Increased sedimentation of a Pseudomonas–Saccharomyces microbial consortium producing medium chain length polyhydroxyalkanoates

Concerns about feasibility,separability,settleability,efficiency once hampered studies on polyhydroxyalkanoates(PHAs)production,which mainly focused on single strain microorganism or activated sludge rather than artificial microbial consortia.Here,a medium chain length PHAs(mcl-PHAs)producing Pseudomonas-Saccharomyces consortium with xylose as the main substrate was studied.Mcl-PHAs accumulation increased from 12.69 mg·L^-1 to 152.3 mg·L^-1 without any optimization method.The presence of Saccharomyces cerevisiae,though in a relatively low concentration,improved the sedimentation of cell mass of the mixed culture by 60%.Reasons for better sedimentation of the consortium were complex:first,the length of Pseudomonas putida increased two to three times in the consortium;second,the positive surface charge of P.putida was neutralized by S.cerevisiae;third,the adhesion proteins on the surface of S.cerevisiae interacted with the P.putida.

Blend of Polyhydroxyalkanoates Synthesized By Lipase Positive Bacteria From Plant Oils

A total of 5 biochemically characterized lipase positive bacterial strains were screened for Polyhydroxyalkanoates(PHA)production by Nile blue staining and confirmation was done by Sudan Black B.PHA production ability for all strains was optimized followed by time profiling calculation and comparison via using glucose and two plant oils i.e.,canola and mustard oil.Quantitative analysis showed that glucose can serve as a carbon source for maximum biomass(2.5 g/L CDW for strain 5)and PHA production(70.3%for strain 2).PHA produced by strain 2 was further analyzed for its chemical composition and type via Fourier Transform Infrared(FT-IR)spectroscopy.It revealed homopolymer(PHB)and copolymer(PHB-co-PHV)production of PHA(peaks at 1743 cm-1 and 2861 cm-1,respectively)with both canola and mustard oil unlike glucose which produced only homopolymer one i.e.,PHB(peaks at 1110 cm-1,1411 cm-1 and 1650 cm-1).Crystallinity of FT-IR analyzed PHA was calculated using mathematical formulas which showed decrease from glucose to canola to mustard oil.This study revealed that plant oils can serve as better carbon source to produce better quality(ductile and copolymer)PHA.Moreover,16S rRNA gene sequencing analysis showed that strain 1,strain 2,strain 3,strain 4 and strain 5 are Stenotrophomonas sp.N3,Exiguobacterium sp.N4,Exiguobacterium sp.Ch3,Cellulosimicrobium sp.A8 and Klebsiella sp.LFSM2,respectively.

Creation of High-Yield Polyhydroxyalkanoates Engineered Strains by Low Energy Ion Implantation

Polyhydroxyalkanoates （PHAs）, as a candidate for biodegradable plastic materials, can be synthesized by numerous microorganisms. However, as its production cost is high in comparison with those of chemically synthesized plastics, a lot of research has been focused on the efficient production of PHAs using different methods. In the present study, the mutation effects of PHAs production in strain pCB4 were investigated with implantation of low energy ions. It was found that under the implantation conditions of 7.8×10^14 N^＋/cm^2 at 10 keV, a high-yield PHAs strain with high genetic stability was generated from many mutants. After optimizing its fermentation conditions, the biomass, PHAs concentration and PHAs content of pCBH4 reached 2.26 g/L, 1.81 g/L, and 80.08% respectively, whereas its wild type controls were about 1.24 g/L, 0.61 g/L, and 49.20%. Moreover, the main constituent of PHAs was identified as poly-3-hydroxybutyrates （PHB） in the mutant stain and the yield of this compound was increased up to 41.33% in contrast to that of 27.78% in the wild type strain.

Characterization of polyhydroxyalkanoates synthesized in activated sludge using three carbon sources

Polyhydroxyalkanoates（PHAs）were synthesized in activated sludge using three types of carbon sources（sodium acetate,sodium propionate and sodium butyrate）,and their characterization were studied.It was shown that the content of PHA synthesized by microorganisms in activated sludge were different.The biggest synthetic amount up to 36.7% of VSS was obtained when sodium acetate was used as carbon source.The polymer yield was lower when using sodium propionate as the carbon source than when using others,with25.1% of VSS,while resulted in an increase of hydroxyvalerate（HV）units produced.The structure and thermal properties of extracted biopolymers were analyzed by scanning electron microscope（SEM）,Fourier transform infrared spectroscopy（FTIR）and themogravimetry（TG）.

Measuring methods for three kinds of polyhydroxyalkanoates from activated sludge by gas chromatography

Gas chromatography determination of polyhydroxyalkanoates has been common;however,the pretreatment steps are often complex,and gas chromatography operation conditions are not given in detail.In this study,gas chromatography is used for analyzing PHB,PHV and PH2MV,three majors of PHAs in activated sludge.The sample was centrifuged at a speed of 4000 r/min for the separation of floc and supernatant,freezen,and dried for 12 h in vacuum freezing drier;and then transferred to the fridge for freezing to ice and drying for 12 h in vacuum freezing drier;then chloroform and a simple composition digestion solution including methanol,sulfuric acid and benzoic acid was added;digested at 105 ℃ for 6 h;cooled to room temperature,the lower solution of the result can be used for analyzing.Samples were analyzed by gas chromatography with FID detector and auto sampler;the standard curve of standard material shows an excellent linear relationship with correlation coefficients larger than 0.99;the relative standard deviation (RSD) of sludge samples is less than 1%.The recovery rates of each sample are between 95%-105%.The GC analysis time of each PHA sample is shorter than 10 minutes.

FUNCTIONAL POLYHYDROXYALKANOATES SYNTHESIZED BY MICROORGANISMS

Many bacteria have been found to synthesize a family of polyesters termed polyhydroxyalkanoate, abbreviated as PHA. Some interesting physical properties of PHAs such as piezoelectricity, non-linear optical activity, biocompatibility and biodegradability offer promising applications in areas such as degradable packaging, tissue engineering and drug delivery. Over 90 PHAs with various structure variations have been reported and the number is still increasing. The mechanical property of PHAs changes from brittle to flexible to elastic, depending on the side-chainlength of PHA. Many attempts have been made to produce PHAs as biodegradable plastics using various microorganisms obtained from screening natural environments, genetic engineering and mutation. Due to the high production cost, PHAs still can not compete with the nondegradable plastics, such as polyethylene and polypropylene. Various processes have been developed using low cost raw materials for fermentation and an inorganic extraction process for PHA purification. However, a super PHA production strain may play the most critical role for any large-scale PHA production. Our recent study showed that PHA synthesis is a common phenomenon among bacteria inhabiting various locations, especially oil-contaminated soils. This is very important for finding a suitable bacterial strain for PHA production. In fact, PHA production strains capable of rapid growth and rapid PHA synthesis on cheap molasses substrate have been found on molasses contaminated soils. A combination of novel properties and lower cost will allow easier commercialization of PHA for many applications.

Hydrogel forming microneedles loaded with VEGF and Ritlecitinib/polyhydroxyalkanoates nanoparticles for mini-invasive androgenetic alopecia treatment

Androgenetic alopecia(AGA),the most prevalent clinical hair loss,lacks safe and effective treatments due to downregulated angiogenic genes and insufficient vascularization in the perifollicular microenvironment of the bald scalp in AGA patients.In this study,a hyaluronic acid(HA)based hydrogel-formed microneedle(MN)was designed,referred to as V-R-MNs,which was simultaneously loaded with vascular endothelial growth factor(VEGF)and the novel hair loss drug Ritlecitinib,the latter is encapsulated in slowly biodegradable polyhydroxyalkanoates(PHAs)nanoparticles(R-PHA NPs)for minimally invasive AGA treatment.The integration of HA based hydrogel alongside PHA nanoparticles significantly bolstered the mechanical characteristics of microneedles and enhanced skin penetration efficiency.Due to the biosafety,mechanical strength,and controlled degradation properties of HA hydrogel formed microneedles,V-R-MNs can effectively penetrate the skin’s stratum corneum,facilitating the direct delivery of VEGF and Ritlecitinib in a minimally invasive,painless and long-term sustained release manner.V-R-MNs not only promoted angiogenesis and improve the immune microenvironment around the hair follicle to promote the proliferation and development of hair follicle cells,but also the application of MNs to the skin to produce certain mechanical stimulation could also promote angiogenesis.In comparison to the clinical drug minoxidil for AGA treatment,the hair regeneration effect of V-R-MN in AGA model mice is characterized by a rapid onset of the anagen phase,improved hair quality,and greater coverage.This introduces a new,clinically safer,and more efficient strategy for AGA treatment,and serving as a reference for the treatment of other related diseases.

Tailoring the Properties of Polyhydroxyalkanoates from Plastics to Elastomers via Stereoselective Copolymerizations of rac-β-Butyrolactone and β-Propiolactone

Poly(3-hydroxybutyrate),a crucial member of the large biodegradable polyhydroxyalkanoate family,suffers from its brittleness.To enhance its performance,we employed a straightforward approach involving the ring-opening copolymerization of racemic-β-butyrolactone(rac-β-BL)andβ-propiolactone(β-PL)using the syndio-selective amino-alkoxy-bis(phenolate)-yttrium complex as a catalyst,thanks to the excellent ductility of poly(3-hydroxypropionate).Control over the rac-β-BL/β-PL feeding ratios and polymerization time yielded random or block copolymers with tunable thermal and mechanical properties comparable to traditional fossil-based plastics.Furthermore,we achieved one-pot synthesis of hard-soft-hard triblock copolymers by exploiting monomers’different copolymerization rates and a bifunctional initiator,thus transforming polyhydroxyalkanoates from hard and tough plastics to soft and ductile thermoplastic elastomers.

Recent advances of medical polyhydroxyalkanoates in musculoskeletal system

Infection and rejection in musculoskeletal trauma often pose challenges for natural healing,prompting the exploration of biomimetic organ and tissue transplantation as a common alternative solution.Polyhydroxyalkanoates(PHAs)are a large family of biopolyesters synthesised in microorganism,demonstrating excellent biocompatibility and controllable biodegradability for tissue remodelling and drug delivery.With different monomer-combination and polymer-types,multi-mechanical properties of PHAs making them have great application prospects in medical devices with stretching,compression,twist in long time,especially in musculoskeletal tissue engineering.This review systematically summarises the applications of PHAs in multiple tissues repair and drug release,encompassing areas such as bone,cartilage,joint,skin,tendons,ligament,cardiovascular tissue,and nervous tissue.It also discusses challenges encountered in their application,including high production costs,potential cytotoxicity,and uncontrollable particle size distribution.In conclusion,PHAs offer a compelling avenue for musculoskeletal system applications,striking a balance between biocompatibility and mechanical performance.However,addressing challenges in their production and application requires further research to unleash their full potential in tackling the complexities of musculoskeletal regeneration.

MEDICAL APPLICATIONS OF BIOPOLYESTERS POLYHYDROXYALKANOATES

Microbial polyhydroxyalkanoates （PHAs） are a family of biopolyesters produced by many wild type and engineered bacteria. PHAs have diverse structures accompanied by flexible thermal and mechanical properties. Combined with their in vitro biodegradation, cell and tissue compatibility, PHAs have been studied for medical applications, especially medical implants applications, including heart valve tissue engineering, vascular tissue engineering, bone tissue engineering, cartilage tissue engineering, nerve conduit tissue engineering as well as esophagus tissue engineering. Most studies have been conducted in the authors＇ lab in the past 20＋ years. Recently, mechanism on PHA promoted tissue regeneration was revealed to relate to cell responses to PHA biodegradation products and cell-material interactions mediated by microRNA. Very importantly, PHA implants were found not to cause carcinogenesis during long-term implantation. Thus, PHAs should have a bright future in biomedical areas.

Comparative study of polyhydroxyalkanoates production from acidified and anaerobically treated brewery wastewater using enriched mixed microbial culture

The production of polyhydroxyalkanoates(PHA) from wastewaters using microbial mixed cultures(MMC) has been attracting increased interest because of PHA's biodegradability characteristics. Production of PHA by an MMC enriched with PHA-accumulating bacteria was compared using anaerobically treated and acidified brewery wastewaters under various feeding strategies, namely pulse and batch feed addition. To obtain an enriched MMC, a sequencing batch reactor was inoculated with activated sludge fed with acetate and subjected to aerobic dynamic feeding. The enriched MMC was able to accumulate PHA up to 72.6% of cell dry weight(CDW) with pulse addition of acetate controlled by the dissolved oxygen(DO) concentration in the reactor. In a batch accumulation experiment with acetate,the PHA content achieved(28.5% CDW) was less than that of the pulse feeding strategy with the same amount of acetate(～2000 mg C/L). Using anaerobically treated and acidified brewery wastewater fed in pulses, the maximum PHA accumulated by the enriched MMC was similar for both wastewaters(45% CDW), in spite of the higher volatile fatty acid concentration in acidified brewery wastewater. The pulse feed addition controlled by the DO concentration was difficult to implement for wastewater as compared to acetate because the difference in DO concentration between substrate availability and depletion was low. For the batch addition of acidified wastewater, a slightly lower PHA content(39%CDW) was obtained. These results show that both brewery wastewaters can be utilized for PHA production with a similar maximum PHA storage capacity.

Engineering bacteria for enhanced polyhydroxyalkanoates(PHA)biosynthesis

Polyhydroxyalkanoates(PHA)have been produced by some bacteria as bioplastics for many years.Yet their commercialization is still on the way.A few issues are related to the difficulty of PHA commercialization:namely,high cost and instabilities on molecular weights(Mw)and structures,thus instability on thermo-mechanical properties.The high cost is the result of complicated bioprocessing associated with sterilization,low conversion of carbon substrates to PHA products,and slow growth of microorganisms as well as difficulty of downstream separation.Future engineering on PHA producing microorganisms should be focused on contamination resistant bacteria especially extremophiles,developments of engineering approaches for the extremophiles,increase on carbon substrates to PHA conversion and controlling Mw of PHA.The concept proof studies could still be conducted on E.coli or Pseudomonas spp.that are easily used for molecular manipulations.In this review,we will use E.coli and halophiles as examples to show how to engineer bacteria for enhanced PHA biosynthesis and for increasing PHA competitiveness.

Benzene containing polyhydroxyalkanoates homo-and copolymers synthesized by genome edited Pseudomonas entomophila

Microbial synthesis of functional polymers has become increasingly important for industrial biotechnology. For the first time, it became possible to synthesize controllable composition of poly（3-hydroxyalkanoate） （P3HA） consisting of 3-hydroxydodec- anoate （3HDD） and phenyl group on the side-chain when chromosome of Pseudomonas entomophila was edited to weaken its t-oxidation. Cultured in the presence of 5-phenylvaleric acid （PVA）, the edited P. entomophila produced only homopolymer poly（3-hydroxy-5-phenylvalerate） or P（3HPhV）. While copolyesters P（3HPhV-co-3HDD） of 3-hydroxy-5-phenylvalerate （3HPhV） and 3-hydroxydodecanoate （3HDD） were synthesized when the strain was grown on mixtures of PVA and dodecanoic acid （DDA）. Compositions of 3HPhV in P（3HPhV-co-3HDD） were controllable ranging from 3% to 32% depending on DDDA/PVA ratios. Nuclear magnetic resonance （NMR） spectra clearly indicated that the polymers were homopolymer of P（3HPhV） and random copolymers of 3HPhV and 3HDD. Their mechanical and thermal properties varied dramatically de- pending on the monomer ratios. Our results demonstrated the possibility to produce tailor-made, novel functional PHA using the chromosome edited P. entomophila.

Polyhydroxyalkanoates： Current applications in the medical field

Polyhydroxyalkanoates （PHAs） are a class of biopolyesters that are synthesized intracellularly by microorganisms, mainly by different genera of eubacteria. These biopolymers have diverse physical and chemical properties that also classify them as biodegradable in nature and make them compatible to living systems. In the last two decades or so, PHAs have emerged as potential useful materials in the medical field for different applications owing to their unique properties. The lower acidity and bioactivity of PHAs confer them with minimal risk compared to other biopolymers such as poly-lactic acid （PLA） and poly-glycolic acid （PGA）. Therefore, the versatility of PHAs in terms of their non-toxic degradation products, biocompatibility, desired surface modifications, wide range of physical and chemical properties, cellular growth support, and attachment without carcinogenic effects have enabled their use as in vivo implants such as sutures, adhesion barriers, and valves to guide tissue repair and in regeneration devices such as cardiovascular patches, articular cartilage repair scaffolds, bone graft substitutes, and nerve guides. Here, we briefly describe some of the most recent innovative research involving the use of PHAs in medical applications. Microbial production of PHAs also provides the opportunity to develop PHAs with more unique monomer compositions economically through metabolic engineering approaches. At present, it is generally established that the PHA monomer composition and surface modifications influence cell responses.PHA synthesis by bacteria does not require the use of a catalyst （used in the synthesis of other polymers）, which further promotes the biocompatibility of PHA-derived polymers.

Research on polyhydroxyalkanoates and glycogen transformations: Key aspects to biologic nitrogen and phosphorus removal in low dissolved oxygen systems

In this paper,a study was conducted on the effect of polyhydroxyalkanoates(PHA)and glycogen transformations on biologic nitrogen and phosphorus removal in low dissolved oxygen(DO)systems.Two laboratory-scale sequencing batch reactors(SBR1 and SBR2)were operating with anaerobic/aerobic(low DO,0.15-0.45 mg·L^(-1))configurations,which cultured a propionic to acetic acid ratio(molar carbon ratio)of 1.0 and 2.0,respectively.Fewer poly-3-hydroxybutyrate(PHB),total PHA,and glycogen transformations were observed with the increase of propionic/acetic acid,along with more poly-3-hydroxyvalerate(PHV)and poly-3-hydroxy-2-methyvalerate(PH2MV)shifts.The total nitrogen(TN)removal efficiency was 68%and 82%in SBR1 and SBR2,respectively.In the two SBRs,the soluble ortho-phosphate(SOP)removal efficiency was 94%and 99%,and the average sludge polyphosphate(poly-P)content(g·g-MLVSS^(-1))was 8.3%and 10.2%,respectively.Thus,the propionic to acetic acid ratio of the influent greatly influenced the PHA form and quantity,glycogen transformation,and poly-P contained in activated sludge and further determined TN and SOP removal efficiency.Moreover,significant correlations between the SOP removal rate and the(PHV+PH2MV)/PHA ratio were observed(R^(2)>0.99).Accordingly,PHA and glycogen transformations should be taken into account as key components for optimizing anaerobic/aerobic(low DO)biologic nitrogen and phosphorus removal systems.

Synthetic biology of microbes synthesizing polyhydroxyalkanoates (PHA)

Microbial polyhydroxyalkanoates(PHA)have been produced as bioplastics for various purposes.Under the support of China National Basic Research 973 Project,we developed synthetic biology methods to diversify the PHA structures into homo-,random,block polymers with improved properties to better meet various application requirements.At the same time,various pathways were assembled to produce various PHA from glucose as a simple carbon source.At the end,Halomonas bacteria were reconstructed to produce PHA in changing morphology for low cost production under unsterile and continuous conditions.The synthetic biology will advance the PHA into a bio-and material industry.

Investigation of polyhydroxyalkanoates （PHAs） biosynthesis from mixed culture enriched by valerate-dominant hydrolysate

The production of polyhydroxyalkanoates （PHAs） with a high fraction of 3-hydroxyvalerate （3HV） and 3-hydroxy-2-methylvalerate （3H2/MV） from mixed culture enriched by valerate-dominant hydrolysate was evaluated in this study. After long-term enrichment, the culture showed strong ability to synthesize 3HV and 3H2MV, even with acetate-dominant substrate. The ultilization of single or mixed iso-/n-valerate by the enriched culture showed that the mixture of iso-valerate and n-valerate was more efficient substrate than any single in tenaas of balancing microbial growth and PHAs synthesis. Besides, through comparing the kinetics and stoichiometry of the tests supplying valerate and propionate, the enriched culture with equivalent valerate and propionate （1 ： 1 molar ratio） exhibited superior PHAs production performances to pure valerate or propionate, attaining more than 70 tool% of 3HVand 3H2MV. The above findings reveal that valerate-dominant hydrolysate is a kind of suitable substrate to enrich PHAs producing culture with great capability to synthesize 3HV and 3H2MV monomers, thus improving product properties than pure poly（3-hydroxybutyrate） （P3HB）; also 3HV and 3H2MV production behaviors can be regulated by the type of odd-carbon VFAs in the substrate.

Biosynthesis of Polyhydroxyalkanoates

Many bacteria are able to synthesize polyhydroxyalkanoates, abbreviated as PHA, as carbon and energy storage compounds. PHA with over 90 different monomer structures has been reported and the number keeps increasing. The piezoelectricity, non linear optical activity, biocompatibility and biodegradability of PHA have offered promising applications in areas such as tissue engineering, drug delivery, smart materials and degradable packaging. Various microorganisms obtained from nature, genetic engineering and mutations have been used for microbial production of PHA as biodegradable plastics. In fact, PHA synthesizing bacteria can be easily found in various locations, especially oil contaminated soils. Many bacterial strains possessing the ability to synthesize PHA with various monomers have been isolated from oil contaminated soils. In molasses contaminated soil, 40% of the isolated bacteria were capable of growing rapidly and synthesizing PHA at the same time. It seems that nature has provided endless numbers of novel PHA synthesizing strains.

Biosynthesis Pathway Related Production of Medium Chain Length Polyhydroxyalkanoates

Pseudomonas flava HBE06 isolated from oil contaminated soil was found to produce polyesters consisting of medium chain length polyhydroxyalkanoates (mcl PHA). The strain mainly synthesized PHA containing 3 hydroxyoctanoate (C 8 or HO), 3 hydroxynonanoate (C 9 or HN), 3 hydroxydecanoate (C 10 or HD), and 3 hydroxyunidecanoate (C 11 or HUD) as monomers when grown on various substrates. It was found that the monomer ratio (especially C 10 /C 8 or C 11 /C 9) was related to the PHA biosynthesis pathway. When PHA was synthesized via the de novo fatty acid pathway, the molar ratio of C 10 /C 8 was greater than 2. If PHA was synthesized from β oxidation of fatty acids, C 10 /C 8 was less than 1. Pseudomonas stutzeri 1317 is another mcl PHA synthesizing bacteria whose C 10 /C 8 ratio is also related to the synthesis pathway. When the two synthesis pathways were used together, the C 10 /C 8 ratio could be adjusted according to the ratio of the mixed substrates.

Production of Biodegradable Copolyesters and Terpolyesters of Polyhydroxyalkanoates by Alcaligenes latus DSM 1124

Copolyesters of 3 hydroxybutyrate and 4 hydroxybutyrate along with terpolyesters of 3 hydroxybutyrate, 3 hydroxyvalerate and 4 hydroxybutyrate were produced using Alcaligenes latus DSM 1124 in growth media with precursors of 1,4 butanediol or 1,4 butanediol and valerate. Monomer mass fraction w in the copolyesters varied from 63% to 78% 3 hydroxybutyrate and 1 5% to 6 5% 4 hydroxybutyrate (cell dry weight), depending on precursor supplied. w(monomer) in the terpolyesters varied from 55% to 69% 3 hydroxybutyrate, 0 5% to 3 4% 4 hydroxybutyrate and 3 4% to 16% 3 hydroxyvalerate.