Polyhydroxyalkanoates Research Articles

Influence of biodegradable microplastics on soil carbon cycling: Insights from soil respiration, enzyme activity, carbon use efficiency and microbial community.

The rising prevalence of biodegradable microplastics (BMPs) in soils has raised concerns about their impacts on soil ecosystems and carbon cycling. This study investigates the effects of different BMPs on soil carbon cycling, focusing on soil respiration, enzyme activities, and carbon use efficiency (CUE) from 13C-labeled dissolved organic carbon (DOC) in an upland soil. The BMPs tested were polybutylene adipate terephthalate (PBAT), polyhydroxyalkanoates (PHA), and polylactic acid (PLA), at high (H, 1% w/w) and low (L, 0.1% w/w) concentrations. Over a 64-day incubation, cumulative CO2 emissions increased in the PHA_L, PHA_H, and PLA_H treatments, with the highest rise of 665% PHA_H treatment. Microbial biomass carbon (MBC) ranged from 97.73±3.03mgC kg⁻1 in the control to 223.09±7.91mgC kg⁻1 in PHA_H, with microbial CUE peaking at 0.26 in PHA_H. Enzymatic activities were notably affected: β-glucosidase (BG) increased by 50% in PLA_H, while cellobiohydrolase (CBH) activity decreased by up to 62% in PBAT_H and PLA_L. N-acetylglucosaminidase (NAG) and phosphatase (AP) activities were highest in PHA_H, indicating enhanced nutrient cycling. Microbial community structure based on PLFAs was significantly altered, with total PLFA content increasing by 191% in PHA_H. Correlation analysis and partial least squares path modeling (PLS-PM) revealed that BMP concentration, DOC content, and microbial diversity were positively correlated with microbial CUE. This study highlights the significant role of BMPs in influencing soil carbon cycling, primarily through their effects on microbial diversity and soil enzyme activities.

Application of Bacillus cereus for synthesis of polyhydroxyalkanoates from industrial corn starch residue.

Starch hydrolysate from corn is typically employed as a carbon source for citric acid production by Aspergillus niger. Enzymatic or acid hydrolysis of corn starch generates a corn starch residue (CSR), which represents a potential bio-fermentation carbon source. In addition, polyhydroxyalkanoates (PHAs) can be used as an alternative to traditional petrochemical plastics. In this study, the potential applications of CSR in microbial PHA production were investigated. Two Bacillus cereus strains, MG1 and MQ1, were isolated based on their ability to grow with CSR as the sole carbon source and their capacity for PHA accumulation. After introducing key genes involved in the PHA synthesis pathway and optimizing fermentation conditions, the PHA content of MG2 reached 61.61 % of the cell dry weight (CDW), representing the highest PHA content (CDW%) in B. cereus. To the best of our knowledge, this is the first report of PHA synthesis from CSR in B. cereus and provides a novel approach for the rational utilization of CSR.

Production of polyhydroxyalkanoate from new isolated bacteria of Acidovorax diaphorobacter ZCH-15 using orange peel and its underlying metabolic mechanisms

Polyhydroxyalkanoate (PHA) is considered a sustainable alternative to traditional petroleum-based plastics due to its biodegradability and biocompatibility. In this study, Acidovorax diaphorobacter ZCH-15, an efficient PHA-producing strain, was isolated from activated sludge. Using food waste-derived orange peel as a substrate, the strain initially achieved a PHA concentration of 0.39 g/L. Under optimal fermentation conditions (30℃, pH 8, 2 % inoculum concentration, and 30 g/L carbon source), the PHA concentration increased by 138 % to reach a maximum of 0.93 g/L. Proton nuclear magnetic resonance spectroscopy and gas chromatography analyses identified the PHA composition as poly(3-hydroxybutyrate-co-3-hydroxyvalerate), which exhibited high crystallinity and structural stability. Metabolomic analysis indicated that the tricarboxylic acid cycle and pentose phosphate pathway were involved in producing succinyl-CoA, a precursor required for PHA synthesis. This study demonstrates the potential for cost-effective industrial PHA production while enabling the high-value utilization of food waste.

The role of carbon and nitrogen ratio on sewage sludge microbiota for producing polyhydroxyalkanoates.

The products of an advanced sewage sludge fermentation process can be used to generate polyhydroxyalkanoates (PHAs), precursors of bioplastics considered excellent candidates for replacing petroleum-derived plastics. The aerobic feast-anoxic famine cycling strategy has proven to be an efficient method for enriching sewage sludge microbiota with PHA-producing microorganisms. This work evaluated the effect of different carbon to nitrogen ratios (C/N) of 3.5, 2, and 1g COD/g N for modulating the structure of sewage sludge microbiota to improve PHA production. The study was executed on a pilot plant scale using wasted activated sludge as an organic carbon source derived from an oxic-settling anaerobic plant that collects wastewater from various facilities at the University of Palermo campus. PHA production performance was monitored over three experimental periods characterized by a different C/N ratio. The results showed that lower C/N ratios reduced PHA production with 20, 24, and 26 % w/w of PHAs for COD/N values of 1, 2, and 3.5g COD/g N, respectively. In parallel, the metataxonomic analysis revealed a higher abundance of PHA-producing microorganisms at the ratio of 3.5g COD/g N, such as Proteobacteria, specifically Betaproteobacteria. In addition, the analysis showed an increase in fungal abundance and diversity as decrease the ratio C/N decreased. Thus, these findings demonstrate the utility of metataxonomics in elucidating the relationships between operating conditions, bacterial and fungal microbiota structure and the achievement of specific outputs. The insights gained from this study demonstrated a positive correlation between C/N ratios, PHA-producing microorganisms, and PHA yields.

Long-chain fatty acids as sole carbon source in polyhydroxyalkanoates production by Cupriavidus necator H16.

Polyhydroxyalkanoates (PHA) are promising eco-friendly alternatives to petrochemical plastics. This study investigated the impact of the main fatty acids present in waste and fresh oils -palmitic, stearic, oleic, and linoleic acid-on PHA production using Cupriavidus necator H16, focusing on production yield, polymer composition, thermal properties, and microbial viability. Experiments were conducted with low (5g/L) and high (15g/L) carbon content for 168h. Oleic acid was the most effective carbon source, yielding higher PHA production rates, especially noticeable at higher concentrations. The monomer composition and thermal properties of PHAs varied with the type and concentration of fatty acid used. Stearic acid produced PHAs with more 3-hydroxyvalerate and medium-chain length monomers. Microbial viability was consistent across all conditions, except for linoleic acid, which had a detrimental effect. These findings provide key insights into optimizing fatty acid selection to enhance PHA production and tailor polymer properties for industrial applications.

Production and Bioseparation Applications of Polyhydroxyalkanoate Nano-Granules Functionalized with Streptavidin

Rapidly growing industrial biotechnology and bio-manufacturing require simple and cost-effective bioseparation tools. A novel strategy of bioseparation based on the streptavidin-decorated polyhydroxyalkanoate (PHA) nano-granules was developed in this study. By fusing to the N-terminus of PHA-associated phasin protein, the streptavidin was one-step immobilized on the surface of PHA nano-granules simultaneously with the accumulation of PHA in recombinant Escherichia coli. About 1.95 g/L of PHA nano-granules (54.51 wt% of cell dry weight) were produced after 48 h bacterial cultivation. The following qualitative and quantitative characterizations demonstrated that the streptavidin accounted for approximately 6.78% of the total weight of the purified PHA nano-granules and confirmed a considerable biotin affinity of 0.1 ng biotin/μg surface protein. As a proof of concept, the nano-granules were further functionalized with biotinylated oligo(dT) for mRNA isolation and about 1.26 μg of mRNA (occupied 2.59%) was purified from 48.45 μg of total RNA, achieving good integrity and high purity with few DNA and rRNA contaminations. Moreover, the nano-granules retained more than 80% of their initial mRNA recovery efficiency after ten cycles of repeated use. The PHA-SAP nano-granules were also functionalized with biotinylated magnetic beads, allowing magnetic recovery of the PHA nano-granules from cell lysates that still needs optimization. Our study provides a novel and expandable platform of PHA nano-granules that can be further functionalized with various biological groups for bioseparation applications. The functional PHA nano-granules have a great potential to serve as bioseparation resin for large-scale purification processes after suitable optimizations for “bench-to-factory” translation, contributing to scalable and sustainable bioprocessing.

Engineering xylose utilization in Cupriavidus necator for enhanced poly(3-hydroxybutyrate) production from mixed sugars.

Lignocellulosic biomass is a promising renewable feedstock for biodegradable plastics like polyhydroxyalkanoates (PHAs). Cupriavidus necator, a versatile microbial host that synthesizes poly(3-hydroxybutyrate) (PHB), the most abundant type of PHA, has been studied to expand its carbon source utilization. Since C. necator NCIMB11599 cannot metabolize xylose, we developed xylose-utilizing strains by introducing synthetic xylose metabolic pathways, including the xylose isomerase, Weimberg, and Dahms pathways. Through rational and evolutionary engineering, the RXI22 and RXW62 strains were able to efficiently utilize xylose as the sole carbon source, producing 64.2 wt% (wt%) and 61.4 wt% PHB, respectively. Among the engineered strains, the xylose isomerase-based RXI22 strain demonstrated the most efficient co-fermentation performance, with a PHB content of 75.7 wt% and a yield of 0.32 (g PHB/g glucose and xylose) from mixed sugars. The strains developed in this study represent an enhanced PHA producer, offering a sustainable route for converting lignocellulosic biomass into bioplastics.

3D printing of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/poly(lactide-co-glycolide) blend loaded with β-tricalcium phosphate for the development of scaffolds to support human mesenchymal stromal cell proliferation.

Polyhydroxyalkanoates (PHAs) are microbially produced aliphatic polyesters investigated for tissue engineering thanks to their biocompatibility, processability, and suitable mechanical properties. Taking advantage of these properties, the present study investigates the development by 3D printing of bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds loaded with β-tricalcium phosphate (β-TCP) for bone tissue regeneration. PHBV blending with poly(lactide-co-glycolide) (PLGA) (30wt%) was exploited to enhance material processability via an optimized computer-aided wet-spinning approach. In particular, PHBV/PLGA blends were loaded with different β-TCP percentages (up to 15wt%) by suspending the ceramic particles into the polymeric solution. The composite materials were successfully fabricated into 3D scaffolds with a fully interconnected porous architecture, as demonstrated by scanning electron microscopy. The ceramic phase had a significant effect on PHBV crystallization as shown by differential scanning calorimetry, as well as on scaffold mechanical properties with a significant increase of compressive modulus in the case of 5wt% β-TCP loading. In addition, in vitro cell culture experiments demonstrated that β-TCP loading led to a significant increase of the viability of human mesenchymal stem/stromal cells grown on the scaffolds. Taken together, our data suggest that microbial PHBV processability, biomechanical performance, and bioactivity can be improved through combined PLGA blending and β-TCP loading.

Valorization of chicken feathers for polyhydroxyalkanoates production using Rohodococcus quinshengii LS18 and the biodegradation studies with the extracted polymer and its blends.

Polyhydroxyalkanoates (PHAs) are promising polymer in the battle against plastic pollution. Here, Rhodococcus qingshengii LS18 bacterial strain recovered from abattoir soil was identified as significant PHA producer (2.16g/L PHA). Amongst five animal wastes tested, chicken feathers (2.2g/L) were found to be best for PHA production from R. qingshengii LS18. Finest conditions for PHA production after statistical optimization were found at 120h with 40g/L chicken feathers and 1g/L urea at 80rpm which showed 2.6 times increment in PHA production as compared to One-factor-at-a-time optimization (OFAT). The extracted PHA was analysed using NMR, FTIR, XRD and GCMS. The melting temperature (Tm) of microbial PHA was recorded at 176.63°C as per DSC analysis and this polymer suffered complete disintegration at ~ 300°C as per the TGA profile which is better than the standard PHA (~ 283.14 ± 10°C). Blending of microbial PHA was done to overcome the brittleness with sole extracted PHA and for better degradation capability. Best PHA blend obtained with gelatin and Egg shell powder (ESP) in ratio 2:1:1 where 94% Elongation at break, 62% Elongation at max load and 9.81MPa Tensile strength was found. Here, biodegradation rate of PHA blend in unaltered garden soil (at 30-40 °C, humidity ~ 30%, pH 7.4) was recorded to be 94% on 60th day of soil burial experiment. The study concludes that R. qingshengii LS18 is a promising PHA producer having the ability to utilize chicken feathers in optimised conditions and can be used for packaging applications in the form of PHA blends.

Simultaneous partial nitrification, endogenous and autotrophic denitrification in a single-stage electrolysis-integrated sequencing batch biofilm reactor (E-SBBR) for stable and enhanced kitchen digested wastewater treatment.

Simultaneous partial nitrification-denitrification (SPND) is a promising process for nitrogen (N) removal from kitchen digested wastewater characterized by a low C/N ratio. However, its widespread application is often restricted due to the unstable partial nitrification and unsatisfactory denitrification performance. This work developed a novel simultaneous partial nitrification, endogenous and autotrophic denitrification process using a single-stage electrolysis-integrated sequencing batch biofilm reactor (E-SBBR) with anoxic/electro-anaerobic/aerobic operating strategy. The novel process considerably enhanced the stability and N removal efficiency (NH4+-N>94.5% and TN>90.8%) of the SPND process. The pre-electro-anaerobic phase achieved alkalinity and H2 generation, and intracellular carbon storage. The increased alkalinity resulted in increased free ammonia (FA) which secured complete suppression of nitrite-oxidizing bacteria (NOB). SPND efficiency in the aerobic phase was dramatically improved using polyhydroxyalkanoates (PHAs) and H2 as electron donors for endogenous and autotrophic denitrification. Microbial community analysis indicated the successful washout of NOB and the enrichment of ammonia-oxidizing bacteria (AOB), denitrifying glycogen accumulating organisms (DGAOs), autotrophic and heterotrophic denitrifiers in the system. This research presents a distinctive SPND process for intensified kitchen digested wastewater treatment and gives insights into the underlying mechanism.

Recent advances in engineering non-native microorganisms for poly(3-hydroxybutyrate) production.

Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that belongs to a group of polymers called polyhydroxyalkanoates (PHAs). PHB can be synthesized from renewable resources, making it a promising alternative to petroleum-derived plastics. It is also considered non-toxic, biodegradable, and biocompatible, which makes it suitable for various applications in the medicine and biomedicine. Many microorganisms biosynthesize and accumulate PHB naturally. However, recent advancements in metabolic engineering and synthetic biology have allowed scientists to engineer non-native microorganisms to produce PHB. This review comprehensively summarizes all non-native microbial hosts used for PHB biosynthesis and discusses different metabolic engineering approaches used to enhance PHB production. These strategies include optimizing the biosynthesis pathway through cofactor engineering, metabolic pathway reconstruction, and cell morphology engineering. Moreover, the CRISPR/Cas9 approach is also used for manipulating the genome of non-host microorganisms to enable them produce PHB. Among non-native microbial hosts, Escherichia coli has been successfully used for industrial-scale PHB production. However, further genetic engineering approaches are needed to make non-native microbial hosts more suitable for large-scale PHB production.

Introduction of acetyl-phosphate bypass and increased culture temperatures enhanced growth-coupled poly-hydroxybutyrate production in the marine cyanobacterium Synechococcus sp. PCC7002.

Polyhydroxyalkanoate (PHA) is an attractive bio-degradable plastic alternative to petrochemical plastics. Photosynthetic cyanobacteria accumulate biomass by fixing atmospheric CO2, making them promising hosts for sustainable PHA production. Conventional PHA production in cyanobacteria requires prolonged cultivation under nutrient limitation to accumulate cellular PHA. In this study, we developed a system for growth-coupled production of the PHA poly-hydroxybutyrate (PHB), using the marine cyanobacterium Synechococcus sp. PCC 7002. A recombinant strain termed KB1 expressing a set of heterologous PHB biosynthesis genes (phaA/phaB from Cupriavidus necator H16 and phaE/phaC from Synechocystis sp. PCC 6803) accumulated substantial PHB during growth (11.4% of dry cell weight). To improve PHB accumulation, we introduced the Pseudomonas aeruginosa phosphoketolase gene (pk) into strain KB1, rewiring intermediates of the Calvin-Benson-Bassham (CBB) cycle (xyluose-5-phosphate, sedoheptulose 7-phosphate, and fructose-6-phosphate) to acetyl-CoA. The pk-expressing strain, KB15, accumulated 2.1-fold enhanced levels of PHB (23.8% of dried cell weight), relative to the parent strain, KB1. The highest PHB titer of KB15 strain supplemented with acetate was about 1.1gL-1 and the yield was further enhanced by 2.6-fold following growth at 38°C (0.21gL-1d-1), relative to growth at 30°C. Metabolome analysis revealed that pool sizes of CBB intermediates decreased, while levels of acetyl-CoA increased in strain KB15 compared with strain KB1, and this increase was further enhanced following growth at 38°C. Our data demonstrate that acetyl-phosphate generated by Pk was converted into acetyl-CoA via acetate by hitherto unidentified enzymes. In conclusion, expression of heterologous PHB biosynthesis genes enabled growth-coupled PHB production in strain PCC 7002, which was increased through acetyl-CoA supplementation by bypassing acetyl-phosphate and elevating culture temperature.

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.

Optimization of polyhydroxyalkanoate (PHA) production from biohythane pilot plant effluent by Cupriavidus necator TISTR 1335.

Bioplastics, particularly polyhydroxyalkanoates (PHAs), are emerging as promising alternatives to traditional materials due to their biodegradability. This study focuses on the production of PHAs as bioplastics using effluent from hydrogen production in a two-stage Biohythane Pilot Plant, which provides a low-cost substrate. The aim is to optimize production conditions, with Cupriavidus necator TISTR 1335 being used as the PHA producer. Utilizing Response Surface Methodology-Central Composite Design, we explored optimal conditions, revealing peak PHA production at a substrate concentration of 33.51 g COD/L and a pH of 6.87. The predicted optimal PHA concentration was at 3.05 g/L within the established model, closely matching the experimentally validated value of 3.02 g/L, with the overall usage rate of reducing sugars approximately 50-60%. This study underscores the importance of optimizing PHA production conditions and paving the way toward large-scale PHA production.

Valorization of waste cooking oil (WCO) for production of polyhydroxyalkanoates (PHAs) copolymers using Bacillus megaterium MTCC 428 and characterization by untargeted metabolomics

Valorization of waste cooking oil (WCO) for production of polyhydroxyalkanoates (PHAs) copolymers using Bacillus megaterium MTCC 428 and characterization by untargeted metabolomics

Exploiting latent microbial potentials for producing polyhydroxyalkanoates: A holistic approach.

Plastics are versatile, however, nonbiodegradable polymers that are primarily derived from fossil fuels and pose notable environmental challenges. However, biopolymers, such as polyhydroxyalkanoates (PHAs), poly(lactic acid), starch, and cellulose have emerged as sustainable alternatives to conventional plastics. Among these, PHAs stand out as strong contenders as they are completely bio-based and biodegradable and are synthesized by microbes as an energy reserve under stress conditions. Despite their limitations, including low mechanical strength, susceptibility to degradation, a restricted scope of application, and high production costs, biopolymers have promising potential. This review explores strategies for enhancing PHA production to address these challenges, emphasizing the need for sustainable PHA production. These strategies include selecting robust microbial strains and feedstock combinations, optimizing cell biomass and biopolymer yields, genetically engineering biosynthetic pathways, and improving downstream processing techniques. Additives such as plasticizers, thermal stabilizers, and antioxidants are crucial for modifying PHA characteristics, and its processing for achieving the desired balance between processability and end-use performance. By overcoming these complications, biopolymers have become more viable, versatile, and eco-friendly alternatives to conventional plastics, offering hope for a more sustainable future.

Advances in polyhydroxyalkanoate (PHA) production from renewable waste materials using halophilic microorganisms: A comprehensive review.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polymers that can replace conventional plastics in different sectors. However, PHA commercialization is hampered due to their high production cost resulting from the use of high purity substrates, their low conversion into PHAs by using conventional microbial chassis and the high downstream processing cost. Taking these challenges into account, researchers are focusing on the use of waste by-products as alternative low-cost feedstocks for fast-growing and contamination-resistant halophilic microorganisms (Bacteria, Archaea…). This is of great importance since these extremophiles can use low-cost substrates, produce high PHA content of copolymers or different PHA monomer compositions. They can present high potential for reducing the costs of PHA fermentation and recovery processes, making their use in commercial applications easier. However, little is known about the potential of halophiles in advancing the PHA production from renewable waste materials at lab-scale and their successful implementation at industrial-scale. This review presents actual advances in PHA production by halophilic pure/engineered species (e.g. Haloferax mediterranei, Halomonas spp.) and mixed microbial consortia (MMC) using organic waste streams. The development of optimal PHA production process involves robust genetic engineering strategies, advanced fermentation processes using mixed microbial consortia versus pure/engineered strains as well as algal biomass as feedstocks.

High-Yield Production of Polyhydroxybutyrate and Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Crude Glycerol by a Newly Isolated Burkholderia Species Oh_219.

Crude glycerol (CG), a major biodiesel production by-product, is the focus of ongoing research to convert it into polyhydroxyalkanoate (PHA). However, few bacterial strains are capable of efficiently achieving this conversion. Here, 10 PHA-producing strains were isolated from various media. Among them, Burkholderia sp. Oh_219 exhibited the highest polyhydroxybutyrate (PHB) production from glycerol and was therefore characterized further. Burkholderia sp. Oh_219 demonstrated significant tolerance to major growth inhibitors in CG and metabolized the fatty acids present as impurities in CG. Furthermore, the Oh_219 strain was genetically engineered using phaCBP-M-CPF4 and phaJPa to enable the fatty acid-based production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a component of CG. The resulting strain produced PHBHHx containing 1.0-1.3 mol% of 3HHx from CG. Further supplementation with capric and lauric acids increased the 3HHx molar fraction to 9.7% and 18%, respectively. In a 5 L fermenter, the Oh_219 strain produced 15.3 g/L PHB from 29.6 g/L biomass using a two-stage fermentation system. This is the highest yield reported for PHA production from glycerol by Burkholderia spp. Additionally, PHB produced from CG had a lower melting point than that from pure glycerol and fructose. Taken together, Burkholderia sp. Oh_219 is a promising new candidate strain for producing PHA from CG.

One-Pot lignin bioconversion to polyhydroxyalkanoates based on hierarchical utilization of heterogeneous compounds.

Pseudomonas putida degraded 35% of compounds in alkali-pretreated lignin liquor under nitrogen-replete conditions but with low polyhydroxyalkanoates (PHA) production, while limiting nitrogen supplement improved PHA content (PHA/dry cell weight) to 43% at the expense of decreased lignin degradation of 22%. Increase of initial cell biomass (0.1-1.5g/L) monotonically improved the lignin degradation from 22% to 33% under nitrogen-limited conditions. Hierarchical utilization of heterogenous compounds under cell growth restricted conditions has been unveiled - simple carbon sources were prioritized for valorization, followed by aromatic compounds bioconversion. Based on the results of hierarchy and leveraging the initial bacterial biomass, acetate was augmented to facilitate one-pot lignin bioconversion under nitrogen-limited conditions. This approach improved lignin bioconversion closer to its upper degradation limit of 35%, concomitant with PHA yield of 39mg/g-lignin. Anaerobic digestion of lignocellulose was redesigned to favor acetate-type fermentation, with acetate constituting 91wt%, providing an economic source of acetate.

The Effectiveness of Polyhydroxyalkanoate (PHA) Extraction Methods in Gram-Negative Pseudomonas putida U.

Bioplastics are emerging as a promising solution to reduce pollution caused by petroleum-based plastics. Among them, polyhydroxyalkanoates (PHAs) stand out as viable biotechnological alternatives, though their commercialization is limited by expensive downstream processes. Traditional PHA extraction methods often involve toxic solvents and high energy consumption, underscoring the need for more sustainable approaches. This study evaluated physical and chemical methods to extract PHAs from Pseudomonas putida U, a bacterium known to produce poly-3-hydroxyoctanoate P(3HO). Lyophilized cells underwent six extraction methods, including the use of the following: boiling, sonication, sodium hypochlorite (NaClO), sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), and chloroform. Physical methods such as boiling and sonication achieved yields of 70% and 60%, respectively, but P(3HO) recovery remained low (30-40%). NaClO extraction provided higher yields (80%) but resulted in significant impurities (70%). NaOH methods offered moderate yields (50-80%), with P(3HO) purities between 50% and 70%, depending on the conditions. Spectroscopic and analytical techniques (FTIR, TGA, NMR, GPC) identified 0.05 M NaOH at 60 °C as the optimal extraction condition, delivering high P(3HO) purity while minimizing environmental impact. This positions NaOH as a sustainable alternative to traditional halogenated solvents, paving the way for more eco-friendly PHA production processes.