Polyhydroxyalkanoate Biosynthesis Research Articles

Biosynthesis of polyhydroxyalkanoates employing a newly isolated Paramecium multimicronucleatum

Biosynthesis of polyhydroxyalkanoates employing a newly isolated Paramecium multimicronucleatum

Biosynthesis of Polyhydroxyalkanoates in Cupriavidus necator B-10646 on Saturated Fatty Acids.

It has been established that the wild-type Cupriavidus necator B-10646 strain uses saturated fatty acids (SFAs) for growth and polyhydroxyalkanoate (PHA) synthesis. It uses lauric (12:0), myristic (14:0), palmitic (16:0) and stearic (18:0) acids as carbon sources; moreover, the elongation of the C-chain negatively affects the biomass and PHA yields. When bacteria grow on C12 and C14 fatty acids, the total biomass and PHA yields are comparable up to 7.5 g/L and 75%, respectively, which twice exceed the values that occur on longer C16 and C18 acids. Regardless of the type of SFAs, bacteria synthesize poly(3-hydroxybutyrate), which have a reduced crystallinity (Cx from 40 to 57%) and a molecular weight typical for poly(3-hydroxybutyrate) (P(3HB)) (Mw from 289 to 465 kDa), and obtained polymer samples demonstrate melting and degradation temperatures with a gap of about 100 °C. The ability of bacteria to assimilate SFAs opens up the possibility of attracting the synthesis of PHAs on complex fat-containing substrates, including waste.

Synergistic effects of peracetic acid and free ammonia pretreatment on anaerobic fermentation of waste activated sludge to promote short-chain fatty acid production for polyhydroxyalkanoate biosynthesis: Mechanisms and optimization

Peracetic acid (PAA) combined with free ammonia (FA) pretreatment can be utilized to promote anaerobic fermentation (AF) of waste activated sludge (WAS) to produce short-chain fatty acids (SCFAs), and the resulting SCFAs are desirable carbon sources (C-sources) for polyhydroxyalkanoate (PHA) biosynthesis. This work aimed to determine the optimum conditions for PAA + FA pretreatment of sludge AF and the feasibility of using anaerobic fermentation liquor (AFL) for PHA production. To reveal the mechanisms of integrated pretreatment, the impacts of PAA + FA pretreatment on different stages of sludge AF and changes in the microbial community structure were explored. The experimental results showed that the maximum SCFA yield reached 491.35 ± 6.02 mg COD/g VSS on day 5 after pretreatment with 0.1 g PAA/g VSS +70 mg FA/L, which was significantly greater than that resulting from PAA or FA pretreatment alone. The mechanism analysis showed that PAA + FA pretreatment promoted sludge solubilization but strongly inhibited methanogenesis. According to the analysis of the microbial community, PAA + FA pretreatment changed the microbial community structure and promoted the enrichment of bacteria related to hydrolysis and acidification, and Proteiniclasticum, Macellibacteroides and Petrimonas became the dominant hydrolytic and acidifying bacteria. Finally, after alkali treatment, the AFL was utilized for batch-mode PHA production, and a maximum PHA yield of 55.05 wt% was achieved after five operation periods.

Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Biorefinery design with combined deacetylation and microwave pretreatment for enhanced production of polyhydroxyalkanoates and efficient carbon utilization from lignocellulose

Lignocellulosic biomass has emerged as a sustainable and economically viable feedstock for polyhydroxyalkanoate (PHA) production. However, it has seldom been used as part of an integrated process for effective carbon utilization. An innovative combined deacetylation and microwave pretreatment strategy (DEA-MW) and a wild-type Cupriavidus necator strain with the potential to utilize a lignocellulose-based carbon source and PHA biosynthesis were selected to set up the module needed to enable a new sustainable platform for biomass pretreatment and waste carbon valorization. DEA-MW pretreatment was introduced to mitigate the concentration of inhibitory acetic acid and phenolic compounds in the biomass hydrolysate. This pretreatment enhanced deacetylation and delignification, resulting in 95.50 % deacetylation and 45.30 % delignification. When DEA-MW pretreated rice husk biomass was coupled with microbial fermentation, C. necator promoted the fermentability of the rice husk hydrolysate. With a 3 g/L of levulinic acid from hydrolysis, as the sole carbon source, we achieved a concentration of PHA 440 mg/L with a content of 65 %. We also successfully recovered 72.22 % of lignin using a combined DEA-MW pretreatment. A detailed structural analysis showed that β-O-4, β–β, and β-5 linkages had been preserved in the recovered lignin. This study has shown that DEA-MW pretreatment plays a role in promoting biomass conversion and enhancing PHA production and opens up new possibilities for efficient and cost-effective lignocellulose-based biorefinery processes.

Valorization of Cabbage Waste as a Feedstock for Microbial Polyhydroxyalkanoate Production: Optimizing Hydrolysis Conditions and Polyhydroxyalkanoate Production.

Establishing a platform for the bioconversion of waste resources into value-added compounds is critical for achieving a sustainable and eco-friendly economy. Herein, we produced polyhydroxyalkanoate via microbial fermentation using cabbage waste as a feedstock and metabolically engineered Escherichia coli. For this, the hydrolysis conditions of cabbage waste were optimized by focusing on parameters such as substrate and enzyme concentrations to enhance the saccharification efficiency. The phaABC operon, which encodes key enzymes responsible for polyhydroxyalkanoate biosynthesis in Ralstonia eutropha H16, was overexpressed in E. coli. Using cabbage hydrolysate as the feedstock, this engineered E. coli strain could produce poly(3-hydroxybutyrate) with a polymer content of 26.0 wt % of dry cell weight. Moreover, malic acid in cabbage hydrolysate significantly enhanced poly(3-hydroxybutyrate) production; the addition of 0.5 g/L malic acid markedly increased poly(3-hydroxybutyrate) content by 59.9%. This study demonstrates the potential of cabbage waste as a promising raw material for the microbial production of polyhydroxyalkanoate.

Exploring polyhydroxyalkanoates biosynthesis using hydrocarbons as carbon source: a comprehensive review.

Environmental pollution caused by petrochemical hydrocarbons (HC) and plastic waste is a pressing global challenge. However, there is a promising solution in the form of bacteria that possess the ability to degrade HC, making them valuable tools for remediating contaminated environments and effluents. Moreover, some of these bacteria offer far-reaching potential beyond bioremediation, as they can also be utilized to produce polyhydroxyalkanoates (PHAs), a common type of bioplastics. The accumulation of PHAs in bacterial cells is facilitated in environments with high C/N or C/P ratio, which are often found in HC-contaminated environments and effluents. Consequently, some HC-degrading bacteria can be employed to simultaneously produce PHAs and conduct biodegradation processes. Although bacterial bioplastic production has been thoroughly studied, production costs are still too high compared to petroleum-derived plastics. This article aims to provide a comprehensive review of recent scientific advancements concerning the capacity of HC-degrading bacteria to produce PHAs. It will delve into the microbial strains involved and the types of bioplastics generated, as well as the primary pathways for HC biodegradation and PHAs production. In essence, we propose the potential utilization of HC-degrading bacteria as a versatile tool to tackle two major environmental challenges: HC pollution and the accumulation of plastic waste. Through a comprehensive analysis of strengths and weaknesses in this aspect, this review aims to pave the way for future research in this area, with the goal of facilitating and promoting investigation in a field where obtaining PHAs from HC remains a costly and challenging process.

Polyhydroxyalkanoate Biosynthesis from Waste Cooking Oils by Cupriavidus necator Strains Harbouring phaCBP-M-CPF4

Polyhydroxyalkanoate Biosynthesis from Waste Cooking Oils by Cupriavidus necator Strains Harbouring phaCBP-M-CPF4

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

De novo assembly and comparative genome analysis for polyhydroxyalkanoates-producing Bacillus sp. BNPI-92 strain

BackgroundCertain Bacillus species play a vital role in polyhydroxyalkanoate (PHA) production. However, most of these isolates did not properly identify to species level when scientifically had been reported. ResultsFrom NGS analysis, 5719 genes were predicted in the de novo genome assembly. Based on genome annotation using RAST server, 5,527,513 bp sequences were predicted with 5679 bp number of protein-coding sequence. Its genome sequence contains 35.1% and 156 GC content and contigs, respectively. In RAST server analysis, subsystem (43%) and non-subsystem coverage (57%) were generated. Ortho Venn comparative genome analysis indicated that Bacillus sp. BNPI-92 shared 2930 gene cluster (core gene) with B. cereus ATCC 14579 T (AE016877), B. paranthracis Mn5T (MACE01000012), B. thuringiensis ATCC 10792 T (ACNF01000156), and B. antrics Amen T (AE016879) strains. For our strain, the maximum gene cluster (190) was shared with B. cereus ATCC 14579 T (AE016877). For Ortho Venn pair wise analysis, the maximum overlapping gene clusters thresholds have been detected between Bacillus s p.BNPI-92 and Ba. cereus ATCC 14579 T (5414). Average nucleotide identity (ANI) such as OriginalANI and OrthoANI, in silicon digital DND-DNA hybridization (isDDH), Type (Strain) Genome Server (TYGS), and Genome-Genome Distance Calculator (GGDC) were more essentially related Bacillus sp. BNPI-92 with B. cereus ATCC 14579 T strain. Therefore, based on the combination of RAST annotation, OrthoVenn server, ANI and isDDH result Bacillus sp.BNPI-92 strain was strongly confirmed to be a B. cereus type strain. It was designated as B. cereus BNPI-92 strain. In B. cereus BNPI-92 strain whole genome sequence, PHA biosynthesis encoding genes such as phaP, phaQ, phaR (PHA synthesis repressor phaR gene sequence), phaB/phbB, and phaC were predicted on the same operon. These gene clusters were designated as phaPQRBC. However, phaA was located on other operons. ConclusionsThis newly obtained isolate was found to be new a strain based on comparative genomic analysis and it was also observed as a potential candidate for PHA biosynthesis.

How temperature shapes the biosynthesis of polyhydroxyalkanoates in mixed microbial cultures.

Three sequential batch reactors were operated for the enrichment in microbial communities able to store polyhydroxyalkanoates (PHAs) using activated sludge as inoculum. They ran simultaneously under the same operational conditions (organic loading rate, hydraulic and solids retention time, cycle length, C/N ratio) just with the solely difference of the working temperature: psychrophilic (15°C), mesophilic (30°C), and thermophilic (48°C). The microbial communities enriched showed different behaviors in terms of consumption and production rates. In terms of PHA accumulation, the psychrophilic community was able to accumulate an average amount of 17.7 ± 5.7wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the mesophilic 40.3 ± 7.0wt% PHBV, and the thermophilic 14.8 ± 0.3wt% PHBV in dry weight over total solids. The average PHBV production yields for each selected community were 0.41 ± 0.12 CmmolPHBV /CmmolVFA at 15°C, 0.64 ± 0.05 CmmolPHBV /CmmolVFA at 30°C, and 0.39 ± 0.14 CmmolPHBV /CmmolVFA at 48°C. The overall performance of the mesophilic reactor was better than the other two, and the copolymers obtained at this temperature contained a higher PHV fraction. The physico-chemical properties of the obtained biopolymers at each temperature were also measured, and major differences were found in the molecular weight, following an increasing trend with temperature. PRACTITIONER POINTS: PHBV molecular weight is influenced by the operational temperature increasing with it. Increasing temperatures promote the production of HB over HV. The best accumulation performance was found at 30°C for the tested operational conditions.

Different lanthanide elements induce strong gene expression changes in a lanthanide-accumulating methylotroph.

Since its discovery, Ln-dependent metabolism in bacteria attracted a lot of attention due to its bio-metallurgical application potential regarding Ln recycling and circular economy. The physiological role of Ln is mostly studied dependent on presence and absence. Comparisons of how different (utilizable) Ln affect metabolism have rarely been done. We noticed unexpectedly pronounced changes in gene expression caused by different Ln supplementation. Our research suggests that strain RH AL1 distinguishes different Ln elements and that the effect of Ln reaches into many aspects of metabolism, for instance, chemotaxis, motility, and polyhydroxyalkanoate metabolism. Our findings regarding Ln accumulation suggest a distinction between individual Ln elements and provide insights relating to intracellular Ln homeostasis. Understanding comprehensively how microbes distinguish and handle different Ln elements is key for turning knowledge into application regarding Ln-centered biometallurgy.

Polyhydroxyalkanoate production by the plant beneficial rhizobacterium Pseudomonas chlororaphis PCL1606 influences survival and rhizospheric performance

Pseudomonas chlororaphis PCL1606 (PcPCL1606) is a model rhizobacterium used to study beneficial bacterial interactions with the plant rhizosphere. Many of its beneficial phenotypes depend on the production of the antifungal compound 2-hexyl, 5-propyl resorcinol (HPR). Transcriptomic analysis of PcPCL1606 and the deletional mutant in HPR production ΔdarB strain, assigned an additional regulatory role to HPR, and allowed the detection of differentially expressed genes during the bacterial interaction with the avocado rhizosphere. Interestingly, the putative genes phaG (PCL1606_46820) and phaI (PCL1606_56560), with a predicted role in polyhydroxyalkanoate biosynthesis, were detected to be under HPR control. Both putative genes were expressed in the HPR-producing wild-type strain, but strongly repressed in the derivative mutant ΔdarB, impaired in HPR production. Thus, a derivative mutant impaired in the phaG gene was constructed, characterized and compared with the wild-type strain PcPCL1606 and with the derivative mutant ΔdarB. The phaG mutant had strongly reduced PHA production by PcPCL1606, and displayed altered phenotypes involved in bacterial survival on the plant roots, such as tolerance to high temperature and hydrogen peroxide, and decreased root survival, in a similar way that the ΔdarB mutant. On the other hand, the phaG mutant does not have altered resistance to desiccation, motility, biofilm formation or adhesion phenotypes, as displayed by the HPR-defective ΔdarB mutant have. Interestingly, the mutant defective in PHA production also lacked a biocontrol phenotype against the soilborne pathogenic fungus Rosellinia necatrix, even when the derivative mutant still produced the antifungal HPR compound, demonstrating that the final biocontrol phenotype of PcPCL1606 first requires bacterial survival and adaptation traits to the soil and rhizosphere environment.

Biosynthesis of polyhydroxyalkanoates (PHAs) from non-edible Cerbera odollam (sea mango) oil

Polyhydroxyalkanoates (PHAs) are biodegradable polymers, which have recently attracted much attention as a promising substitute for petroleum-based plastics. For circumventing the financial barrier of PHA production, non-edible Cerbera odollam oil was proposed as a novel PHA feedstock. The objective of this study was to investigate the feasibility of C. odollam oil for PHA production using Cupriavidus necator and Pseudomonas resinovorans. The PHA production performance of P. resinovorans on different oil conditions (i.e., crude, hydrolyzed, and emulsified oil) was also compared. Direct fermentation of crude C. odollam oil by P. resinovorans showed the most promising results with a biomass accumulation of 1.32 g/L and a maximum PHA concentration of 0.25 g/L (corresponding to 18.8 % polymer content). The polymer was composed of 3-hydroxybutyrate (3HB), 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), and 3-hydroxydecanoate (3HD). For the first time, C. odollam oil has been proven to be a feasible feedstock for an economical production of PHAs.

Biosynthesis, characterization, and biodegradation of elastomeric polyhydroxyalkanoates consisting of α-dimethylated monomer units

Although poly[(R)-3-hydroxybutyrate)] [P(3HB)] is a microbial polyester that can be used as a sustainable and biodegradable plastic, its use has been limited because of poor material properties due to its stiff and brittle nature. To overcome this issue, it is critical to diversify the monomeric composition of 3HB-based polymers through the introduction of new repeating units that can improve material properties. In this study, a new 3HB-based copolymer was biosynthesized with 3-hydroxypivalate (3HPi) units containing signature dimethyl groups on the α-carbon. To biosynthesize poly[(R)-3-hydroxybutyrate-co-3-hydroxypivalate] {P(3HB-co-3HPi)}, recombinant Escherichia coli LSBJ was cultured with glucose and 3-hydroxypivalic acid (3HPiA) as the carbon source and 3HPi precursor, respectively. This resulted in the successful synthesis of P(3HB-co-3HPi) copolymers with a 3HPi fraction in the range of 2–50 mol% by adjusting the precursor concentration to the microbial catalyst. Interestingly, the biosynthesized polymers exhibited an increasing molecular weight with increasing 3HPi fractions, up to 424 × 104 in weight average molecular weight. The thermal properties of P(3HB-co-3HPi) were similar to those of conventional 3HB-based copolymers, but the copolymers displayed extraordinary mechanical performance compared to other 3HB homo- and copolymers. P(3HB-co-32 mol% 3HPi) displayed an elongation at break of 1919 %, the highest value ever reported for bacterial polyesters. Given its high flexibility, P(3HB-co-3HPi) copolymers exhibited reversible deformation during tension testing, unlike other 3HB-based copolymers. Furthermore, P(3HB-co-3HPi) copolymers and monomeric 3HPiA were both biodegradable. This study has demonstrated that the incorporation of 3HPi monomers into 3HB-based copolymers results in superior mechanical properties and has introduced a promising new class of materials for use as sustainable and biodegradable plastics.

Polyhydroxyalkanoates Production from Cacao Fruit Liquid Residues Using a Native Bacillus megaterium Strain: Preliminary Study

Cacao mucilage exudates (CMEs), byproducts of cacao beans fermentation, are effluents rich in sugars and low-molecular-weight organic acids. In this study, we utilized CMEs to formulate culture media for the biosynthesis of polyhydroxyalkanoates (PHAs) using a native Bacillus megaterium strain (B2). Our investigation followed the adaptation process of B2 to the CMEs-based media, and we closely monitored the development of the microorganism in terms of consumption of acids and sugars. Upon successful adaptation to the CMEs-based media, we transferred the strain to a 4-L batch bioreactor to evaluate the impact of operational variables on PHAs production. We examined the microorganisms’ yield, productivity, and growth kinetics using the CME-based media. Our results showed a product yield of 0.98 g PHA/g carbon source, with an accumulation percentage of 57% and a productivity increase of 33% compared to PHA produced from residual glycerol using the same microorganism. The biopolymer's structure was confirmed to be of a polyhydroxybutyrate homopolymer (PHB) type using various molecular characterization techniques, including Matrix-Assisted Laser Desorption/Ionization mass spectrometry (MALDI-TOF-MS), infrared spectroscopy (IR), elemental composition (EC), ultraviolet–visible spectroscopy (UV–VIS), and thermal analysis (TGA, DSC).Graphical

Genetic engineering of low-temperature polyhydroxyalkanoate production by Acidovorax sp. A1169, a psychrophile isolated from a subglacial outflow

In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2 g/L) at a temperature of 15 °C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5 °C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10 g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles.

Upcycling of PET oligomers from chemical recycling processes to PHA by microbial co-cultivation

Polyethylene terephthalate (PET) is the most widely consumed polyester plastic and can be recycled by many chemical processes, of which glycolysis is most cost-effective and commercially viable. However, PET glycolysis produces oligomers due to incomplete depolymerization, which are undesirable by-products and require proper disposal. In this study, the PET oligomers from chemical recycling processes were completely bio-depolymerized into monomers and then used for the biosynthesis of biodegradable plastics polyhydroxyalkanoates (PHA) by co-cultivation of two engineered microorganisms Escherichia coli BL21 (DE3)-LCCICCG and Pseudomonas putida KT2440-ΔRDt-ΔZP46C-M. E. coli BL21 (DE3)-LCCICCG was used to secrete the PET hydrolase LCCICCG into the medium to directly depolymerize PET oligomers. P. putida KT2440-ΔRDt-ΔZP46C-M that mastered the metabolism of aromatic compounds was engineered to accelerate the hydrolysis of intermediate products mono-2-(hydroxyethyl) terephthalate (MHET) by expressing IsMHETase, and biosynthesize PHA using ultimate products terephthalate and ethylene glycol depolymerized from the PET oligomers. The population ratios of the two microorganisms during the co-cultivation were characterized by fluorescent reporter system, and revealed the collaboration of the two microorganisms to bio-depolymerize and bioconversion of PET oligomers in a single process. This study provides a biological strategy for the upcycling of PET oligomers and promotes the plastic circular economy.

Valorization of food waste derived anaerobic digestate into polyhydroxyalkanoate (PHA) using Thauera mechernichensis TL1

Polyhydroxyalkanoate (PHA) is a biopolymer that can be used as a bioplastic, offering a green alternative to petroleum-based plastics. In this study, we investigated PHA production using Thauera mechernichensis TL1. The optimal molar C/N ratio was determined to be 20 from among the ratios of 4, 20, 40, 80, and 200 and in the absence of nitrogen. Food waste anaerobic digestate, mainly comprised of acetate and propionate, was used as the carbon source for PHA production by T. mechernichensis TL1, resulting in a maximum PHA content of 23.98 ± 0.52 wt% (0.52 ± 0.02 g/L PHA) with a PHA productivity of 0.043 g/L-h PHA. In addition, when using acetate and propionate, T. mechernichensis TL1 produced PHA with a maximum PHA content of 57.43 ± 2.84 wt% (2.04 ± 0.11 g/L PHA) and 50.94 ± 1.61 wt% (2.62 ± 0.16 g/L PHA), with a PHA productivity of 0.092 g/L-h PHA and 0.070 g/L-h PHA, respectively. Proton nuclear magnetic resonance spectroscopy (1H NMR) confirmed polyhydroxybutyrate (PHB) production using acetate as a carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production using propionate or food waste anaerobic digestate as the carbon source. The whole-genome analysis of T. mechernichensis TL1 confirmed the existence of a PHA biosynthesis pathway, with the presence of phaA, phaB, phaC (Class I and Class II), and phaJ genes. This study was the first to demonstrate Thauera sp.’s ability to produce PHA from food waste anaerobic digestate, rendering it as a promising candidate for PHA-producing bacteria for the valorization of food waste.