Recent developments in short- and medium-chain- length Polyhydroxyalkanoates: Production, properties, and applications.

Developing renewable resource-based plastics with complete biodegradability and a minimal carbon footprint can open new opportunities to effectively manage the end-of-life plastics waste and achieve a low carbon society. Polyhydroxyalkanoates (PHAs) are biobased and biodegradable thermoplastic polyesters that accumulate in microorganisms (e.g., bacterial, microalgal, and fungal species) as insoluble and inert intracellular inclusion. The PHAs recovery from microorganisms, which typically involves cell lysis, extraction, and purification, provides high molecular weight and purified polyesters that can be compounded and processed using conventional plastics converting equipment. The physio-chemical, thermal, and mechanical properties of the PHAs are comparable to traditional synthetic polymers such as polypropylene and polyethylene. As a result, it has attracted substantial applications interest in packaging, personal care, coatings, agricultural and biomedical uses. However, PHAs have certain performance limitations (e.g. slow crystallization), and substantially more expensive than many other polymers. As such, more research and development is required to enable them for extensive use. This review provides a critical review of the recent progress achieved in PHAs production using different microorganisms, downstream processing, material properties, processing avenues, recycling, aerobic and anaerobic biodegradation, and applications.

Comparative analysis of various extraction processes based on economy, eco-friendly, purity and recovery of polyhydroxyalkanoate: A review.

Bioplastics have been an interesting area of research and development in the last few decades. Normal plastics are made out of petroleum products, which is a non-renewable resource. Apart from that, its non-biodegradable nature makes it a serious threat to the environment, and hence a better alternative is needed. Bioplastics are synthesized by microorganisms and are biodegradable; this property makes them a promising alternative to normal plastic. However, the major drawback related to bioplastic is the high cost of its production. Polyhydroxyalkanoate (PHA) is a very popular biopolymer produced by different types of microbes. The review focuses on the different methods of extraction of PHA based on the percentage of purity, recovery, eco-friendly, and cost-effectiveness. There is a wide array of extraction methods reported to date, wherein there is the involvement of different types of solvents (like halogenated, non-halogenated, and green solvents) or mechanical or enzymatic methods. Each extraction process has its advantages and disadvantages. In this study, we have attempted to present a structured comparison of these different methods and highlight the factors that can be addressed for better extraction of PHA thereby making it a feasible alternative to commercial synthetic plastic.

Rapid analysis of polyhydroxyalkanoate contents and its monomer compositions by pyrolysis-gas chromatography combined with mass spectrometry (Py-GC/MS).

Here, we report an analysis method for determining PHA (polyhydroxyalkanoates) contents and their monomer composition in microbial cells based on pyrolysis gas chromatography combined with mass spectrometry (Py-GC/MS). Various kinds of microbial cells accumulating different PHA contents and monomer compositions were prepared through the cultivation of Ralstonia eutropha and recombinant Escherichia coli. Py-GC/MS could analyse these samples in a short time without complicated pretreatment steps. Characteristic peaks such as 2-butenoic acid, 2-pentenoic acid, and hexadecanoic acid regarding PHA compositions and cell components were identified. Considering constituents of cells and ratios of peak areas of dehydrated monomers to hexadecanoic acid, a simple equation for estimation of PHA contents in microbial cells was derived. Also, monomer compositions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in R. eutropha could be successfully determined based on peak area of 2-butenoic acid and 2-pentenoic acid of Py-GC/MS, which are the corresponding species of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in PHBV. Correlation of results between GC-FID and Py-GC/MS could be fitted very well. This method shows similar results for the samples obtained from same experimental conditions, allowing rapid and reliable analysis. Py-GC/MS can be a promising tool to rapidly screen PHA-positive strains based on polymer contents along with monomer compositions.

Production of medium-chain-length polyhydroxyalkanoates by Pseudomonas chlororaphis subsp. aurantiaca: Cultivation on fruit pulp waste and polymer characterization.

Pseudomonas chlororaphis subsp. aurantiaca DSM 19603 was cultivated on apple pulp, a glucose- and fructose-rich waste generated during juice production, to produce medium-chain length polyhydroxyalkanoates. A cell dry mass of 8.74 ± 0.20 g/L, with a polymer content of 49.25 ± 4.08% were attained. The produced biopolymer was composed of 42.7 ± 0.1 mol% 3-hydroxydecanoate, 17.9 ± 1.0 mol% 3-hydroxyoctanoate, 14.5 ± 1.1 mol% 3-hydroxybutyrate, 11.1 ± 0.6 mol% 3-hydroxytetradecanoate, 10.1 ± 0.5 mol% 3-hydroxydodecanoate and 3.7 ± 0.2 mol% 3-hydroxyhexanoate. It presented low glass transition and melting temperatures (-40.9 ± 0.7 °C and 42.0 ± 0.1 °C, respectively), and a degradation temperature of 300.0 ± 0.1 °C, coupled to a low crystallinity index (12.7 ± 2.7%), a molecular weight (Mw) of 1.34 × 105 ± 0.18 × 105 Da and a polydispersity index of 2.70 ± 0.03. The biopolymer's films were dense and had a smooth surface, as demonstrated by Scanning Electron Microscopy. They presented a tension at break of 5.21 ± 1.09 MPa, together with an elongation of 400.5 ± 55.8% and an associated Young modulus of 4.86 ± 1.49 MPa, under tensile tests. These attractive filming properties of this biopolymer could potentially be valorised in several areas such as the fine chemicals industry, biomedicine, pharmaceuticals, or food packaging.

Polyhydroxyalkanoates (PHA) from Halomonas pacifica ASL10 and Halomonas salifodiane ASL11 isolated from Mariout salt lakes.

Two novel PHA producing bacterial strains were chosen out of 12 strains collected from Mariout salt lakes. Analysis of 16srRNA gene sequence of the two new strains revealed 95.38% and 98.78% similarity to that of Halomonas pacifica and Halomonas salifodiane, respectively. A maximum polymer productivity of 6.9 g/l and 7.1 g/l was recorded by ASL10 and ASL11, respectively. Furthermore, a pH of 7 contributed to the highest polymer production for both strains. Interestingly, both ASL10 and ASL11showed a great ability to tolerate salinity up to 17 g/l NaCL. Moreover, both promising isolates were able to degrade crude oil efficiently by degradation percentages of 69.2% and 67.3% for ASL10 and ASL11, respectively. GCMS, FTIR, NMR, XRD and thermal properties were performed for poly (3 HV-co-3HB) characterization.

Recent advances in polyhydroxyalkanoate production: Feedstocks, strains and process developments.

Polyhydroxyalkanoates (PHAs) have been actively studied in academia and industry for their properties comparable to petroleum-derived plastics and high biocompatibility. However, the major limitation for commercialization is their high cost. Feedstock costs, especially carbon costs, account for the majority of the final cost. Finding cheap feedstocks for PHA production and associated process development are critical for a cost-effective PHA production. In this study, waste materials from different sources, particularly lignocellulosic biomass, were proposed as suitable feedstocks for PHA production. Strains involved in the conversion of these feedstocks into PHA were reviewed. Newly isolated strains were emphasized. Related process development, including the factors that affect PHA production, fermentation modes and downstream processing, was elaborated upon.

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.

Novel sodium alkyl-1,3-disulfates, anionic biosurfactants produced from microbial polyesters.

A sodium alkyl disulfate mixture (SADM) synthesised from microbially produced 3-hydroxy fatty acids methyl esters (HFAMEs), showed 13-fold surface tension decrease when compared with the reference surfactant sodium dodecyl sulfate (SDS). Polyhydroxyalkanoates, accumulated by bacteria intracellularly when supplied with a mixture of fatty acids derived from hydrolysed rapeseed oil, were isolated, depolymerised and methylated to produce HFAMEs in very high yield (90%). A sequential chemical reduction and sulfation of the HFAMEs produced the sodium alkyl disulfates in high yields (>65%). SADM performs also 1.3-times better than dodecyl (1,3) disulfate, in surface tension tests. SADM shows also the formation of a specific critical micelle concentration (CMC) at a concentration 21-fold lower than SDS. The wettability of the SADM mixture is similar to SDS but the foaming volume of SADM is 1.5-fold higher. The foam is also more stable with its volume decreasing 3 times slower over time compared to SDS at their respective CMC values. Established sulfation technologies in chemical manufacturing could use the 3-hydroxy fatty acids methyl esters moiety (3-HFAME) given its origin from rapeseed oil and the extra OH residue on 3-position in the molecule, which affords the opportunity to produce disulfate surfactants with a proven superior performance to monosulphated surfactants. Thus, not only addressing environmental issues by avoiding threats of deforestation and monocultivation associated with palm oil use but also achieve a higher performance with lower use of surfactants.

An investigation for recovery of polyhydroxyalkanoates (PHA) from Bacillus sp. BPPI-14 and Bacillus sp. BPPI-19 isolated from plastic waste landfill.

Bio-plastic synthesis from renewable and cheap agro-based materials is a sustainable solution for replacing conventionally produced plastic with environmental contamination. The current study was aimed at screening and characterization of Polyhydroxyalkanoates (PHA) producing bacterial isolates, evaluation of their potential and recovery of PHA using the isolates. The PHA compounds were characterized using FT-IR. Based on 16SrRNA sequence analyses the isolates were designated as Bacillus sp. BPPI-14 and Bacillus sp. BPPI-19. The isolates were gram-positive, rod-shaped, endospore former, and citrate test positive. Intracellular PHA granules were observed when these isolates were stained with Sudan black B (SBB) and Nile blue A (NBA) preliminary and specific staining dyes, respectively. Effect of pH, temperature and carbon sources on the PHA production by the isolates BPPI-14 and BPPI-19 was studied. Maximum PHA production was recorded for Glucose (49.46±2.79%) by Bacillus sp. BPPI-14 and followed by molasses (45.86±2.17%) by Bacillus sp. BPPI-19, respectively at 37°C and pH7. The obtained PHA polymers were confirmed by preparation of plastic films for both the isolates. Fourier transform infrared spectrum for BPPI-14 and BPPI-19 showed the peak (carboxylic acid group) at 1706-1719.39cm-1 was a characteristic feature of PHA and corresponds functional group (C=O).

Polyhydroxyalkanoate production using waste vegetable oil and filtered digestate liquor of chicken manure.

The production of polyhydroxyalkanoates (PHA) using digestate of chicken manure combined with waste sunflower oil as no-cost feedstocks in a multi-stage process was investigated. Using Cupriavidus necator H16 in combined culture media, a maximum PHA accumulation of 4.6 ± 0.2 g/L at 75.1 ± 1.4% of cell dry matter and a residual cell matter yield of 1.5 ± 0.1 g/L were obtained after 96 hr of cultivation (30 °C, 160 rpm, pH 7.0) in flask-based experiments. Manure was acidogenically fermented in a continuous stirring tank reactor in fed-batch mode. The bioreactor was operated at varying organic loading rates (OLR) and hydraulic retention times (HRT) ranging from 1-4 g volatile solids (VS)/L/d and 4-8 days, respectively. Optimal operation was observed at an OLR of 4 g VS/L/d and an HRT of 4 days. Analysis showed the presence of significant amounts of ammonia, potassium, magnesium, calcium, and trace elements, i.e. Fe, Cu, Ni, Mn, Co, Zn, Cr in the digestate. The micro-filtered digestate was utilized as a complex culture media base while waste oil served as an additional carbon source and supplemented for effective PHA production. The total volatile fatty acid content of digestate greatly affected the growth performance of the PHA-producing microorganism Cupriavidus necator H16.

Bioplastic recovery from wastewater: A new protocol for polyhydroxyalkanoates (PHA) extraction from mixed microbial cultures.

A new protocol for polyhydroxyalkanoates (PHA) extraction from mixed microbial cultures (MMCs) is proposed. PHA-accumulating capacity of the MMC was selected in a sequencing batch reactor (SBR) fed with a synthetic effluent emulating a fermented oil mill wastewater (OMW). The highest recovery yield and purity (74 ±â€¯8% and 100 ±â€¯5%, respectively) was obtained when using NH4-Laurate for which operating conditions of the extraction process such as temperature, concentration and contact time were optimized. Best conditions for PHA extraction from MMC turned to be: i) a pre-treatment with NaClO at 85 °C with 1 h of contact time, followed by ii) a treatment with lauric acid in a ratio acid lauric to biomass of 2:1 and 3 h of contact time.

Microalgae as source of polyhydroxyalkanoates (PHAs) - A review.

Polyhydroxyalkanoates (PHA) are biopolymers synthesized by different microorganisms and considered substitute powers for petroleum-based plastics because they have similar mechanical properties as synthetic polymers, can be processed in a similar way and are fully biodegradable. Currently commercial PHAs are produced in fermenters using bacteria and large amounts of organic carbon sources and salts in the culture media, accounting for approximately 50% of the total production costs. A greater commercial application of the PHA is limited to a decrease in the cost of production. Several studies suggest that microalgae are a type of microorganisms that can be used to obtain PHAs at a lower cost because they have minimum nutrient requirements for growth and are photoautotrophic in nature, i.e. they use light and CO2 as their main sources of energy. Thus, this work aims to provide a review on the production of PHAs of different microalgae, focusing on the properties and composition of biopolymers, verifying the potential of using these bioplastics instead of petroleum based plastics. Studies of stimulation PHA synthesis by microalgae are still considered incipient. Still, it is clear that microalgae have the potential to produce biopolymers with lower cost and can play a vital role in the environment.

Organic removal and synthesis of biopolymer from synthetic oily bilge water using the novel mixed bacterial consortium.

Synthetic oily bilge water (OBW) treatment and subsequent production of biopolymer were studied by using a sequential batch reactor (SBR). The effect of various influencing parameters such as solids retention time (SRT), cycle time (CT), substrate concentration, pH level on the organic removal and synthesis of polyhydroxyalkanoates (PHA) was examined by novel soil bacteria isolated from hydrocarbon contaminated site near Karaikal port, India. The isolates were identified as Pseudomonas tuomuerensis and Pseudomonas nitroreducens using 16S rRNA. Sudan Black B staining was performed to visualize the presence of PHA. The experimental results showed that a decrease in substrate concentration to 5000 mg/L of soluble COD (CODs) showed maximum organic removal (81%) and maximum PHA yields of its cell dry mass (81%). The PHA yield was maximum at SRT of 5 d, pH = 7 and CT of 24 h. The produced PHA was characterized by using FTIR, XRD and SEM analysis.

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.

Fed-Batch Strategies for Production of PHA Using a Native Isolate of Halomonas venusta KT832796 Strain.

In this study, polyhydroxyalkanoates (PHA) accumulation by Halomonas venusta KT832796, a moderate halophilic bacteria isolated from marine source was studied. Both nutritional requirements and process parameters for submerged cultivation of the organism in bioreactor have been standardized. From the shake flask studies, glucose and ammonium citrate as carbon and nitrogen source produced maximum PHA at a ratio 20 with 3.52 g/L of dry cell weight and 70.56% of PHA content. However, ammonium sulfate as the nitrogen source was found to be more suitable for fed-batch cultivation. Several feeding strategies including pH-based fed-batch and variants of pulse feeding were studied to improve the PHA levels. pH-based feeding, although improved PHA level to 26 g/L, most of the carbon flux was diverted towards biomass formation; hence, the percent PHA was only 39.15% of the dry cell weight. Maximum PHA of 33.4 g/L, which corresponded to 88.12% of the dry cell, was obtained from high concentration single pulse method. There was a net 8.65-fold increase in PHA using this feeding strategy when compared to batch studies. According to our knowledge, this is the highest amount of PHA reported for a Halomonas venusta strain.

Production enhancement and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10.

Application of halophiles can decrease the cost of polyhydroxyalkanoate (PHA) production or bioplastic which are an alternative to the petroleum-derived plastic. Extremely halophilic archaeon, Natrinema ajinwuensis RM-G10 accumulated 61.02±0.68% PHA of its cell dry mass at 72h in repeated batch cultures yielding 0.210±0.001gL-1h-1 volumetric productivity after selection of the best cultivation conditions. Transmission electron microscopy showed the presence of PHA granules inside the archaeal cells. Characterization by gas chromatographic analysis, gas chromatographic- mass spectrophotometric analysis, thermogravimetric analysis, differential scanning calorimetric analysis, X-ray diffraction analysis, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy revealed the polymer to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 13.93mol% 3-hydroxyvalerate content and having 35.45% crystallinity, -12.3°C glass transition temperature, 143°C and 157.5°C melting temperatures and 284°C degradation temperature. This is the first report on production enhancement (on a small scale) and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10 and the Natrinema genus in general.

Co-synthesis of medium-chain-length polyhydroxyalkanoates and CdS quantum dots nanoparticles in Pseudomonas putida KT2440.

Microbial polymers and nanomaterials production is a promising alternative for sustainable bioeconomics. To this end, we used Pseudomonas putida KT2440 as a cell factory in batch cultures to coproduce two important nanotechnology materials- medium-chain-length (MCL)-polyhydroxyalkanoates (PHAs) and CdS fluorescent nanoparticles (i.e. quantum dots [QDots]). Due to high cadmium resistance, biomass and PHA yields were almost unaffected by coproduction conditions. Fluorescent nanocrystal biosynthesis was possible only in presence of cysteine. Furthermore, this process took place exclusively in the cell, displaying the classical emission spectra of CdS QDots under UV-light exposure. Cell fluorescence, zeta potential values, and particles size of QDots depended on cadmium concentration and exposure time. Using standard PHA-extraction procedures, the biosynthesized QDots remained associated with the biomass, and the resulting PHAs presented no traces of CdS QDots. Transmission electron microscopy located the synthesized PHAs in the cell cytoplasm, whereas CdS nanocrystals were most likely located within the periplasmic space, exhibiting no apparent interaction. This is the first report presenting the microbial coproduction of MCL-PHAs and CdS QDots in P. putida KT2440, thus constituting a foundation for further bioprocess developments and strain engineering towards the efficient synthesis of these highly relevant bioproducts for nanotechnology.

Low frequency sonic waves assisted cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator.

Low frequency sonic waves, less than 10kHz were introduced to assist cloud point extraction of polyhydroxyalkanoate from Cupriavidus necator present within the crude broth. Process parameters including surfactant system variables and sonication parameters were studied for their effect on extraction efficiency. Introduction of low frequency sonic waves assists in the dissolution of microbial cell wall by the surfactant micelles and release of cellular content, polyhydroxyalkanoate granules released were encapsulated by the micelle core which was confirmed by crotonic acid assay. In addition, sonic waves resulted in the separation of homogeneous surfactant and broth mixture into two distinct phases, top aqueous phase and polyhydroxyalkanoate enriched bottom surfactant rich phase. Mixed surfactant systems showed higher extraction efficiency compared to that of individual Triton X-100 concentrations, owing to increase in the hydrophobicity of the micellar core and its interaction with polyhydroxyalkanoate. Addition of salts to the mixed surfactant system induces screening of charged surfactant head groups and reduces inter-micellar repulsion, presence of ammonium ions lead to electrostatic repulsion and weaker cation sodium enhances the formation of micellar network. Addition of polyethylene glycol 8000 resulted in increasing interaction with the surfactant tails of the micelle core there by reducing the purity of polyhydroxyalkanoate.

Simple and rapid method for isolation and quantitation of polyhydroxyalkanoate by SDS-sonication treatment.

We developed a new method for isolation and quantitation of polyhydroxyalkanoate (PHA) from culture broth. In this method, the cells were sonicated in sodium dodecyl sulfate (SDS) solution and centrifuged to recover PHA. The recovered PHA was rinsed with deionized water and ethanol, and then weighed after drying. Hazardous chemicals such as chloroform, methanol, and sulfuric acid were not used, and no expensive analytical instruments were needed. We applied this method to Cupriavidus necator culture broths that included various amounts of poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) from flasks and jar fermentors. The quantitation by this method was practical for use with a wide range of production amounts and PHA monomer compositions compared to the conventional whole-cell methanolysis method with gas chromatographic analysis, and besides, the recovered PHAs were adequately pure (≥96% purity). Therefore, this new method would be valuable not only for quantitation of PHA but also for preparation of samples to characterize their mechanical properties.

Specific character of bacterial biodegradation of polyhydroxyalkanoates with different chemical structure in soil.

The study addresses the influence of the physicochemical properties of the reserve cellular macromolecules (polyhydroxyalkanoates, PHA) with different chemical composition on their biodegradation in the agro-transformed field soil of the Siberian region (Krasnoyarsk Territory, Russia). It was shown that the degradation of the PHA samples depends on the degree of polymer crystallinity (Cx). For the first time, it was shown that the range of PHA-degrading microorganisms differs for each of PHA types. The study defines the primary degraders specific to each PHA type and common to all types of examined polymers.