Polyhydroxybutyrate Synthesis Research Articles

Effects of methane to oxygen ratio on cell growth and polyhydroxybutyrate synthesis in high cell density cultivation of Methylocystis sp. MJC1.

Polyhydroxybutyrate (PHB) production through CH4 conversion by methanotrophs offers a solution for greenhouse gas emissions and plastic waste concerns. In this study, we aimed to achieve high cell density cultivation of Methylocystis sp. MJC1 for efficient PHB production. Cultivating MJC1 using CH4 and air (3:7, v/v) yielded a final cell density of 52.9 g/L with a 53.7% (28.4 g/L) PHB content after 210 h, showcasing PHB mass production potential. However, long-term cultivation led to a low volumetric productivity of 0.200g/L/h. To address this, we conducted cultivation at various O2/CH4 ratios using O2 instead of air, which significantly improved the PHB productivity. Under high O2 conditions (O2/CH4 ratio of 1.5), biomass productivity increased 1.51-fold compared to that under low O2 conditions in the same time frame; however, PHB accumulation was delayed. Using an equal ratio of CH4 and O2 induced active cell growth and selective PHB production, achieving the highest PHB productivity (0.365 g/L/h) with a final cell density of 55.9 g/L and PHB content of 61.7% (34.5 g/L) in 162 h. This study highlighted the significance of the O2/CH4 ratio in CH4 conversion and PHB production by M. sp. MJC1.

The stringent response regulates the poly-β-hydroxybutyrate (PHB) synthesis in Azotobacter vinelandii.

The stringent response exerted by (p)ppGpp and RNA-polymerase binding protein DksA regulates gene expression in diverse bacterial species. To control gene expression (p)ppGpp, synthesized by enzymes RelA and SpoT, interacts with two sites within the RNA polymerase; site 1, located in the interphase between subunits β' and ω (rpoZ), and site 2 located in the secondary channel that is dependent on DksA protein. In Escherichia coli, inactivation of dksA results in a reduced sigma factor RpoS expression. In Azotobacter vinelandii the synthesis of polyhydroxybutyrate (PHB) is under RpoS regulation. In this study, we found that the inactivation of relA or dksA, but not rpoZ, resulted in a negative effect on PHB synthesis. We also found that the dksA, but not the relA mutation reduced both rpoS transcription and RpoS protein levels, implying that (p)ppGpp and DksA control PHB synthesis through different mechanisms. Interestingly, despite expressing rpoS from a constitutive promoter in the dksA mutant, PHB synthesis was not restored to wild type levels. A transcriptomic analysis in the dksA mutant, revealed downregulation of genes encoding enzymes needed for the synthesis of acetyl-CoA, the precursor substrate for PHB synthesis. Together, these data indicate that DksA is required for optimal expression of RpoS which in turn activates transcription of genes for PHB synthesis. Additionally, DksA is required for optimal transcription of genes responsible for the synthesis of precursors for PHB synthesis.

Poly(3-hydroxybutyrate) production from cassava processing wastes by Paracoccus sp. through high cell density cultivation: Effects of substrates limitation and kinetic analysis

Polyhydroxybutyrate (PHB) is bioplastic that has been recognized as a viable alternative to petroleum-based ones, owing to its biodegradability and biocompatibility. However, its use has long been hampered by its high selling price, caused mainly by using costly fermentation feedstock. In the present study, cassava pulp (CP) was investigated as an alternative low-cost carbon source for a high cell density cultivation of Paracoccus sp. KKU01 for PHB production. CP was hydrolyzed enzymatically, and the resulting hydrolysate was used as the base medium to optimize the bacterial growth conditions, i.e., medium compositions and dissolved oxygen setpoints, attaining as high as 40.9 ± 0.0 g/L of biomass. The optimum growth conditions were subsequently used in 4 fed-batch fermentation regimes, and Regime 2, where complete medium (CP hydrolysate supplemented with other nutrients) was fed twice followed by twice feedings of concentrated CP hydrolysate (CPH), was found to give highest biomass and PHB production of 114.5 ± 1.3 and 47.8 ± 3.9 g/L, respectively, with 41.8 ± 2.9% PHB content and yield of 0.12 kg/kg-CP. PHB productivity was 0.80 ± 0.06 g/(L·h). Logistic and Luedeking-Piret models were used successfully to describe the profiles of bacterial growth and PHB production, respectively. Curve fitting using the Luedeking-Piret model revealed that PHB synthesis and PHB turnover occurred simultaneously during the process. Overall, the results demonstrated that cassava processing wastes are feasible feedstock for PHB production by Paracoccus sp. KKU01.

Sustainable Synthesis of Biopolymer Polyhydroxybutyrate (PHB) from Agro-residue by Brevibacterium casei with Emphasis on Degradation Analysis

The Polyhydroxybutyrate (PHB) polymer is a biodegradable microbial polyester that is intracellularly accruing due to the depletion of nitrogen and phosphorous resources and an increase in carbon supply. As part of this research investigation, Sudan Black B staining, fermentation, chloroform-sodium hypochlorite solvent-based extraction, and characterization of extracted PHB were used to isolate and identify organisms capable of producing PHB. Brevibacterium casei (OQ519751) was used to synthesize PHB biopolymer from agro-residues (orange peel, mangosteen peel, sugarcane bagasse, water hyacinth, and jackfruit peel). Using the Response Surface Methodology (RSM) and the Central Composite Design (CCD) has proven to be highly effective for optimizing PHB synthesis. The optimal conditions determined through RSM allowed Brevibacterium casei to produce significant amounts of PHB when compared to an unoptimized medium. The model demonstrated statistical significance, as indicated by the F-value of 19.96 with an associated p-value of <0.0001. Furthermore, with an optimized pH level of 7, temperature of 37°C, and yeast extract as the nitrogen source, the carbon source water hyacinth was found to synthesize an enhanced quantity of a PHB yield of 1.29 g/L from 2.2 g/L of dry biomass (58.63%). PHB characterization was done with the aid of FTIR (Fourier-transform infrared spectroscopy), XRD (X-ray diffraction), and TGA (Thermogravimetric analysis) analysis. The degradation study of PHB films was performed by soil burial method and morphological changes were scrutinized by SEM analysis. The results reveal that utilizing water hyacinth as a feedstock employs an enhanced production of PHB. This is the first report to synthesize maximum yield of PHB from Brevibacterium casei using water hyacinth as a substrate for production.

Screening and optimization of polyhydroxybutyrate (PHB) by Lysinibacillus fusiformis from diverse environmental sources

The need to explore microbial strains inhabiting ecosystems capable of yielding bioplastics with high production capacity and environmental sustainability is particularly of noteworthy due to the negative impacts of petrochemical plastics on humans and the environment. This study thus focused on the identification of novel polyhydroxybutyrate (PHB) producers, coupled with the screening and optimization of PHB or bioplastics for maximum yield. Bacterial strains were isolated from incinerator dump-site soil, hydrocarbon soil, rhizosphere and bulk soil samples of tomato and okra biochar plants. The identification of isolates was carried out based on biochemical and molecular methods. Screening of bacterial isolates for PHB was conducted using Sudan stain and Bromothymol techniques. Optimization analysis for maximum PHB production conditions by potential PHB strains was carried out via response surface methodology using Box-Behnken design at pH (2–11); substrates: rice bran (1–5 g); and time (24–96 h). Results showed that 112 bacterial isolates were recovered from all the samples collected. Tomato plant biochar soil recorded the highest number of bacterial isolates (37), while hydrocarbon soil yielded the lowest number of bacterial isolates (16). Predominant genera are Micrococcus, Bacillus and Staphylococcus. Lysinibacillus fusiformis was revealed to be the most potent PHB producer. Among the 112 bacteria isolated, nine isolates (5 isolates from tomato plant biochar soil, 2 bacteria from okra plant biochar soil and a single bacterial isolate from an incinerator dump site and hydrocarbon soil) exhibited positive results in both Sudan black and bromothymol tests. Box-Behnken design indicated the optimal experimental conditions for PHB synthesis to be pH 7, 72 h and substrates of rice bran of 3 g. In conclusion, Lysinibacillus fusiformis obtained from a tomato biochar plant demonstrates a high potential for bioplastic synthesis utilizing agricultural waste (rice bran) as a cost-effective as well as sustainable carbon source and could be utilized for commercial-scale PHB production.

Biosynthesis of polyhydroxybutyrate by Methylorubrum extorquens DSM13060 is essential for intracellular colonization in plant endosymbiosis.

Methylorubrum extorquens DSM13060 is an endosymbiont that lives in the cells of shoot tip meristems. The bacterium is methylotrophic and consumes plant-derived methanol for the production of polyhydroxybutyrate (PHB). The PHB provides protection against oxidative stress for both host and endosymbiont cells through its fragments, methyl-esterified 3-hydroxybutyrate (ME-3HB) oligomers. We evaluated the role of the genes involved in the production of ME-3HB oligomers in the host colonization by the endosymbiont M. extorquens DSM13060 through targeted genetic mutations. The strains with deletions in PHB synthase (phaC), PHB depolymerase (phaZ1), and a transcription factor (phaR) showed altered PHB granule characteristics, as ΔphaC had a significantly low number of granules, ΔphaR had a significantly increased number of granules, and ΔphaZ1 had significantly large PHB granules in the bacterial cells. When the deletion strains were exposed to oxidative stress, the ΔphaC strain was sensitive to 10 mM HO· and 20 mM H2O2. The colonization of the host, Scots pine (Pinus sylvestris L.), by the deletion strains varied greatly. The deletion strain ΔphaR colonized the host mainly intercellularly, whereas the ΔphaZ1 strain was a slightly poorer colonizer than the control. The deletion strain ΔphaC lacked the colonization potential, living mainly on the surfaces of the epidermis of pine roots and shoots in contrast to the control, which intracellularly colonized all pine tissues within the study period. In earlier studies, deletions within the PHB metabolic pathway have had a minor effect on plant colonization by rhizobia. We have previously shown the association between ME-3HB oligomers, produced by PhaC and PhaZ1, and the ability to alleviate host-generated oxidative stress during plant infection by the endosymbiont M. extorquens DSM13060. Our current results show that the low capacity for PHB synthesis leads to poor tolerance of oxidative stress and loss of colonization potential by the endosymbiont. Altogether, our findings demonstrate that the metabolism of PHB in M. extorquens DSM13060 is an important trait in the non-rhizobial endosymbiosis.

Coupling of CO2 Electrolysis with Parallel and Semi-Automated Biopolymer Synthesis - Ex-Cell and without Downstream Processing.

Important improvements have been achieved in developing the coupling of electrochemical CO2 reduction to formate with its subsequent microbial conversion to polyhydroxybutyrate (PHB) by Cupriavidus necator. The CO2 based formate electrosynthesis was optimised by electrolysis parameter adjustment and application of Sn based gas diffusion electrodes reaching almost 80 % Faradaic efficiency at 150 mA cm-2. Thereby, catholyte with the high formate concentration of 441±9 mmol L-1 was generated as feedstock without intermediate downstream processing for semi-automated formate feeding into a fed-batch reactor system. Moreover, microbial formate conversion to PHB was studied further, optimised, and successfully scaled from shake flasks to semi-automated bioreactors. Therein, a PHB per formate ratio of 16.5±4.0 mg g-1 and a PHB synthesis rate of 8.4±2.1 mg L-1 OD-1 h-1 were achieved. By this process combination, an almost doubled overall process yield of 22.3±5.5 % was achieved compared to previous reports. The findings allow a detailed evaluation of the overall CO2 to PHB conversion, providing the basis for potential technical exploitation.

Production of polyhydroxybutyrate (PHB), a biodegradable polymer from seaweed biomass using novel bacterial isolates

Poly-(3-hydroxybutyrate) (PHB), is a partially crystalline polymer naturally synthesized by microorganisms, and the production of PHB has attracted much attention recently due to its exceptional biodegradable and biocompatible nature. By serving as a copolymer and by being a block copolymer, PHB enhances the mechanical properties of the polymer materials. The materials developed using PHB as a block copolymer are used in numerous applications such as polymer aging resistance, controlled drug delivery, cardiovascular engineering, thin film formation, etc. This study focuses on synthesizing PHB from microbial sources as cell factories. From distinct natural sources two novel bacterial isolates, Bacillus pacificus NAA2 (NAA2) and Klebsiella quasipneumonia NAA4 (NAA4) were screened based on their high PHB synthesizing abilities. To improve the PHB levels, a series of process optimization studies were conducted. The process conditions such as different carbon and nitrogen sources, concentrations, pH levels, temperatures, and incubation periods have been explored and optimized. Under optimized conditions, NAA2 produced 2.92 g/L of PHB (79 %) with glycerol and peptone as carbon and nitrogen sources respectively at pH 7.5 and 35 °C at 48 h. The other isolate, NAA4, produced 2.89 g/L of PHB (73 %) from xylose and urea as equivalent carbon and nitrogen sources respectively with the same pH and temperature after 36 h of incubation. In addition, the replacement of conventional carbohydrates (glucose and/or xylose) with seaweed hydrolysate exhibited almost equivalent levels of PHB 72.7 and 70.7 % of PHB in NAA2 and NAA4 respectively. Fourier Transform Infrared (FTIR) spectroscopy and FE-SEM analysis confirmed the production of a polymer. Though there are several studies reported with other Bacillus sp and Klebsiella sp., this is the only study that used novel strains Bacillus pacificus (NAA2) and Klebsiella quasipneumonia (NAA4) as the microbial cell factories for the effective synthesis of PHB using seaweed as a substrate. Thus, the technology for bioconversion of harmful and waste seaweed biomass into PHB with dual sustainability was developed.

The adc1 knockout with proC overexpression in Synechocystis sp. PCC 6803 induces a diversion of acetyl-CoA to produce more polyhydroxybutyrate

BackgroundLack of nutrients, in particular nitrogen and phosphorus, has been known in the field to sense glutamate production via 2-oxoglutarate and subsequently accelerate carbon storage, including glycogen and polyhydroxybutyrate (PHB), in cyanobacteria, but a few studies have focused on arginine catabolism. In this study, we first time demonstrated that gene manipulation on proC and adc1, related to proline and polyamine syntheses in arginine catabolism, had a significant impact on enhanced PHB production during late growth phase and nutrient-modified conditions. We constructed Synechocystis sp. PCC 6803 with an overexpressing proC gene, encoding Δ1pyrroline-5-carboxylate reductase in proline production, and adc1 disruption resulted in lower polyamine synthesis.ResultsThree engineered Synechocystis sp. PCC 6803 strains, including a ProC-overexpressing strain (OXP), adc1 mutant, and an OXP strain lacking the adc1 gene (OXP/Δadc1), certainly increased the PHB accumulation under nitrogen and phosphorus deficiency. The possible advantages of single proC overexpression include improved PHB and glycogen storage in late phase of growth and long-term stress situations. However, on day 7 of treatment, the synergistic impact created by OXP/Δadc1 increased PHB synthesis by approximately 48.9% of dry cell weight, resulting in a shorter response to nutrient stress than the OXP strain. Notably, changes in proline and glutamate contents in engineered strains, in particular OXP and OXP/Δadc1, not only partially balanced the intracellular C/N metabolism but also helped cells acclimate under nitrogen (N) and phosphorus (P) stress with higher chlorophyll a content in comparison with wild-type control.ConclusionsIn Synechocystis sp. PCC 6803, overexpression of proC resulted in a striking signal to PHB and glycogen accumulation after prolonged nutrient deprivation. When combined with the adc1 disruption, there was a notable increase in PHB production, particularly in situations where there was a strong C supply and a lack of N and P.

Metabolic response to a heterologous poly-3-hydroxybutyrate (PHB) pathway in Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is an emerging host for metabolic engineering, but little is known about how introduced pathways are integrated into the existing metabolic framework of the host or influence transgene expression. In this study, we expressed the heterologous poly-3-hydroxybutyrate (PHB) pathway using episomal expression, which draws on the precursor acetyl coenzyme-A (AcCoA). By experimentally perturbing cultivation conditions, we gained insight into the regulation of the endogenous metabolism in transgenic lines under various environmental scenarios, as well as on alterations in AcCoA flux within the host cell. Biosynthesis of PHB led to distinct shifts in the metabolome of the host, and further analysis revealed a condition-dependent relationship between endogenous and transgenic metabolic pathways. Under N limitation, which induced a significant increase in neutral lipid content, both metabolic and transcriptomic data suggest that AcCoA was preferably shunted into the endogenous pathway for lipid biosynthesis over the transgenic PHB pathway. In contrast, supply of organic carbon in the form of glycerol supported both fatty acid and PHB biosynthesis, suggesting cross-talk between cytosolic and plastidial AcCoA precursors. This is the first study to investigate the transcriptomic and metabolomic response of diatom cell lines expressing a heterologous multi-gene pathway under different environmental conditions, providing useful insights for future engineering attempts for pathways based on the precursor AcCoA. KEY POINTS: • PHB expression had minimal effects on transcription of adjacent pathways. • N limitation favoured native lipid rather than transgenic PHB synthesis. • Glycerol addition allowed simultaneous lipid and PHB accumulation.

A comprehensive investigation to the fate of phosphorus in full-scale wastewater treatment plants using aluminum salts for enhanced phosphorus removal

This study investigated the fate of phosphorus (P) in 8 full-scale municipal wastewater treatment plants (WWTPs) in Shanghai, China, in which both biological nutrient removal and aluminum-based chemical phosphorus removal were used. The results showed that 83.8–98.9 % P was transferred to the sludge in the 8 WWTPs by both chemical and biological reactions. P speciation analysis indicated that chemical P precipitates accounted for 84.3 % in the activated sludge, of which crystalline AlPO4 and amorphous iron‑phosphorus compounds (FePs) were the main components. Sludge with more water-soluble and weakly adsorbed P was generated in the anaerobic-anoxic-oxic (A/A/O) process than in other processes. Among the 8 WWTPs, the one with the largest flow rate and relatively short sludge retention time (SRT) had the best potential to release P from all types of sludge. The recovery potential of P from thickened sludge can be improved by separately thickening the sludge produced in the high-efficiency sedimentation tank or feeding it into the dewatering process directly. Different P removal chemicals and dosing points changed the amount of chemical precipitate formed but had little effect on the composition of P accumulating organisms (PAOs) at the genus level. Although aluminum-based coagulants were applied in the investigated WWTPs, Fe in wastewater had the most positive effect on the proliferation of PAOs. The synthesis of polyphosphate was also related to the metabolism of PAOs as it affected transmembrane inorganic phosphate (Pi) transport and polyhydroxybutyrate (PHB) synthesis. The in-depth understanding of the fate of P is beneficial to improve P recovery efficiency in WWTPs.

Metagenomics, metatranscriptomics, and proteomics reveal the metabolic mechanism of biofilm sequencing batch reactor with higher phosphate enrichment capacity under low phosphorus load

The biofilm sequencing batch reactor (BSBR) process has higher phosphate recovery efficiency and enrichment multiple when the phosphorus load is lower, but the mechanism of phosphate enrichment at low phosphorus load remains unclear. In this study, we operated two BSBR operating under low and high phosphorus load (0.012 and 0.032 kg/(m3·d)) respectively, and used metagenomic, metatranscriptomic, and proteomics methods to analyze the community structure of the phosphorus accumulating organisms (PAOs) in the biofilm, the transcription and protein expression of key functional genes and enzymes, and the metabolism of intracellular polymers. Compared with at high phosphorus load, the BSBR at low phosphorus load have different PAOs and fewer types of PAOs, but in both cases the PAOs must have the PHA, PPX, Pst, and acs genes to become dominant. Some key differences in the metabolism of PAOs from the BSBR with different phosphorus load can be identified as follows. When the phosphorus load is low, the adenosine triphosphoric acid (ATP) and NAD(P)H in the anaerobic stage come from the TCA cycle and the second half of the EMP pathway. The key genes that are upregulated include GAPDH, PGK, ENO, ppdk in the EMP pathway, actP in acetate metabolism, phnB in polyhydroxybutyrate (PHB) synthesis, and aceA, mdh, sdhA, and IDH1 in the TCA cycle. In the meantime, the ccr gene in the PHV pathway is inhibited. As a result, the metabolism of the PAOs features low glycogen with high PHB, Pupt, Prel, and low PHV. That is, more ATP and NAD(P)H flow to phosphorus enrichment metabolism, thus allowing the highly efficient enrichment of phosphorus from low concentration phosphate thanks to the higher abundance of PAOs. The current results provide theoretical support and a new technical option for the enrichment and recovery of low concentrations of phosphate from wastewater by the BSBR process.

Eliminating genes for a two-component system increases PHB productivity in Cupriavidus basilensis 4G11 under PHB suppressing, nonstress conditions.

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two-component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions. We observe average increases in PHB productivity of 56% and 41% relative to the wildtype parent strain upon deleting each gene individually from the genome. The increased PHB phenotype disappears, however, in nitrogen-free unbalanced growth conditions suggesting the phenotype is specific to fast-growing, replete, nonstress growth. Bioproduction modeling suggests this phenotype could be due to a decreased reliance on metabolic stress induced by nitrogen limitation to initiate PHB production in the mutant strains. Due to uncertainty in the two-component system's input signal and regulon, the mechanism by which these genes impart this phenotype remains unclear. Such strains may allow for the use of single-stage, continuous bioreactor systems, which are far simpler than many PHB bioproduction schemes used previously, given a similar product yield to batch systems in such a configuration. Bioproductivity modeling suggests that omitting this regulation in the cells may increase PHB productivity up to 24% relative to the wildtype organism when using single-stage continuous systems. This work expands our understanding of the regulation of PHB accumulation in Cupriavidus, in particular the initiation of this process upon transition into unbalanced growth regimes.

Polyhydroxybutyrate synthesis in Camelina: Towards coproduction of renewable feedstocks for bioplastics and fuels.

Plant-based co-production of polyhydroxyalkanoates (PHAs) and seed oil has the potential to create a viable domestic source of feedstocks for renewable fuels and plastics. PHAs, a class of biodegradable polyesters, can replace conventional plastics in many applications while providing full degradation in all biologically active environments. Here we report the production of the PHA poly[(R)-3-hydroxybutyrate] (PHB) in the seed cytosol of the emerging bioenergy crop Camelina sativa engineered with a bacterial PHB biosynthetic pathway. Two approaches were used: cytosolic localization of all three enzymes of the PHB pathway in the seed, or localization of the first two enzymes of the pathway in the cytosol and anchoring of the third enzyme required for polymerization to the cytosolic face of the endoplasmic reticulum (ER). The ER-targeted approach was found to provide more stable polymer production with PHB levels up to 10.2% of the mature seed weight achieved in seeds with good viability. These results mark a significant step forward towards engineering lines for commercial use. Plant-based PHA production would enable a direct link between low-cost large-scale agricultural production of biodegradable polymers and seed oil with the global plastics and renewable fuels markets.

Energy storage efficiency in artificial photosynthesis – An evaluation method in engineering perspective

In engineering perspective, energy storage efficiency is a crucial indicator for assessing economic feasibility of artificial photosynthetic energy storage systems, as it determines not only the investment return but also the life cycle of renewable energy. However, few models for evaluating the efficiency are available in the open literature. In this work, a framework for evaluating the efficiency of artificial photosynthetic energy storage systems was proposed using the synthesis of PHB (polyhydroxy-butyrate) by Cupriavidus necator and hydrogen as an example, and models for the reaction kinetics and thermodynamics of the conversion and mass transfer in the biochemical processes were proposed and checked with published experimental results. An optimization method was developed to achieve the maximum energy efficiency. It was found in the present study that continuous reactors are more efficient than batch reactors, and improving mass transfer in the reactor is a key to increasing the maximum energy storage power, which should be compromised with energy efficiency for an engineering design. It was also found that altering reaction paths with higher thermodynamic efficiency is another key to improving energy efficiency of an artificial photosynthesis energy storage system.

A unique small RNA, sSp_p4 governs nutrition stress response and plant-growth promoting traits in Azospirillum baldaniorum Sp245

Azospirillum baldaniorum Sp245 is a well-studied plant growth-promoting bacterium and has recently been shown to possess ∼468 small, non-coding RNAs (sRNA), many of which exhibited differential expression under nutrient stress conditions. Plant-microbe interactions are usually a part of a multigenic response involving a multitude of regulators such as sRNAs and their associated mRNA targets. We report the functional characterization of the sRNA, sSp_p4, from A. baldaniorum Sp245 and confirm its differential expression in nutrient stress conditions. sSp_p4 was overexpressed, knocked down and complemented to characterize its physiological functions and contribution to plant growth promoting (PGP) traits of strain Sp245. Expression analysis of sSp_p4 confirmed that it influenced and regulates PGP traits such as polyhydroxybutyrate synthesis, indole acetic acid production, and biofilm formation. sSp_p4 is likely involved in the regulation of essential bacterial biological networks in this agriculturally relevant bacterial strain and may impact plant growth.

Electron uptake from solid electrodes promotes the more efficient conversion of CO2 to polyhydroxybutyrate by using Rhodobacter sphaeroides

Microbial electrosynthesis (MES) is a promising strategy for the conversion of CO2 to useful chemicals. Nevertheless, the characteristics of electrode-associated cells in MES and their metabolic pathway regulation in CO2 fixation have not been elucidated. This study examined the electrode-driven polyhydroxybutyrate (PHB) production from CO2 in Rhodobacter sphaeroides. The electron uptake and regulation of the metabolic pathways differed in electrode-associated and suspended R. sphaeroides. The electrode-associated cells produced PHB at concentrations up to 23.50 ± 2.8% of the dry cell weight (DCW), whereas the suspended cells grew faster but with a lower cellular PHB content. Gene expression analyses showed that phaA expression was upregulated in electrode-associated R. sphaeroides, whereas phaB expression was downregulated in suspended cells. The electrode-associated cells expressed unconventional CO2 fixation enzymes, such as isocitrate dehydrogenase and formate dehydrogenase, with more PHB synthesis. These results show that CO2 can be upcycled to polymeric substances and provide novel insights into the genetic regulation of electrode-associated cells in MES.

Structure analysis and thermal stability of PHB recovered from sugar industry waste

ABSTRACT The current article emphasized on cultural conditions for Alcaligenes sp. NCIM 5085 to synthesize Polyhydroxybutyrate (PHB) from sugar industry waste during batch fermentation. Alcaligenes sp. NCIM 5085 was found to grow best in conditions that included 40 g l−1 of cane molasses, 1 g l−1 of ammonium sulphate, 10% inoculum with neutral pH and 48 h of incubation time. Sudan Black B staining was employed to verify the PHB synthesis initially, and further TEM analysis was performed to confirm it. The structural analysis of recovered PHB was carried out by using GC-MS, FTIR, 1H NMR and 13C NMR analysis. The absorption peak at 1724.56 cm−1 revealed the presence of C=O (carbonyl) group by FTIR, which is an indicator of PHB presence. Furthermore, results of NMR and GC-MS analysis confirmed the recovered polymer was PHB. The thermal properties of recovered polymer were analyzed by TGA, DTG and DSC and showed thermal stability of PHB. The observed glass transient temperature (Tg) −2.8°C was within the normal PHB range of Tg. However, melting temperature of recovered PHB was 161.7°C, where the degree of crystallinity was lower than standard PHB that widens the application possibilities.

Characterization and Process Optimization for Enhanced Production of Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Bacillus flexus Isolated from Municipal Solid Waste Landfill Site.

In recent years, there has been a growing interest in bio-based degradable plastics as an alternative to synthetic plastic. Polyhyroxybutyrate (PHB) is a macromolecule produced by bacteria as a part of their metabolism. Bacteria accumulate them as reserve materials when growing under different stress conditions. PHBs can be selected as alternatives for the production of biodegradable plastics because of their fast degradation properties when exposed to natural environmental conditions. Hence, the present study was undertaken in order to isolate the potential PHB-producing bacteria isolated from the municipal solid waste landfill site soil samples collected from the Ha'il region of Saudi Arabia to assess the production of PHB using agro-residues as a carbon source and to evaluate the growth of PHB production. In order to screen the isolates for producing PHB, a dye-based procedure was initially employed. Based on the 16S rRNA analysis of the isolates, Bacillus flexus (B. flexus) accumulated the highest amount of PHB of all the isolates. By using a UV-Vis spectrophotometer and Fourier-transform infrared spectrophotometer (FT-IR), in which a sharp absorption band at 1721.93 cm-1 (C=O stretching of ester), 1273.23 cm-1 (-CH group), multiple bands between 1000 and 1300 cm-1 (stretching of the C-O bond), 2939.53 cm-1 (-CH3 stretching), 2880.39 cm-1 (-CH2 stretching) and 3510.02 cm-1 (terminal -OH group), the extracted polymer was characterized and confirmed its structure as PHB. The highest PHB production by B. flexus was obtained after 48 h of incubation (3.9 g/L) at pH 7.0 (3.7 g/L), 35 °C (3.5 g/L) with glucose (4.1 g/L) and peptone (3.4 g/L) as carbon and nitrogen sources, respectively. As a result of the use of various cheap agricultural wastes, such as rice bran, barley bran, wheat bran, orange peel and banana peel as carbon sources, the strain was found to be capable of accumulating PHB. Using response surface methodology (RSM) for optimization of PHB synthesis using a Box-Behnken design (BBD) proved to be highly effective in increasing the polymer yield of the synthesis. With the optimum conditions obtained from RSM, PHB content can be increased by approximately 1.3-fold when compared to an unoptimized medium, resulting in a significant reduction in production costs. Thus, isolate B. flexus is a highly promising candidate for the production of industrial-size quantities of PHB from agricultural wastes and is capable of removing the environmental concerns associated with synthetic plastics from the industrial production process. Moreover, the successful production of bioplastics using a microbial culture provides a promising avenue for the large-scale production of biodegradable and renewable plastics with potential applications in various industries, including packaging, agriculture and medicine.

Cost effective media optimization for PHB production by Bacillus badius MTCC 13004 using the statistical approach

Polyhydroxybutyrate (PHB) has significant potential for replacing non-biodegradable traditional plastic, which is responsible for several global environmental issues. The main problem with switching to bio-based alternatives for petrochemical plastics is the large price gap on the market. To overcome this problem, the present research was focused on the utilization of inexpensive substrates i.e. agricultural residues for cost-effective PHB production by endospore-forming bacteria Bacillus badius MTCC 13004. For efficient PHB production, Box-Behnken Design (BBD) was selected for media optimization and to observe the interactive effects of four variables i.e. pH, Na acetate, Banana peel, and mustard cake. PHB yield of 2.11 g/L was attained under optimized conditions compared to non-optimized conditions (0.72 g/L). FTIR spectra analysis of PHB extracted from Bacillus badius was found to be similar to commercial PHB. NMR data was also matched with the chemical shift signals CH, CH2, and CH3 of PHB. The melting temperature (Tm) and glass transition temperature (Tg) of PHB from Bacillus badius was found to be 165.14 and 2.68 °C, respectively. Further, PCR protocol was also designed to amplify key enzymes of the PHB synthesis pathway i.e. PHB synthase (phb C gene).