Degradation Of Polyhydroxyalkanoates Research Articles

Carbon Cycle of Polyhydroxyalkanoates (CCP): Biosynthesis and Biodegradation.

Carbon neutrality of bioactive materials is vital in promoting sustainable development for human society. Polyhydroxyalkanoates (PHAs) is a class of typical carbon-cycle bio-polyesters synthesized by microorganisms using sugars, organic acids, and even carbon dioxide. PHAs first degrade into 3-hydroxybutyrate (3HB) before further breaking down into carbon dioxide and water, aligning with carbon-neutral goals. Due to their diverse molecular structures and material properties, excellent biocompatibility, and controlled biodegradability, PHAs have found widespread applications in environmental protection and biomedicine. However, challenges persist in achieving cost-effective PHA production and reusing degradation products. Additionally, understanding the carbon pathways in PHA synthesis and degradation remains limited. In this review, we first introduce the concept of the Carbon Cycle of Polyhydroxyalkanoates (CCP) and describe the biosynthetic pathways of aromatic monomers, carbon conversion processes, and PHA degradation in compost, soil, and marine environments. This will help us fully understand the sustainable utilization value of PHA as a biomaterial. Future trends point to integrating synthetic biology with emerging technologies to produce low-cost, high-value PHAs, supporting global green and low-carbon development.

Cloning, Overexpression and Application of Lipase from Thermotolerant Bacillus subtilis TTP-06 in the Degradation of Polyhydroxyalkanoate.

The online version contains supplementary material available at 10.1007/s12088-024-01329-z.

Elucidating regulation of polyhydroxyalkanoate metabolism in Ralstonia eutropha: Identification of transcriptional regulators from phasin and depolymerase genes

Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by LC-MS/MS, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using β-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli and their interaction with the promoters investigated in vitro by Electrophoretic Mobility Shift Assays. We could show that R. eutropha DNA-binding XRE-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a KD of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators.

ДОСЛІДЖЕННЯ ГІДРОЛІТИЧНОЇ ДЕГРАДАЦІЇ ПОЛІГІДРОКСІАЛКАНОАТІВ І ЇХ СУМІШЕЙ З ПОЛІЛАКТИДАМИ

The hydrolytic degradation of polyhydroxyalkanoates, polylactide and their mixtures in vitro in physiological solution and phosphate-salt buffer as well was researched. The hydrolysis intensity of biopolymers was evaluated via the mass loss, change in molecular weight as well as the water absorption applying the methods of infrared spectroscopy and complex thermal analysis. It was determined that films based on the researched biodegradable polymers thermostated in a phosphate-salt buffer have been degrading faster than in physiological solution.

Characterization of Ralstonia insidiosa C1 isolated from Alpine regions: Capability in polyhydroxyalkanoates degradation and production

This study ventures into the exploration of potential poly-3-hydroxybutyrate (PHB) degradation in alpine environments. PHB-degrading bacteria were identified in both campus soil, representing a residential area, and Mt. Kurodake soil, an alpine region in Hokkaido, Japan. Next-generation sequencing analysis indicated that the campus soil exhibited higher microbial diversity, while Ralstonia insidiosa C1, isolated from Mt. Kurodake soil, displayed the highest proficiency in PHB degradation. R. insidiosa C1 efficiently degraded up to 3% (w/v) of PHB and various films composed of other biopolymers at 14 °C. This bacterium synthesized homopolymers using substrates such as 3-hydroxybutyric acid, sugars, and acetic acid, while also produced copolymers using a mixture of fatty acids. The analysis results confirmed that the biopolymer synthesized by strain C1 using glucose was PHB, with physical properties comparable to commercial products. The unique capabilities of R. insidiosa C1, encompassing both the production and degradation of bioplastics, highlight its potential to establish a novel material circulation model.

Kinetic models for water sorption and a hydrolysis reaction with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH)

Biodegradable polymers, including polyhydroxyalkanoate (PHA), have attracted attention because plastic waste is entering the ocean and causing problems. Controlling the rate of PHA degradation is essential for expanding its application, and fundamental studies on the reaction mechanisms are needed. The purpose of this study is to experimentally verify the mechanisms of water sorption and hydrolysis reactions involving poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), a type of PHA, and construct a model. In this study, the water sorption process was measured by near-infrared (NIR) spectroscopy and Fourier transform infrared spectroscopic (FTIR) imaging. From the results, we found that water sorption on PHBH proceeds by non-Fickian diffusion, two kinetic processes involving adsorption and diffusion. The reaction rate of PHBH hydrolysis was determined by gel permeation chromatography (GPC) analysis, and a model was developed to predict PHBH degradation during hydrolysis from the experimental results. Furthermore, we proposed a reaction-diffusion model that combines water sorption and hydrolysis reactions. As a result, the degradation of PHBH plates with different thicknesses can be predicted for this first time.

Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (Alloalcanivorax, Alteromonas, Arenicella, Microbacterium, and Pseudoalteromonas) based on 16S rRNA analysis, including two novel genera (Arenicella and Microbacterium) as marine PHA-degrading bacteria. Microbacterium schleiferi (DSM 20489) and Alteromonas macleodii (NBRC 102226), the type strains closest to the several isolates, have an extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase homolog that does not fit a marine-type domain composition. However, A. macleodii exhibited no PHA degradation ability, unlike M. schleiferi. This result demonstrates that the isolated Alteromonas spp. are different species from A. macleodii. P(3HB) depolymerase homologs in the genus Alteromonas should be scrutinized in the future, particularly about which ones work as the depolymerase.

Expression, thermal stability modification and application in PHB degradation of polyhydroxyalkanoate depolymerase from Thermomonospora umbrina

Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.

Production of Polyhydroxybutyrate by Genetically Modified Pseudomonas sp. phDV1: A Comparative Study of Utilizing Wine Industry Waste as a Carbon Source.

Pseudomonas sp. phDV1 is a polyhydroxyalkanoate (PHA) producer. The presence of the endogenous PHA depolymerase (phaZ) responsible for the degradation of the intracellular PHA is one of the main shortages in the bacterial production of PHA. Further, the production of PHA can be affected by the regulatory protein phaR, which is important in accumulating different PHA-associated proteins. PHA depolymerase phaZ and phaR knockout mutants of Pseudomonas sp. phDV1 were successfully constructed. We investigate the PHA production from 4.25 mM phenol and grape pomace of the mutants and the wild type. The production was screened by fluorescence microscopy, and the PHA production was quantified by HPLC chromatography. The PHA is composed of Polydroxybutyrate (PHB), as confirmed by 1H-nuclear magnetic resonance analysis. The wildtype strain produces approximately 280 μg PHB after 48 h in grape pomace, while the phaZ knockout mutant produces 310 μg PHB after 72 h in the presence of phenol per gram of cells, respectively. The ability of the phaZ mutant to synthesize high levels of PHB in the presence of monocyclic aromatic compounds may open the possibility of reducing the costs of industrial PHB production.

Insight into polyhydroxyalkanoate (PHA) production from xylose and extracellular PHA degradation by a thermophilic Schlegelella thermodepolymerans

Accumulation of non-degradable plastic waste in the environment might be prevented by the use of biodegradable polyhydroxyalkanoate (PHA). In this study, the thermophile Schlegelella thermodepolymerans produced up to 80 wt% PHA based on dry cell mass. The largest PHA granules were found in the cells within 48 h using 20 g/L xylose, a C/N ratio of 100, an initial pH of 7, at 50 °C. The substrate consumption, pH changes, and cell growth were monitored, revealing the time dependency of PHA production in S. thermodepolymerans. The metabolic pathways from xylose to PHA were identified based on proteomic analysis, revealing involvement of classic phaCAB, de novo fatty acid biosynthesis, and fatty acid β-oxidation. In addition, it was shown that S. thermodepolymerans degraded extracellular PHA with a high efficiency at 50 °C.

Long-term in situ ruminal degradation of biodegradable polymers in Holstein dairy cattle

Using biodegradable materials such as polyhydroxyalkanoates (PHA) and poly(butylene succinate-co-adipate) (PBSA) to develop single-use agricultural plastics like bale netting may reduce the negative effects of plastic accumulation in the rumens of cattle. The objective of this research was to assess the long-term degradation of PHA, PBSA, and a PBSA:PHA blend (Blend) compared with a low-density polyethylene (LDPE) control. Polyhydroxyalkanoate, PBSA, Blend, and LDPE films were incubated in the rumens of 3 cannulated, nonlactating Holsteins for up to 150 d. In situ disappearance (ISD) and residue length were assessed after every incubation time. Data were analyzed with PROC MIXED in SAS and adjusted by Tukey's method to determine least squares differences between polymer treatments, incubation time, and their interaction. By 30 d, PHA achieved 100% degradation, with initiation occurring at 14 d indicated by ISD and a reduction in residue length. Poly(butylene succinate-co-adipate) and Blend did not achieve any significant ISD, but fragmentation of PBSA occurred at 60 d and fragmentation of Blend at just 1 d, likely due to abiotic hydrolysis. Low-density polyethylene achieved no ISD, and residue length did not change over incubation time. We propose that a PBSA:PHA blend is a valid alternative to polyethylene single-use agricultural plastic products based on its fragmentation within 1 d of incubation.

Recovery of polyhydroxyalkanoates (PHAs) polymers from a mixed microbial culture through combined ultrasonic disruption and alkaline digestion

PHAs are a form of cellular storage polymers with diverse structural and material properties, and their biodegradable and renewable nature makes them a potential green alternative to fossil fuel-based plastics. PHAs are obtained through extraction via various mechanical, physical and chemical processes after their intracellular synthesis. Most studies have until now focused on pure cultures, while information on mixed microbial cultures (MMC) remains limited. In this study, ultrasonic (US) disruption and alkaline digestion by NaOH were applied individually and in combination to obtain PHAs products from an acclimated MMC using phenol as the carbon source. Various parameters were tested, including ultrasonic sound energy density, NaOH concentration, treatment time and temperature, and biomass density. US alone caused limited cell lysis and resulted in high energy consumption and low efficiency. NaOH of 0.05–0.2 M was more efficient in cell disruption, but led to PHAs degradation under elevated temperature and prolonged treatment. Combining US and NaOH significantly improved the overall process efficiency, which could reduce energy consumption by 2/3rds with only minimal PHAs degradation. The most significant factor was identified to be NaOH dosage and treatment time, with US sound energy density playing a minor role. Under the semi-optimized condition (0.2 M NaOH, 1300 W L-1, 10 min), over 70% recovery and 80% purity were achieved from a 3 g L−1 MMC slurry of approximately 50% PHAs fraction. The material and thermal properties of the products were analyzed, and the polymers obtained from US + NaOH treatments showed comparable or higher molecular weight to previously reported results. The products also exhibited good thermal stability and rheological properties, compared to the commercial standard. In conclusion, the combined US and NaOH method has the potential in real application as an efficient process to obtain high quality PHAs from MMC, and cost-effectiveness can be further optimized.

Thauera sp. Sel9, a new bacterial strain for polyhydroxyalkanoates production from volatile fatty acids

Thauera is one of the main genera involved in polyhydroxyalkanoate (PHA) production in microbial mixed cultures (MMCs) from volatile fatty acids (VFAs). However, no Thauera strains involved in PHA accumulation have been obtained in pure culture so far. This study is the first report of the isolation and characterization of a Thauera sp. strain, namely Sel9, obtained from a sequencing batch reactor (S-SBR) set up for the selection of PHA storing biomass. The 16S rRNA gene evidenced a high sequence similarity with T. butanivorans species. Genome sequencing identified all genes involved in PHA synthesis, regulation and degradation. The strain Sel9 was able to grow with an optimum of chemical oxygen demand-to-nitrogen (COD:N) ratio ranging from 4.7 to 18.9. Acetate, propionate, butyrate and valerate were used as sole carbon and energy sources: a lag phase of 72 h was observed in presence of propionate. Final production of PHAs, achieved with a COD:N ratio of 75.5, was 60.12 ± 2.60 %, 49.31 ± 0.7 %, 37.31 ± 0.43 % and 18.06 ± 3.81 % (w/w) by using butyrate, acetate, valerate and propionate as substrates, respectively. Also, the 3-hydroxybutyrate/3-hydroxyvalerate ratio reflected the type of carbon sources used: 12.30 ± 0.82 for butyrate, 3.56 ± 0.02 for acetate, 0.93 ± 0.03 for valerate and 0.76 ± 0.02 for propionate. The results allow a better elucidation of the role of Thauera in MMCs and strongly suggest a possible exploitation of Thauera sp. Sel9 for a cost-effective and environmentally friendly synthesis of PHAs using VFAs as substrate.

Recovering phosphorus as struvite from the concentrated solution produced by the alternating aerobic/anaerobic biofilm system

The recovery of phosphorus is so far limited to recovering phosphorus as struvite or vivianite from the supernatant of digester, but this process is not economically viable. In this study, a biofilm reactor was operated to directly enrich phosphorus from sewage to investigate whether phosphorus harvest affected the phosphorus uptake/release of phosphorus accumulating organisms (PAOs) and evaluated the potential of phosphorus recovery from concentrated liquid. The results showed that phosphorus was quickly released in the new recirculated solution by PAOs with saturated poly-phosphate after phosphorus harvest, which promoted the synthesis or degradation of polyhydroxyalkanoate (PHA) and glycogen in PAOs, restoring its ability to absorb phosphorus. Although the results of crystallization experiments demonstrated that there were traces of K-struvite and Na-struvite (<0.1 wt%) in precipitate, the simultaneous precipitation of compounds could not significantly affect the purity of products. Using the concentrated solution with a phosphorus concentration of 168.5 ± 2.7 mg·L−1 as phosphorus source for struvite precipitation achieved phosphorus recovery efficiency of 74.5–95.8% and obtained struvite crystals with a size of 83.5–114.3 µm, whereas the quantity of fine products (<10 µm) was rised due to an increase in saturation. Especially, the highest phosphorus recovery efficiency (95.8 ± 3.2%) was obtained at pH= 8.5 and Mg: P = 1.5. Moreover, life cycle assessment (LCA) analysis indicated that the CO2 equivalent emission of struvite production was 0.64–1.01 kg CO2 equivalent/kg struvite, and the phosphorus recovery efficiency and environmental impact could be balanced at pH= 8.5 and Mg: P = 1.5:1.

Elucidating degradation properties, microbial community, and mechanism of microplastics in sewage sludge under different terminal electron acceptors conditions

This study is designed to investigate the roles of five key terminal electron acceptors (TEAs): O2, NO3−, Fe3+, SO42−, and CH2O, typically existing in the sludge on the degradation rates and pathways of three representative MPs: polylactic acid (PLA), polyvinyl chloride (PVC), and polyhydroxyalkanoate (PHA). The results revealed that approximately 51.46 ∼ 52.70% of PHA was degraded within 43 days, despite PLA and PVC being degraded insignificantly. Different TEAs significantly affected the end-products of PHA. The production rate of acetate gradually decreased from 90.48, 42.67, 38.30, and 17.56 to 3.30% when the TEAs were tested with CH2O, O2, SO42-, NO3– and Fe3+, respectively. The main functional bacteria involved in the PHA degradation were hydrolysis bacteria Burkholderiaceae and homo-acetogenic bacteria Clostridiacea, which accounted for 0.83% and 18.91% of the microbes. The current investigation could help improve understanding of MPs degradation pathways and mechanisms and minimize their risks in practice.

Valorization of lipid-rich wastewaters: A theoretical analysis to tackle the competition between polyhydroxyalkanoate and triacylglyceride-storing populations

The lipid fraction of the effluents generated in several food-processing activities can be transformed into polyhydroxyalkanoates (PHAs) and triacylglycerides (TAGs), through open culture biotechnologies. Although competition between storing and non-storing populations in mixed microbial cultures (MMCs) has been widely studied, the right selective environment allowing for the robust enrichment of a community when different types of accumulators coexist is still not clear. In this research, comprehensive metabolic analyses of PHA and TAG synthesis and degradation, and concomitant respiration of external carbon, were used to understand and explain the changes observed in a laboratory-scale bioreactor fed with the lipid-rich fraction (mainly oleic acid) of a wastewater stream produced in the fish-canning industry. It was concluded that the mode of oxygen, carbon, and nitrogen supply determines the enrichment of the culture in specific populations, and hence the type of intracellular compounds preferentially accumulated. Coupled carbon and nitrogen feeding regime mainly selects for TAG producers whereas uncoupled feeding leads to PHA or TAG production function of the rate of carbon supply under specific aeration rates and feast and famine phases lengths.

PHA granule formation and degradation by Cupriavidus necator under different nutritional conditions.

Polyhydroxyalkanoates (PHA) are polymers produced by microorganisms with increasing commercialization potential; Cupriavidus necator has been the model microorganism to research PHA production. Despite many contributions concerning the formation and degradation of PHA granules, as well as the morphological changes in cells, these phenomena have not been univocally explained yet. Thus, this study aims to integrate the microscopic and analytical analysis to characterize changes in bacterial cell/PHA granules morphology, PHA content, and yield coefficients under different cultivation strategies of C. necator ATCC 17697. The cell size and morphology, granule size and amount, residual biomass, and PHA concentration along the fermentation and degradation depend greatly on nutritional conditions and cultivation time of C. necator. It was proposed to calculate a yield coefficient for the residual biomass production in the PHA utilization stage, related to the bacteria's ability to survive without a carbon source in the culture medium by utilizing the accumulated PHA previously. Maximum granule length reached 1.07 µm after 72 h of PHA accumulation stage under optimum nutritional conditions. This value is twice the values previously reported for C. necator. It is important since the larger PHA granules facilitate the recovery of PHA and different application development.

Effects of antibiotics on enhanced biological phosphorus removal and its mechanisms

Many kinds of antibiotics are continuously discharged into wastewater and typically cause a great decrease in sewage treatment performance, whereas mechanisms of differences in the impacts of commonly used antibiotics on phosphate removal are still elusive. Thus, an enhanced biological phosphorus removal (EBPR) system, as an effective method of phosphate removal, was developed, and its performance in the treatment of artificial wastewater containing antibiotics at short- (8 h) and long-term (15 days) exposure was investigated. The results show that phosphorus removal was consistently inhibited by the addition of antibiotics with a significant difference (P < 0.05). To interpret the phenomena, mechanistic equations were developed, and the results indicate that for short-term tests, the difference was mainly caused by the suppression of polyhydroxyalkanoate (PHA) degradation and the activity of polyphosphate kinase (PPK), resulting in the different inhibition of the soluble orthophosphorus (SOP) uptake process. For long-term tests, the difference in SOP uptake was principally caused by the inhibition of PHA degradation and the activity of PPK, whereas the difference in SOP release resulted from the inhibition of activities of exopolyphosphatase (PPX) and adenylate kinase (ADK). Moreover, micro-mechanisms of such inhibition were identified from molecular docking and electrostatic potential.

Development and evaluation of controlled release fertilizer using P(3HB-co-3HHx) on oil palm plants (nursery stage) and soil microbes

Abstract The scope of this study was to formulate polyhydroxyalkanoate (PHA)-based controlled release fertilizer (PHA-CRF) for oil palm (nursery stage) by using biologically recovered poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and oil palm trunk vascular bundles combined with commercial NPK fertilizer. The efficiency of PHA-CRF was measured based on plant growth parameters such as plant height, number of leaves, chlorophyll content, stem thickness and fresh biomass. The release of the fertilizer in soil was monitored by electrical conductivity measurement. As expected, the highest electrical conductivity was recorded for the soil amended with immediate release fertilizer (IRF) which was 847 μS/cm compared to the soil amended with various combinations of CRF which were all less than 90 μS/cm. In terms of plant growth, PHA-CRF resulted in plants with similar heights to those amended with commercial CRF and IRF. In addition, the total fresh plant biomass was higher when PHA-CRF was used to amend the oil palm planting soil. Moreover, the degradation of PHA observed for PGVFB, PRVBF and PRVB was 61%, 47% and 27% after three months of application. The PHA degradation results confirm the enrichment of bacterial and fungal communities in oil palm rhizosphere with the addition of fertilizer in the formulations.

Influence of Depolymerases and Lipases on the Degradation of Polyhydroxyalkanoates Determined in Langmuir Degradation Studies

AbstractMicrobially produced polyhydroxyalkanoates (PHAs) are polyesters that are degradable by naturally occurring enzymes. Albeit PHAs degrade slowly when implanted in animal models, their disintegration is faster compared to abiotic hydrolysis under simulated physiological environments. Ultrathin Langmuir‐Blodgett (LB) films are used as models for fast in vitro degradation testing, to predict enzymatically catalyzed hydrolysis of PHAs in vivo. The activity of mammalian enzymes secreted by pancreas and liver, potentially involved in biomaterials degradation, along with microbial hydrolases is tested toward LB‐films of two model PHAs, poly(3‐R‐hydroxybutyrate) (PHB) and poly[(3‐R‐hydroxyoctanoate)‐co‐(3‐R‐hydroxyhexanoate)] (PHOHHx). A specific PHA depolymerase from Streptomyces exfoliatus, used as a positive control, is shown to hydrolyze LB‐films of both polymers regardless of their side‐chain‐length and phase morphology. From amorphous PHB and PHOHHx, ≈80% is eroded in few hours, while mass loss for semicrystalline PHB is 25%. Surface potential and interfacial rheology measurements show that material dissolution is consistent with a random‐chain‐scission mechanism. Degradation‐induced crystallization of semicrystalline PHB LB‐films is also observed. Meanwhile, the surface and the mechanical properties of both LB‐films remain intact throughout the experiments with lipases and other microbial hydrolases, suggesting that non‐enzymatic hydrolysis could be the predominant factor for acceleration of PHAs degradation in vivo.