Production of eco-friendly PHB-based bioplastics by Pseudomonas aeruginosa CWS2020 isolate using poultry (chicken feather) waste.

Nowadays, the accumulation of non-degradable plastics and other disposed wastes leads to environmental pollution across the world. The production of eco-friendly and cost-effective poly-ß-hydroxybutyrate (PHB) could be a better alternative to conventional petroleum-based plastics and prevent environmental pollution. Besides, the area in and around Namakkal, Tamil Nadu, India is well known for poultries, currently facing the number of environmental issues due to the accumulation of chicken feather waste. This study focused on the production of eco-friendly PHB by recycling poultry (chicken feather) waste as the substrate. The native PHB producers were screened from the chicken waste disposal site in Namakkal by Sudan black B staining method. Further, the potent bacterial isolate was identified as Pseudomonas aeruginosa (NCBI accession MF18889) by phenotypic and genotypic characteristics. The PHB production media with chicken feather waste was statistically optimized by response surface methodology. The dry weight of PHB produced under optimized condition (15.96 g/L chicken feather waste, 37 °C temperature, 19.8 g/L glucose and 6.85 pH) was found to be 4.8 g/L. Besides, PHB was characterized and confirmed by thin-layer chromatography, Fourier-transform infrared spectroscopy and Gas chromatography-mass spectrometry analysis. Thus, this study concludes that poultry waste could be a complex nitrogen source for improving the growth of PHB producers and substantially increasing the yield of PHB, and it will be an eco-friendly and low-cost production in bioprocess technology.

Analysis of PBT and PET cyclic oligomers in extracts of coffee capsules and food simulants by a HPLC-UV/FLD method.

A HPLC-UV/FLD method was validated for the quantification of six polyethylene terephthalate (PET) and four polybutylene terephthalate (PBT) oligomers. PBT oligomers are EU regulated, while the PET ones are considered non-intentionally added substances (NIAS). LOQs were higher than 0.4 and 3.5 µg kg-1 for the simulants and in the polymer extracts, respectively. Recoveries ranged from 95 to 114 % with RSDs below 12%. Migration testing of PBT and polypropylene coffee capsules were performed with H2O and simulant C, and extracts were obtained with accelerated solvent extraction (ASE). For the latter legislative limits weren't surpassed. As no migration limits are existing for the analytes, both EFSA's toxicological threshold of concern (TTC) and sum of oligomers approaches were applied. The majority of oligomers were below the TTC (90 µg/person/day), but the limit value of 50 µg/kg food was surpassed for some capsules, which indicates a significant intake in both single and multiple consumption.

Factors affecting microplastic retention and emission by a wastewater treatment plant on the southern coast of Caspian Sea.

Understanding how wastewater treatment plants (WWTPs) process microplastics (MPs) will help informing management practices to reduce MP emissions to the environment. We show that composite 24 h samples taken at three replications from the outflow of the grit chamber, primary settling tank and clarifier of the WWTP of Sari City, on the southern coast of the Caspian Sea, contained 12667 ± 668, 3514 ± 543 and 423 ± 44.9 MP/m3, respectively. Fibers accounted for 94.9%, 89.9% and 77.5% of the total number of MPs, respectively. The MP removal efficiency was 96.7%. MP shape (fiber, particle), size and structure were the most important factors determining their removal in different steps of the wastewater treatment process. The structure of microfibers (polyester, acrylic and nylon) and the consequent higher density than water explained their high removal (72.3%) in the primary settling tank. However, size was more important in microparticle removal with particles ≥500 µm being removed in the primary settling tank and <500 µm in the clarifier unit. The smallest particles (37-300 µm) showed the lowest removal efficiency. The predominant types of fibers and particles were polyester and polyethylene, respectively, which are likely to originate from the washing of synthetic textiles and from microbeads in toothpaste and cosmetics. Despite the efficiency of the Sari WWTP in removing MPs, it remains a major emission source of MPs to the Caspian Sea due to its high daily discharge load.

An untargeted liquid chromatography-mass spectrometry-based workflow for the structural characterization of plant polyesters.

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant-specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography-mass spectrometry (LC-MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC-MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi-stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo-esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.

Preparation and characterization of semi-IPNs of polycaprolactone/poly (acrylic acid)/cellulosic nanowhisker as artificial articular cartilage.

Cartilage is a semi-solid resilient and smooth elastic connective tissue and upon damage, its repair is almost impossible or occurs with a very slow recovery process. Polycaprolactone (PCL), used as a biocompatible polymer, withholds all required mechanical properties, except suitable cell adhesion due to its hydrophobicity. In order to resolve this issue, we sought to introduce appropriate semi-IPNs into the system to regain its hydrophilicity base on increasing of the hydrophilic polymer. PCL and Cellulose nanowhiskers (CNWs) were entrapped in a network of poly (acrylic acid) that had been crosslinked via a novel acrylic-urethane crosslinker. The influential synthetic parameters on the preparation of artificial articular cartilages were investigated based on the Taguchi test design. The prepared CNW, acrylic-urethane crosslinker and semi-IPNs were studied via 1H NMR, FTIR, SEM, TEM, TGA, water swelling, water contact angle, tensile, and MTT analyses. According to the results, the optimal amount of monomer was about 46%. Incorporation of an optimized amount of CNW, which was 0.5%, improved the mechanical properties of artificial cartilage. After a 30 h time period, semi-IPNs showed the water absorption of about 30%. MTT on days 1, 3 and 5, as well as cell attachment, confirmed the biocompatibility of the semi-IPNs.

Production and recovery of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from biodiesel liquid waste (BLW).

Polyhydroxyalkanoates (PHAs) has been paid great attention because of its useful thermoplastic properties and complete degradation in various natural environments. But, at industrial level, the successful commercialization of PHAs is limited by the high production cost due to the expensive carbon source and recovery processes. Pseudomonas mendocina PSU cultured for 72 h in mineral salts medium (MSM) containing 2% (v/v) biodiesel liquid waste (BLW) produced 79.7 wt% poly(3-hydroxybutyrate) (PHB) at 72 h. In addition, this strain produced 43.6 wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 8.6 HV mol% at 60 h when added with 0.3% sodium propionate. The synthesized intracellular PHA granules were recovered and purified by the recently reported biological method using mealworms. The weight average molecular weight (Mw ) and number average molecular weight (Mn ) of the biologically extracted PHA were higher than that from the chloroform extraction with comparable melting temperature (Tm ) and high purity. This study has successfully established a low-cost process to synthesize PHAs from BLW and subsequently confirmed the ability of mealworms to extract PHAs from various kinds of bacterial cells.

Assessment of poly(3-hydroxybutyrate) synthesis from a novel obligate alkaliphilic Bacillus marmarensis and generation of its composite scaffold via electrospinning.

In this study, poly(3-hydroxybutyrate) (PHB) production from a newly isolated obligate alkaliphilic Bacillus marmarensis DSM 21297 was investigated to evaluate the ability of obligate alkaliphilic strain to produce a biopolymer. Additionally, electrospun nanofibers from B. marmarensis PHB (Bm-PHB) were generated using Bm-PHB/polycaprolactone (PCL) blend to evaluate the applicability of Bm-PHB. According to the experimental results, the metabolic activity of B. marmarensis decreased the pH of the medium by generating H+ ions to initiate Bm-PHB production, which was achieved at pH below 9.0. Regarding medium components, the addition of MgSO4.7H2O and KH2PO4 to the medium containing 1% glucose enhanced the amount of Bm-PHB synthesis, and an approximately 60% increase in PHB concentration was obtained in the presence of mineral salts. Based on FTIR analysis, the chemical structures of Bm-PHB and commercial PHB were found to be highly similar. Additionally, the Tg and Tm values of Bm-PHB were determined to be 17.77 °C and 165.17 °C, respectively. Moreover, Bm-PHB/PCL composite scaffold was generated by electrospinning method that produced nanofibers between 150 and 400 nm in diameter, with an average of 250 nm. To our knowledge, this is the first report to produce PHB from an obligate alkaliphilic Bacillus strain and PHB scaffold.

Exploring multi potential uses of marine bacteria; an integrated approach for PHB production, PAHs and polyethylene biodegradation.

There are copious of bacteria exist in marine environment and it is very important to screen the potential microbes that has the ability to produce biopolymer polyhydroxybutyrate (PHB) as well as polycyclic aromatic hydrocarbons (PAHs) degradation and conventional plastic high density polyethylene (HDPE) biodegradation. Numerous studies have been investigated individually on either one of characteristic feature like PHB production, PAHs and high density polyethylene (HDPE) degradation, but not all together. Hence, in this study, we tried to screen potential marine microbes that have the ability to perform all three features. We have isolated 203 phenotyphicaly different colonies from 19 different sites (marine soil sediments, marine water and oil spilled marine water) which cover the north east to down south seashore regions of Tamilnadu, India. Of the 203 microbial isolates, the best PHB producing (Micrococcus luteus), PAHs degradation (Klebsiella pneumonia) and HDPE degradation (Brevibacillus borstelensis) microorganisms were identified through 16S rRNA sequencing. Analytical studies confirmed PHB production by fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (1H &13C NMR); PAHs degradation by high performance liquid chromatography (HPLC), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM); HDPE degradation by CLSM, FT-IR and SEM which cover the spectroscopy studies on biological systems.

A novel autolysis system controlled by magnesium and its application to poly (3-hydroxypropionate) production in engineered Escherichia coli.

The release of intracellular products, especially polyhydroxyalkanoates, is still a great challenge in industry. To solve this bottleneck, a novel autolysis system strictly controlled with magnesium was constructed and applied to poly(3-hydroxypropionate) production in engineered Escherichia coli. The autolysis system was constructed by inserting the 5'untranslated region (5'UTR) behind promoter PmgtA with lysis genes (S, R, and Rz, from E. coli) overexpressed. The autolysis system functioned well (lysis efficiency of more than 90%) in the P3HP producer with double plasmids containing lysis genes and P3HP biosynthesis genes, whereas the P3HP production was reduced due to plasmid losses. After the autolysis genes and P3HP biosynthesis genes were integrated into one plasmid, the P3HP content of 72.7% (2.4 times of the control) and the plasmid stability of 79.8 ± 3.1% were achieved in strain Q2646 with promoter PmgtA-UTR. However, the strain Q2647 with promoter PmgtA could not accumulate P3HP because of rapid cell lysis. The novel autolysis system activated in Mg2+-depleted conditions proves to be feasible for polyhydroxyalkanoates production, which may have great application potential for other intracellular products.

Improving metabolite production in microbial co-cultures using a spatially constrained hydrogel.

Microbial co-cultures promise the development of more efficient bioproductions. However, the design of obligate mutualisms is complicated when using organisms that possess differing growth rates or incompatible media requirements. In this work, we investigate sucrose production by cscB Synechococcus elongatus PCC 7942 within a polyacrylate hydrogel matrix. This system secretes sucrose only when the hydrogel is spatially constrained, demonstrating a new utilization of hydrogel swelling pressure to control the osmotic strength of a microbial microenvironment. The sucrose produced via the constrained microbial hydrogel is used to grow the diazotrophic organism, Azotobacter vinelandii, in a mutually dependent fashion. The growth of this hydrogel-based coculture has several advantages over batch cultures, including better growth over a longer period of time and decreased salt stress on A. vinelandii. Biotechnol. Bioeng. 2017;114: 1195-1200. © 2016 Wiley Periodicals, Inc.

Processing and size range separation of pristine and magnetic poly(l-lactic acid) based microspheres for biomedical applications.

Biodegradable poly(l-lactic acid) (PLLA) and PLLA/CoFe2O4 magnetic microspheres with average sizes ranging between 0.16-3.9µm and 0.8-2.2µm, respectively, were obtained by an oil-in-water emulsion method using poly(vinyl alcohol) (PVA) solution as the emulsifier agent. The separation of the microspheres in different size ranges was then performed by centrifugation and the colloidal stability assessed at different pH values. Neat PLLA spheres are more stable in alkaline environments when compared to magnetic microspheres, both types being stable for pHs higher than 4, resulting in a colloidal suspension. On the other hand, in acidic environments the microspheres tend to form aggregates. The neat PLLA microspheres show a degree of crystallinity of 40% whereas the composite ones are nearly amorphous (17%). Finally, the biocompatibility was assessed by cell viability studies with MC3T3-E1 pre-osteoblast cells.

Dihydro-ß-agarofuran sesquiterpene polyesters isolated from the stems of Tripterygium regelii.

Twelve new dihydro-ß-agarofuran polyesters, triptregelines A-J (1-3 and 5-11) and triptregelols A, B (4, 12), together with five known ones (13-17) were isolated from the stems of Tripterygium regelii. The structures were determined on the basis of extensive analysis of spectroscopic data (1D, 2D NMR, and UV etc.) and HRMS data. Cytotoxic effects of these compounds were evaluated against a pair of cancer cell lines, A549 and taxol-resistant A549T. Triptofordin B (14) showed cytotoxicity against both A549 and A549T cells with IC50 value of 21.2 and 10.8µM, respectively. In addition, triptregeline B (2), triptregeline C (3), triptregeline H (9) and 1α, 6ß, 15-triacetoxy-8α-benzoyloxy-4ß-hydroxy-9α-(3-nicotinoyloxy)-dihydro-ß-agarofuran (17) exhibited weak cytotoxic effects on taxol-resistant A549T with IC50 values ranged from 29.4 to 54.4µM. The cytotoxic effect of triptofordin B (14) was much less than taxol with IC50 value of 0.08µM on A549 cancer cell.

Effective recovery of poly-ß-hydroxybutyrate (PHB) biopolymer from Cupriavidus necator using a novel and environmentally friendly solvent system.

This work demonstrates a significant advance in bioprocessing for a high-melting lipid polymer. A novel and environmental friendly solvent mixture, acetone/ethanol/propylene carbonate (A/E/P, 1:1:1 v/v/v) was identified for extracting poly-hydroxybutyrate (PHB), a high-value biopolymer, from Cupriavidus necator. A set of solubility curves of PHB in various solvents was established. PHB recovery of 85% and purity of 92% were obtained from defatted dry biomass (DDB) using A/E/P. This solvent mixture is compatible with water, and from non-defatted wet biomass, PHB recovery of 83% and purity of 90% were achieved. Water and hexane were evaluated as anti-solvents to assist PHB precipitation, and hexane improved recovery of PHB from biomass to 92% and the purity to 93%. A scale-up extraction and separation reactor was designed, built and successfully tested. Properties of PHB recovered were not significantly affected by the extraction solvent and conditions, as shown by average molecular weight (1.4 × 10(6) ) and melting point (175.2°C) not being different from PHB extracted using chloroform. Therefore, this biorenewable solvent system was effective and versatile for extracting PHB biopolymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:678-685, 2016.

Unexpected talaroenamine derivatives and an undescribed polyester from the fungus Talaromyces stipitatus ATCC10500.

Chemical investigation of the fungus Talaromyces stipitatus ATCC 10500, whose genome has been sequenced, led to the isolation of four undescribed talaroenamines B-E along with the known talaroenamine A. Their structures were elucidated on the basis of spectroscopic studies including mass spectrometry, extensive 2D NMR and electronic circular dichroism (ECD). Interestingly, talaroenamine A had previously been isolated from the strain of T. stipitatus Δtrop C, a strain knocked out for the gene encoding a non-heme Fe(II)-dependent dioxygenase catalyzing the oxidative ring expansion leading to the tropolone, but never from a wild-type strain. All talaroenamines were evaluated for their antiplasmodial activity and Talaroenamine D exhibited the best inhibition against the chloroquine-resistant Plasmodium falciparum (FcB1 strain) without noticeable toxicity on HeLa and preadipose cell lines. In the course of the chemical investigation of T. stipitatus, an undescribed polyester was also isolated and its absolute configuration was determined by hydrolysis and transesterification followed by gas chromatography on chiral column.

An Environmentally Friendly and Efficient Method for Extraction of PHB Biopolymer with Non-Halogenated Solvents.

The present study developed an efficient and environmentally friendly method for recovering polyhydroxybutyrate (PHB) from Cupriavidus necator. Several non-halogenated solvents were tested and it was found that butyl acetate and ethyl acetate are powerful solvents for the biopolymer. Testing was performed to examine the effects of temperature (25 °C until temperature below solvent boiling points) and heating incubation time (0-60 min) on the two solvents. Butyl acetate had a higher recovery level (96%) and product purity (up to 99%) than ethyl acetate at 103 °C and a heating incubation time of 30 min. Under these conditions, PHB recorded the highest molecular weight of 1.4 × 10(6) compared with the standard procedure (i.e., recovery using chloroform). The proposed strategy showed that butyl acetate is a good alternative to halogenated solvents such as chloroform for recovery of PHB.

Poly-3-hydroxybutyrate (PHB) production from alkylphenols, mono and poly-aromatic hydrocarbons using Bacillus sp. CYR1: A new strategy for wealth from waste.

In the present study five different types of alkylphenols, each of the two different types of mono and poly-aromatic hydrocarbons were selected for degradation, and conversion into poly-3-hydroxybutyrate (PHB) using the Bacillus sp. CYR1. Strain CYR1 showed growth with various toxic organic compounds. Degradation pattern of all the organic compounds at 100 mg/l concentration with or without addition of tween-80 were analyzed using high pressure liquid chromatography (HPLC). Strain CYR1 showed good removal of compounds in the presence of tween-80 within 3 days, but it took 6 days without addition of tween-80. Strain CYR1 showed highest PHB production with phenol (51 ± 5%), naphthalene (42 ± 4%), 4-chlorophenol (32 ± 3%) and 4-nonylphenol (29 ± 3%). The functional groups, structure, and thermal properties of the produced PHB were analyzed. These results denoted that the strain Bacillus sp. CYR1 can be used for conversion of different toxic compounds persistent in wastewaters into useable biological polyesters.

Effective production of low crystallinity Poly(3-hydroxybutyrate) by recombinant E. coli strain JM109 using crude glycerol as sole carbon source.

Utilization of bio-diesel by-products (glycerol) for microbial polymer production has created a novel biorefinery concept. In the present study, recombinant Escherichia coli JM109 was used for the production of P(3 HB) from glycerol as carbon source. Batch fermentation in a 7.5L bioreactor with the statistically optimized culture condition (pre-treated glycerol: 27.5 g/L and casein hydrolysate: 5.25 g/L) scaled up the P3HB production to 65% (∼ 8 g/L). FTIR, (1)H and (13)C NMR analysis proved the polymer produced to be P(3 HB). Gel permeation chromatography, Differential Scanning Calorimetry (DSC) and thermogravimetric analysis (TGA) demonstrated the produced P(3 HB) to have high molecular weight (2.84 × 10(6)) and lowered crystallinity (∼ 30%) compared to commercial polymer. Integrating the production efficiency and the thermal characteristics of the polymer produced by recombinant E. coli, the viability and sustainability of biofuels and biopolymers for economic human need could be enhanced.

Influence of nutritional and physicochemical variables on PHB production from raw glycerol obtained from a Colombian biodiesel plant by a wild-type Bacillus megaterium strain.

Biodegradable polymers are currently viable alternatives to traditional synthetic polymers. For instance, polyhydroxybutyrate (PHB) is intracellularly produced and accumulated by Bacillus species, among others. This study reports several wild-type Bacillus strains with the ability to accumulate PHB using raw glycerol from biodiesel production as the sole carbon source. Out of 15 strains from different sources, B. megaterium B2 was selected as the most promising strain for further statistical optimization of the medium composition. Plackett-Burman and central composite designs were used to establish key variables and optimal culture conditions for PHB production using both 250-mL shake flasks and a 7.5-L bioreactor. Temperature and concentrations of glycerol and Na2HPO4 are the experimental variables with the most significant influence on PHB production by B2. After 14 hours of fermentation in shake flasks with optimized medium, B2 produced 0.43 g/L of PHB with a 34% accumulation in the cells. In contrast, under the same conditions, a maximum PHB concentration of 1.20 g/L in the bioreactor was reached at 11 hours. These values correspond to a 48% and 314% increase in PHB production compared to the initial culture conditions. These results suggest the potential of B2 as a PHB producer using raw glycerol, which is an inexpensive, abundant and readily available carbon source.

A new myrsinol-type diterpene polyester from Euphorbia dracunculoides Lam.

A new myrsinol-type diterpene polyester, 14-deoxo-3ß-O-propinoyl-2α,5α,7ß,15ß-tetra-O-acetyl-14α-O-benzoyl-myrsinol (1), and its known analogue, 14-deoxo-3ß-O-prorionyl-5α,15ß-di-O-acetyl-7ß-O-nicotinoyl-myrsinol-14ß-acetate (2), together with a monoterpenoid, pubinernoid A (3), two indole alkaloids, neoechinulin A (4) and dihydroxyisoechinulin A (5), two benzene derivatives, siringin (6) and (3-methoxyphenyl) acetic acid (7), were isolated from the 70% acetone extract of the aerial parts of Euphorbia dracunculoides Lam. Their structures were elucidated on the basis of spectroscopic evidence and comparison with literature reports. The absolute configuration of 1 was deduced by comparing experimental and calculated ECD spectra. Among them, compounds 4 and 5 were first obtained from the plant source. In addition, the (13)C NMR data of compound 2 was reported for the first time.