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Fermentation Strategies for PHB Production in a Novel Membrane Bioreactor: Investigating Batch and Fed-Batch Operations

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Fungi and their natural products, like secondary metabolites, have gained a huge demand in the last decade due to their increasing applications in healthcare, environmental cleanup, and biotechnology-based industries. The fungi produce these secondary metabolites WSMs) during the different phases of their growth, which are categorized into terpenoids, alkaloids, polyketides, and non-ribosomal peptides. These SMs exhibit significant biological activity, which contributes to the formulation of novel pharmaceuticals, biopesticides, and environmental bioremediation agents. Nowadays, these fungal-derived SMs are widely used in food and beverages, for fermentation, preservatives, protein sources, and in dairy industries. In healthcare, it is being used as an antimicrobial, anticancer, anti-inflammatory, and immunosuppressive drug. The usage of modern tools of biotechnology can achieve an increase in demand for these SMs and large-scale production. The present review comprehensively analyses the diversity of fungal SMs along with their emerging applications in healthcare, agriculture, environmental sustainability, and nutraceuticals. Here, the authors have reviewed the recent advancements in genetic engineering, metabolic pathway manipulation, and synthetic biology to improve the production and yield of these SMs. Advancement in fermentation techniques, bioprocessing, and co-cultivation approaches for large-scale production of SMs. Investigators further highlighted the importance of omics technologies in understanding the regulation and biosynthesis of SMs, which offers an understanding of novel applications in drug discovery and sustainable agriculture. Finally, the authors have addressed the potential for genetic manipulation and biotechnological innovations for further exploitation of fungal SMs for commercial and environmental benefits.
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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.
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The utilization of waste cooking oil (WCO) or waste fish oil (WFO) as inexpensive carbon substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16 was investigated. Fed-batch cultivation mode in bioreactor was applied in this study. High cell dry weight (CDW) of 135.1 g/L, PHB content of 76.9 wt%, PHB productivity of 1.73 g/L/h, and PHB yield of 0.8 g/g were obtained from WCO. In the case of WFO, the CDW, PHB content, PHB productivity, and PHB yield were 114.8 g/L, 72.5 wt%, 1.73 g/L/h, and 0.92 g/g, respectively. The PHB productivity and yield obtained in the current study from WCO or WFO are among the highest reported so far for PHA production using oils as sole carbon substrate, suggesting that both WCO and WFO can be used as inexpensive carbon substrates for the production of PHA on an industrial scale.
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Over the past decade, formic acid and acetic acid have gained increasing attention as alternative feedstocks for poly-3-hydroxybutyrate (PHB) production as these potentially CO2-derived molecules are naturally assimilated by Cupriavidus necator. Both organic acids were individually evaluated in fed-batch fermentations at bioreactor scale. Acetic acid was revealed as the most promising carbon source yielding 42.3 g L⁻¹ PHB, whereas no significant amount of PHB was produced from formic acid. Hence, acetic acid was further used as the substrate during process intensification. Key performance characteristics, including process stability, PHB titer, and productivity were optimized by introducing NH4-acetate as the nitrogen source, extending the growth phase, and implementing a repeated fed-batch procedure, respectively. These advanced fermentation strategies resulted in the establishment of a stable fermentation process reaching 58.5 g L⁻¹ PHB, while doubling the productivity to 0.93 g L⁻¹ h⁻¹ PHB.
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Poly(3-hydroxybutyrate) (PHB) belongs to the family of polyhydroxyalkanoates, biopolymers used for agricultural, industrial, or even medical applications. However, scaling up the production is still an issue due to the myriad of parameters involved in the fermentation processes. The present work seeks, firstly, to scale up poly(3-hydroxybutyrate) (PHB) production by wild type C. necator ATCC 17697 from shaken flasks to a stirred-tank bioreactor with the optimized media and fructose as carbon source. The second purpose is to improve the production of PHB by applying both the batch and fed-batch fermentation strategies in comparison with previous works of wild type C. necator with fructose. Furthermore, thinking of biomedical applications, physicochemical, and cytotoxicity analyses of the produced biopolymer, are presented. Fed-batch fermentation with an exponential feeding strategy enabled us to achieve the highest values of PHB concentration and productivity, 25.7 g/l and 0.43 g/(l h), respectively. The PHB productivity was 3.3 and 7.2 times higher than the one in batch strategy and shaken flask cultures, respectively. DSC, FTIR, ¹H, and ¹³C NMR analysis led to determine that the biopolymer produced by C. necator ATCC 17697 has a molecular structure and characteristics in agreement with the commercial PHB. Additionally, the biopolymer does not induce cytotoxic effects on the NIH/3T3 cell culture. Due to the improved fermentation strategies, PHB concentration resulted in 40 % higher of the already reported one for wild type C. necator using other fed-batch modes and fructose as a carbon source. Thus the produced PHB could be attractive for biomedical applications, which generate a rising interest in polyhydroxyalkanoates during recent years.
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Even though extensive research has been directed toward optimization of the strain, media, and the process, yet there is limited knowledge on the performance and viability of large-scale PHB production lines exploiting cyanobacteria. In this study, the scale-up challenges associated with photosynthetic PHB production are listed. The high PHB producing cyanobacterial mutant, MT_a24, a randomly mutated strain of Synechocystis sp. PCC 6714, has been tested in pilot-scale trials for photosynthetic PHB production under non-sterile conditions. The MT_a24 obtained PHB content of 0.356 g L⁻¹ and 1.7 g L⁻¹ of glycogen from CO2 after 10 days of cultivation using a self-limiting media and non-optimized cultivation parameters. The results obtained here suggest that in order to achieve high PHB productivity values of the lab and to overcome the existing scalability issues reassessment of the optimized parameters needs to be performed during the pilot-scale trials.
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The biosynthesis of poly(3-hydroxybutyrate) (PHB) directly from carbon dioxide (CO2), is a sustainable alternative for non-renewable, petroleum-based polymer production. The conversion of CO2 implies a reduction of greenhouse gas emissions. Hydrogen oxidizing bacteria such as Cupriavidus necator have the ability to store PHB using CO2 as a carbon source, i.e., through an autotrophic conversion. In this study, a mathematical model based on mass balances was set up to describe autotrophic PHB production. The model takes into account the stoichiometry and kinetics of biomass growth and PHB formation as well as physical transfer from the gas phase to the liquid fermentation broth. The developed model was calibrated and validated based on independent experimental datasets from literature, obtained for C. necator. The obtained simulation results accurately described the dynamics of autotrophic biomass growth and PHB production. The effect of oxygen (O2) and/or nitrogen stress conditions, as well as of the gas mixture composition in terms of O2 and hydrogen (H2) was investigated through scenario analysis. As major outcome, a higher maximum PHB concentration was obtained under oxygen stress conditions compared to nitrogen stress conditions. At high O2 fractions in the gas mixture, which would result in H2 limitation before O2 limitation, PHB production can be increased by applying nitrogen stress. The effect of the reactor type was assessed through comparing a continuous stirred tank reactor (CSTR) with an air-lift fermentor. The developed model forms the basis for future design with minimum experimentation of suitable control strategy aiming at a high PHB production.
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In this study, poly(3-hydroxybutyrate) (PHB) production was investigated for the first time using high cell density cultivation of Methylocystis hirsuta from natural gas. Various carbon sources including acetate, ethanol and glucose were used in a loop bioreactor. Then, effects of magnesium and phosphorus contents on PHB production were investigated. Results revealed that the bacterial strain had the capability to grow on various substrates. Furthermore, the cell dry mass (CDM) reached to 4.5 g/L with a PHB content of 85% (w/w) using a ratio of 1:1 of methanol:ethanol. Deficiencies of magnesium and phosphorus were shown to play a key role to achieve high cell densities in bioreactors. In addition, PHB was produced in a bubble column bioreactor at optimum conditions, resulting in the production of 8 g/L of CDM with a PHB content of 73.4% (w/w).
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Current gas/liquid membrane contactors are classified into tubular hollow fiber contactors and tube-shell cross flow hollow fiber contactors. They are usually built with a closed and integrated structure, which reduces the maintainability of the contactor and makes the scaling-up of the contactor inconvenient. In this paper, a novel gas/liquid hollow fiber membrane contactor is proposed. It is consisted of many changeable and standard small contactors (elements), in which the fibers are randomly packed. These randomly packed small elements are then serially and orderly arranged to form the scaled up contactor for industrial applications. A two-dimensional predictive model is proposed to study the performance of the contactor, which is validated by air humidification experiments. The effects of inter-elements and intra-element flow maldistributions are investigated. Correlations are proposed to estimate the performance of the contactor from the parameters of the elements. It is found that for the contactors built with elements of high packing densities (0.5), the inter-elements effect is dominant for flow maldistribution, but for contactors built with elements of low packing densities (0.35), the collaborative effect of inter-elements and intra-element is dominant. It could maximally decrease the average air side Sherwood numbers by about 83%, with a pressure drop reduction of about 50%. The scaled up contactor has a comparable performance to the small elements when the elements are optimized, which shows the good scalability of this novel contactor.
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Implementation of biofuels as an alternative to fossil fuels has been established as an answer to climate change by limiting GHG emissions. Syngas fermentation has emerged as a promising process for the conversion of waste biomasses to valuable products with bioethanol being on the main focus. However, the bottleneck of the mass transfer of syngas compounds H2 and CO along with low production yields has set barriers to the development of an industrial scale plant. Recent research indicates that many different methodologies spring up in order to face this important challenge. The aim of this review is to assemble all these techniques applied in syngas fermentation, focusing on the different bioreactor configurations operated in continuous mode for the production of liquid and gas biofuels. This article also outlines the so far entrepreneurial initiatives and the progress made towards the commercialization of the process.
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The membrane-biofilm reactor (MBfR), sometimes known as the membrane-aerated biofilm reactor (MABR), is an emerging treatment technology based on gas-transferring membranes. The membranes typically supply a gaseous electron donor or acceptor substrate, such as oxygen, hydrogen, and methane. The substrate diffuses through the membrane to a biofilm naturally forming on the membrane outer surface. The complementary substrate (electron donor or acceptor) typically diffuses from the bulk liquid into the biofilm, making MBfR counter diffusional. This paper reviews the unique behavior of counter-diffusional biofilms and highlights recent research on the MBfR. Key advances include insights into the microbial community structure of MBfRs, applying the MBfR to novel contaminants, providing a better understanding of biofilm morphology and its effects on MBfR behavior, and the development of methane-based MBfR applications. These advances are likely to further the development of the MBfR for environmental applications, such as energy-efficient wastewater treatment and advanced water treatment.
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Biosynthesis of poly-3-hydroxybutyrate (PHB) by Methylocystis hirsuta from natural gas in two different media was studied for the first time. After selection of the suitable medium, the effect of two key parameters such as methane to air ratio and nitrogen content on the PHB production was determined in a bubble column bioreactor using a full factorial design. It was found that both of these factors had a significant effect on the PHB accumulation (42.5% w/w of cell dry weight). PHB production by M. hirsuta, unlike other methanotrophic bacteria, was found to be a growth associated metabolite. Subsequently, the PHB production was carried out in a forced-liquid vertical tubular loop bioreactor (VTLB) at optimum condition determined in the bubble column bioreactor. The PHB content of biomass was 51.6% w/w of cell dry weight (CDW) in the VTLB. These results indicated that the loop bioreactors specially are suitable candidates for PHB production from natural gas.
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Trickle-bed reactor (TBR), hollow fiber membrane reactor (HFR) and stirred tank reactor (STR) can be used in fermentation of sparingly soluble gasses such as CO and H(2) to produce biofuels and bio-based chemicals. Gas fermenting reactors must provide high mass transfer capabilities that match the kinetic requirements of the microorganisms used. The present study compared the volumetric mass transfer coefficient (K(tot)A/V(L)) of three reactor types; the TBR with 3mm and 6mm beads, five different modules of HFRs, and the STR. The analysis was performed using O(2) as the gaseous mass transfer agent. The non-porous polydimethylsiloxane (PDMS) HFR provided the highest K(tot)A/V(L) (1062h(-1)), followed by the TBR with 6mm beads (421h(-1)), and then the STR (114h(-1)). The mass transfer characteristics in each reactor were affected by agitation speed, and gas and liquid flow rates. Furthermore, issues regarding the comparison of mass transfer coefficients are discussed.
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Synthesis gas is readily obtained by gasifying coal, oil, biomass, or waste organics and represents an abundant, potentially inexpensive, feedstock for bioprocessing. The primary components of synthesis gas, carbon monoxide and hydrogen, can be converted into methane, organic acids, and alcohols via anaerobic fermentations. Bioconversion of synthesis gas is an attractive alternative to catalytic processing because the biological catalysts are highly specific and often more tolerant of sulfur contaminants than inorganic catalysts. However, because the aqueous solubilities of carbon monoxide and hydrogen are low, synthesis-gas fermentations are typically limited by the rate of gas-to-liquid mass transfer. Consequently, a major engineering challenge in commercial development of synthesis-gas fermentations is to provide sufficient gas mass transfer in an energy-efficient manner. This paper reviews recent progress in the development of synthesis-gas fermentations, with emphasis on efforts to increase the efficiency of gas mass transfer. Metabolic properties of several microbes able to ferment synthesis gas are described. Results of synthesis-gas fermentations conducted in various bioreactor configurations are summarized. Recent results showing enhancement of synthesis-gas fermentations using microbubble dispersions are presented, and studies of the mass-transfer and coalescence properties of microbubbles are described.
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In this study, the volumetric mass transfer coefficients (Ka) for CO were examined in a composite hollow fiber (CHF) membrane bioreactor. The mass transfer experiments were conducted at various inlet gas pressures (from 5 to 30psig (34.5-206.8kPa(g))) and recirculation flow rates (300, 600, 900, 1200 and 1500mL/min) through CHF module. The highest Ka value of 946.61/h was observed at a recirculation rate of 1500mL/min and at an inlet gas pressure of 30psig(206.8kPa(g)). The findings of this study confirm that the use of CHF membranes is effective and improves the efficiency CO mass transfer into the aqueous phase.
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Diffusion of the electron acceptor is the rate controlling step in virtually all biofilm reactors employed for aerobic wastewater treatment. The membrane-aerated biofilm reactor (MABR) is a technology that can deliver oxygen at high rates and transfer efficiencies, thereby enhancing the biofilm activity. This paper provides a comparative performance rate analysis of the MABR in terms of its application for carbonaceous pollutant removal, nitrification/denitrification and xenobiotic biotreatment. We also describe the mechanisms influencing process performance in the MABR and the inter-relationships between these factors. The challenges involved in scaling-up the process are discussed with recommendations for prioritization of research needs.
Production of Lactic Acid by Ralstonia Eutropha H16, a Gram-Negative Chemolithoautotroph, Using Tools of Synthetic Biology, MSc
  • A Holohan
A. Holohan, Production of Lactic Acid by Ralstonia Eutropha H16, a Gram-Negative Chemolithoautotroph, Using Tools of Synthetic Biology, MSc, Biomolecular and Biomedical Science, University College Dublin, 2021.
How to Scale-Out and Scale-Up Cell Therapy Production
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Salatul Islam Mozumder, Eveline Heleen DeWevera, I.P. Volcke, Poly(3-hydroxybutyrate) (PHB) production from CO 2 : model development and process optimization
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L.G. Md. Salatul Islam Mozumder, Eveline Heleen DeWevera, I.P. Volcke, Poly(3-hydroxybutyrate) (PHB) production from CO 2 : model development and process optimization, Biochem. Eng. J. vol. 98 (2015) 107-116.