Intracellular Polyhydroxyalkanoates Research Articles

Genome-wide analysis of quorum sensing regulon in marine fish pathogen Vibrio scophthalmi.

Opportunistic fish pathogen Vibrio scophthalmi frequently infects olive flounder and turbot, which are primary marine species cultured for seafood production in Far East Asia. These infections cause substantial yield reductions and significant economic losses. Although quorum sensing (QS) genes were previously reported in V. scophthalmi, the impacts of QS on genome-wide gene expression and consequent behaviors and physiological traits have remained largely unexplored. In this study, we conducted genomic and transcriptomic analyses to uncover the global regulatory network governed by LuxRVs, a QS master regulator in V. scophthalmi. By comparing the wild-type strain and a luxRVs deletion mutant strain, we found that LuxRVs positively regulates biosynthetic genes for poly-hydroxyalkanoate (PHA) while negatively controlling genes for biofilm formation. Quantification of intracellular PHAs and biofilm biomass on borosilicate tubes confirmed these results. Gene set enrichment analyses further demonstrated that LuxRVs also governs genes related to osmoprotection and defense against reactive oxygen species. Overall, these findings indicate that LuxRVs acts as a global transcriptional regulator, controlling a wide range of physiological processes in V. scophthalmi. Targeting LuxRVs could therefore be a promising strategy for improving seafood production by disrupting diverse physiological and pathogenic traits in this fish pathogen.

Uncovering novel polyhydroxyalkanoate biosynthesis genes and unique pathway in yeast hanseniaspora valbyensis for sustainable bioplastic production

This study delves into the exploration of polyhydroxyalkanoate (PHA) biosynthesis genes within wild-type yeast strains, spotlighting the exceptional capabilities of isolate DMG-2. Through meticulous screening, DMG-2 emerged as a standout candidate, showcasing vivid red fluorescence indicative of prolific intracellular PHA granules. Characterization via FTIR spectroscopy unveiled a diverse biopolymer composition within DMG-2, featuring distinct functional groups associated with PHA and polyphosphate. Phylogenetic analysis placed DMG-2 within the Hanseniaspora valbyensis NRRL Y-1626 group, highlighting its distinct taxonomic classification. Subsequent investigation into DMG-2’s PHA biosynthesis genes yielded promising outcomes, with successful cloning and efficient PHA accumulation confirmed in transgenic E. coli cells. Protein analysis of ORF1 revealed its involvement in sugar metabolism, supported by its cellular localization and identification of functional motifs. Genomic analysis revealed regulatory elements within ORF1, shedding light on potential splice junctions and transcriptional networks influencing PHA synthesis pathways. Spectroscopic analysis of the biopolymer extracted from transgenic E. coli DMG2-1 provided insights into its co-polymer nature, comprising segments of PHB, PHV, and polyphosphate. GC-MS analysis further elucidated the intricate molecular architecture of the polymer. In conclusion, this study represents a pioneering endeavor in exploring PHA biosynthesis genes within yeast cells, with isolate DMG-2 demonstrating remarkable potential. The findings offer valuable insights for advancing sustainable bioplastic production and hold significant implications for biotechnological applications.

Partial least squares regression based rapid quantification of intracellular biopolymers from a Sudan black absorption assay

Rapid quantitation of intracellular polyhydroxyalkanoate (PHA) is essential for monitoring and possibly intervening in ongoing bioprocesses. The traditional multistep protocol involving cell separation, lysis, organic solvent extraction, and purification of intracellular polymers is time intensive. In this study, indirect Sudan black (ISB) adsorption data combined with culture optical density and intracellular protein measurements were used to develop partial least squares (PLS) regression models to predict the intracellular PHA concentration in Cupriavidus necator. ISB analysis supported the hypothesized partitioning of Sudan black into residual biomass, PHA, and abiotic compartments. The PLS regression model developed with autoscaled data offered excellent fit on cross-validation (R2CV of 0.987) and independent prediction (R2P of 0.994) sets. The method limit of detection for the PLS model (0.036 g l−1) was reasonably low to track biopolymer accumulation in microbial culture, even in early growth phase. The conservative estimate suggests a significantly shorter processing time with the machine learning-based approach (190 min) than with the traditional protocol (940 min) while retaining analytical interchangeability.

Multi-stage process for mixed microbial culture production of polyhydroxyalkanoates from sugarcane stillage: Assessment of external nutrient supplementation

Sugarcane stillage is an abundant wastewater from ethanol production. It has drawn considerable interest as a potential feedstock for biotechnological processes aiming at the recovery of energy and value-added products. In this study we explored the use of stillage for the production of polyhydroxyalkanoates (PHA), which are microbial bioplastics with potential to replace conventional plastics in specific applications. As stillage is nutrient deficient, we investigated the impact of external nutrient supplementation on the selection of PHA-producing mixed cultures. Cultures selected under four different total-chemical-oxygen-demand-to-nitrogen ratios (COD.t/N) (15, 30, 40 and 80) were evaluated according to their PHA production performance and microbial composition. The experimental results demonstrated that the most favorable PHA production was achieved for the biomass selected under strict carbon-limiting conditions, with a COD.t/N ratio of 15. This resulted in an overall PHA volumetric productivity of 2.10 g PHA L−1 d−1, a maximum intracellular PHA content of 0.65 g PHA g VSS−1 and a PHA storage yield of 0.71 g CODPHA g CODH.Org−1. The resulting PHA was a copolymer of 3-hydroxybutyrate (73 %mol) and 3-hydroxyvalerate (27 %mol) monomers. The microbial consortium was enriched by members of the Brevundimonas genus, known PHA producers, with a relative abundance of 26.4 %.

Graphene quantum dots enhance the biological nitrogen fixation by up-regulation of cellular metabolism and electron transport

Graphene quantum dots (GQDs) are useful nanomaterials of excellent water solubility, biocompatibility and optical stability for various applications. In this study, we achieved the nitrogen fixation activity enhancement of autogenous azotobacters (Azotobacter vinelandii) by GQDs and revealed its mechanism. GQDs (1–10 mg/L) accelerated the logarithmic growth phase of A. vinelandii, making bacteria enter the plateau earlier. Biocompatible GQDs did not cause cell death and oxidative damage of A. vinelandii. The expression levels of metabolism and nitrogen fixation related genes were significantly up-regulated by GQDs, thereby enhancing the activity of nitrogenase. The nitrogen fixation activity of A. vinelandii reached 471.7 % of the control upon the exposure to GQDs. The intracellular polyhydroxyalkanoate vesicles were consumed to provide energy for nitrogen fixation, which involved with a chain reaction of carbohydrate metabolism, lipid metabolism, and energy metabolism pathways accompanied by significant increases in electron transfer rate. The synergistic effect of GQDs on A. vinelandii further facilitated the augmentation of soil nitrogen content in Qinghai-Tibet Plateau. Our results provided an effective approach to enhance the biological nitrogen fixation by GQDs.

Sequential recovery of extracellular alginate and intracellular polyhydroxyalkanoate (PHA) from mixed microbial culture PHA production system

Polyhydroxyalkanoate (PHA) is a biodegradable plastic that exhibits properties similar to petroleum-based plastics. The mixed microbial culture (MMC) process utilized a waste carbon source to produce PHA, reducing the raw material costs associated with pure bacterial technology. However, the high cost of PHA recovery and environmental pollution are significant challenges to the sustainable development of MMC technology. In this study, a sequential recovery strategy was proposed. This strategy involved the use of Na2CO3 to break down the structural extracellular polymeric substances and rhamnolipid to digest non-PHA cell material (NPCM). The recovery rate of PHA in this strategy achieved 61.29 ± 5.92% and a purity of 97.55 ± 0.92%. Additionally, the strategy allowed the sequential recovery of the by-product alginate-like extracellular polymers. FT-IR analysis confirmed that the recovered biopolymer was PHA material, possessing mechanical and thermal properties similar to those obtained through CHCl3 extraction. However, it was important to note that this method had the potential to alter the distribution structure of the PHA polymer, although it successfully avoids the issue of reducing the molecular weight of PHA during NPCM digestion. A comprehensive evaluation demonstrates that the cost of PHA production using this method was only 14.61 CNY/kg, with a more positive environmental impact in terms of global warming potential and carbon emission reduction. Therefore, the sequential recovery strategy presents a feasible alternative for the downstream recovery process and overall economy of the MMC PHA process.

Osmoregulation in freshwater anaerobic methane-oxidizing archaea under salt stress.

Climate change-driven sea level rise threatens freshwater ecosystems and elicits salinity stress in microbiomes. Methane emissions in these systems are largely mitigated by methane-oxidizing microorganisms. Here, we characterized the physiological and metabolic response of freshwater methanotrophic archaea to salt stress. In our microcosm experiments, inhibition of methanotrophic archaea started at 1%. However, during gradual increase of salt up to 3% in a reactor over 12weeks, the culture continued to oxidize methane. Using gene expression profiles and metabolomics, we identified a pathway for salt-stress response that produces the osmolyte of anaerobic methanotrophic archaea: N(ε)-acetyl-β-L-lysine. An extensive phylogenomic analysis on N(ε)-acetyl-β-L-lysine-producing enzymes revealed that they are widespread across both bacteria and archaea, indicating a potential horizontal gene transfer and a link to BORG extrachromosomal elements. Physicochemical analysis of bioreactor biomass further indicated the presence of sialic acids and the consumption of intracellular polyhydroxyalkanoates in anaerobic methanotrophs during salt stress.

An efficient and eco-friendly approach for the sustainable recovery and properties characterization of polyhydroxyalkanoates produced by methanotrophs

Synthetic biodegradable and bio-based polymers have emerged as sustainable alternatives to nonrenewable petroleum-derived polymers which cause serious environmental issues. In particular, polyhydroxyalkanoates (PHA) are promising biopolymers owing to their outstanding biodegradability and biocompatibility. The production of the homopolymer poly(3-hydroxybutyrate) (PHB) and copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from type II methanotrophs via microbial fermentation was presented. For the efficient extraction and recovery of intracellular PHA from methanotrophs, different extraction approaches were investigated including solvent extraction using 1,3-dioxolane as a green solvent, integrated cell lysis and solvent extraction, and cell digestion without the use of organic solvents. Among various extraction approaches, the integrated method exhibited the highest extraction performance, with PHA recovery and purity exceeding 91 % and 93 %, respectively, even when the PHA content of the cells was low. Furthermore, the molecular weight, thermal stability, and mechanical properties of the recovered PHA were comprehensively analyzed to suggest its suitable practical applications. The obtained properties were comparable to that of the commercial PHA products and PHA produced from other microbial species, indicating an efficient recovery of high-quality PHA produced from methanotrophs.

Scalable and Sustainable Processing of Intracellular Polyhydroxyalkanoates with Biobased Solvents

Scalable and Sustainable Processing of Intracellular Polyhydroxyalkanoates with Biobased Solvents

Soft sensor based on Raman spectroscopy for the in-line monitoring of metabolites and polymer quality in the biomanufacturing of polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHA) are among the most promising bio-based alternatives to conventional petroleum-based plastics. These biodegradable polyesters can in fact be produced by fermentation from bacteria like Cupriavidus necator, thus reducing the environmental footprint of the manufacturing process. However, ensuring consistent product quality attributes is a major challenge of biomanufacturing. To address this issue, the implementation of real-time monitoring tools is essential to increase process understanding, enable a prompt response to possible process deviations and realize on-line process optimization. In this work, a soft sensor based on in situ Raman spectroscopy was developed and applied to the in-line monitoring of PHA biomanufacturing. This strategy allows the collection of quantitative information directly from the culture broth, without the need for sampling, and at high frequency. In fact, through an optimized multivariate data analysis pipeline, this soft sensor allows monitoring cell dry weight, as well as carbon and nitrogen source concentrations with root mean squared errors (RMSE) equal to 3.71, 7 and 0.03 g/L, respectively. In addition, this tool allows the in-line monitoring of intracellular PHA accumulation, with an RMSE of 14 gPHA/gCells. For the first time, also the number and weight average molecular weights of the polymer produced could be monitored, with RMSE of 8.7E4 and 11.6E4 g/mol, respectively. Overall, this work demonstrates the potential of Raman spectroscopy in the in-line monitoring of biotechnology processes, leading to the simultaneous measurement of several process variables in real time without the need of sampling and labor-intensive sample preparations.

Polyhydroxyalkanoates production by an advanced food-on-demand strategy: The effect of operational conditions

Despite the increasing number of studies related to the polyhydroxyalkanoate (PHA) production from sewage sludge of wastewater treatment plants, there is still a gap in the correlation between the operational conditions, such as the organic loading rate (OLR), and the intracellular polyhydroxyalkanoate (PHA) content, productivity and final recovery of the polymer. Therefore, this work aims to provide experimental data on PHA productivity and purity in view of scaling up the process to an industrial level taking into account process parameters. In view of that, three OLR were applied during the selection of PHA-accumulating bacteria in sewage sludge. Then, the biomass was harvested and subjected to batch accumulation experiments at two organic loads per dosage by employing a tailor-made software to adopt an automated feed-on-demand strategy, which allowed for 30–56 h of accumulation tests in stand-alone mode. Finally, an improved protocol for PHA extraction has been applied. Experimental results show that the maximum PHA content (60% w/w) was achieved using the highest organic load per dosage during the accumulation test with the biomass selected at the highest OLR (1.8 g COD L−1 d−1). Also, the extraction protocol efficiency was proven with four samples with different PHA content, achieving recovery yield as high as 78 ± 3 % with a purity of 89 ± 2 %, thus demonstrating that the adopted strategy might be beneficial for industrial use.

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

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

Polymer-Degrading Enzymes of Pseudomonas chloroaphis PA23 Display Broad Substrate Preferences.

Although many bacterial lipases and PHA depolymerases have been identified, cloned, and characterized, there is very little information on the potential application of lipases and PHA depolymerases, especially intracellular enzymes, for the degradation of polyester polymers/plastics. We identified genes encoding an intracellular lipase (LIP3), an extracellular lipase (LIP4), and an intracellular PHA depolymerase (PhaZ) in the genome of the bacterium Pseudomonas chlororaphis PA23. We cloned these genes into Escherichia coli and then expressed, purified, and characterized the biochemistry and substrate preferences of the enzymes they encode. Our data suggest that the LIP3, LIP4, and PhaZ enzymes differ significantly in their biochemical and biophysical properties, structural-folding characteristics, and the absence or presence of a lid domain. Despite their different properties, the enzymes exhibited broad substrate specificity and were able to hydrolyze both short- and medium-chain length polyhydroxyalkanoates (PHAs), para-nitrophenyl (pNP) alkanoates, and polylactic acid (PLA). Gel Permeation Chromatography (GPC) analyses of the polymers treated with LIP3, LIP4, and PhaZ revealed significant degradation of both the biodegradable as well as the synthetic polymers poly(ε-caprolactone) (PCL) and polyethylene succinate (PES).

Recirculation factor as a key parameter in continuous-flow biomass selection for polyhydroxyalkanoates production

The effectiveness of polyhydroxyalkanoates (PHA) production with mixed microbial cultures (MMC) largely depends on the selection of PHA-storing microorganisms, conventionally performed in sequencing batch reactors (SBR). These, although easily allow the establishment of the required feast and famine (FF) regime, can represent a factor of cost increase when the process is scaled up. Here, a novel continuous-flow process for MMC selection under FF conditions has been developed by using two sequentially operated reactors. The feast reactor, having a tubular configuration, was continuously fed with a synthetic mixture of acetic and propionic acids (at an organic loading rate of 2.12 gCOD/L d) and the effluent of this reactor was in part sent to the CSTR famine reactor. The recirculation factor (RC), that is the ratio between the recirculation flow rate and the feeding flow rate to the feast reactor, was the main parameter investigated. Four different runs were performed with the RC varying from 1 to 8 and the increase in its value caused a decrease of the biomass residence time in each reactor. The intracellular PHA content in the feast reactor almost linearly increased up to RC 4 (with a value of 34 ± 2 %, wt/wt) and dropped at the RC 8 condition that, however, showed the maximum PHA content (58 ± 5 %, wt/wt) during the accumulation tests. Indeed, the relative abundance of sequences affiliated with putative PHA-storing bacteria increased up to 90.5 % at RC 8 and were dominated by members of the Alphaproteobacteria class mostly represented by the genus Meganema (74 %).

Capturing influent organic substrate for endogenous denitrification to enhance nitrogen removal in low C/N ratio municipal wastewater

Intending to reduce aerobic oxidation of carbon sources and leave more carbon resources for denitrification to improve nitrogen removal in low C/N ratio wastewater, batch tests were conducted by allowing sludge to directly capture carbon sources from the influent for feeding denitrifier. The capture effect of three pretreatments and capture modes, i.e., (a) oxic famine (Ofam) pretreatment and then anoxic feast (Afea) mode (Ofam-Afea); (b) anoxic famine (Afam) pretreatment and then oxic feast (Ofea) mode (Afam-Ofea); (c) Afam pretreatment then aerobic feeding of treated sludge in continuous stirred tank reactor (CSTR) with side-stream push-flow reaction (PFR) zone (Afam with side-stream PFR), were investigated. The storage of organic substances and accumulation of intracellular polymers by the sludge in Ofam-Afea mode was compared with that of the sludge in Afam-Ofea mode. The results show that the total capture efficiency (TCE) of the sludge in the Ofam-Afea mode was around 60 %, the amount of glycogen (Gly) remained essentially unchanged, and the intracellular polyhydroxyalkanoates (PHA) accumulated significantly without apparent “stagnation” for carbon source switching. In Afam-Ofea mode, the TCE increased to 60 %–70 %. The amount of Gly increased by 31.2 %–50.0 %, PHA accumulated considerably, and high denitrification efficiency was achieved, whereas denitrification stopped dramatically with the stage of carbon sources switching. The Afam with side-stream PFR allowed high-speed denitrification and hydrolytic fermentation, eliminating the “stagnation” of switching carbon sources. The optimal VPFR/VCSTR values were 1–1.5. A shorter side-stream time was equivalent to CSTR, whereas a longer side-stream time caused the sludge to synthesize fewer intracellular polymers.

Characterization of the polyhydroxyalkanoate (PHA) synthase from Ideonella sakaiensis, a bacterium that is capable of degrading and assimilating poly(ethylene terephthalate)

• Characterization of PhaC from Ideonella sakaiensis 201-F6, a bacterium that is capable of degrading and assimilating PET. • PhaC Is polymerizes five types of monomers: 3HB, 3HV, 4HB, 5HV and 3H4MV. • Provides insights into the substrate specificity of PhaC Is , which is important for the metabolic engineering of I. sakaiensis that produces various types of PHA from PET. Ideonella sakaiensis 201-F6 is a bacterium capable of degrading and assimilating poly(ethylene terephthalate) (PET). A recent study revealed its capability in accumulating intracellular polyhydroxyalkanoate (PHA) at high levels using PET as a carbon source. The finding demonstrated that I. sakaiensis can mediate the direct conversion of non-biodegradable PET into environment-friendly PHA, providing a new approach for PET recycling. The PHA synthase gene of I. sakaiensis ( phaC Is ) was characterized in this study. Phylogenetic analysis of the PhaC protein sequences indicated PhaC Is belong to class I PhaC. To carry out in vivo characterization of PhaC Is , phaC Is was cloned and heterologously expressed in a PHA-negative mutant strain Cupriavidus necator PHB⁻4. The heterologous expression of the PhaC Is in PHB⁻4/pBBR1- C Is resulted in poly(3-hydroxybutyrate) [P(3HB)] accumulation of up to 71 wt% of the cell dry weight. In addition, the PhaC Is was shown to be able to polymerize 5 types of monomers, 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV), and 3-hydroxy-4-methylvalerate (3H4MV). In vitro enzymatic assay showed that PhaC Is was capable of catalyzing ( R )-3HB-CoA with a specific activity of 11.6 ± 0.6 nmol/min/mg and displayed an initial lag-phase. This study provides a basic information of PhaC Is that allows us to create variants applied for the metabolic engineering of I. sakaiensis that produces various types of PHA from PET.

Sludge retention time effects on recovery of soluble organic matters via polyhydroxyalkanoate storage in contact stabilization process under feast/famine regime

AbstractBACKGROUNDContact stabilization (CS) can be used to capture organic matters from wastewater. However, CS is less efficient in capturing soluble organic matters (SOMs). Recovery of SOMs is required to minimize carbon emission and to maximize organic recovery from wastewater.RESULTSSOMs were recovered by applying a feast/famine (F/F) regime in a CS process. Sludge retention time (SRT) was found to regulate the recovery efficiency of SOMs. At a SRT of 4 d, 44.7% of the removed soluble chemical oxygen demand (SCOD) in wastewater was recovered and stored into intracellular polyhydroxyalkanoates (PHA), leading to 12.8% decrease of CO2 emission in comparison with the conventional activated sludge process. The storage of PHA in waste sludge harvested at a SRT of 4 d significantly increased the methane yield (187.3 ± 5.9 mL (g VSS)−1). The dominant bacteria performing SOM recovery in CS belonged to the genera Tessaracoccus, norank_f__Caldilineaceacea, unclassified_f__Microbacteriaceae and Flavobacterium.CONCLUSIONSThe study demonstrated that applying F/F regime in a CS process can recover SOMs via PHA storage. The regulation of SRT can enhance SCOD recovery. The study provides a solution to enhance SOM recovery from wastewater instead of being oxidized into CO2. © 2022 Society of Chemical Industry (SCI).Highlights SCOD can be recovered via PHA by applying F/F regime in CS mode. The optimum SRT of 4 d resulted 44.7% SCOD recovery via PHA storage. PHA‐accumulating bacteria belonged to four genera. Anaerobic digestion of sludge resulted in a 17.6% higher CH4 yield. CO2 emission decreased 12.8% compared with conventional activated sludge process.

Polyhydroxyalkanoate recovery from newly screened Bacillus sp. LPPI-18 using various methods of extraction from Loktak Lake sediment sample

BackgroundNowadays, the conventional plastic wastes are very challenging to environments and its production cost also creates an economic crisis due to petrochemical-based plastic. In order to solve this problem, the current studies were aimed at screening and characterizing these polyhydroxyalkanoate (PHA)-producing isolates and evaluating the suitability of some carbon source for newly screened PHA-producing isolates. Material and methodsSome carbon sources such as D-fructose, glucose, molasses, D-ribose and sucrose were evaluated for PHA production. Data were analyzed using SPSS version 20. The 16SrRNA gene sequence of these isolates was performed. These newly isolated taxa were related to Bacillus species. It was designated as Bacillus sp. LPPI-18 and affiliated Bacillus cereus ATCC 14577T (AE01687) (99.10%). Paenibacillus sp. 172 (AF273740.1) was used as an outgroup. ResultsBacillus sp. LPPI-18 is a gram-positive, rod-shaped, endospore former, and citrate test positive. This isolate showed positive for amylase, catalase, pectinase, and protease test. They produced intracellular PHA granules when this isolate was stained with Sudan Black B (SBB) and Nile blue A (NBA) preliminary and specific staining dyes, respectively. Both temperature and pH used to affect polyhydroxyalkanoates (PHA) productivity. Bacteria are able to reserve PHA in the form of granules during stress conditions. This isolate produces only when supplied with carbon sources. More PHA contents (PCs) were obtained from glucose, molasses, and D-fructose. In this regard, the maximum mean value of PC was obtained from glucose (40.55±0.7%) and the minimum was obtained from D-ribose (12.4±1.4%). Great variations (P≤0.05) of PCs were observed among glucose and sucrose, molasses and sucrose, and D-fructose and sucrose carbon sources for PHA productivity (PP) of cell dry weight (CDW) g/L. After extraction, PHA film was produced for this typical isolate using glucose as a sole carbon source. Fourier transform infrared spectrum was performed for this isolate and showed the feature of polyester at 1719.64 to 1721.16 wavelengths for these extracted samples. The peak of fingerprinting (band of carboxylic acid group) at this wavelength is a characteristic feature of polyhydroxybutyrate (PHB) and corresponds to the ester functional group (C=O). ConclusionIn this study, newly identified Bacillus sp. LPPI-18 is found to be producing biodegradable polymers that are used to replace highly pollutant conventional plastic polymers. This isolate is also used to employ certain cost-effective carbon sources for the production of PHA polymers. Graphical Abstract▪

Polyhydroxyalkanoates from a Mixed Microbial Culture: Extraction Optimization and Polymer Characterization.

Polyhydroxyalkanoates (PHA) are biopolymers with potential to replace conventional oil-based plastics. However, PHA high production costs limit their scope of commercial applications. Downstream processing is currently the major cost factor for PHA production but one of the least investigated aspects of the PHA production chain. In this study, the extraction of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) produced at pilot scale by a mixed microbial culture was performed using sodium hydroxide (NaOH) or sodium hypochlorite (NaClO) as digestion agents of non-PHA cellular mass. Optimal conditions for digestion with NaOH (0.3 M, 4.8 h) and NaClO (9.0%, 3.4 h) resulted in polymers with a PHA purity and recovery of ca. 100%, in the case of the former and ca. 99% and 90%, respectively, in the case of the latter. These methods presented higher PHA recoveries than extraction by soxhlet with chloroform, the benchmark protocol for PHA extraction. The polymers extracted by the three methods presented similar PHA purities, molecular weights and polydispersity indices. Using the optimized conditions for NaOH and NaClO digestions, this study analyzed the effect of the initial intracellular PHA content (40–70%), biomass concentration (20–100 g/L) and biomass pre-treatment (fresh vs. dried vs. lyophilized) on the performance of PHA extraction by these two methods.

Extremophilic Bacterium Halomonas desertis G11 as a Cell Factory for Poly-3-Hydroxybutyrate-co-3-Hydroxyvalerate Copolymer's Production.

Microbial polyhydroxyalkanoates (PHA) are biodegradable and biocompatible bio-based polyesters, which are used in various applications including packaging, medical and coating materials. In this study, an extremophilic hydrocarbonoclastic bacterium, previously isolated from saline sediment in the Tunisian desert, has been investigated for PHA production. The accumulation of intracellular PHA granules in Halomonas desertis G11 was detected by Nile blue A staining of the colonies. To achieve maximum PHA yield by the strain G11, the culture conditions were optimized through response surface methodology (RSM) employing a Box-Behnken Design (BBD) with three independent variables, namely, substrate concentration (1–5%), inoculum size (1–5%) and incubation time (5–15 days). Under optimized conditions, G11 strain produced 1.5 g/L (68% of DCW) of PHA using glycerol as a substrate. Application of NMR (1H and 13C) and FTIR spectroscopies showed that H. desertis accumulated PHA is a poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV). The genome analysis revealed the presence of typical structural genes involved in PHBV metabolism including phaA, phaB, phaC, phaP, phaZ, and phaR, coding for acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase, class I polyhydroxyalkanoates synthases, phasin, polyhydroxyalkanoates depolymerase and polyhydroxyalkanoates synthesis repressor, respectively. Glycerol can be metabolized to 1) acetyl-CoA through the glycolysis pathway and subsequently converted to the 3HB monomer, and 2) to propionyl-CoA via the threonine biosynthetic pathway and subsequently converted to the 3HV monomer. In silico analysis of PhaC1 from H. desertis G11 indicated that this enzyme belongs to Class I PHA synthase family with a “lipase box”-like sequence (SYCVG). All these characteristics make the extremophilic bacterium H. desertis G11 a promising cell factory for the conversion of bio-renewable glycerol to high-value PHBV.