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Cupriavidus necator is a facultative chemolithoautotrophic bacterium able to convert carbon dioxide into poly-3-hydroxybutyrate. This is highly promising as the conversion process allows the production of sustainable and biodegradable plastics. Poly-3-hydroxybutyrate accumulation is known to be induced by nutrient starvation, but information regarding the optimal stress conditions controlling the process is still heterogeneous and fragmentary. This study presents a comprehensive comparison of the effects of nutrient stress conditions, namely nitrogen, hydrogen, phosphorus, oxygen, and magnesium deprivation, on poly-3-hydroxybutyrate accumulation in C. necator DSM545. Nitrogen starvation exhibited the highest poly-3-hydroxybutyrate accumulation, achieving 54% of total cell dry weight after four days of nutrient stress, and a carbon conversion efficiency of 85%. The gas consumption patterns indicated flexible physiological mechanisms underlying polymer accumulation and depolymerization. These findings provide insights into strategies for efficient carbon conversion into bioplastics, and highlight the key role of C. necator for future industrial-scale applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Recycling carbon-rich wastes into high-value platform chemicals through biological processes provides a sustainable alternative to petrochemicals. Cupriavidus necator, known for converting carbon dioxide (CO 2 ) into polyhydroxyalkanoates (PHA) was studied for the first time using biogas streams as the sole carbon source. The bacterium efficiently consumed biogenic CO 2 from raw biogas with methane at high concentrations (50%) proving non-toxic. Continuous addition of H 2 and O 2 enabled growth trends comparable to glucose-based heterotrophic growth. Transcriptomic analysis revealed CO 2 -adaptated cultures exhibited upregulation of hydrogenases and Calvin cycle enzymes, as well as genes related to electron transport, nutrient uptake, and glyoxylate cycle. Non-adapted samples displayed activation of stress response mechanisms, suggesting potential lags in large-scale processes. These findings showcase the setting of growth parameters for a pioneering biological biogas upgrading strategy, emphasizing the importance of inoculum adaptation for autotrophic growth and providing potential targets for genetic engineering to push PHA yields in future applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Single-use bioplastic items pose new challenges for a circular plastics economy as they require different processing than petroleum-based plastics items. Microbial and enzymatic recycling approaches could address some of the pitfalls created by the influx of bioplastic waste. In this study, the recombinant expression of a cutinase-like-enzyme (CLE1) was improved in the yeast Saccharomyces cerevisiae to efficiently hydrolyse several commercial single-use bioplastic items constituting blends of poly(lactic acid), poly(1,4-butylene adipate-co-terephthalate), poly(butylene succinate) and mineral fillers. The hydrolysis process was optimised in controlled bioreactor configurations to deliver substantial monomer concentrations and, ultimately, 29 to 78% weight loss. Product inhibition studies and molecular docking provided insights into potential bottlenecks of the enzymatic hydrolysis process, while FT-IR analysis showed the preferential breakdown of specific polymers in blended commercial bioplastic items. This work constitutes a step towards implementing enzymatic hydrolysis as a circular economy approach for the valorisation of end-of-life single-use bioplastic items., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO 2 and H 2 into PHAs, offer an additional strategy. Typically, the preferred source for CO 2 and H 2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H 2 and CO 2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H 2 (26.7 %) and CO 2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H 2 and CO 2 from fruit waste to produce PHAs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The implementation of consolidated bioprocessing for converting starch to ethanol relies on a robust yeast that produces enough amylases for rapid starch hydrolysis. Furthermore, using low-cost substrates will assist with competitive ethanol prices and support a bioeconomy, especially in developing countries. This paper addresses both challenges with the expression of additional glucoamylase gene copies in an efficient amylolytic strain (Saccharomyces cerevisiae ER T12) derived from the industrial yeast, Ethanol Red™. Recombinant ER T12 was used as a host to increase ethanol productivity during raw starch fermentation; the ER T12.7 variant, selected from various transformants, displayed enhanced raw starch conversion and a 36% higher ethanol concentration than the parental strain after 120 h. Unripe rice, rice bran, potato waste and potato peels were evaluated as alternative starchy substrates to test ER T12.7's fermenting ability. ER T12.7 produced high ethanol yields at significantly improved ethanol productivity, key criteria for its industrial application., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rosemary Cripwell reports financial support, equipment, drugs, or supplies, and travel were provided by National Research Foundation. Favaro Lorenzo reports travel was provided by Government of Italy Ministry of Foreign Affairs and International Cooperation. Willem H. van Zy reports financial support, equipment, drugs, or supplies, and travel were provided by National Research Foundation. Rosemary Cripwell and Willem H. van Zy has patent RECOMBINANT YEAST AND USE THEREOF issued to Assignee., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Polylactic acid (PLA) is a major contributor to the global bioplastic production capacity. However, post-consumer PLA waste is not fully degraded during non-optimal traditional organic waste treatment processes and can persist in nature for many years. Efficient enzymatic hydrolysis of PLA would contribute to cleaner, more energy-efficient, environmentally friendly waste management processes. However, high costs and a lack of effective enzyme producers curtail the large-scale application of such enzymatic systems. This study reports the recombinant expression of a fungal cutinase-like enzyme (CLE1) in the yeast Saccharomyces cerevisiae, which produced a crude supernatant that efficiently hydrolyses different types of PLA materials. The codon-optimised Y294[CLEns] strain delivered the best enzyme production and hydrolysis capabilities, releasing up to 9.44 g/L lactic acid from 10 g/L PLA films with more than 40% loss in film weight. This work highlights the potential of fungal hosts producing PLA hydrolases for future commercial applications in PLA recycling., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M Viljoen-Bloom reports financial support was provided by National Research Foundation, South Africa. Results reported in this paper were included in an international patent application filed by Stellenbosch University and Padova University., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Starch-rich by-products could be efficiently exploited for polyhydroxyalkanoates (PHAs) production. Unfortunately, Cupriavidus necator DSM 545, one of the most efficient PHAs producers, is not able to grow on starch. In this study, a recombinant amylolytic strain of C. necator DSM 545 was developed for the one-step PHAs production from starchy residues, such as broken rice and purple sweet potato waste. The glucodextranase G1d from Arthrobacter globiformis I42 and the α-amylase amyZ from Zunongwangia profunda SM-A87 were co-expressed into C. necator DSM 545. The recombinant C. necator DSM 545 #11, selected for its promising hydrolytic activity, produced high biomass levels with noteworthy PHAs titers: 5.78 and 3.65 g/L from broken rice and purple sweet potato waste, respectively. This is the first report on the engineering of C. necator DSM 545 for efficient amylase production and paves the way to the one-step conversion of starchy waste into PHAs., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Amylolytic Saccharomyces cerevisiae derivatives of Ethanol Red™ Version 1 (ER T12) and M2n (M2n T1) were assessed through enzyme assays, hydrolysis trials, electron microscopy and fermentation studies using broken rice. The heterologous enzymes hydrolysed broken rice at a similar rate compared to commercial granular starch-hydrolysing enzyme cocktail. During the fermentation of 20% dw/v broken rice, the amylolytic strains converted rice starch to ethanol in a single step and yielded high ethanol titers. The best-performing strain (ER T12) produced 93% of the theoretical ethanol yield after 96 h of consolidated bioprocessing (CBP) fermentation at 32 °C. Furthermore, the addition of commercial enzyme cocktail (10% of the recommended dosage) in combination with ER T12 did not significantly improve the maximum ethanol concentration, confirming the superior ability of ER T12 to hydrolyse raw starch. The ER T12 strain was therefore identified as an ideal candidate for the CBP of starch-rich waste streams., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Industrial manufacturing of polyhydroxyalkanoates (PHAs) requires purification of PHAs granules from high-cell-density cultures. Cells are broken by homogenization and PHAs granules are cleansed and treated to obtain PHAs latexes. However, cell lysis releases large amounts of DNA which results in an increasing viscosity of the medium, hampering the following downstream steps. Drop in viscosity is generally achieved by costly procedures such as heat treatment or the supplementation of hypochlorite and commercially available nucleases. Searching for a cost-effective solution to this issue, a nuclease gene from Staphylococcus aureus has been integrated into two efficient PHAs-producing bacteria: Cupriavidus necator DSM 545 and Delftia acidovorans DSM 39. Staphylococcal nuclease has been proficiently expressed in both microbial hosts without affecting PHAs production. Moreover, the viscosity of the lysates of recombinant C. necator cells was greatly reduced, indicating that the engineered strain is expected to yield large reduction cost in PHAs downstream processing., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Carbon dioxide (CO 2 ) injection in anaerobic digestion has recently been proposed as an interesting possibility to boost methane (CH 4 ) recovery from sludge and organic waste by converting a greenhouse gas into a renewable resource. This research assessed the effects of exogenous CO 2 injection on performance and process stability of single-phase continuous anaerobic digesters. Two pilot scale reactors treating sewage sludge were operated for 130days. One reactor was periodically injected with CO 2 while the other acted as control. Two injection frequencies and injection devices were tested. The results indicated that CO 2 enrichment allowed an increase in CH 4 production of ca. 12%, with a CH 4 production rate of 371±100L/(kgVS fed ·d) and a CH 4 concentration of ca. 60% when dissolved CO 2 levels inside the test reactor were increased up to 1.9-fold. Results also indicated an improvement in process resilience to temporary overloads and no impacts on stability parameters., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This work describes the feasibility of using rice milling by-products as feedstock for bioethanol. Starch-rich residues (rice bran, broken, unripe and discolored rice) were individually fermented (20%w/v) through Consolidated Bioprocessing by two industrial engineered yeast secreting fungal amylases. Rice husk (20%w/v), mainly composed by lignocellulose, was pre-treated at 55°C with alkaline peroxide, saccharified through optimized dosages of commercial enzymes (Cellic® CTec2) and fermented by the recombinant strains. Finally, a blend of all the rice by-products, formulated as a mixture (20%w/v) according to their proportions at milling plants, were co-processed to ethanol by optimized pre-treatment, saccharification and fermentation by amylolytic strains. Fermenting efficiency for each by-product was high (above 88% of the theoretical) and further confirmed on the blend of residues (nearly 52g/L ethanol). These results demonstrated for the first time that the co-conversion of multiple waste streams is a promising option for second generation ethanol production., (Copyright © 2017 Elsevier Ltd. All rights reserved.)