Polyhydroxyalkanoate Composition Research Articles

Polyhydroxyalkanoate (PHA) production with acid or alkali pretreated sludge acidogenic liquid as carbon source: Substrate metabolism and monomer composition

The organic acid rich acidogenic liquid produced from waste sludge anaerobic fermentation was used as a potential feedstock for polyhydroxyalkanoates (PHA) production, which is a cost-effective and environmentally sustainable PHA production system. To analyze the effect of substrate on monomer composition of PHA, different acidogenic liquid generated from waste sludge fermentation in varied initial pH were used as external carbon source. The maximal PHA content accounted for 60.3 % of the dry cell with initial fermentation pH 10.0 sludge acidogenic liquid and the recovered polymer composition was 98.3 % polyhydroxybutyrate (PHB) and 1.7 % polyhydroxyvalerate (PHV) by mass. The utilization efficiency of soluble chemical oxygen demand (SCOD), protein, NH4+-N and volatile fatty acids (VFAs) was 95.4 %, 90.5 %, 98.0 %, 99.3 %, respectively. The tyrosine-like protein, tryptophan-like protein, fulvic acid-like organics and soluble microbial by-products were primary used for PHA production, while the humic acid-like organics used little. Furthermore, mass balance analysis was introduced to further illustrate the technology reliability.

Community profile governs substrate competition in polyhydroxyalkanoate (PHA)-producing mixed cultures

Polyhydroxyalkanoates (PHAs) are biobased, biodegradable polymers that can be produced efficiently by mixed microbial cultures (MMCs) at low cost. Substrate preferences have been found to vary depending on the microbial composition in the MMCs. In this study, the competition between the uptake of different substrates has been related for the first time to the abundance of certain microbial communities. With a higher abundance of Plasticicumulans enriched in the culture, the inhibitory effect of butyrate and valerate on the uptake of acetate and propionate increased and the hydroxybutyrate:hydroxyvalerate (HB:HV) ratio in the final PHA polymer approached the theoretical value calculated from the volatile fatty acid (VFA) composition. This finding can help understanding of the metabolism of PHA accumulating microorganisms and thus predict better the VFA consumption and PHA composition.

Mechanical Stability of Polyhydroxyalkanoate (PHA)-Based Wood Plastic Composites (WPCs)

A growing common public interest in a more sustainable society drives efforts into the development of bio-based and biodegradable polymers. Such renewable polymers can replace conventional non-degradable polymers as the matrix for wood plastic composites (WPCs). However, despite ongoing advancements towards the implementation of these biocomposites, experience with biocomposite mechanical durability is limited. This comparative study investigated the mechanical stability of biocomposites based on polyhydroxyalkanoate (PHA) and wood flour at 50 wt% wood content in real-life in-service conditions, including indoor, outdoor and in soil environments. Polylactic acid (PLA) and polyethylene (PE) wood composites served as comparative reference materials. Aging studies over a 12-month period showed that PHA composites were mechanically stable under indoor conditions. A reduction in mechanical stability was observed for outdoor exposure but the composite property deterioration was independent of the polymer type. In soil, the biocomposites steadily degraded towards a complete loss in mechanical properties, in contrast to the PLA and PE based composites. Overall, the rate of water absorption and the rate of polymer biodegradation are principal factors controlling mechanical stability over time. These factors promote the formation of cracks and voids which in turn weaken the material performance under mechanical loading. Without such crack formation, the composites retained mechanical properties over the aging time. The results provide a basis for consideration of strategic applications of PHA-based WPCs in cases where sustained or time-constrained mechanical performance is required.

Determination of the potential of pickle wastewater as feedstock for biopolymer production.

Food industry wastewater (FIWW) streams with high organic content are among the most suitable and inexpensive candidates for polyhydroxyalkanoate (PHA) biopolymer production. Due to its high organic acid content, pickle industry wastewater (PIWW), can be considered as one of the prospective alternatives to petroleum-based polymers for PHA production. In this context, this study aimed to investigate the production of PHA with enriched microbial culture using PIWW. Two laboratory scale sequencing batch reactors (SBRs) were operated under aerobic dynamic feeding conditions at a sludge retention time of 8 days, with a total cycle duration of 24 hours. SBRs were fed with peptone mixture and PIWW. In-cycle analysis and batch respirometric tests were performed to evaluate PHA storage together with biodegradation kinetics. In-cycle analysis showed that maximum PHA content was 1,820 mgCOD/L, corresponding to 44% in the biomass (ratio of chemical oxygen demand (COD) to volatile suspended solids) for PIWW. Experimental results were also confirmed with activated sludge model simulations. As for the PHA composition, hydroxybutyrate was the major fraction. Model simulations proposed a unique conversion-degradation-storage pathway for the organic acid mixture. This paper presents a novel insight for better understanding of PHA biopolymer production using high saline FIWW.

Toward flexible regulation of polyhydroxyalkanoate composition based on substrate feeding strategy: Insights into microbial community and metabolic features

The suitable feeding strategy considering both substrate preference (enrichment stage) and flexible regulation (PHA accumulation stage) were investigated, respectively, based on intracellular polymers synthesis peculiarities of the three types of cultures (M-Ac, M-Pr and M-Bu), which were enriched correspondingly using acetic type, propionic type and butyric type substrate. Compared to M-Ac and M-Bu cultures, maximum PHA content (PHAm) of M-Pr exhibited the most stable responses to varying fractions of propionic acid (fPr) of the substrate. The substrate composed of acetic acid and propionic acid (Mix-AP) demonstrated higher efficiency in regulation of polymer composition than that composed of butyric acid and propionic acid (Mix-BP). For the whole process of three-stage MC PHA production, propionic acid-dominated acidification products should be used for the long-term enrichment of PHA producers, and acidification products mainly composed of propionic and acetic acid are preferred considering the regulation of polymer composition in PHA accumulation stage.

Populated collagen hydrogel and polyhydroxyalkanoate composites: novel matrices for cartilage repair and regeneration?

Purpose Articular cartilage is an avascular connective tissue present in joints which covers the ends of opposing bones providing a smooth, shock absorbing surface to promote normal joint function. Normal cartilage structure is maintained by chondrocytes, the only cell type present. Abnormal function/death of these cells, due to long- term “wear and tear” and other risk factors results in cartilage loss and osteoarthritis (OA). There is currently no cure for OA, the most common treatments simply involve the management of joint pain, or surgical treatments, which are not able to restore cartilage function. In recent years, cartilage repair has been approached with engineered scaffolds, able to support the growth of chondrocytes or progenitor cells to generate a neo-tissue, but the neo tissue generated is often fibrocartilage, mechanically inferior and less–durable than the hyaline cartilage normally found in healthy articular joints, indicating that further research is required to develop a more suitable scaffolds. The aim of this study has been to assess the properties of collagen I hydrogel 3D scaffolds, populated with the phenotypically stable chondrocytes cell line C20/A4, strengthened with solvent cast and 3D printed Polyhydroxyalkanoate (PHA) polymer. PHAs are a family of biopolyesters synthesized by bacteria with monomers of varying chain lengths (C4-C16) which can be blended to produce products with different mechanical properties. PHAs are biodegradable, non-toxic and biocompatible. It has been already established that PHA scaffolds are able to support chondrocyte growth and activity but despite that, they do not represent the natural environment for chondrocyte metabolism, collagen production and cartilage regeneration. A combination collagen hydrogel and PHA scaffold that addresses this issue would be highly desirable. Methods Polymer production and preparation PHAs were produced by Pseudomonas mendocina CH50 in batch fermentation conditions using a 15 L bioreactor with an operating fermenter volume of 10 L. with glucose as the sole carbon source under nitrogen limiting conditions. PHA film and scaffolds were either prepared using the solvent casting method incorporating large NaCl crystals (which were later dissolved out) to form a porous structure or with a 3D melt printing method. Collagen gel preparation and seeding: Type I collagen constructs were prepared with chondrocytes by mixing type I collagen polymer suspension with 10modified Eagle’s medium (MEM) and 1M NaOH solution was added dropwise, to neutralise the acidic gel mixture. This gel solution was then mixed with a cell suspension of C-20/A4 cells in Dulbecco’s MEM (DMEM)-based medium containing 1% foetal calf serum (FCS) . 1.2 ml constructs were cast in 24 well plates either on their own or with the addition of solvent cast PHA polymers or 3D printed PHA polymers. Gels were then allowed to solidify by incubation at 37 C for 5-10 minutes, overlaid with DMEM medium (1% FCS) and cultured for 1 or 7 days, at 37 C and 5% CO2. Compression Studies: Gels were subjected to compression, at 0,6KN/m2 for 5 minutes in a custom built apparatus. The displacement of gels was measured over time. Results The compression testing data demonstrates that type I collagen gels, seeded with C-20/A4 chondrocytes (control gels), exhibit changes in their mechanical properties over time, with an increased resistance and smaller displacement at Day 7 compare to Day 1. The addition of solvent cast and 3D printed scaffolds increase the mechanical resistance of the construct when compared to the populated control gels (collagen matix only) at DAY 1 and is even more evident at DAY 7. 3D printed scaffolds also show higher stiffness of the construct compared to the control and solvent cast polymer samples. Cytotoxicity testing performed with Alamar blue staining, on chondrocyte populations grown on PHA films, indicates that there are no cytotoxic effects of the polymer on cell viability at any of time points analysed over 7 days. Trypan Blue staining, after 5 minutes compression test, reveals no significant difference in viability in any of the samples containing PHA polymer (solvent cast or 3D printed) compare to control samples. Conclusions In conclusion, these studies suggest that a composite collagen gel-PHA construct provides a supportive environment for the maintenance of chondrocyte activity increasing the initial resistance of the structure to compression, whilst the embedded chondrocytes remodel the Type I collagen gel to form a more cartilage like structure. This may offer a potential application of collagen hydrogel-PHA scaffolds for clinical use in support of cartilage regeneration in small and contained osteoarthritic lesions.

Microbial Synthesis and Characterization of Biodegradable Polyester Copolymers from Burkholderia Cepacia and Cupriavidus Necator Strains Using Crude Glycerol as Substrate

Abstract Polymers are materials of pronounced importance in the modern world, since they are massively present in everyday life, especially in the form of packaging. However, most of these materials are derived from non-renewable sources and their disposal generates large volumes of waste, which is extremely damaging to natural environments. In this context, microbial biopolymers appear as a powerful alternative in the substitution of several applications of synthetic plastics, causing less harm to the environment, as they are biodegradable and produced from renewable sources. In this study, evaluation and characterization of polyhydroxyalkanoates (PHAs) produced by Cupriavidus necator (IPT 026 and IPT 027) and Burkholderia cepacia (IPT 119 and IPT 400), using crude glycerol as substrate, were carried out (crude glycerol 15 g L-1, pH 7.0, 150 rpm, 72h). The substrate chemical composition was determined and all microorganisms tested were able to utilize it to synthesize PHA. C. necator IPT 026 exhibited the highest polymer production (1.52 ± 0.03 g L-1). B. cepacia strains produced low crystallinity PHA. All polyesters synthesized exhibited long chain length polymers with low polydispersity and initial thermal degradation temperatures superior to 300°C. The microorganism strains and the substrate composition highly affected PHAs synthesis, composition and thermochemical characteristics.

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis.

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.

Metabolic modeling of the substrate competition among multiple VFAs for PHA production by mixed microbial cultures

Mixed microbial cultures (MMCs) are an effective method for polyhydroxyalkanoates (PHA) production. There are several models established to describe the metabolism of this process. Substrate competition was commonly found in the uptake of multiple volatile fatty acids (VFAs), thus this behavior should be considered in process modeling. In this study, a metabolic model is developed to describe for the first time the substrate competition among four commonly used VFAs (acetate, propionate, butyrate and valerate) in PHA production by MMCs. An inhibition parameter was introduced to describe the inhibition of butyrate and valerate on the uptake of acetate and propionate. The modeled results showed good agreement with experimental data within 95% confidence interval. This model enhanced the understanding of the metabolism of substrate uptake in PHA production, providing a successful approach to predict PHA composition and optimize the process efficiency when four competing VFAs are supplied.

Efficacy of Spirulina sp. polyhydroxyalkanoates extraction methods and influence on polymer properties and composition

The objective of this study was to evaluate the efficacy of methods used to extract polyhydroxyalkanoates from Spirulina sp. LEB-18 microalgae and to examine the influence of these methods on the purity, properties, and composition of the polymers. Polyhydroxyalkanoates were characterized by Fourier transform infrared spectroscopy (FTIR), molecular mass, degree of crystallinity, and monomer composition. The efficacy of the extraction methods varied, with polyhydroxyalkanoate accumulation.between 6.10 and 9.80%, and degrees of purity between 63.51 and 93.62%. Using sodium hypochlorite in the initial stage of the extraction increased accumulation, while using methanol at the end of the process increased the purity of the polymers. The molecular mass and crystallinity index of the polyhydroxyalkanoates varied, showing that the extraction methods interfered with polymer properties. The composition of polyhydroxyalkanoates was also influenced by the extraction, with varying percentages of monomers identified. The copolymers of the polyhydroxyalkanoates obtained are formed by the monomers 11-hydroxyhexadecanoate, in a higher proportion, hydroxyheptanoate and hydroxytetradecanoate, demonstrating that Spirulina sp. LEB-18 is capable of producing medium and long chain polymers. The detection of these monomer blocks in the polyhydroxyalkanoate structure of this microalga is an important scientific novelty because these monomer blocks are constituents of new polymers. An indirect relationship (R2 = 0.8044) was observed between the percentage of the 11-hydroxyhexadecanoate monomer and the degree of crystallinity of polyhydroxyalkanoates obtained by the different methods. This suggests that obtaining the polymer with medium- and long-chain monomers contributes to reduction of crystallinity.

Biosynthesis of 2-Hydroxyacid-Containing Polyhydroxyalkanoates by Employing butyryl-CoA Transferases in Metabolically Engineered Escherichia coli.

The authors previously reported the production of polyhydroxyalkanoates (PHAs) containing 2-hydroxyacid monomers by expressing evolved Pseudomonas sp. 6-19 PHA synthase and Clostridium propionicum propionyl-CoA transferase in engineered microorganisms. Here, the authors examined four butyryl-CoA transferases from Roseburia sp., Eubacterium hallii, Faecalibacterium prausnitzii, and Anaerostipes caccae as potential CoA-transferases to support synthesis of polymers having 2HA monomer. In vitro activity analyses of the four butyryl-CoA transferases suggested that each butyryl-CoA transferase has different activities towards 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB), and lactate (LA). When Escherichia coli XL1-Blue expressing Pseudomonas sp. 6-19 PhaC1437 along with one butyryl-CoA transferase is cultured in chemically defined MR medium containing 20 g L-1 of glucose, 2 g L-1 of sodium 3-hydroxybutyrate, and various concentrations of sodium 2-hydroxybutyrate, PHAs consisting of 3HB, 2HB, and LA are produced. The monomer composition of PHAs agreed well with the substrate specificities of butyryl-CoA transferases from E. hallii, F. prausnitzii, and A. caccae, but not Roseburia sp. When E. coli XL1-Blue expressing PhaC1437 and E. hallii butyryl-CoA transferase is cultured in MR medium containing 20 g L-1 of glucose and 2 g L-1 of sodium 2-hydroxybutyrate, P(65.7 mol% 2HB-co-34.3 mol% LA) is produced with the highest PHA content of 30 wt%. Butyryl-CoA transferases also supported the production of P(3HB-co-2HB-co-LA) from glucose as the sole carbon source in E. coli XL1-Blue strains when one of these bct genes is expressed with phaC1437, cimA3.7, leuBCD, panE, and phaAB genes. Butyryl-CoA transferases characterized in this study can be used for engineering of microorganisms that produce PHAs containing novel 2-hydroxyacid monomers.

Enhanced polyhydroxyalkanoate (PHA) production from the organic fraction of municipal solid waste by using mixed microbial culture

BackgroundIn Europe, almost 87.6 million tonnes of food waste are produced. Despite the high biological value of food waste, traditional management solutions do not consider it as a precious resource. Many studies have reported the use of food waste for the production of high added value molecules. Polyhydroxyalkanoates (PHAs) represent a class of interesting bio-polyesters accumulated by different bacterial cells, and has been proposed for production from the organic fraction of municipal solid waste (OFMSW). Nevertheless, until now, no attention has been paid to the entire biological process leading to the transformation of food waste to organic acids (OA) and then to PHA, getting high PHA yield per food waste unit. In particular, the acid-generating process needs to be optimized, maximizing OA production from OFMSW. To do so, a pilot-scale Anaerobic Percolation Biocell Reactor (100 L in volume) was used to produce an OA-rich percolate from OFMSW which was used subsequently to produce PHA.ResultsThe optimized acidogenic process resulted in an OA production of 151 g kg−1 from fresh OFMSW. The subsequent optimization of PHA production from OA gave a PHA production, on average, of 223 ± 28 g kg−1 total OA fed. Total mass balance indicated, for the best case studied, a PHA production per OFMSW weight unit of 33.22 ± 4.2 g kg−1 from fresh OFMSW, corresponding to 114.4 ± 14.5 g kg−1 of total solids from OFMSW. PHA composition revealed a hydroxybutyrate/hydroxyvalerate (%) ratio of 53/47 and Mw of 8∙105 kDa with a low polydispersity index, i.e. 1.4.ConclusionsThis work showed how by optimizing acidic fermentation it could be possible to get a large amount of OA from OFMSW to be then transformed into PHA. This step is important as it greatly affects the total final PHA yield. Data obtained in this work can be useful as the starting point for considering the economic feasibility of PHA production from OFMSW by using mixed culture.

Polyhydroxyalkanoate Production on Waste Water Treatment Plants: Process Scheme, Operating Conditions and Potential Analysis for German and European Municipal Waste Water Treatment Plants.

This work describes the production of polyhydroxyalkanoates (PHA) as a side stream process on a municipal waste water treatment plant (WWTP) and a subsequent analysis of the production potential in Germany and the European Union (EU). Therefore, tests with different types of sludge from a WWTP were investigated regarding their volatile fatty acids (VFA) production-potential. Afterwards, primary sludge was used as substrate to test a series of operating conditions (temperature, pH, retention time (RT) and withdrawal (WD)) in order to find suitable settings for a high and stable VFA production. In a second step, various tests regarding a high PHA production and stable PHA composition to determine the influence of substrate concentration, temperature, pH and cycle time of an installed feast/famine-regime were conducted. Experiments with a semi-continuous reactor operation showed that a short RT of 4 days and a small WD of 25% at pH = 6 and around 30 °C is preferable for a high VFA production rate (PR) of 1913 mgVFA/(L×d) and a stable VFA composition. A high PHA production up to 28.4% of cell dry weight (CDW) was reached at lower substrate concentration, 20 °C, neutral pH-value and a 24 h cycle time. A final step a potential analysis, based on the results and detailed data from German waste water treatment plants, showed that the theoretically possible production of biopolymers in Germany amounts to more than 19% of the 2016 worldwide biopolymer production. In addition, a profound estimation regarding the EU showed that in theory about 120% of the worldwide biopolymer production (in 2016) could be produced on European waste water treatment plants.

Effect of Triglyceride Addition on Oxygen Uptake, Carbon Metabolism, and Polyhydroxyalkanoate Accumulation in Aerobic Granules

The effect of triglyceride on polyhydroxyalkanoate (PHA) accumulation in aerobic granules used for treating palm oil mill effluent (POME) is unaddressed to date. Triglyceride was added (between 3.7 and 18.5 g/L) to a batch reactor containing aerobic granules and POME. The chemical oxygen demand (COD) removal, PHA content, and PHA composition were analyzed at different triglyceride concentrations. The COD removal decreased to 19% when 18.5 g/L triglyceride was added. Meanwhile, the PHA content decreased from 0.62 to 0.16 g PHA/g cell dry weight (CDW) as the triglyceride concentration increased from 0 to 18.5 g/L. The composition of PHA did not change significantly with the increase in triglyceride concentrations. Accumulated PHA was composed of hydroxybutyrate (HB) and hydroxyvalerate (HV) with a ratio of approximately 77:23. Eventually, the triglyceride was found to limit oxygen uptake, carbon metabolism, and PHA accumulation, in aerobic granules.

Characterisation, biodegradability and application of palm fibre-reinforced polyhydroxyalkanoate composites

The biodegradability, mechanical properties and fabrication of three-dimensional (3D) printing filaments of composite materials made from maleic anhydride-grafted polyhydroxyalkanoate (PHA-g-MA) and coupling agent-treated palm fibre (TPF) were evaluated. Composites of PHA-g-MA and TPF (PHA-g-MA/TPF) exhibited noticeably superior tensile strength and interfacial adhesion compared with those of PHA/PF because of a greater compatibility of PHA-g-MA with TPF. The dispersion of TPF in the PHA-g-MA matrix was highly homogeneous as a result of condensation reactions. The water resistance of the PHA-g-MA/TPF membranes was greater than that of the PHA/PF membranes, and a cell viability evaluation with human foreskin fibroblasts (FBs) indicated that both materials were nontoxic. Additionally, the weight loss of composites buried in soil compost indicated that the biodegradation rate can be enhanced with the increase of the PF or TPF content.

Combining molecular and bioprocess techniques to produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with controlled monomer composition by Burkholderia sacchari

Biopolymers as polyhydroxyalkanoates (PHA) composed by different co-monomers 3-hydroxybutyrate and 3-hydroxyhexanoate [P(3HB-co-3HHx)] has attracted interest since its properties are similar to low density polyethylene. Burkholderia sacchari produces this copolymer with a very low 3HHx molar fraction, about 2 mol%. B. sacchari mutant (unable to produce polymer) was engineered to host PHA biosynthesis genes (phaPCJ) from Aeromonas sp. In addition, a two-step bioprocess to increase biopolymer production was developed. The combination of these techniques resulted in the production of P(3HB-co-3HHx) with 3HHx content up to 20 mol%. The PHA content was about 78% of dry biomass, resulting in PHA volumetric productivities around 0.45gl−1h−1. The P(3HB-co-3HHx) containing 20 mol% of 3HHx presented an elongation at brake of 945%, higher than reported before for this PHA composition. Here we have described an approach to increase 3HHx content into the copolymer, allowing the precise control of the 3HHx molar fractions.

Effect of acid-digested rice straw waste feeding methods on the 3HV fraction of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production

Abstract The effect of different feeding methods of the acid-digested rice straw hydrolysate on the contents and composition of polyhydroxyalkanoates (PHAs) accumulated by Cupriavidus necator was studied. The results showed that the inhibitory effects of furanic compounds on bacterial PHAs synthesis can be alleviated by dilution, and the hydrolysate-to-media (H/M) ratio of 2:8 was appropriate for poly-3-hydroxybutyrate-co-3-hydroxyvalerate [P(3HB-co-3HV)] synthesis. At the H/M ratio of 2:8, the accumulated PHAs content could be increased by using the split-fed method, and the rice straw hydrolysate fed in 4-pulses resulted in the highest P(3HB-co-3HV) concentration in this study. However, the 3-hydroxyvalerate (3HV) mol% decreased with increasing PHAs concentration, thus, the repeatedly-fed method was used to achieve a desirable 3HV fraction range of 20–30% for medical applications. The repeated feeding of the hydrolysate increased the 3HV fraction of the accumulated P(3HB-co-3HV) to the 20–30% range. Overall, this study demonstrates that the feeding methods of rice straw hydrolysates containing multiple carbon sources can be optimized for synthesis of desired PHAs.

Novel interpretations of in vitro polyhydroxyalkanoate polymerization phenomena

Abstract Polyhydroxyalkanoates (PHAs) are gaining popularity as a viable biological alternative to petroleum-derived polyolefin plastic materials, especially because their material properties can be tuned through monomer composition and configuration, an uncommon phenomena in enzymatic polymer products. Because the native function of these polymers is chemical energy storage, the metabolic synthesis routes can vary dramatically from one microorganism to the next, save one crucial enzyme – the PHA synthase, responsible for the direct polymerization and composition of PHAs. For this reason, the field of PHA synthase mechanics is blooming as more variants of this enzyme are discovered. However, due to the complexity associated with this surface-driven micelle growth reaction, the initial reaction conditions dictate a variety of observed behaviors during in vitro kinetic analysis. In this work, we discuss the importance of these initial conditions and their associated behaviors and propose minimal kinetic models to help elucidate the significant mechanics of this complex in vitro reaction. The theory and inferences of this work provide a more complete understanding of in vitro PHA synthesis and aid in the design of future in vitro PHA synthase enzymatic assays.

Effects of carbon sources on the enrichment of halophilic polyhydroxyalkanoate-storing mixed microbial culture in an aerobic dynamic feeding process.

Microbial polyhydroxyalkanoate (PHA) production serves as a substitute for petroleum-based plastics. Enriching mixed microbial cultures (MMCs) with the capacity to store PHA is a key precursor for low-cost PHA production. This study investigated the impact of carbon types on enrichment outcomes. Three MMCs were separately fed by acetate sodium, glucose, and starch as an enriching carbon source, and were exposed to long-term aerobic dynamic feeding (ADF) periods. The PHA production capacity, kinetics and stoichiometry of the enrichments, the PHA composition, and the microbial diversity and community composition were explored to determine carbon and enrichment correlations. After 350-cycle enriching periods under feast-famine (F-F) regimes, the MMCs enriched by acetate sodium and glucose contained a maximum PHA content of 64.7% and 60.5% cell dry weight (CDW). The starch-enriched MMC only had 27.3% CDW of PHA. High-throughput sequencing revealed that non-PHA bacteria survived alongside PHA storing bacteria, even under severe F-F selective pressure. Genus of Pseudomonas and Stappia were the possible PHA accumulating bacteria in acetate-enriched MMC. Genus of Oceanicella, Piscicoccus and Vibrio were found as PHA accumulating bacteria in glucose-enriched MMC. Vibrio genus was the only PHA accumulating bacteria in starch-enriched MMC. The community diversity and composition were regulated by the substrate types.

Dynamic change of pH in acidogenic fermentation of cheese whey towards polyhydroxyalkanoates production: Impact on performance and microbial population

Polyhydroxyalkanoates (PHA) are a sustainable alternative to conventional plastics that can be obtained from industrial wastes/by-products using mixed microbial cultures (MMC). MMC PHA production is commonly carried out in a 3-stage process of acidogenesis, PHA culture selection and accumulation. This research focused on the possibility of tailoring PHA by controlling the acidogenic reactor operating conditions, namely pH, using cheese whey as model feedstock. The objective was to investigate the impact that dynamically varying the acidogenic pH, when targeting different PHA monomer profiles, had on the performance and microbial community profile of the anaerobic reactor. To accomplish this, an anaerobic reactor was continuously operated under dynamic pH changes, ranging from pH 4 to 7, turning to pH 6 after each change of pH. At pH 6, lactate and acetate were the dominant products (41–48% gCOD basis and 22–44% gCOD basis, respectively). At low pH, lactate production was higher while at high pH acetate production was favoured. Despite the dynamic change of pH, the fermentation product composition at pH 6 was always similar, showing the resilience of the process, i.e. when the same pH value was imposed, the culture produced the same metabolic products independently of the history of changes occurring in the system. The different fermentation product fractions led to PHAs of different compositions. The microbial community, analysed by high throughput sequencing of bacterial 16S rRNA gene fragments, was dominated by Lactobacillus, but varied markedly when subjected to the highest and lowest pH values of the tested range (4 and 7), with increase in the abundance of Lactococcus and a member of the Candidate Division TM7. Different bacterial profiles obtained at pH 6 during this dynamic operation were able to produce a consistent profile of fermentation products (and consequently a constant PHA composition), demonstrating the community’s functional redundancy.