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Bioengineering
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Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition
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Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition

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

Deadline for manuscript submissions: closed (30 September 2024) | Viewed by 15264

Special Issue Editor

Dr. Martin Koller

E-Mail Website
Guest 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
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Especially during the last couple of months, humankind became tremendously aware of the threats committed by a shortage of and insecure supply chains for fossil resources needed for the generation of energy carriers and goods like heavily utilized plastics. Together with the climate catastrophe that is becoming more and more apparent, soaring amounts of plastic waste, and ever more profound knowledge about the mechanisms of formation and effects of micro- and nanoplastics, this leads to intensified efforts in science, research and industry to focus on alternatives. In the field of plastics, such alternatives are biodegradable materials with versatile plastic-like characteristics. Such alternatives shall be intrinsically circular: biobased—originating from renewable carbon feedstock; biodegradable—amenable to degradability under aerobic and anaerobic conditions by biological factors, in both terrestrial and aquatic habitats; compostable both in industrial composting facilities and the compost heap in the garden; biocompatible—such materials do not exert any harm to the biosphere.

You, as an expert, are familiar with the fact that microbial polyhydroxyalkanoate (PHA) biopolyesters are the group of choice of alternative materials fulfilling these requirements. Produced predominately by prokaryotes as reserve- and stress-protectant materials, these versatile biopolymers have been demonstrated to mimic the properties and processability of various well-established petroplastics. Indeed, switching from limited fossil resources to PHA for plastic production excellently matches today's initiatives to reach the global Climate Goals, and, if accomplished in an appropriate manner, even fits into the patterns of the 17 United Nations Sustainable Development Goals (SDGs), e.g., by saving food resources, fostering industrial innovation, sustainable consumption and production, and by protecting the oceans and land. Novel inexpensive feedstocks, advanced process engineering, optimized and robust microbial production strains, and smart processing with different compatible (nano)materials to high-tech products are currently lifting PHA to industrial maturity, as witnessed by “a new wave of industrialization of PHA biopolyesters”, which is currently pending worldwide. Indeed, an increasing number of companies of different sizes are already considering PHA as the way to go toward a circular bioeconomy!

However, the end of the road for these seminal materials has by no means been reached. Discovery and development of even more powerful production strains, optimized cultivation regimes, next-generation production strategies, finetuning of the nano-, micro- and macrostructure of PHA and follow-up products, truly sustainable protocols for PHA recovery, sound integration of PHA production into diverse biorefinery schemes, etc., are the directions that still need to be pursued in order not only to be able to produce PHA cost-effectively but also to adapt their properties to constantly increasing consumer expectations. This is exactly where your profound expertise is requested: Present Bioengineering Special Issue on “Polyhydroxyalkanoates: Production, Recent Developments and Applications”, already the fourth edition of this PHA series which I have the honor to guest-edit, is envisioned to invite contributions from all relevant fields of PHA research, encompassing microbiology, synthetic biology, genome editing, bioreactor technology, process design, tapping new raw material sources, mathematic modelling of diverse complexity, novel approaches for product recovery, PHA processing and modification, end of life and post-use options for PHA and follow-up materials, life cycle and sustainability considerations, and holistic circularity aspects.

We are very much looking forward to the participation of both emerging and long-established research teams active in the field of biopolymers. Moreover, we are confident that this Special Issue will provide similar seminal inspiration to the scientific community as the first three issues:

Volume 1 (15 papers): https://www.mdpi.com/journal/bioengineering/special_issues/PHA

Volume 2 (12 papers): https://www.mdpi.com/journal/bioengineering/special_issues/PHA2

Volume 3 (16 papers): https://www.mdpi.com/journal/bioengineering/special_issues/PHA3

Dr. Martin Koller
Guest Editor

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Published Papers (7 papers)

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Research

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17 pages, 3036 KiB  
Article
Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei Using Candy Industry Waste as Raw Materials
by Lorena Simó-Cabrera, Salvador García-Chumillas, Sergio J. Benitez-Benitez, Verónica Cánovas, Fuensanta Monzó, Carmen Pire and Rosa María Martínez-Espinosa
Bioengineering 2024, 11(9), 870; https://doi.org/10.3390/bioengineering11090870 - 27 Aug 2024
Viewed by 774
Abstract
The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical [...] Read more.
The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical properties of biotechnological and biomedical interest and can be used as an alternative to plastics made from chemical synthesis (which are not environmentally friendly). The versatile metabolism of H. mediterranei makes the use of waste as a carbon source for cellular growth and PHA synthesis possible. In this work, cellular growth and the production and characterization of PHBV using two different types of confectionery waste were analyzed and compared with cellular growth and PHBV synthesis in a standard culture media with glucose of analytical grade as a carbon source. The PHBV granules produced were analyzed by TEM and the biopolymer was isolated and characterized by GC-MS, FTIR NMR, and DSC. The results reveal that H. mediterranei can use these two residues (R1 and R2) for pure PHBV production, achieving 0.256 and 0.983 g PHBV/L, respectively, which are among the highest yields so far described using for the first-time waste from the candy industry. Thus, a circular economy-based process has been designed to optimize the upscaling of PHBV production by using haloarchaea as cell factories and valorizing confectionery waste. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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14 pages, 2236 KiB  
Article
Polyhydroxyalkanoate Copolymer Production by Recombinant Ralstonia eutropha Strain 1F2 from Fructose or Carbon Dioxide as Sole Carbon Source
by Chih-Ting Wang, Ramamoorthi M Sivashankari, Yuki Miyahara and Takeharu Tsuge
Bioengineering 2024, 11(5), 455; https://doi.org/10.3390/bioengineering11050455 - 2 May 2024
Cited by 1 | Viewed by 1557
Abstract
Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO2 as its sole carbon source for this process. A PHA-negative mutant of [...] Read more.
Ralstonia eutropha strain H16 is a chemoautotrophic bacterium that oxidizes hydrogen and accumulates poly[(R)-3-hydroxybutyrate] [P(3HB)], a prominent polyhydroxyalkanoate (PHA), within its cell. R. eutropha utilizes fructose or CO2 as its sole carbon source for this process. A PHA-negative mutant of strain H16, known as R. eutropha strain PHB−4, cannot produce PHA. Strain 1F2, derived from strain PHB−4, is a leucine analog-resistant mutant. Remarkably, the recombinant 1F2 strain exhibits the capacity to synthesize 3HB-based PHA copolymers containing 3-hydroxyvalerate (3HV) and 3-hydroxy-4-methyvalerate (3H4MV) comonomer units from fructose or CO2. This ability is conferred by the expression of a broad substrate-specific PHA synthase and tolerance to feedback inhibition of branched amino acids. However, the total amount of comonomer units incorporated into PHA was up to around 5 mol%. In this study, strain 1F2 underwent genetic engineering to augment the comonomer supply incorporated into PHA. This enhancement involved several modifications, including the additional expression of the broad substrate-specific 3-ketothiolase gene (bktB), the heterologous expression of the 2-ketoacid decarboxylase gene (kivd), and the phenylacetaldehyde dehydrogenase gene (padA). Furthermore, the genome of strain 1F2 was altered through the deletion of the 3-hydroxyacyl-CoA dehydrogenase gene (hbdH). The introduction of bktB-kivd-padA resulted in increased 3HV incorporation, reaching 13.9 mol% from fructose and 6.4 mol% from CO2. Additionally, the hbdH deletion resulted in the production of PHA copolymers containing (S)-3-hydroxy-2-methylpropionate (3H2MP). Interestingly, hbdH deletion increased the weight-average molecular weight of the PHA to over 3.0 × 106 on fructose. Thus, it demonstrates the positive effects of hbdH deletion on the copolymer composition and molecular weight of PHA. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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13 pages, 1854 KiB  
Article
Characterization of Poly(3-hydroxybutyrate) (P3HB) from Alternative, Scalable (Waste) Feedstocks
by Rogerio Ramos de Sousa Junior, Fabiano Eduardo Marques Cezario, Leonardo Dalseno Antonino, Demetrio Jackson dos Santos and Maximilian Lackner
Bioengineering 2023, 10(12), 1382; https://doi.org/10.3390/bioengineering10121382 - 30 Nov 2023
Cited by 2 | Viewed by 1278
Abstract
Bioplastics hold significant promise in replacing conventional plastic materials, linked to various serious issues such as fossil resource consumption, microplastic formation, non-degradability, and limited end-of-life options. Among bioplastics, polyhydroxyalkanoates (PHA) emerge as an intriguing class, with poly(3-hydroxybutyrate) (P3HB) being the most utilized. The [...] Read more.
Bioplastics hold significant promise in replacing conventional plastic materials, linked to various serious issues such as fossil resource consumption, microplastic formation, non-degradability, and limited end-of-life options. Among bioplastics, polyhydroxyalkanoates (PHA) emerge as an intriguing class, with poly(3-hydroxybutyrate) (P3HB) being the most utilized. The extensive application of P3HB encounters a challenge due to its high production costs, prompting the investigation of sustainable alternatives, including the utilization of waste and new production routes involving CO2 and CH4. This study provides a valuable comparison of two P3HBs synthesized through distinct routes: one via cyanobacteria (Synechocystis sp. PCC 6714) for photoautotrophic production and the other via methanotrophic bacteria (Methylocystis sp. GB 25) for chemoautotrophic growth. This research evaluates the thermal and mechanical properties, including the aging effect over 21 days, demonstrating that both P3HBs are comparable, exhibiting physical properties similar to standard P3HBs. The results highlight the promising potential of P3HBs obtained through alternative routes as biomaterials, thereby contributing to the transition toward more sustainable alternatives to fossil polymers. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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12 pages, 2546 KiB  
Article
Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO2 via pH-Stat Jar Cultivation of an Engineered Hydrogen-Oxidizing Bacterium Cupriavidus necator
by Kenji Tanaka, Izumi Orita and Toshiaki Fukui
Bioengineering 2023, 10(11), 1304; https://doi.org/10.3390/bioengineering10111304 - 10 Nov 2023
Cited by 2 | Viewed by 1527
Abstract
The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a biodegradable plastic characterized by high flexibility, softness, a wide process window, and marine biodegradability. PHBHHx is usually produced from structurally related carbon sources, such as vegetable oils or fatty acids, but not [...] Read more.
The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is a biodegradable plastic characterized by high flexibility, softness, a wide process window, and marine biodegradability. PHBHHx is usually produced from structurally related carbon sources, such as vegetable oils or fatty acids, but not from inexpensive carbon sources such as sugars. In previous studies, we demonstrated that engineered strains of a hydrogen-oxidizing bacterium, Cupriavidus necator, synthesized PHBHHx with a high cellular content not only from sugars but also from CO2 as the sole carbon source in the flask culture. In this study, the highly efficient production of PHBHHx from CO2 was investigated via pH-stat jar cultivation of recombinant C. necator strains while feeding the substrate gas mixture (H2/O2/CO2 = 80:10:10 v/v%) to a complete mineral medium in a recycled-gas, closed-circuit culture system. As a result, the dry cell mass and PHBHHx concentration with the strain MF01/pBPP-ccrMeJAc-emd reached up to 59.62 ± 3.18 g·L−1 and 49.31 ± 3.14 g·L−1, respectively, after 216 h of jar cultivation with limited addition of ammonia and phosphate solutions. The 3HHx composition was close to 10 mol%, which is suitable for practical applications. It is expected that the autotrophic cultivation of the recombinant C. necator can be feasible for the mass production of PHBHHx from CO2. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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12 pages, 1528 KiB  
Article
Co-Production of Poly(3-hydroxybutyrate) and Gluconic Acid from Glucose by Halomonas elongata
by Tânia Leandro, M. Conceição Oliveira, M. Manuela R. da Fonseca and M. Teresa Cesário
Bioengineering 2023, 10(6), 643; https://doi.org/10.3390/bioengineering10060643 - 25 May 2023
Cited by 4 | Viewed by 1628
Abstract
Polyhydroxyalkanoates (PHA) are biopolyesters regarded as an attractive alternative to petroleum-derived plastics. Nitrogen limitation and phosphate limitation in glucose cultivations were evaluated for poly(3-hydroxybutyrate) (P(3HB)) production by Halomonas elongata 1H9T, a moderate halophilic strain. Co-production of P(3HB) and gluconic acid was [...] Read more.
Polyhydroxyalkanoates (PHA) are biopolyesters regarded as an attractive alternative to petroleum-derived plastics. Nitrogen limitation and phosphate limitation in glucose cultivations were evaluated for poly(3-hydroxybutyrate) (P(3HB)) production by Halomonas elongata 1H9T, a moderate halophilic strain. Co-production of P(3HB) and gluconic acid was observed in fed-batch glucose cultivations under nitrogen limiting conditions. A maximum P(3HB) accumulation of 53.0% (w/w) and a maximum co-production of 133 g/L of gluconic acid were attained. Fed-batch glucose cultivation under phosphate limiting conditions resulted in a P(3HB) accumulation of only 33.3% (w/w) and no gluconic acid production. As gluconic acid is a valuable organic acid with extensive applications in several industries, this work presents an interesting approach for the future development of an industrial process aiming at the co-production of an intracellular biopolymer, P(3HB), and a value-added extracellular product, gluconic acid. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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11 pages, 1596 KiB  
Article
Quantification of the Monomer Compositions of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and Poly(3-hydroxyvalerate) by Alkaline Hydrolysis and Using High-Performance Liquid Chromatography
by Kyo Saito, M. Venkateswar Reddy, Omprakash Sarkar, A. Naresh Kumar, DuBok Choi and Young-Cheol Chang
Bioengineering 2023, 10(5), 618; https://doi.org/10.3390/bioengineering10050618 - 20 May 2023
Cited by 3 | Viewed by 2109
Abstract
With the growing interest in bioplastics, there is an urgent need to develop rapid analysis methods linked to production technology development. This study focused on the production of a commercially non-available homopolymer, poly(3-hydroxyvalerate) (P(3HV)), and a commercially available copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), [...] Read more.
With the growing interest in bioplastics, there is an urgent need to develop rapid analysis methods linked to production technology development. This study focused on the production of a commercially non-available homopolymer, poly(3-hydroxyvalerate) (P(3HV)), and a commercially available copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), through fermentation using two different bacterial strains. The bacteria Chromobacterium violaceum and Bacillus sp. CYR1 were used to produce P(3HV) and P(3HB-co-3HV), respectively. The bacterium Bacillus sp. CYR1 produced 415 mg/L of P(3HB-co-3HV) when incubated with acetic acid and valeric acid as the carbon sources, whereas the bacterium C. violaceum produced 0.198 g of P(3HV)/g dry biomass when incubated with sodium valerate as the carbon source. Additionally, we developed a fast, simple, and inexpensive method to quantify P(3HV) and P(3HB-co-3HV) using high-performance liquid chromatography (HPLC). As the alkaline decomposition of P(3HB-co-3HV) releases 2-butenoic acid (2BE) and 2-pentenoic acid (2PE), we were able to determine the concentration using HPLC. Moreover, calibration curves were prepared using standard 2BE and 2PE, along with sample 2BE and 2PE produced by the alkaline decomposition of poly(3-hydroxybutyrate) and P(3HV), respectively. Finally, the HPLC results obtained by our new method were compared using gas chromatography (GC) analysis. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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Review

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16 pages, 1005 KiB  
Review
Microbial PolyHydroxyAlkanoate (PHA) Biopolymers—Intrinsically Natural
by Anindya Mukherjee and Martin Koller
Bioengineering 2023, 10(7), 855; https://doi.org/10.3390/bioengineering10070855 - 19 Jul 2023
Cited by 15 | Viewed by 5408
Abstract
Global pollution from fossil plastics is one of the top environmental threats of our time. At their end-of-life phase, fossil plastics, through recycling, incineration, and disposal result in microplastic formation, elevated atmospheric CO2 levels, and the pollution of terrestrial and aquatic environments. [...] Read more.
Global pollution from fossil plastics is one of the top environmental threats of our time. At their end-of-life phase, fossil plastics, through recycling, incineration, and disposal result in microplastic formation, elevated atmospheric CO2 levels, and the pollution of terrestrial and aquatic environments. Current regional, national, and global regulations are centered around banning plastic production and use and/or increasing recycling while ignoring efforts to rapidly replace fossil plastics through the use of alternatives, including those that occur in nature. In particular, this review demonstrates how microbial polyhydroxyalkanoates (PHAs), a class of intrinsically natural polymers, can successfully remedy the fossil and persistent plastic dilemma. PHAs are bio-based, biosynthesized, biocompatible, and biodegradable, and thus, domestically and industrially compostable. Therefore, they are an ideal replacement for the fossil plastics pollution dilemma, providing us with the benefits of fossil plastics and meeting all the requirements of a truly circular economy. PHA biopolyesters are natural and green materials in all stages of their life cycle. This review elaborates how the production, consumption, and end-of-life profile of PHAs are embedded in the current and topical, 12 Principles of Green Chemistry, which constitute the basis for sustainable product manufacturing. The time is right for a paradigm shift in plastic manufacturing, use, and disposal. Humankind needs alternatives to fossil plastics, which, as recalcitrant xenobiotics, contribute to the increasing deterioration of our planet. Natural PHA biopolyesters represent that paradigm shift. Full article
(This article belongs to the Special Issue Advances in Polyhydroxyalkanoate (PHA) Production, 4th Edition)
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