Biocompatible PHB Production from Bacillus Species Under Submerged and Solid-State Fermentation and Extraction Through Different Downstream Processing.

Catastrophic global accumulation of non-biodegradable plastic has led to efforts for production of alternative eco-friendly biopolymer. Here, we attempted to produce a biodegradable, cytocompatible and eco-friendly polyhydroxy-butyrate (PHB) from a pigmented Bacillus sp. C1 (2013) (KF626477) through submerged (SmF) and solid-state fermentation (SSF). Under SmF and SSF, 0.60 g l-1 and 1.56 g l-1 of PHB with 0.497 g l-1 of yellow fluorescent pigment (YFP) was produced. Fourier transform infrared (FTIR) absorption bands at 1719-1720 cm-1 indicate the presence of C=O group of PHB. Nuclear magnetic resonance (NMR) exhibited the typical chemical shift patterns of PHB, and crystallinity was confirmed from X-ray diffraction (XRD). The melting temperature (Tm), degradation temperature (Td) and crystallinity (Xc) of extracted PHB were found to be 171 °C, 288 °C and 35%, respectively. FACS (Fluorescence-activated cell sorting) confirmed cytocompatibility of PHB at 400 µg ml-1 in mouse fibroblast line. Moreover, biodegradability and elevated cytocompatibility of the PHB produced through SSF make them highly potential biomaterials to be used as a drug delivery carrier in future.

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.

A simple recovery process for biodegradable plastics accumulated in cyanobacteria treated with ionic liquids.

Here, we proposed a simple recovery process for poly(3-hydroxybutyrate) (PHB) accumulated in cyanobacteria by using ionic liquids (ILs), which dissolve cyanobacteria but not PHB. First, we investigated the effects of IL polarity on hydrogen-bonding receipt ability (ß value) and hydrogen-bonding donating ability (α value) and evaluated the subsequent dissolution of cyanobacteria. We found that ILs having α values higher than approximately 0.4 and ß values of approximately 0.9 were suitable for dissolution of cyanobacteria. In particular, 1-ethyl-3-methylimidazolium methylphosphonate ([C2mim][MeO(H)PO2]) was found to dissolve cyanobacteria components, but not PHB. Thus, we verified that PHB produced in cyanobacteria could be separated and recovered by simple filtering after dissolution of cyanobacteria in [C2mim][MeO(H)PO2]. Using this technique, more than 98 % of PHB was obtained on the filter as residues separated from cyanobacteria. Furthermore, [C2mim][MeO(H)PO2] maintained the ability to dissolve cyanobacteria after a simple recycling procedure.

Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme.

Xylose, which is a major constituent of lignocellulosic biomass, was utilized for the production of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], having transparent and flexible properties. The recombinant Escherichia coli JW0885 (pflA(-)) expressing LA-polymerizing enzyme (LPE) and monomer supplying enzymes grown on xylose produced a copolymer having a higher LA fraction (34mol%) than that grown on glucose (26mol%). This benefit of xylose was further enhanced by combining it with an evolved LPE (ST/FS/QK), achieving a copolymer production having 60mol% LA from xylose, while glucose gave a 47mol% LA under the same condition. The overall carbon yields from the sugars to the polymer were similar for xylose and glucose, but the ratio of the LA and 3HB units in the copolymer was different. Notably, the P(LA-co-3HB) yield from xylose (7.3gl(-1)) was remarkably higher than that of P(3HB) (4.1gl(-1)), indicating P(LA-co-3HB) as a potent target for xylose utilization.

High production of poly(3-hydroxybutyrate) from a wild Bacillus megaterium Bolivian strain.

AIM: Taking into account that a novel strain of Bacillus megaterium was isolated from Uyuni salt lake (Bolivia) in a previous work, the objectives of this new study were to determine the maximal Poly-3-hydroxybutyrate production potential of B. megaterium strain uyuni S29 in an industrial conventional media, the possibility that the strain accumulates different types of polyhydroxyalkanoates, the cellular morphology during the biosynthesis process and the characterization of the produced biopolymers. METHODS AND RESULTS: The micro-organism was first tested in a 3-L bioreactor obtaining a high specific growth rate of 1·64 h(-1). A second fed-batch experiment was carried out in shaking flasks, reaching up to 70% PHB of cell dry mass. The biosynthesized polymers were extracted by two different extraction procedures and characterized. The results showed that all of them were PHB with thermal properties different to the conventional PHB. The micrographs taken by TEM show the different cell morphology during the fermentation process. CONCLUSIONS: In this previous study, the strain not only grew properly in the industrial conditions proposed without spore formation, but also produced and accumulated a large content of PHB, never reached before for its genus. Therefore, if the culture conditions can be optimized, the biopolymer production could be increased. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of the study has related to the area of the biomaterials and their production. The study provides new data related to the high production of PHB from the wild novel strain B. megaterium uyuni S29, the highest polymer accumulation for the genus Bacillus without spores formation.

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.

Production and characterization of PHB from two novel strains of Bacillus spp. isolated from soil and activated sludge.

The present study reports two bacteria, designated 87I and 112A, which were isolated from soil and activated sludge samples from Hyderabad, India, and that are capable of producing poly-3-hydroxybutyrate (PHB). Based on phenotypical features and genotypic investigations, these microorganisms were identified as Bacillus spp. Their optimal growth occurred between 28 degrees C and 30 degrees C and pH 7. Bacillus sp. 87I yielded a maximum of 70.04% dry cell weight (DCW) PHB in medium containing glucose as carbon source, followed by 55.5% DCW PHB in lactose-containing medium, whereas Bacillus sp. 112A produced a maximum of 67.73% PHB from glucose, 58.5% PHB from sucrose, followed by 50.5% PHB from starch as carbon substrates. The viscosity average molecular mass (M (v)) of the polymers from Bacillus sp. 87I was 513 kDa and from Bacillus sp. 112A was 521 kDa. All the properties of the biopolymers produced by the two strains 87I and 112A were characterized.

Characterization of short-chain poly3-hydroxybutyrate in baker's yeast.

A short-chain poly3-hydroxybutyrate including four comonomers, originating from a complex with calcium polyphosphate, was isolated from commercial baker's yeast cells (Saccharomyces cerevisiae) and characterized as the second complexed poly(3-hydroxyalkanoate) (cPHA) in eukaryotes. The number-average molecular weight of 4982.5 Da with a polydispersity index of 1.11 was much lower than that of beet cPHA previously isolated. End-group analysis suggested that at least 60% of the molecules form the cyclic structures. Here, the organism-dependent structural diversity of cPHAs was completely established. It was also found that a change of culture medium influences the molecular weight but not the polydispersity of baker's yeast cPHA.

Production of Poly(3-hydroxybutyrate) from waste potato starch.

There has been a considerable interest in using low cost carbon substrates for the production of Poly(3-hydroxybutyrate) (PHB). We have shown that saccharified waste potato starch can be used as a viable alternative carbon source in high cell density PHB production. Using Ralstonia eutropha NCIMB 11599 with phosphate limitation, 179 g/l biomass, 94 g/l PHB, Y(biomass/starch) = 0.46 g/g, Y(PHB/starch) = 0.22 g/g, and PHB productivity = 1.47 g/(l*h) were achieved. Residual maltose accumulated in the fed-batch reactor but caused no noticeable inhibition. Performance with saccharified starch was virtually identical to that with glucose.

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.

Large-scale production and efficient recovery of PHB with desirable material properties, from the newly characterised Bacillus cereus SPV.

A newly characterised Bacillus strain, Bacillus cereus SPV was found to produce PHB at a concentration of 38% of its dry cell weight in shaken flask cultures, using glucose as the main carbon source. Polymer production was then scaled up to 20 L batch fermentations where 29% dry cell weight of PHB was obtained within 48 h. Following this, a simple glucose feeding strategy was developed and the cells accumulated 38% dry cell weight of PHB, an increase in the overall volumetric yield by 31% compared to the batch fermentation. Sporulation is the cause of low PHB productivity from the genus Bacillus [Wu, Q., Huang, H., Hu, G.H., Chen, J., Ho, K.P., Chen, G.Q., 2001. Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media. Antonie van Leeuwenhoek 80, 111-118]. However, in this study, acidic pH conditions (4.5-5.8) completely suppress sporulation, in accordance with Kominek and Halvorson [Kominek, L.A., Halvorson, H.O., 1965. Metabolism of poly-beta-hydroxybutyrate and acetoin in Bacillus cereus. J. Bacteriol. 90, 1251-1259], and result in an increase in the yield of PHB production. This observation emphasises the potential of the use of Bacillus in the commercial production of PHB and other PHAs. The recovery of the PHB produced was optimised and the isolated polymer characterised to identify its material properties. The polymer extracted, was found to have similar molecular weight, polydispersity index and lower crystallinity index than others reported in literature. Also, the extracted polymer was found to have desirable material properties for potential tissue engineering applications.

Production of poly-beta-hydroxybutyrate (PHB) by Vibrio spp. isolated from marine environment.

Bacteria isolated from marine sediments were screened for their ability to accumulate polyhydroxyalkanoates. Among the isolates, four Vibrio spp. (strain M11, M14, M20 and M31) were studied in detail. All synthesized intracellular lipid inclusions during growth on diverse carbon sources including acetate, glycerol, succinate, glucose and sucrose. The inclusions were identified to be poly-beta-hydroxybutyrate (PHB) using gas chromatography and nuclear magnetic resonance analysis. No other type of polyhydroxyalkanoates (PHAs) was found to be accumulated by these marine isolates, suggesting that the diversity of PHAs produced in marine environments may be not as versatile as found in other environments. Strain M11 accumulated PHB in concentrations as high as 41% of cell dry weight when grown in medium containing 4% of sodium chloride. One of the Vibrio spp. was identified to be closely related to Vibrio natriegens (98% identity) by partial 16S rDNA sequence homology. V. natriegens has the shortest generation time (9.8 min) of any bacterium and this characteristic may be an exploitable trait for the industrial production of PHB.

Enzymatic recovery and purification of polyhydroxybutyrate produced by Ralstonia eutropha.

Polyhydroxybutyrate (PHB) is the most studied among a wide variety of polyhydroxyalkanoates, bacterial biodegradable polymers known as potential substitutes for conventional plastics. This work aimed at evaluating the use of enzymes to recover and purify the PHB produced by Ralstonia eutropha DSM545. Screening experiments allowed the selection of trypsin, bromelain and lysozyme among six enzymes, based on their efficiency in lysing cells of a non-PHB producing R. eutropha strain. Then, process conditions for high efficiency in PHB purification from the DSM545 cells were searched for the enzymes previously selected. The best result was achieved with 2.0% of bromelain (enzyme mass per biomass), equivalent to 14.1 U ml(-1), at 50 degrees C and pH 9.0, resulting in 88.8% PHB purity. Aiming at improving the process efficiency and reducing the enzyme cost, experiments were carried out with pancreatin, leading to 90.0% polymer purity and an enzyme cost three times lower than the one obtained with bromelain. The molecular mass analysis of PHB showed no polymer degradation. Therefore, this work demonstrates the potential of using enzymes in order to recover and purify PHB and bacterial biopolymers in general.

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.

Intracellular delivery of peptide cargos using polyhydroxybutyrate based biodegradable nanoparticles: Studies on antitumor efficacy of BCL-2 converting peptide, NuBCP-9.

Faster biodegradation, immunogenicity and lack of cell penetrative capabilities are hurdles in development of peptidyl drugs for cancer therapy. Polymeric carriers can be used to overcome these problems. The present study is focused on the use of polyhydroxybutyrate as a potential nanovehicle for the delivery of anticancer peptides. PHB (72kDa) was produced by thermal treatment of high molecular weight PHB (300kDa) under melt conditions and then conjugated with PEG (4kDa) by Steglich esterification reaction. Anticancer peptide NuBCP-9 (FSRSLHSLL) encapsulated PHB(72K)-PEG(4K) NPs were prepared by double emulsion solvent evaporation method. PHB(72K)-PEG(4K) NPs showed encapsulation efficiency of 61% and exhibited sustained release of peptide over a period of 26days at physiological pH. NuBCP-9 loaded PHB(72K)-PEG(4K) NPs showed an IC50 value of 2.2µM & 1.6µM in MCF-7 cells in 48h and 72h respectively. Confocal laser microscopy confirmed efficient cellular uptake and induction of apoptosis by peptide loaded NPs in a time dependent manner. In vivo intraperitonial administration of 20mg/kg NuBCP-9/NPs twice a week for three weeks triggered 90% tumor regression in Ehrlich syngeneic mouse model. Our results illustrated the potential of PHB(72K)-PEG(4K) based nanoformulation as a tool for targeting intracellular proteins.

Surface-modified bacterial nanofibrillar PHB scaffolds for bladder tissue repair.

The aim of the study is in vitro investigation of the feasibility of surface-modified bacterial nanofibrous poly [(R)-3-hydroxybutyrate] (PHB) graft for bladder reconstruction. In this study, the surface of electrospun bacterial PHB was modified with PEG- or EDA via radio frequency glow discharge method. After plasma modification, contact angle of EDA-modified PHB scaffolds decreased from 110 ± 1.50 to 23 ± 0.5 degree. Interestingly, less calcium oxalate stone deposition was observed on modified PHB scaffolds compared to that of non-modified group. Results of this study show that surface-modified scaffolds not only inhibited calcium oxalate growth but also enhanced the uroepithelial cell viability and proliferation.

Isolation and structure determination of complexed poly(3-hydroxyalkanoate) from beet (Beta vulgaris L.).

Complexed poly(3-hydroxyalkanoate)s (cPHAs), one of two types of natural PHAs, occur in both prokaryotes and eukaryotes as a complex with biomacromolecules and could be involved in various physiological functions. In this study, a cPHA-component derived from a complex with calcium polyphosphate was isolated from sugar beet (Beta vulgaris L.) and determined to be a homopolymer composed of 3-hydroxybutyrate. MALDI MS provided the number-average molecular weight (Mn = 9,124 Da) and polydispersity index (PDI = 1.01), showing that beet cPHA has a slightly lower molecular mass than the known Escherichia coli cPHA. In addition, the structural analysis of both end groups showed that (i) 100 mol-% of the carboxyl end is free, while about 30 mol-% of the hydroxyl end is free and about 70 mol-% masked and (ii) the end hydroxyl group is masked by at least six identified short-chain alkanoic and alkanedioic acids. Based on such end-group characteristics, the polymerization mechanism of beet cPHA is discussed.

Accumulation of poly-beta-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources.

Accumulation of poly-beta-hydroxybutyrate (PHB) in Nostoc muscorum was studied. Cells harvested at stationary phase of growth depicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. In contrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such as NH(4)Cl and KNO(3), were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbon sources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accumulation in N. muscorum was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected to dark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniques to improve the expression level of PHB photoproduction in cyanobacteria.

Synthesis of polyhydroxyalkanoate (PHA) from excess activated sludge under various oxidation-reduction potentials (ORP) by using acetate and propionate as carbon sources.

Accumulation of poly hydroxyalkanoate (PHA) from excess activated sludge (EAS) was monitored and controlled via the oxidation-reduction potential (ORP) adjusting process. The ORP was adjusted and controlled by only regulating the gas-flow rate pumped into the cultural broth in which sodium acetate (C2) and propionate (C3) were used as carbon sources. Productivity of PHA and the PHA compositions at various C2 to C3 ratios were also investigated. When ORP was maintained at +30 mV, 35% (w/w) of PHA of cell dry weight obtained when C2 was used as sole carbon source. The PHA copolymer, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), accumulated by EAS with different 3-hydroxyvalarate (3HV) molar fractions ranged from 8% to 78.0% when C2 and C3 was used as sole carbon source, By using ORP to monitor and control the fermentation process instead DO meter, the ORP system provided more precise control to the PHA accumulation process from EAS under low dissolved oxygen (DO) concentrations. Adjusting the C2 to C3 ratios in the media could control the composition such as the 3HV/3HB ratios of the PHBV. Furthermore, it might be an effective way to adjust the 3HV molar fractions in PHBV by controlling the DO concentration via the ORP monitoring system. The 3HV molar fractions in the PHBV declined with increasing ORP from -30 mV to +100 mV by adjusting the gas-flow rate (i.e. the DO concentration). It is concluded that the DO plays a very important role in the synthesis of 3HV subunits in PHBV co-polymer from the EAS. Therefore, a hypothetic metabolic model for PHA synthesis from EAS was proposed to try to explain the results in this study.