Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3

A special issue of Bioengineering (ISSN 2306-5354). This special issue belongs to the section "Biochemical Engineering".

Deadline for manuscript submissions: closed (30 April 2022) | Viewed by 68843

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Special Issue Editor

Office of Research Management and Service, c/o Institute of Chemistry, University of Graz, 8010 Graz, Austria
Interests: polyhydroxyalkanoates (PHA); biopolymers; fermentation technology; downstream processing; sustainable process development
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Special Issue Information

Dear Colleagues,       

Nowadays, We are witnessing highly dynamic research activities related to the intriguing field of biodegradable materials with plastic-like properties. These activities are provoked by a strengthened public awareness of prevailing ecological issues connected to growing piles of plastic waste and increasing greenhouse gas emissions; this goes hand-in-hand with the ongoing depletion of fossil feedstocks, which are traditionally used to produce full carbon backbone polymers. To a steadily increasing extend, polyhydroxyalkanoate (PHA) biopolyesters, a family of plastic-like materials with versatile material properties, are considered a future-oriented solution for diminishing these concerns. PHA production is based on renewable resources, and occurs in a bio-mediated fashion by the action of living organisms. If accomplished in an optimized way, PHA production and the entire PHA lifecycle are embedded into nature´s closed cycles of carbon.

You as a highly recognized expert in this field are aware of the fact that sustainable and efficient PHA production requires the understanding and improvement of all individual process steps. Holistic improvement of PHA production, applicable on an industrially relevant scale, calls for inter alia: consolidated knowledge about the enzymatic and genetic particularities of PHA accumulating organisms, in-depth understanding of the kinetics of the bioprocess, the selection of appropriate inexpensive fermentation feedstocks, tailoring the composition of PHA on the level of the monomeric constituents, optimized biotechnological engineering, and novel strategies for PHA recovery from biomass characterized by minor energy and chemical requirement.

In order to provide a comprehensive compilation of articles addressing all these individual aspects, we are contacting globally recognized experts like you. We are convinced that a contribution based on your special expertise in the PHA arena will be of major benefit to make this Special Issue even more attractive to the scientific community.

You may like to read the papers in our precious issues:

Volume 1 (15 papers): https://0-www-mdpi-com.brum.beds.ac.uk/journal/bioengineering/special_issues/PHA

Volume 2 (12 papers): https://0-www-mdpi-com.brum.beds.ac.uk/journal/bioengineering/special_issues/PHA2

Dr. Martin Koller
Guest Editor

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Published Papers (16 papers)

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Editorial

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8 pages, 241 KiB  
Editorial
Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3
by Martin Koller
Bioengineering 2022, 9(7), 328; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9070328 - 19 Jul 2022
Cited by 4 | Viewed by 2671
Abstract
Steadily increasing R&D activities in the field of microbial polyhydroxyalkanoate (PHA) biopolyesters are committed to growing global threats from climate change, aggravating plastic pollution, and the shortage of fossil resources. These prevailing issues paved the way to launch the third Special Issue of [...] Read more.
Steadily increasing R&D activities in the field of microbial polyhydroxyalkanoate (PHA) biopolyesters are committed to growing global threats from climate change, aggravating plastic pollution, and the shortage of fossil resources. These prevailing issues paved the way to launch the third Special Issue of Bioengineering dedicated to future-oriented biomaterials, characterized by their versatile plastic-like properties. Fifteen individual contributions to the Special Issue, written by renowned groups of researchers from all over the world, perfectly mirror the current research directions in the PHA sector: inexpensive feedstock like carbon-rich waste from agriculture, mitigation of CO2 for PHA biosynthesis by cyanobacteria or wild type and engineered “knallgas” bacteria, powerful extremophilic PHA production strains, novel tools for rapid in situ determination of PHA in photobioreactors, modelling of the dynamics of PHA production by mixed microbial cultures from inexpensive raw materials, enhanced bioreactor design for high-throughput PHA production by sophisticated cell retention systems, sustainable and efficient PHA recovery from biomass assisted by supercritical water, enhanced processing of PHA by application of novel antioxidant additives, and the development of compatible biopolymer blends. Moreover, elastomeric medium chain length PHA (mcl-PHA) are covered in-depth, inter alia, by introduction of a novel class of bioactive mcl-PHA-based networks, in addition to the first presentation of the new rubber-like polythioester poly(3-mercapto-2-methylpropionate). Finally, the present Special Issue is concluded by a critical essay on past, ongoing, and announced global endeavors for PHA commercialization. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)

Research

Jump to: Editorial, Review

12 pages, 1038 KiB  
Article
Subcritical Water as a Pre-Treatment of Mixed Microbial Biomass for the Extraction of Polyhydroxyalkanoates
by Liane Meneses, Asiyah Esmail, Mariana Matos, Chantal Sevrin, Christian Grandfils, Susana Barreiros, Maria A. M. Reis, Filomena Freitas and Alexandre Paiva
Bioengineering 2022, 9(7), 302; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9070302 - 08 Jul 2022
Cited by 2 | Viewed by 1791
Abstract
Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical [...] Read more.
Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical water (SBW), was evaluated as a method for the dissolution of the NPCM of a mixed microbial culture (MMC) biomass. A temperature of 150 °C was found as a compromise to reach NPCM solubilization while mostly preventing the degradation of the biopolymer during the procedure. Such conditions yielded a polymer with a purity of 77%. PHA purity was further improved by combining the SBW treatment with hypochlorite digestion, in which a significantly lower hypochlorite concentration (0.1%, v/v) was sufficient to achieve an overall polymer purity of 80%. During the procedure, the biopolymer suffered some depolymerization, as evidenced by the lower molecular weight (Mw) and higher polydispersity of the extracted samples. Although such changes in the biopolymer’s molecular mass distribution impact its mechanical properties, impairing its utilization in most conventional plastic uses, the obtained PHA can find use in several applications, for example as additives or for the preparation of graft or block co-polymers, in which low-Mw oligomers are sought. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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15 pages, 3889 KiB  
Article
Poly(3-mercapto-2-methylpropionate), a Novel α-Methylated Bio-Polythioester with Rubber-like Elasticity, and Its Copolymer with 3-hydroxybutyrate: Biosynthesis and Characterization
by Lucas Vinicius Santini Ceneviva, Maierwufu Mierzati, Yuki Miyahara, Christopher T. Nomura, Seiichi Taguchi, Hideki Abe and Takeharu Tsuge
Bioengineering 2022, 9(5), 228; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9050228 - 23 May 2022
Cited by 7 | Viewed by 4471
Abstract
A new polythioester (PTE), poly(3-mercapto-2-methylpropionate) [P(3M2MP)], and its copolymer with 3-hydroxybutyrate (3HB) were successfully biosynthesized from 3-mercapto-2-methylpropionic acid as a structurally-related precursor. This is the fourth PTE of biological origin and the first to be α-methylated. P(3M2MP) was biosynthesized using an engineered Escherichia [...] Read more.
A new polythioester (PTE), poly(3-mercapto-2-methylpropionate) [P(3M2MP)], and its copolymer with 3-hydroxybutyrate (3HB) were successfully biosynthesized from 3-mercapto-2-methylpropionic acid as a structurally-related precursor. This is the fourth PTE of biological origin and the first to be α-methylated. P(3M2MP) was biosynthesized using an engineered Escherichia coli LSBJ, which has a high molecular weight, amorphous structure, and elastomeric properties, reaching 2600% elongation at break. P(3HB-co-3M2MP) copolymers were synthesized by expressing 3HB-supplying enzymes. The copolymers were produced with high content in the cells and showed a high 3M2MP unit incorporation of up to 77.2 wt% and 54.8 mol%, respectively. As the 3M2MP fraction in the copolymer increased, the molecular weight decreased and the polymers became softer, more flexible, and less crystalline, with lower glass transition temperatures and higher elongations at break. The properties of this PTE were distinct from those of previously biosynthesized PTEs, indicating that the range of material properties can be further expanded by introducing α-methylated thioester monomers. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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18 pages, 1157 KiB  
Article
Lab-Scale Cultivation of Cupriavidus necator on Explosive Gas Mixtures: Carbon Dioxide Fixation into Polyhydroxybutyrate
by Vera Lambauer and Regina Kratzer
Bioengineering 2022, 9(5), 204; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9050204 - 10 May 2022
Cited by 18 | Viewed by 4433
Abstract
Aerobic, hydrogen oxidizing bacteria are capable of efficient, non-phototrophic CO2 assimilation, using H2 as a reducing agent. The presence of explosive gas mixtures requires strict safety measures for bioreactor and process design. Here, we report a simplified, reproducible, and safe cultivation [...] Read more.
Aerobic, hydrogen oxidizing bacteria are capable of efficient, non-phototrophic CO2 assimilation, using H2 as a reducing agent. The presence of explosive gas mixtures requires strict safety measures for bioreactor and process design. Here, we report a simplified, reproducible, and safe cultivation method to produce Cupriavidus necator H16 on a gram scale. Conditions for long-term strain maintenance and mineral media composition were optimized. Cultivations on the gaseous substrates H2, O2, and CO2 were accomplished in an explosion-proof bioreactor situated in a strong, grounded fume hood. Cells grew under O2 control and H2 and CO2 excess. The starting gas mixture was H2:CO2:O2 in a ratio of 85:10:2 (partial pressure of O2 0.02 atm). Dissolved oxygen was measured online and was kept below 1.6 mg/L by a stepwise increase of the O2 supply. Use of gas compositions within the explosion limits of oxyhydrogen facilitated production of 13.1 ± 0.4 g/L total biomass (gram cell dry mass) with a content of 79 ± 2% poly-(R)-3-hydroxybutyrate in a simple cultivation set-up with dissolved oxygen as the single controlled parameter. Approximately 98% of the obtained PHB was formed from CO2. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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29 pages, 5119 KiB  
Communication
PHB Producing Cyanobacteria Found in the Neighborhood—Their Isolation, Purification and Performance Testing
by Katharina Meixner, Christina Daffert, Lisa Bauer, Bernhard Drosg and Ines Fritz
Bioengineering 2022, 9(4), 178; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9040178 - 18 Apr 2022
Cited by 4 | Viewed by 3105
Abstract
Cyanobacteria are a large group of prokaryotic microalgae that are able to grow photo-autotrophically by utilizing sunlight and by assimilating carbon dioxide to build new biomass. One of the most interesting among many cyanobacteria cell components is the storage biopolymer polyhydroxybutyrate (PHB), a [...] Read more.
Cyanobacteria are a large group of prokaryotic microalgae that are able to grow photo-autotrophically by utilizing sunlight and by assimilating carbon dioxide to build new biomass. One of the most interesting among many cyanobacteria cell components is the storage biopolymer polyhydroxybutyrate (PHB), a member of the group of polyhydroxyalkanoates (PHA). Cyanobacteria occur in almost all habitats, ranging from freshwater to saltwater, freely drifting or adhered to solid surfaces or growing in the porewater of soil, they appear in meltwater of glaciers as well as in hot springs and can handle even high salinities and nutrient imbalances. The broad range of habitat conditions makes them interesting for biotechnological production in facilities located in such climate zones with the expectation of using the best adapted organisms in low-tech bioreactors instead of using “universal” strains, which require high technical effort to adapt the production conditions to the organism‘s need. These were the prerequisites for why and how we searched for locally adapted cyanobacteria in different habitats. Our manuscript provides insight to the sites we sampled, how we isolated and enriched, identified (morphology, 16S rDNA), tested (growth, PHB accumulation) and purified (physical and biochemical purification methods) promising PHB-producing cyanobacteria that can be used as robust production strains. Finally, we provide a guideline about how we managed to find potential production strains and prepared others for basic metabolism studies. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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20 pages, 1260 KiB  
Article
Sugar Beet Molasses as a Potential C-Substrate for PHA Production by Cupriavidus necator
by Evgeniy G. Kiselev, Aleksey V. Demidenko, Natalia O. Zhila, Ekaterina I. Shishatskaya and Tatiana G. Volova
Bioengineering 2022, 9(4), 154; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9040154 - 04 Apr 2022
Cited by 15 | Viewed by 4664
Abstract
To increase the availability and expand the raw material base, the production of polyhydroxyalkanoates (PHA) by the wild strain Cupriavidus necator B-10646 on hydrolysates of sugar beet molasses was studied. The hydrolysis of molasses was carried out using β-fructofuranosidase, which provides a [...] Read more.
To increase the availability and expand the raw material base, the production of polyhydroxyalkanoates (PHA) by the wild strain Cupriavidus necator B-10646 on hydrolysates of sugar beet molasses was studied. The hydrolysis of molasses was carried out using β-fructofuranosidase, which provides a high conversion of sucrose (88.9%) to hexoses. We showed the necessity to adjust the chemical composition of molasses hydrolysate to balance with the physiological needs of C. necator B-10646 and reduce excess sugars and nitrogen and eliminate phosphorus deficiency. The modes of cultivation of bacteria on diluted hydrolyzed molasses with the controlled feeding of phosphorus and glucose were implemented. Depending on the ratio of sugars introduced into the bacterial culture due to the molasses hydrolysate and glucose additions, the bacterial biomass concentration was obtained from 20–25 to 80–85 g/L with a polymer content up to 80%. The hydrolysates of molasses containing trace amounts of propionate and valerate were used to synthesize a P(3HB-co-3HV) copolymer with minor inclusions of 3-hydroxyvlaerate monomers. The introduction of precursors into the medium ensured the synthesis of copolymers with reduced values of the degree of crystallinity, containing, in addition to 3HB, monomers 3HB, 4HB, or 3HHx in an amount of 12–16 mol.%. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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26 pages, 1102 KiB  
Article
Modelling Mixed Microbial Culture Polyhydroxyalkanoate Accumulation Bioprocess towards Novel Methods for Polymer Production Using Dilute Volatile Fatty Acid Rich Feedstocks
by Alan Werker, Laura Lorini, Marianna Villano, Francesco Valentino and Mauro Majone
Bioengineering 2022, 9(3), 125; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9030125 - 21 Mar 2022
Cited by 4 | Viewed by 2468
Abstract
Volatile fatty acid (VFA) rich streams from fermentation of organic residuals and wastewater are suitable feedstocks for mixed microbial culture (MMC) Polyhydroxyalkanoate (PHA) production. However, many such streams have low total VFA concentration (1–10 gCOD/L). PHA accumulation requires a flow-through bioprocess if the [...] Read more.
Volatile fatty acid (VFA) rich streams from fermentation of organic residuals and wastewater are suitable feedstocks for mixed microbial culture (MMC) Polyhydroxyalkanoate (PHA) production. However, many such streams have low total VFA concentration (1–10 gCOD/L). PHA accumulation requires a flow-through bioprocess if the VFAs are not concentrated. A flow through bioprocess must balance goals of productivity (highest possible influent flow rates) with goals of substrate utilization efficiency (lowest possible effluent VFA concentration). Towards these goals, dynamics of upshift and downshift respiration kinetics for laboratory and pilot scale MMCs were evaluated. Monod kinetics described a hysteresis between the upshift and downshift responses. Substrate concentrations necessary to stimulate a given substrate uptake rate were significantly higher than the concentrations necessary to sustain the attained substrate uptake rate. A benefit of this hysteresis was explored in Monte Carlo based PHA accumulation bioprocess numerical simulations. Simulations illustrated for a potential to establish continuous flow-through PHA production bioprocesses even at a low (1 gCOD/L) influent total VFA concentration. Process biomass recirculation into an engineered higher substrate concentration mixing zone, due to the constant influent substrate flow, enabled to drive the process to maximal possible PHA production rates without sacrificing substrate utilization efficiency. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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11 pages, 1569 KiB  
Article
Cell Retention as a Viable Strategy for PHA Production from Diluted VFAs with Bacillus megaterium
by Milos Kacanski, Lukas Pucher, Carlota Peral, Thomas Dietrich and Markus Neureiter
Bioengineering 2022, 9(3), 122; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9030122 - 16 Mar 2022
Cited by 13 | Viewed by 2926
Abstract
The production of biodegradable and biocompatible materials such as polyhydroxyalkanoates (PHAs) from waste-derived volatile fatty acids (VFAs) is a promising approach towards implementing a circular bioeconomy. However, VFA solutions obtained via acidification of organic wastes are usually too diluted for direct use in [...] Read more.
The production of biodegradable and biocompatible materials such as polyhydroxyalkanoates (PHAs) from waste-derived volatile fatty acids (VFAs) is a promising approach towards implementing a circular bioeconomy. However, VFA solutions obtained via acidification of organic wastes are usually too diluted for direct use in standard batch or fed-batch processes. To overcome these constraints, this study introduces a cell recycle fed-batch system using Bacillus megaterium uyuni S29 for poly(3-hydroxybutyrate) (P3HB) production from acetic acid. The concentrations of dry cell weight (DCW), P3HB, acetate, as well as nitrogen as the limiting substrate component, were monitored during the process. The produced polymer was characterized in terms of molecular weight and thermal properties after extraction with hypochlorite. The results show that an indirect pH-stat feeding regime successfully kept the strain fed without prompting inhibition, resulting in a dry cell weight concentration of up to 19.05 g/L containing 70.21% PHA. After appropriate adaptations the presented process could contribute to an efficient and sustainable production of biopolymers. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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15 pages, 3237 KiB  
Article
Improved Processability and Antioxidant Behavior of Poly(3-hydroxybutyrate) in Presence of Ferulic Acid-Based Additives
by Lionel F. Longé, Laurent Michely, Antoine Gallos, Agustin Rios De Anda, Henri Vahabi, Estelle Renard, Michel Latroche, Florent Allais and Valérie Langlois
Bioengineering 2022, 9(3), 100; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9030100 - 28 Feb 2022
Cited by 5 | Viewed by 2609
Abstract
Poly(3-hydroxybutyrate), PHB, has gathered a lot of attention for its promising properties—in particular its biobased nature and high biodegradability. Although PHB is prime candidate for the packaging industry, the applications are still limited by a narrow processing window and thermal degradation during melt [...] Read more.
Poly(3-hydroxybutyrate), PHB, has gathered a lot of attention for its promising properties—in particular its biobased nature and high biodegradability. Although PHB is prime candidate for the packaging industry, the applications are still limited by a narrow processing window and thermal degradation during melt processing. In this work, three novel additives based on ferulic acid esterified with butanediol, pentanediol, and glycerol (BDF, PDF, and GTF, respectively) were used as plasticizers and antioxidative additives to improve mechanical properties of PHB. Elongation at break up to 270% was obtained in presence of BDF and the processing window was improved nearly 10-fold. The Pawley method was used to identify the monoclinic space group P2 of the BDF. The estimated crystallite size (71 nm) agrees with a crystalline additive. With PHB70BDF30 blends, even higher elongations at break were obtained though dwindled with time. However, these properties could be recovered after thermal treatment. The high thermal stability of this additive leads to an increase in the fire retardancy property of the material, and the phenolic structure induced antioxidant properties to the samples as demonstrated by radical scavenging tests, further highlighting the possibilities of the PHB/additive blends for packaging applications. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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10 pages, 991 KiB  
Article
Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO2 by a Recombinant Cupriavidusnecator
by Kenji Tanaka, Kazumasa Yoshida, Izumi Orita and Toshiaki Fukui
Bioengineering 2021, 8(11), 179; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering8110179 - 07 Nov 2021
Cited by 26 | Viewed by 3462
Abstract
The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars [...] Read more.
The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through β-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO2 as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H2/O2/CO2 = 8:1:1). The results of GC and 1H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO2 with high cellular content. When 1.0 g/L (NH4)2SO4 was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccrMeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccrMeJAc-emd harboring short-chain-length-specific PhaJ. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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23 pages, 5060 KiB  
Article
Two-Stage Polyhydroxyalkanoates (PHA) Production from Cheese Whey Using Acetobacter pasteurianus C1 and Bacillus sp. CYR1
by Young-Cheol Chang, Motakatla Venkateswar Reddy, Kazuma Imura, Rui Onodera, Natsumi Kamada and Yuki Sano
Bioengineering 2021, 8(11), 157; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering8110157 - 24 Oct 2021
Cited by 22 | Viewed by 4305
Abstract
Cheese whey (CW) can be an excellent carbon source for polyhydroxyalkanoates (PHA)-producing bacteria. Most studies have used CW, which contains high amounts of lactose, however, there are no reports using raw CW, which has a relatively low amount of lactose. Therefore, in the [...] Read more.
Cheese whey (CW) can be an excellent carbon source for polyhydroxyalkanoates (PHA)-producing bacteria. Most studies have used CW, which contains high amounts of lactose, however, there are no reports using raw CW, which has a relatively low amount of lactose. Therefore, in the present study, PHA production was evaluated in a two-stage process using the CW that contains low amounts of lactose. In first stage, the carbon source existing in CW was converted into acetic acid using the bacteria, Acetobacter pasteurianus C1, which was isolated from food waste. In the second stage, acetic acid produced in the first stage was converted into PHA using the bacteria, Bacillus sp. CYR-1. Under the condition of without the pretreatment of CW, acetic acid produced from CW was diluted at different folds and used for the production of PHA. Strain CYR-1 incubated with 10-fold diluted CW containing 5.7 g/L of acetic acid showed the higher PHA production (240.6 mg/L), whereas strain CYR-1 incubated with four-fold diluted CW containing 12.3 g/L of acetic acid showed 126 mg/L of PHA. After removing the excess protein present in CW, PHA production was further enhanced by 3.26 times (411 mg/L) at a four-fold dilution containing 11.3 g/L of acetic acid. Based on Fourier transform infrared spectroscopy (FT-IR), and 1H and 13C nuclear magnetic resonance (NMR) analyses, it was confirmed that the PHA produced from the two-stage process is poly-β-hydroxybutyrate (PHB). All bands appearing in the FT-IR spectrum and the chemical shifts of NMR nearly matched with those of standard PHB. Based on these studies, we concluded that a two-stage process using Acetobacter pasteurianus C1 and Bacillus sp. CYR-1 would be applicable for the production of PHB using CW containing a low amount of lactose. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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13 pages, 2567 KiB  
Article
Biotechnological Conversion of Grape Pomace to Poly(3-hydroxybutyrate) by Moderately Thermophilic Bacterium Tepidimonas taiwanensis
by Xenie Kourilova, Iva Pernicova, Michaela Vidlakova, Roman Krejcirik, Katerina Mrazova, Kamila Hrubanova, Vladislav Krzyzanek, Jana Nebesarova and Stanislav Obruca
Bioengineering 2021, 8(10), 141; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering8100141 - 14 Oct 2021
Cited by 20 | Viewed by 2762
Abstract
Polyhydroxyalkanoates (PHA) are microbial polyesters that have recently come to the forefront of interest due to their biodegradability and production from renewable sources. A potential increase in competitiveness of PHA production process comes with a combination of the use of thermophilic bacteria with [...] Read more.
Polyhydroxyalkanoates (PHA) are microbial polyesters that have recently come to the forefront of interest due to their biodegradability and production from renewable sources. A potential increase in competitiveness of PHA production process comes with a combination of the use of thermophilic bacteria with the mutual use of waste substrates. In this work, the thermophilic bacterium Tepidimonas taiwanensis LMG 22826 was identified as a promising PHA producer. The ability to produce PHA in T. taiwanensis was studied both on genotype and phenotype levels. The gene encoding the Class I PHA synthase, a crucial enzyme in PHA synthesis, was detected both by genome database search and by PCR. The microbial culture of T. taiwanensis was capable of efficient utilization of glucose and fructose. When cultivated on glucose as the only carbon source at 50 °C, the PHA titers reached up to 3.55 g/L, and PHA content in cell dry mass was 65%. The preference of fructose and glucose opens the possibility to employ T. taiwanensis for PHA production on various food wastes rich in these abundant sugars. In this work, PHA production on grape pomace extracts was successfully tested. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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10 pages, 2266 KiB  
Communication
In Situ Quantification of Polyhydroxybutyrate in Photobioreactor Cultivations of Synechocystis sp. Using an Ultrasound-Enhanced ATR-FTIR Spectroscopy Probe
by Philipp Doppler, Christoph Gasser, Ricarda Kriechbaum, Ardita Ferizi and Oliver Spadiut
Bioengineering 2021, 8(9), 129; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering8090129 - 21 Sep 2021
Cited by 5 | Viewed by 3089
Abstract
Polyhydroxybutyrate (PHB) is a very promising alternative to most petroleum-based plastics with the huge advantage of biodegradability. Biotechnological production processes utilizing cyanobacteria as sustainable source of PHB require fast in situ process analytical technology (PAT) tools for sophisticated process monitoring. Spectroscopic probes supported [...] Read more.
Polyhydroxybutyrate (PHB) is a very promising alternative to most petroleum-based plastics with the huge advantage of biodegradability. Biotechnological production processes utilizing cyanobacteria as sustainable source of PHB require fast in situ process analytical technology (PAT) tools for sophisticated process monitoring. Spectroscopic probes supported by ultrasound particle traps provide a powerful technology for in-line, nondestructive, and real-time process analytics in photobioreactors. This work shows the great potential of using ultrasound particle manipulation to improve spectroscopic attenuated total reflection Fourier-transformed mid-infrared (ATR-FTIR) spectra as a monitoring tool for PHB production processes in photobioreactors. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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11 pages, 2162 KiB  
Article
Antioxidant Network Based on Sulfonated Polyhydroxyalkanoate and Tannic Acid Derivative
by Laura Brelle, Estelle Renard and Valerie Langlois
Bioengineering 2021, 8(1), 9; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering8010009 - 08 Jan 2021
Cited by 3 | Viewed by 3019
Abstract
A novel generation of gels based on medium chain length poly(3-hydroxyalkanoate)s, mcl-PHAs, were developed by using ionic interactions. First, water soluble mcl-PHAs containing sulfonate groups were obtained by thiol-ene reaction in the presence of sodium-3-mercapto-1-ethanesulfonate. Anionic PHAs were physically crosslinked by [...] Read more.
A novel generation of gels based on medium chain length poly(3-hydroxyalkanoate)s, mcl-PHAs, were developed by using ionic interactions. First, water soluble mcl-PHAs containing sulfonate groups were obtained by thiol-ene reaction in the presence of sodium-3-mercapto-1-ethanesulfonate. Anionic PHAs were physically crosslinked by divalent inorganic cations Ca2+, Ba2+, Mg2+ or by ammonium derivatives of gallic acid GA-N(CH3)3+ or tannic acid TA-N(CH3)3+. The ammonium derivatives were designed through the chemical modification of gallic acid GA or tannic acid TA with glycidyl trimethyl ammonium chloride (GTMA). The results clearly demonstrated that the formation of the networks depends on the nature of the cations. A low viscoelastic network having an elastic around 40 Pa is formed in the presence of Ca2+. Although the gel formation is not possible in the presence of GA-N(CH3)3+, the mechanical properties increased in the presence of TA-N(CH3)3+ with an elastic modulus G’ around 4200 Pa. The PHOSO3/TA-N(CH3)3+ gels having antioxidant activity, due to the presence of tannic acid, remained stable for at least 5 months. Thus, the stability of these novel networks based on PHA encourage their use in the development of active biomaterials. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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Review

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24 pages, 2187 KiB  
Review
Review of the Developments of Bacterial Medium-Chain-Length Polyhydroxyalkanoates (mcl-PHAs)
by V. Uttej Nandan Reddy, S. V. Ramanaiah, M. Venkateswar Reddy and Young-Cheol Chang
Bioengineering 2022, 9(5), 225; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9050225 - 21 May 2022
Cited by 28 | Viewed by 7143
Abstract
Synthetic plastics derived from fossil fuels—such as polyethylene, polypropylene, polyvinyl chloride, and polystyrene—are non-degradable. A large amount of plastic waste enters landfills and pollutes the environment. Hence, there is an urgent need to produce biodegradable plastics such as polyhydroxyalkanoates (PHAs). PHAs have garnered [...] Read more.
Synthetic plastics derived from fossil fuels—such as polyethylene, polypropylene, polyvinyl chloride, and polystyrene—are non-degradable. A large amount of plastic waste enters landfills and pollutes the environment. Hence, there is an urgent need to produce biodegradable plastics such as polyhydroxyalkanoates (PHAs). PHAs have garnered increasing interest as replaceable materials to conventional plastics due to their broad applicability in various purposes such as food packaging, agriculture, tissue-engineering scaffolds, and drug delivery. Based on the chain length of 3-hydroxyalkanoate repeat units, there are three types PHAs, i.e., short-chain-length (scl-PHAs, 4 to 5 carbon atoms), medium-chain-length (mcl-PHAs, 6 to 14 carbon atoms), and long-chain-length (lcl-PHAs, more than 14 carbon atoms). Previous reviews discussed the recent developments in scl-PHAs, but there are limited reviews specifically focused on the developments of mcl-PHAs. Hence, this review focused on the mcl-PHA production, using various carbon (organic/inorganic) sources and at different operation modes (continuous, batch, fed-batch, and high-cell density). This review also focused on recent developments on extraction methods of mcl-PHAs (solvent, non-solvent, enzymatic, ultrasound); physical/thermal properties (Mw, Mn, PDI, Tm, Tg, and crystallinity); applications in various fields; and their production at pilot and industrial scales in Asia, Europe, North America, and South America. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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29 pages, 6363 KiB  
Review
A New Wave of Industrialization of PHA Biopolyesters
by Martin Koller and Anindya Mukherjee
Bioengineering 2022, 9(2), 74; https://0-doi-org.brum.beds.ac.uk/10.3390/bioengineering9020074 - 15 Feb 2022
Cited by 95 | Viewed by 12412
Abstract
The ever-increasing use of plastics, their fossil origin, and especially their persistence in nature have started a wave of new innovations in materials that are renewable, offer the functionalities of plastics, and are biodegradable. One such class of biopolymers, polyhydroxyalkanoates (PHAs), are biosynthesized [...] Read more.
The ever-increasing use of plastics, their fossil origin, and especially their persistence in nature have started a wave of new innovations in materials that are renewable, offer the functionalities of plastics, and are biodegradable. One such class of biopolymers, polyhydroxyalkanoates (PHAs), are biosynthesized by numerous microorganisms through the conversion of carbon-rich renewable resources. PHA homo- and heteropolyesters are intracellular products of secondary microbial metabolism. When isolated from microbial biomass, PHA biopolymers mimic the functionalities of many of the top-selling plastics of petrochemical origin, but biodegrade in soil, freshwater, and marine environments, and are both industrial- and home-compostable. Only a handful of PHA biopolymers have been studied in-depth, and five of these reliably match the desired material properties of established fossil plastics. Realizing the positive attributes of PHA biopolymers, several established chemical companies and numerous start-ups, brand owners, and converters have begun to produce and use PHA in a variety of industrial and consumer applications, in what can be described as the emergence of the “PHA industry”. While this positive industrial and commercial relevance of PHA can hardly be described as the first wave in its commercial development, it is nonetheless a very serious one with over 25 companies and start-ups and 30+ brand owners announcing partnerships in PHA production and use. The combined product portfolio of the producing companies is restricted to five types of PHA, namely poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), even though PHAs as a class of polymers offer the potential to generate almost limitless combinations of polymers beneficial to humankind. To date, by varying the co-monomer type and content in these PHA biopolymers, their properties emulate those of the seven top-selling fossil plastics, representing 230 million t of annual plastics production. Capacity expansions of 1.5 million t over the next 5 years have been announced. Policymakers worldwide have taken notice and are encouraging industry to adopt biodegradable and compostable material solutions. This wave of commercialization of PHAs in single-use and in durable applications holds the potential to make the decisive quantum leap in reducing plastic pollution, the depletion of fossil resources, and the emission of greenhouse gases and thus fighting climate change. This review presents setbacks and success stories of the past 40 years and the current commercialization wave of PHA biopolymers, their properties, and their fields of application. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, Volume 3)
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