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In-Line Monitoring of Polyhydroxyalkanoate (PHA) Production during High-Cell-Density Plant Oil Cultivations Using Photon Density Wave Spectroscopy.

ABSTRACT: Polyhydroxyalkanoates (PHAs) are biodegradable plastic-like materials with versatile properties. Plant oils are excellent carbon sources for a cost-effective PHA production, due to their high carbon content, large availability, and comparatively low prices. Additionally, efficient process development and control is required for competitive PHA production, which can be facilitated by on-line or in-line monitoring devices. To this end, we have evaluated photon density wave (PDW) spectroscopy as a new process analytical technology for Ralstonia eutropha (Cupriavidus necator) H16 plant oil cultivations producing polyhydroxybutyrate (PHB) as an intracellular polymer. PDW spectroscopy was used for in-line recording of the reduced scattering coefficient µs' and the absorption coefficient µa at 638 nm. A correlation of µs' with the cell dry weight (CDW) and µa with the residual cell dry weight (RCDW) was observed during growth, PHB accumulation, and PHB degradation phases in batch and pulse feed cultivations. The correlation was used to predict CDW, RCDW, and PHB formation in a high-cell-density fed-batch cultivation with a productivity of 1.65 gPHB·L-1·h-1 and a final biomass of 106 g·L-1 containing 73 wt% PHB. The new method applied in this study allows in-line monitoring of CDW, RCDW, and PHA formation.

SUBMITTER: Gutschmann B

PROVIDER: S-EPMC6783927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Microbial Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoate (mcl-PHA) from Waste Cooking Oil.

Project description:Waste cooking oil is a common byproduct in the culinary industry, often posing disposal challenges. This study explores its conversion into the valuable bioplastic material, medium-chain-length polyhydroxyalkanoate (mcl-PHA), through microbial biosynthesis in controlled bioreactor conditions. Twenty-four bacterial isolates were obtained from oil-contaminated soil and waste materials in Mahd Ad-Dahab, Saudi Arabia. The best PHA-producing isolates were identified via 16S rDNA analysis as Neobacillus niacini and Metabacillus niabensis, with the sequences deposited in GenBank (accession numbers: PP346270 and PP346271). This study evaluated the effects of various carbon and nitrogen sources, as well as environmental factors, such as pH, temperature, and shaking speed, on the PHA production titer. Neobacillus niacini favored waste cooking oil and yeast extract, achieving a PHA production titer of 1.13 g/L, while Metabacillus niabensis preferred waste olive oil and urea, with a PHA production titer of 0.85 g/L. Both strains exhibited optimal growth at a neutral pH of 7, under optimal shaking -flask conditions. The bioreactor performance showed improved PHA production under controlled pH conditions, with a final titer of 9.75 g/L for Neobacillus niacini and 4.78 g/L for Metabacillus niabensis. Fourier transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) confirmed the biosynthesized polymer as mcl-PHA. This research not only offers a sustainable method for transforming waste into valuable materials, but also provides insights into the optimal conditions for microbial PHA production, advancing environmental science and materials engineering.

| S-EPMC11314287 | biostudies-literature

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

Project description:Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of ?-proteobacteria, ?-proteobacteria and ?-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.

| S-EPMC4879537 | biostudies-literature

Advances in Polyhydroxyalkanoate (PHA) Production, Volume 2.

Project description:During the two years that have passed since the first volume of "Advances in Polyhydroxyalkanoate (PHA) production" was published, the progress in PHA-related research was indeed tremendous, calling for the next, highly bioprocess- and bioengineering-oriented volume. This editorial paper summarizes and puts into context the contributions to this second volume of the Bioengineering Special Issue; it covers highly topical fields of PHA-related R&D activities, covering, beside the pronounced bioengineering-related articles, the fields of the microbiology of underexplored, but probably emerging, PHA production strains from the groups of Pseudomonas, cyanobacteria, methanotrophs, and from the extremophilic domain of haloarchaea. Moreover, novel second-generation lignocellulose feedstocks for PHA production from agriculture to be used in biorefinery concepts, new approaches for fine-tuning the composition of PHA co- and terpolyesters, process simulation for PHA production from methane-rich natural gas, the challenges associated with rheology-governed oxygen transfer in high cell density cultivations, rapid spectroscopic in-line analytics for process monitoring, and the biomedical application of PHA biopolyesters after appropriate advanced processing are the subjects of the presented studies.

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Process Characterization of Polyvinyl Acetate Emulsions Applying Inline Photon Density Wave Spectroscopy at High Solid Contents.

Project description:The high solids semicontinuous emulsion polymerization of polyvinyl acetate using poly (vinyl alcohol-co-vinyl acetate) as protective colloid is investigated by optical spectroscopy. The suitability of Photon Density Wave (PDW) spectroscopy as inline Process Analytical Technology (PAT) for emulsion polymerization processes at high solid contents (>40% (w/w)) is studied and evaluated. Inline data on absorption and scattering in the dispersion is obtained in real-time. The radical polymerization of vinyl acetate to polyvinyl acetate using ascorbic acid and sodium persulfate as redox initiator system and poly (vinyl alcohol-co-vinyl acetate) as protective colloid is investigated. Starved-feed radical emulsion polymerization yielded particle sizes in the nanometer size regime. PDW spectroscopy is used to monitor the progress of polymerization by studying the absorption and scattering properties during the synthesis of dispersions with increasing monomer amount and correspondingly decreasing feed rate of protective colloid. Results are compared to particle sizes determined with offline dynamic light scattering (DLS) and static light scattering (SLS) during the synthesis.

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A novel LED-based 2D-fluorescence spectroscopy system for in-line monitoring of Chinese hamster ovary cell cultivations - Part I.

Project description:A new two-dimensional fluorescence sensor system was developed for in-line monitoring of mammalian cell cultures. Fluorescence spectroscopy allows for the detection and quantification of naturally occurring intra- and extracellular fluorophores in the cell broth. The fluorescence signals correlate to the cells' current redox state and other relevant process parameters. Cell culture pretests with twelve different excitation wavelengths showed that only three wavelengths account for a vast majority of spectral variation. Accordingly, the newly developed device utilizes three high-power LEDs as excitation sources in combination with a back-thinned CCD-spectrometer for fluorescence detection. This setup was first tested in a lab design of experiments study with process relevant fluorophores proving its suitability for cell culture monitoring with LOD in the μg/L range. The sensor was then integrated into a CHO-K1 cell culture process. The acquired fluorescence spectra of several batches were evaluated using multivariate methods. The resulting batch evolution models were challenged in deviating and "golden batch" validation runs. These first tests showed that the new sensor can trace the cells' metabolic state in a fast and reliable manner. Cellular distress is quickly detected as a deviation from the "golden batch".

| S-EPMC6999564 | biostudies-literature

A novel LED-based 2D-fluorescence spectroscopy system for in-line bioprocess monitoring of Chinese hamster ovary cell cultivations-Part II.

Project description:This study was performed in order to evaluate a new LED-based 2D-fluorescence spectrometer for in-line bioprocess monitoring of Chinese hamster ovary (CHO) cell culture processes. The new spectrometer used selected excitation wavelengths of 280, 365, and 455 nm to collect spectral data from six 10-L fed-batch processes. The technique provides data on various fluorescent compounds from the cultivation medium as well as from cell metabolism. In addition, scattered light offers information about the cultivation status. Multivariate data analysis tools were applied to analyze the large data sets of the collected fluorescence spectra. First, principal component analysis was used to accomplish an overview of all spectral data from all six CHO cultivations. Partial least square regression models were developed to correlate 2D-fluorescence spectral data with selected critical process variables as offline reference values. A separate independent fed-batch process was used for model validation and prediction. An almost continuous in-line bioprocess monitoring was realized because 2D-fluorescence spectra were collected every 10 min during the whole cultivation. The new 2D-fluorescence device demonstrates the significant potential for accurate prediction of the total cell count, viable cell count, and the cell viability. The results strongly indicated that the technique is particularly capable to distinguish between different cell statuses inside the bioreactor. In addition, spectral data provided information about the lactate metabolism shift and cellular respiration during the cultivation process. Overall, the 2D-fluorescence device is a highly sensitive tool for process analytical technology applications in mammalian cell cultures.

| S-EPMC6999457 | biostudies-literature

Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase.

Project description:In this study, we performed in vitro and in vivo activity assays of polyhydroxyalkanoate (PHA) synthases (PhaCs) in the presence of phasin proteins (PhaPs), which revealed that PhaPs are activators of PhaC derived from Aeromonas caviae (PhaCAc). In in vitro assays, among the three PhaCs tested, PhaCAc was significantly activated when PhaPs were added at the beginning of polymerization (prepolymerization PhaCAc), whereas the prepolymerization PhaCRe (derived from Ralstonia eutropha) and PhaCDa (Delftia acidovorans) showed reduced activity with PhaPs. The PhaP-activated PhaCAc showed a slight shift of substrate preference toward 3-hydroxyhexanoyl-CoA (C6). PhaPAc also activated PhaCAc when it was added during polymerization (polymer-elongating PhaCAc), while this effect was not observed for PhaCRe. In an in vivo assay using Escherichia coli TOP10 as the host strain, the effect of PhaPAc expression on PHA synthesis by PhaCAc or PhaCRe was examined. As PhaPAc expression increased, PHA production was increased by up to 2.3-fold in the PhaCAc-expressing strain, whereas it was slightly increased in the PhaCRe-expressing strain. Taken together, this study provides evidence that PhaPs function as activators for PhaCAc both in vitro and in vivo but do not activate PhaCRe. This activating effect may be attributed to the new role of PhaPs in the polymerization reaction by PhaCAc.

| S-EPMC3993283 | biostudies-literature

Role of genetic redundancy in polyhydroxyalkanoate (PHA) polymerases in PHA biosynthesis in Rhodospirillum rubrum.

Project description:This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C(5) and C(6) monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.

| S-EPMC3458679 | biostudies-literature

Inhibitors of polyhydroxyalkanoate (PHA) synthases: synthesis, molecular docking, and implications.

Project description:Polyhydroxyalkanoate (PHA) synthases (PhaCs) catalyze the formation of biodegradable PHAs that are considered to be ideal alternatives to non-biodegradable synthetic plastics. However, study of PhaCs has been challenging because the rate of PHA chain elongation is much faster than that of initiation. This difficulty, along with lack of a crystal structure, has become the main hurdle to understanding and engineering PhaCs for economical PHA production. Here we report the synthesis of two carbadethia CoA analogues--sT-CH2-CoA (26 a) and sTet-CH2-CoA (26 b)--as well as sT-aldehyde (saturated trimer aldehyde, 29), as new PhaC inhibitors. Study of these analogues with PhaECAv revealed that 26 a/b and 29 are competitive and mixed inhibitors, respectively. Both the CoA moiety and extension of PHA chain will increase binding affinity; this is consistent with our docking study. Estimation of the Kic values of 26 a and 26 b predicts that a CoA analogue incorporating an octameric hydroxybutanoate (HB) chain might facilitate the formation of a kinetically well-behaved synthase.

| S-EPMC4391825 | biostudies-literature

The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases.

Project description:BACKGROUND: Polyhydroxyalkanoates (PHAs) can be degraded by many microorganisms using intra- or extracellular PHA depolymerases. PHA depolymerases are very diverse in sequence and substrate specificity, but share a common alpha/beta-hydrolase fold and a catalytic triad, which is also found in other alpha/beta-hydrolases. RESULTS: The PHA Depolymerase Engineering Database (DED, http://www.ded.uni-stuttgart.de) has been established as a tool for systematic analysis of this enzyme family. The DED contains sequence entries of 587 PHA depolymerases, which were assigned to 8 superfamilies and 38 homologous families based on their sequence similarity. For each family, multiple sequence alignments and profile hidden Markov models are provided, and functionally relevant residues are annotated. CONCLUSION: The DED is a valuable tool which can be applied to identify new PHA depolymerase sequences from complete genomes in silico, to classify PHA depolymerases, to predict their biochemical properties, and to design enzyme variants with improved properties.

| S-EPMC2666664 | biostudies-literature

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