfonate. Anionic PHAs were physically crosslinked by divalent inorganic cations Ca2+, Ba2+, Mg2+ or by ammonium derivatives of gallic acid GA-N(CH3)3+ or tannic acid TA-N(CH3)3+. The ammonium derivatives were designed through the chemical modification of gallic acid GA or tannic acid TA with glycidyl trimethyl ammonium chloride (GTMA). The results clearly demonstrated that the formation of the networks depends on the nature of the cations. A low viscoelastic network having an elastic around 40 Pa is formed in the presence of Ca2+. Although the gel formation is not possible in the presence of GA-N(CH3)3+, the mechanical properties increased in the presence of TA-N(CH3)3+ with an elastic modulus G&rsquo, around 4200 Pa. The PHOSO3&minus, /TA-N(CH3)3+ gels having antioxidant activity, due to the presence of tannic acid, remained stable for at least 5 months. Thus, the stability of these novel networks based on PHA encourage their use in the development of active biomaterials.

Background Efficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy—co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440—for the first time, which simultaneously improved both cell biomass and PHA production. Results Co-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4–16.1% and PHA content by 29.0–63.2%, respectively, compared with feeding glycerol alone. GC–MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4–4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8–3.5%. The 1H–13C HMBC, 1H–13C HSQC, and 1H–1H COSY NMR analysis confirmed that the PHA monomers (C6–C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD+/NADH and NADPH/NADP+ ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner–Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis. Conclusions This work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

Photosynthetic microorganisms are promising candidates in a strategy to replace fossil fuels as feedstock for production of bioplastic and hydrogen (H2). These microorganisms have advantages over chemical synthesis since they grow using renewable energy sources (sun) and operate at ambient temperature and pressure. However, oxygen inhibition of the enzyme responsible for H2 production and the high cost of photobioreactors are preventing practical implementation of the process. This chapter presents experimental data of our group and some suggestions concerning photosynthetic microorganisms (cyanobacteria and purple non-sulfur bacteria) growth in outdoor photobioreactors for the biological H2 production and the synthesis of poly-3-hydroxybutyrate, a common type of polyhydroxyalkanoate (PHA) bioplastic.

Wound dressings are high performance and high value products which can improve the regeneration of damaged skin. In these products, bioresorption and biocompatibility play a key role. The aim of this study is to provide progress in this area via nanofabrication and antimicrobial natural materials. Polyhydroxyalkanoates (PHAs) are a bio-based family of polymers that possess high biocompatibility and skin regenerative properties. In this study, a blend of poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxyoctanoate-co-3-hydroxy decanoate) (P(3HO-co-3HD)) was electrospun into P(3HB))/P(3HO-co-3HD) nanofibers to obtain materials with a high surface area and good handling performance. The nanofibers were then modified with silver nanoparticles (AgNPs) via the dip-coating method. The silver-containing nanofiber meshes showed good cytocompatibility and interesting immunomodulatory properties in vitro, together with the capability of stimulating the human beta defensin 2 and cytokeratin expression in human keratinocytes (HaCaT cells), which makes them promising materials for wound dressing applications.

Because of availability and inexpensive, glycerol can be considered as a suitable raw material for polyhydroxyalkanoate (PHA) production with bacterial fermentation. Nevertheless, compared to the production of glucose as a raw precursor, PHA produced from glycerol by Cupriavidus necator was found to produce lower PHA with low bacterial growth rates. According to our study, C. necator was able to synthesize glucose-like intermediates from glycerol via gluconeogenesis. This resulted in a decrease of the cell dry weight and the yield of PHA polymers, especially in the active cell growth phase. It was indicated that glycerol used as a carbon source of the PHA synthesis pathway has glucogenesis-shift, which causes a decrease of the PHA content and productivity. In this research, we investigated the proteins that were closely expressed with the increase of the intracellular PHA and glucose content. For solving the above problem, the proteins inside the bacterial cells were analyzed and compared to the database proteins via mass spectrometry. The proteins were isolated by 1-D SDS-polyacrylamide gel electrophoresis (PAGE) technique and identified by the liquid chromatography mass spectrometry (LC-MS) technique. By using bioinformatics validation, a total number of 1361 proteins were examined and found in the culture bacterial cells. Selective protein expression was correlated with the amount of PHA at each cultivation time and generating glucose by studying the 1361 proteins was elucidated in proteomic information. The results of the cluster of proteins were found to contain 93 proteins using the multiple array viewer (MEV) program with the KMS data analysis model. Protein species with the same expression pattern for PHA and six proteins with similar expression patterns were found to be correlated with generating glucose content. The associations of the two protein groups were then determined through a Stitch program. The protein and chemical associations were analyzed both directly and indirectly through different databases. The proteins of interest were found with research data linked between glycerol and glucose. Five protein types are connecting to glucose and glycerol shift pathway, two of which are glycosyl hydrolase (H16_B1563) and short-chain dehydrogenase (H16_B0687), both of which are enzymes used to break the bonds of complex sugars, possibly related to the partial conversion of glycerol to glucose. The two proteins found in the strains used in the Cupriavidus necator H16 experiment give rise to the break down the bonds of &alpha, &alpha, 1,1-glucoside of malto-oligosyltrehalose and short-chain sugar molecules such as mannitol (C6H14O6), respectively. In this research, finding the associated expression proteins which is involved in changing the pathway of gluconeogenesis shift to PHA synthesis will be useful information on genetically modifying microorganisms to produce PHA more efficiently, leading to reduction of the production costs.

We isolated the Cobetia sp. strains IU 180733JP01 (5-11-6-3) and 190790JP01 (5-25-4-2) from seaweeds and showed that both strains accumulate poly(3-hydroxybutyrate) [P(3HB)] homopolymer in a nitrogen-limiting mineral salt medium containing alginate as a sole carbon source. Genome sequence analysis of the isolated strains showed that they have putative genes which encode enzymes relevant to alginate assimilation and P(3HB) synthesis, and the putative alginate-assimilating genes formed a cluster. Investigation of the optimum culture conditions for high accumulation of P(3HB) showed that when the 5-11-6-3 strain was cultured in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and 3% (w/v) alginate as a sole carbon source for 2 days, the P(3HB) content and P(3HB) production reached 62.1 ± 3.4 wt% and 3.11 ± 0.16 g/L, respectively. When the 5-25-4-2 strain was cultured in a nitrogen-limiting mineral salt medium (pH 4.0) containing 5% NaCl and 3% (w/v) alginate for 2 days, the P(3HB) content and P(3HB) production reached 56.9 ± 2.1 wt% and 2.67 ± 0.11 g/L, respectively. Moreover, the 5-11-6-3 strain also produced P(3HB) in a nitrogen-limiting mineral salt medium (pH 5.0) containing 6% NaCl and freeze-dried and crushed waste Laminaria sp., which is classified into brown algae and contains alginate abundantly. The resulting P(3HB) content and P(3HB) productivity were 13.5 ± 0.13 wt% and 3.99 ± 0.15 mg/L/h, respectively. Thus, we demonstrated the potential application of the isolated strains to a simple P(3HB) production process from seaweeds without chemical hydrolysis and enzymatic saccharification.

Liquid cultures of Vibrio sp. SI9, isolated from the outer tissue of the sea anemone Radianthus crispus, was found to produce three new O-isocrotonyl-3-hydroxybutyric acid derivatives, O-isocrotonyl-3-hydroxypentanoic acid (1), O-isocrotonyl-3-hydroxyhexanoic acid (2), and O-(Z)-2-hexenoyl-3-hydroxybutyric acid (3), together with the known O-isocrotonyl-3-hydroxybutyric acid (4). The structures of 1–3 were established by NMR spectroscopy and mass spectrometry, coupled with anisotropy-based chiral analysis, revealing the same R-configuration for all congeners 1–4. The compounds 1–4 were weakly growth-inhibitory against a marine fish ulcer pathogenic bacterium, Tenacibaculum maritimum NBRC16015. Structural similarities among 1–4, the O-isocrotonylated 3-hydroxybutyrate oligomers 5, and microbial biopolymer polyhydroxyalkanoates (PHA) suggest the presence of a common biosynthetic machinery, and hence a possible dehydrative modification at the hydroxy terminus of PHA.

Rhodovulum sulfidophilum DSM-1374 is a potential producer of polyester when growing in phototrophic conditions. The present study investigated on a polyester product (P3HB) by culturing Rhodovulum sulfidophilum DSM-1374 in two different photobioreactors (PBR-1 and PBR-2) both with 4-L working volumes. PBR-1 is equipped with an internal rotor having 4 paddles to mix the bacterial culture while PBR-2 has an internal coil-shaped rotor. After selecting PBR-1, which best performed in the preliminary experiment, the effect of different stressing growth conditions as pH (7.0, 8.0, and 9.0), temperature (25, 30, and 35 °C), and medium salinity (1.5, 2.5, 3.5, and 4.5%) were tested. When the pH of the culture was set to 8.0, the capability of the bacterium to synthetize the polyester increased significantly reaching a concentration of 412 mg (P3HB)/L; the increase of the pH at 9.0 caused a reduction of the P3HB concentration in the culture. The medium salinity of 4.5% was the best stress-growth condition to reach the highest concentration of polyester in the culture (820 ± 50 mg (P3HB)/L) with a P3HB mass fraction in the dry biomass of 33 ± 1.5%. Stresses caused by culture temperature are another potential parameter that could increase the synthesis of P3HB.

International audience; Within the current increasing environmental restrictions, biopolymers tend to replace common materials in many applications, from daily life items to process engineering facilities. Synthetic filtration membranes are also of concern. Herein, biopolymer based microfiltration (MF) membranes were produced with a polyhydroxyalkanoate (PHA), the poly(hydoxybutyrate-co-hydroxyvalerate) (PHBHV). The membranes were made by evaporation induced phase separation (EIPS) and the influence of the dope solution composition was studied by adding additives, polyvinylpyrrolidones (PVPs) and polyethylene glycols (PEGs). The nature, molecular weight and concentration of the additives were linked to the obtained microstructures. Both types of additives can increase membrane porosity by acting as pore former agent. However, interesting opposite effects were obtained in case of PEGs from 300 to 4000 g mol−1 where the additives were observed to act as plasticizers. The membranes performances were evaluated with pure water permeability and E. coli bacteria rejection and correlated to the microstructure analyses. The performances were greatly improved by selecting the proper additive. This study leads to promising results for the consideration of PHA as new potential biomaterial intended for membrane fabrication. © 2020

The interest for biodegradable electronic devices is rapidly increasing for application in the field of wearable electronics, precision agriculture, biomedicine, and environmental monitoring. Energy storage devices integrated on polymeric substrates are of particular interest to enable the large-scale on field use of complex devices. This work presents a novel class of eco-friendly supercapacitors based on biodegradable poly(3-hydroxybutyrrate) PHB, ionic liquids, and cluster-assembled gold electrodes. By electrochemical characterization, we demonstrate the possibility of tuning the supercapacitor energetic performance according to the type and amount of the ionic liquid employed. Our devices based on hydrophobic plastic materials are stable under cyclic operation and resistant to moisture exposure.

Background Lignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysaccharide-based biorefinery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to find the important enzymes for polymer utilization. Results Genomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profile on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase–peroxidase for lignin degradation. A strong stress response and detoxification mechanism was discovered. The two important gene clusters for lignin degradation and three PHA polymerase spanning gene clusters were identified and all the clusters were functionally active on KL–VA. Conclusions The unusual aerobic ‘-CoA’-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4–5% of total sequenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chemistry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profile. Genomic and proteomic approaches provided validation to this strain’s robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future. Electronic supplementary material The online version of this article (10.1186/s13068-018-1148-2) contains supplementary material, which is available to authorized users.

Systems biology examines organisms and processes by a holistic approach instead of the traditional reductionist approach. This can be beneficial to researchers and bioengineers as it allows for the formulation of the “big picture” where visualization of the influences and interactions of system components can occur. Significant amounts of data about the system are required, however, for this approach to be successful. Here, the polyhydroxyalkanoate (PHA) homeostasis process in Ralstonia eutropha is examined with a systems biology slant. Copious amounts of data exist about the enzymes, structural proteins and other players that make up the PHA production/mobilization process in R. eutropha. These data are utilized to create an interaction map of the PHA granule (and PHA homeostasis enzymes) that can inform PHA production strain engineering by providing information about key interactions

RESUMEN: Actualmente, el desarrollo de implantes o procedimientos médicos para resolver lesiones óseas o cartilaginosas no cumple satisfactoriamente con los requerimientos funcionales del tejido afectado; una solución alternativa es el uso de la ingeniería de tejidos (IT), que busca regenerar el tejido con plataformas de crecimiento celular fabricadas, por ejemplo, con polímeros naturales, como el polihidroxialcanoato (PHA), que permite la reconstrucción del tejido gracias a su capacidad de degradación, y cuyo origen bacteriano permite la producción a gran escala y el control de las propiedades finales. En este proyecto se desarrollaron scaffolds de PHA comercial, mediante la técnica de liofilización, con un diseño experimental factorial, utilizando diclorometano como solvente, Tergitol como surfactante y nitrógeno líquido (N2) como congelante. El PHA se caracterizó con espectroscopia de infrarrojo (FTIR) y análisis termogravimétrico (TGA). Los scaffolds obtenidos se caracterizaron con microscopia electrónica de barrido (SEM), ensayos mecánicos de compresión y ensayos de degradación hidrolítica. Los análisis sobre el PHA indicaron que el material es una mezcla de dicho polímero y ácido poliláctico (PLA). Los scaffolds mostraron una distribución de poros adecuada para la migración de condrocitos a través de ellos y presentaron un comportamiento similar al cartílago articular, pero una menor resistencia mecánica; también se encontró que la pérdida de masa está relacionada con el porcentaje de PHA presente en la muestra de una forma no lineal. En conclusión, los scaffolds de PHA tienen un potencial uso en la ingeniería de tejidos para la restauración de cartílago articular ABSTRACT: Tissue engineering (TE) seeks to improve the unsatisfactory development of implants and medical procedures to solve bone and cartilage injuries. TE aims at regenerating tissues using cell growth platforms (scaffolds), which may consist of natural polymers such as polyhydroxyalkanoate (PHA). PHA is an innovative material useful in medical applications due to its degradation capability and bacterial origin that allows large-scale production and control final properties. In this research, we developed commercial PHA scaffolds using the lyophilization technique with a factorial experimental design. We used dichloromethane as PHA solvent, tergitol as surfactant, and liquid nitrogen (N2) for the freezing process. We characterized the PHA by Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA); and the scaffolds by scanning electron microscopy (SEM) and mechanical compression and hydrolysis degradation tests. The characterization of the PHA indicated that the material is a mixture of PHA and polylactic acid (PLA). The results showed a suitable pore distribution for migration of chondrocytes through the scaffold, in addition to a behavior similar to that of the articular cartilage, although it presented lower mechanical strength. Also, the scaffolds displayed mass loss in a non-linear way related to the percentage of PHA present in the sample. In conclusion, PHA scaffolds have a potential use in tissue engineering for restoring articular cartilage RESUMO: Atualmente, o desenvolvimento de implantes ou procedimentos médicos para resolver lesões ósseas ou cartilaginosas não cumpre satisfatoriamente com os requerimentos funcionais do tecido afetado; uma solução alternativa é o uso da engenharia de tecidos (IT), que busca regenerar o tecido com plataformas de crescimento celular fabricadas, por exemplo, com polímeros naturais, como o polihidroxialcanoato (PHA), que permite a reconstrução do tecido graças a sua capacidade de degradação, e cuja origem bacteriana permite a produção a grande escala e o controle das propriedades finais. Neste projeto desenvolveram-se scaffolds de PHA comercial, mediante a técnica de liofilização, com um desenho experimental fatorial, utilizando diclorometano como solvente, Tergitol como surfactante e nitrogênio líquido (N2) como congelante. O PHA caracterizou-se com espectroscopia de infravermelho (FTIR) e análise termogravimétrica (TGA). Os scaffolds obtidos caracterizaramse com microscopia eletrônica de varredura (SEM), ensaios mecânicos de compressão e ensaios de degradação hidrolítica. As análises sobre o PHA indicaram que o material é uma mistura deste polímero e ácido poliláctico (PLA). Os scaffolds mostraram uma distribuição de poros adequada para a migração de condrócitos através deles e apresentaram um comportamento similar à cartilagem articular, mas uma menor resistência mecânica; também se encontrou que a perda de massa está relacionada com a porcentagem de PHA presente na amostra de uma forma não lineal. Em conclusão, os scaffolds de PHA têm um potencial uso na engenharia de tecidos para a restauração de cartilagem articular. COL0055049

This work deals with the thermal stability of poly[(R)-3-hydroxybutyrate] (PHB) obtained by different isolation methods. PHB was recovered from bacteria by digestion with three different reagents (acid, base, surfactant) and by chloroform extraction as a reference. Thermal stability of the samples was investigated by differential scanning calorimetry, isothermal degradation with hot press, and gel permeation chromatography and was compared with the stability of commercially available PHB. PHBs recovered by digestion with alkaline reagents were found to be thermally unstable, compared to the extracted polymer and the commercially available one. Two different techniques for stabilization of PHB obtained by digestion methods were tested wash with acid solution or addition of carboxylic acid or anhydride before heating. Both methods proved to significantly increase the thermal stability. (C) 2021 Elsevier Ltd. All rights reserved.

Despite numerous efforts to develop an effective vaccine against Pseudomonas aeruginosa, no vaccine has yet been approved for human use. This study investigates the utility of the P. aeruginosa inherently produced polyhydroxyalkanaote (PHA) inclusions and associated host–cell proteins (HCP) as a particulate vaccine platform. We further engineered PHA inclusions to display epitopes derived from the outer membrane proteins OprF/OprI/AlgE (Ag) or the type III secretion system translocator PopB. PHA and engineered PHA beads induced antigen-specific humoral, cell-mediated immune responses, anti-HCP and anti-polysaccharide Psl responses in mice. Antibodies mediated opsonophagocytic killing and serotype-independent protective immunity as shown by 100% survival upon challenge with P. aeruginosa in an acute pneumonia murine model. Vaccines were stable at 4 °C for at least one year. Overall, our data suggest that vaccination with subcellular empty PHA beads was sufficient to elicit multiple immune effectors that can prevent P. aeruginosa infection.

Escherichia coli strain LS5218 is a useful host for the production of fatty acid derived products, but the genetics underlying this utility have not been fully investigated. Here, we report the genome sequence of LS5218 and a list of large mutations and single nucleotide permutations (SNPs) relative to E. coli K-12 strain MG1655. We discuss how genetic differences may affect the physiological differences between LS5218 and MG1655. We find that LS5218 is more closely related to E. coli strain NCM3722 and suspect that small genetic differences between K-12 derived strains may have a significant impact on metabolic engineering efforts., Highlights • Escherichia coli LS5218 genome sequence is presented. • E. coli LS5218 is more closely related to E. coli NCM3722 than MG1655. • E. coli LS5218 has 17 large and 74 small differences from MG1655.

Polyhydroxyalkanoates (PHA) are a key constituent of excess sludge produced by Aerobic Sewage Sludge Treatment plants. The accumulation of significant amount of PHA inside aerobic microbial cells occurs when a surplus of an easily degradable carbon source (e.g., volatile fatty acids, VFA) is found in combination with other nutrients limitation. Herein, hydrothermal treatment (HT) of PHA-containing sludge at 300 and 375 °C was demonstrated to be effective in converting most (>70% w/w) of the bacterial PHA stored inside microbial cells into alkene/CO2 gas mixtures. Simultaneously, most of non-PHA biomass was converted into water-soluble compounds (50% carbon yield) that were acidogenic fermented to produce volatile fatty acids, ideal substrate to feed aerobic bacteria and produce more PHA. According to results here presented, HT of excess sludge with moderate (13%) PHA content can produce about 50 kg of alkenes per tonne of suspended solids treated, with a significant reduction of sludge mass (80% reduction of wet sludge volume) and consequent disposal cost.

Organo- and hydrogels have been proposed in the restoration field to treat different types of surfaces. The possibility to retain solvents and to have a controlled and superficial action allowed to use these materials for the removal of very thin layers applied on ancient historical objects, when the under paint layers are particularly delicate and water sensitive. In the last years, an increased attention has been devoted to the proposal of more healthy products to guarantee the safeguard of the operators. Few attention has been devoted to the development of green methods which foresee the use of renewable and biodegradable materials. The aim of this paper is to test a green organo-gel for the cleaning of water sensitive surfaces like varnished egg tempera paintings. The gel has been tested experimented on mock ups varnished with natural and synthetic materials and has been validated on a small portion of a Cimabue painting for the removal of two varnishes applied on two different test areas of the painting.

Agro-industry by-products are abundant in various valuable compounds. Some of these raw materials are considered as a cheaper carbon source for polyhydroxyalkanoate (PHA) production when compared with pure substrates. It is however often a costly affair for industries to recover the residual carbon components. In this study, poly(3-hydroxybutyrate) [P(3HB)] was produced using a marine Bacillus megaterium UMTKB-1 strain from sweetwater, a by-product from cane sugar refining process. The bioconversion was initiated in shaken-flasks and fermenter experiments. Applications of seawater and sweetwater mixture as well as sweetwater only as PHA culture media were investigated. The produced polymer was characterized using Gas Chromatography (GC) and Gel Permeation Chromatography (GPC). P(3HB) accumulation from tested carbon sources ranged from 3 to 49 wt%. The strain could accumulate P(3HB) in saline conditions when minimal salts medium was replaced with seawater and sweetwater. The P(3HB) content was between 7 and 27 wt%. This strain was also able to grow and accumulate up to 14 wt% P(3HB) when sweetwater was the sole PHA biosynthesis medium. The weights of P(3HB) produced was in the range 3-12 × 105 with polydispersity index values ranging from 2.7 to 3.8. The agroindustry by-products have proven to be potential carbon feedstocks for P(3HB) production. The tested strain was able to grow and accumulate P(3HB) in a novel carbon substrate medium, the sweetwater. This by-product could be used as a raw material for P(3HB) production without any pretreatment.

The need for cost-effectively produced and improved biocatalysts for industrial, pharmaceutical and environmental processes is steadily increasing. While enzyme properties themselves can be improved via protein engineering, immobilization by attachment to carrier materials remains a critical step for stabilization and process implementation. A new emerging immobilization approach, the in situ immobilization, enables simultaneous production of highly active enzymes and carrier materials using bioengineering/synthetic biology of microbial cells. In situ enzyme immobilization holds the promise of cost-effective production of highly functional immobilized biocatalysts for uses such as in bioremediation, drug synthesis, bioenergy and food processing.

Photosynthetic microorganisms can serve as the ideal hosts for the sustainable production of high-value compounds. Purple photosynthetic bacteria are typical anoxygenic photosynthetic microorganisms and are expected to be one of the suitable microorganisms for industrial production. Purple photosynthetic bacteria are reported to produce polyhydroxyalkanoate (PHA), extracellular nucleic acids and hydrogen gas. We characterized PHA production as a model compound in purple photosynthetic bacteria, especially focused on marine strains. PHA is a family of biopolyesters synthesized by a variety of microorganisms as carbon and energy storage materials. PHA have recently attracted attention as an alternative to conventional petroleum-based plastics. Production of extracellular nucleic acids have been studied in Rhodovulum sulfidophilum, a marine purple non-sulfur bacterium. Several types of artificial RNAs have been successfully produced in R. sulfidophilum. Purple photosynthetic bacteria produce hydrogen via nitrogenase, and genetic engineering strategies have been investigated to enhance the hydrogen production. This mini review describes the microbial production of these high-value compounds using purple photosynthetic bacteria as the host microorganism.

Enzymes function as biocatalysts and are extensively exploited in industrial applications. Immobilization of enzymes using support materials has been shown to improve enzyme properties, including stability and functionality in extreme conditions and recyclability in biocatalytic processing. This review focuses on the recent advances utilizing the design space of in vivo self-assembled polyhydroxyalkanoate (PHA) particles as biocatalyst immobilization scaffolds. Self-assembly of biologically active enzyme-coated PHA particles is a one-step in vivo production process, which avoids the costly and laborious in vitro chemical cross-linking of purified enzymes to separately produced support materials. The homogeneous orientation of enzymes densely coating PHA particles enhances the accessibility of catalytic sites, improving enzyme function. The PHA particle technology has been developed into a remarkable scaffolding platform for the design of cost-effective designer biocatalysts amenable toward robust industrial bioprocessing. In this review, the PHA particle technology will be compared to other biological supramolecular assembly-based technologies suitable for in vivo enzyme immobilization. Recent progress in the fabrication of biological particulate scaffolds using enzymes of industrial interest will be summarized. Additionally, we outline innovative approaches to overcome limitations of in vivo assembled PHA particles to enable fine-tuned immobilization of multiple enzymes to enhance performance in multi-step cascade reactions, such as those used in continuous flow bioprocessing.

Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient &micro, s&rsquo, and the absorption coefficient &micro, a at 638 nm. A correlation of &micro, with the cell dry weight (CDW) and &micro, a with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB&middot, L&minus, 1&middot, h&minus, 1 and a final biomass of 106 g&middot, 1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.

Sugar esters are bioactive compounds derived from renewable resources. They consist of a sugar moiety with attached non-polar part &ndash, usually a fatty acid. These compounds find uses in cosmetic, food and pharmaceutical industries as surfactants due to their physicochemical and antimicrobial activities. In this study we have produced fatty acids for sugar ester synthesis from bacterially derived polyesters, namely polyhydroxyalkanoates (PHAs). We have developed methodology to decorate PHA monomers with a fluorinated moiety. With aid of biocatalysis a series of glucose esters was created with unmodified and modified PHA monomers. All synthesised compounds showed moderate antimicrobial activity.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that a variety of microorganisms accumulate as carbon and energy storage molecules under starvation conditions in the presence of excess carbon. Anoxygenic photosynthetic bacteria exhibit a variety of growth styles and high PHA production activity. Here, we characterized PHA production by four marine purple non-sulfur bacteria strains (Rhodovulum sulfidophilum, Rhodovulum euryhalinum, Rhodovulum imhoffii, and Rhodovulum visakhapatnamense) under different growth conditions. Unlike the well-studied PHA-producing bacteria, nutrient limitation is not appropriate for PHA production in marine purple non-sulfur bacteria. We found that marine purple non-sulfur bacteria did not accumulate PHA under aerobic conditions in the presence of malate and pyruvate. Interestingly, PHA accumulation was observed upon the addition of acetate under aerobic conditions but was not observed upon the addition of reductants, suggesting that an acetate-dependent pathway is involved in PHA accumulation. Gene expression analysis revealed that the expression of isocitrate dehydrogenase in the tricarboxylic acid (TCA) cycle decreased under aerobic conditions and increased with the addition of acetate, indicating that TCA cycle activity is involved in PHA production under aerobic conditions. We also found that expression of PdhRrs, which belongs to the GntR family of transcription regulators, in Rhodovulum sulfidophilum was upregulated upon the addition of acetate. Taken together, the results show that the changes in the metabolic state upon the addition of acetate, possibly regulated by PdhR, are important for PHA production under aerobic conditions in marine purple non-sulfur bacteria.