Gas supply apparatus using rotational motion of shaking incubator for flask culture of aerobic microorganisms.

Shake flask cultivation, a cornerstone in bioprocess research encounters limitations in supplying sufficient oxygen and exchanging gases, restricting its accuracy in assessing microbial growth and metabolic activity. In this communication, we introduce an innovative gas supply apparatus that harnesses the rotational motion of a shaking incubator to facilitate continuous air delivery, effectively overcoming these limitations. We measured the mass transfer coefficient (kLa) and conducted batch cultures of Corynebacterium glutamicum H36LsGAD using various working volumes to assess its performance. Results demonstrated that the gas supply apparatus significantly outperforms conventional silicone stoppers regarding oxygen delivery, with kLa values of 2531.7 h-1 compared to 20.25 h-1 at 230 rpm. Moreover, in batch cultures, the gas supply apparatus enabled substantial improvements in microbial growth, maintaining exponential growth even at larger working volumes. Compared to the existing system, an increase in final cell mass by a factor of 3.4-fold was observed when utilizing 20% of the flask's volume, and a remarkable 9-fold increase was achieved when using 60%. Furthermore, the gas supply apparatus ensured consistent oxygen supply and efficient gas exchange within the flask, overcoming challenges associated with low working volumes. This approach offers a simple yet effective solution to enhance gas transfer in shake flask cultivation, bridging the gap between laboratory-scale experiments and industrial fermenters. Its broad applicability holds promise for advancing research in bioprocess optimization and scale-up endeavors.

Microbial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), from lab to the shelf: A review.

Polyhydroxyalkanoates (PHAs) are natural biopolyesters produced by microorganisms that represent one of the most promising candidates for the replacement of conventional plastics due to their complete biodegradability and advantageous material properties which can be modulated by varying their monomer composition. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has received particular research attention because it can be synthesized based on the same microbial platform developed for poly(3-hydroxybutyrate) [P(3HB)] without much modification, with as high productivity as P(3HB). It also offers more useful mechanical and thermal properties than P(3HB), which broaden its application as a biocompatible and biodegradable polyester. However, a significant commercial disadvantage of P(3HB-co-3HV) is its rather high production cost, thus many studies have investigated the economical synthesis of P(3HB-co-3HV) from structurally related and unrelated carbon sources in both wild-type and recombinant microbial strains. A large number of metabolic engineering strategies have also been proposed to tune the monomer composition of P(3HB-co-3HV) and thus its material properties. In this review, recent metabolic engineering strategies designed for enhanced production of P(3HB-co-3HV) are discussed, along with their current status, limitations, and future perspectives.

ß-Ketoadipic acid production from poly(ethylene terephthalate) waste via chemobiological upcycling.

The upcycling of poly(ethylene terephthalate) (PET) waste can simultaneously produce value-added chemicals and reduce the growing environmental impact of plastic waste. In this study, we designed a chemobiological system to convert terephthalic acid (TPA), an aromatic monomer of PET, to ß-ketoadipic acid (ßKA), a C6 keto-diacid that functions as a building block for nylon-6,6 analogs. Using microwave-assisted hydrolysis in a neutral aqueous system, PET was converted to TPA with Amberlyst-15, a conventional catalyst with high conversion efficiency and reusability. The bioconversion process of TPA into ßKA used a recombinant Escherichia coli ßKA expressing two conversion modules for TPA degradation (tphAabc and tphB) and ßKA synthesis (aroY, catABC, and pcaD). To improve bioconversion, the formation of acetic acid, a deleterious factor for TPA conversion in flask cultivation, was efficiently regulated by deleting the poxB gene along with operating the bioreactor to supply oxygen. By applying two-stage fermentation consisting of the growth phase in pH 7 followed by the production phase in pH 5.5, a total of 13.61 mM ßKA was successfully produced with 96% conversion efficiency. This efficient chemobiological PET upcycling system provides a promising approach for the circular economy to acquire various chemicals from PET waste.

Customized valorization of waste streams by Pseudomonas putida: State-of-the-art, challenges, and future trends.

Preventing catastrophic climate events warrants prompt action to delay global warming, which threatens health and food security. In this context, waste management using engineered microbes has emerged as a long-term eco-friendly solution for addressing the global climate crisis and transitioning to clean energy. Notably, Pseudomonas putida can valorize industry-derived synthetic wastes including plastics, oils, food, and agricultural waste into products of interest, and it has been extensively explored for establishing a fully circular bioeconomy through the conversion of waste into bio-based products, including platform chemicals (e.g., cis,cis-muconic and adipic acid) and biopolymers (e.g., medium-chain length polyhydroxyalkanoate). However, the efficiency of waste pretreatment technologies, capability of microbial cell factories, and practicability of synthetic biology tools remain low, posing a challenge to the industrial application of P. putida. The present review discusses the state-of-the-art, challenges, and future prospects for divergent biosynthesis of versatile products from waste-derived feedstocks using P. putida.

Recent advances in microbial production of diamines, aminocarboxylic acids, and diacids as potential platform chemicals and bio-based polyamides monomers.

Recently, bio-based manufacturing processes of value-added platform chemicals and polymers in biorefineries using renewable resources have extensively been developed for sustainable and carbon dioxide (CO2) neutral-based industry. Among them, bio-based diamines, aminocarboxylic acids, and diacids have been used as monomers for the synthesis of polyamides having different carbon numbers and ubiquitous and versatile industrial polymers and also as precursors for further chemical and biological processes to afford valuable chemicals. Until now, these platform bio-chemicals have successfully been produced by biorefinery processes employing enzymes and/or microbial host strains as main catalysts. In this review, we discuss recent advances in bio-based production of diamines, aminocarboxylic acids, and diacids, which has been developed and improved by systems metabolic engineering strategies of microbial consortia and optimization of microbial conversion processes including whole cell bioconversion and direct fermentative production.

Bioupgrading of the aqueous phase of pyrolysis oil from lignocellulosic biomass: a platform for renewable chemicals and fuels from the whole fraction of biomass.

Pyrolysis, a thermal decomposition without oxygen, is a promising technology for transportable liquids from whole fractions of lignocellulosic biomass. However, due to the hydrophilic products of pyrolysis, the liquid oils have undesirable physicochemical characteristics, thus requiring an additional upgrading process. Biological upgrading methods could address the drawbacks of pyrolysis by utilizing various hydrophilic compounds as carbon sources under mild conditions with low carbon footprints. Versatile chemicals, such as lipids, ethanol, and organic acids, could be produced through microbial assimilation of anhydrous sugars, organic acids, aldehydes, and phenolics in the hydrophilic fractions. The presence of various toxic compounds and the complex composition of the aqueous phase are the main challenges. In this review, the potential of bioconversion routes for upgrading the aqueous phase of pyrolysis oil is investigated with critical challenges and perspectives.

Production of γ-Aminobutyrate (GABA) in Recombinant Corynebacterium glutamicum by Expression of Glutamate Decarboxylase Active at Neutral pH.

γ-Aminobutyrate (GABA) is an important chemical by itself and can be further used for the production of monomer used for the synthesis of biodegradable polyamides. Until now, GABA production usingCorynebacterium glutamicum harboring glutamate decarboxylases (GADs) has been limited due to the discrepancy between optimal pH for GAD activity (pH 4.0) and cell growth (pH 7.0). In this study, we developed recombinant C. glutamicum strains expressing mutated GAD from Escherichia coli (EcGADmut) and GADs from Lactococcus lactis CICC20209 (LlGAD) and Lactobacillus senmaizukei (LsGAD), all of which showed enhanced pH stability and adaptability at a pH of approximately 7.0. In shake flask cultivations, the GABA productions of C. glutamicum H36EcGADmut, C. glutamicum H36LsGAD, and C. glutamicum H36LlGAD were examined at pH 5.0, 6.0, and 7.0, respectively. Finally, C. glutamicum H36EcGADmut (40.3 and 39.3 g L-1), H36LlGAD (42.5 and 41.1 g L-1), and H36LsGAD (41.6 and 40.2 g L-1) produced improved GABA titers and yields in batch fermentation at pH 6.0 and pH 7.0, respectively, from 100 g L-1 glucose. The recombinant strains developed in this study could be used for the establishment of sustainable direct fermentative GABA production from renewable resources under mild culture conditions, thus increasing the availability of various GADs.

Valorization of lignocellulosic biomass for polyhydroxyalkanoate production: Status and perspectives.

With the increasing concerns regarding climate, energy, and plastic crises, bio-based production of biodegradable polymers has become a dire necessity. Significant progress has been made in biotechnology for the production of biodegradable polymers from renewable resources to achieve the goal of zero plastic waste and a net-zero carbon bioeconomy. In this review, an overview of polyhydroxyalkanoate (PHA) production from lignocellulosic biomass (LCB) was presented. Having established LCB-based biorefinery with proper pretreatment techniques, various PHAs could be produced from LCB-derived sugars, hydrolysates, and/or aromatic mixtures employing microorganisms. This provides a clue for addressing the current environmental crises because "biodegradable polymers" could be produced from one of the most abundant resources that are renewable and sustainable in a "carbon-neutral process". Furthermore, the potential future of LCB-to-non-natural PHA production was discussed with particular reference to non-natural PHA biosynthesis methods and LCB-derived aromatic mixture biofunnelling systems.

Microbial production of 2-pyrone-4,6-dicarboxylic acid from lignin derivatives in an engineered Pseudomonas putida and its application for the synthesis of bio-based polyester.

Lignin valorization depends on microbial upcycling of various aromatic compounds in the form of a complex mixture, including p-coumaric acid and ferulic acid. In this study, an engineered Pseudomonas putida strain utilizing lignin-derived monomeric compounds via biological funneling was developed to produce 2-pyrone-4,6-dicarboxylic acid (PDC), which has been considered a promising building block for bioplastics. The biosynthetic pathway for PDC production was established by introducing the heterologous ligABC genes under the promoter Ptac in a strain lacking pcaGH genes to accumulate a precursor of PDC, i.e., protocatechuic acid. Based on the culture optimization, fed-batch fermentation of the final strain resulted in 22.7 g/L PDC with a molar yield of 1.0 mol/mol and productivity of 0.21 g/L/h. Subsequent purification of PDC at high purity was successfully implemented, which was consequently applied for the novel polyester.

Efficient Production of Naringin Acetate with Different Acyl Donors via Enzymatic Transesterification by Lipases.

Naringin, one of the citrus flavonoids and known as a natural antioxidant, has limited bioavailability owing to its low stability and solubility. However, naringin esters formed via acylation have recently been reported to possess improved physical and chemical properties. The development of these compounds has a great potential in the food, cosmetic and pharmaceutical industries, but low conversion and productivity are barriers to industrial applications. This study aimed to improve the conversion of naringin acetate, which is formed via the enzymatic reaction between naringin and an acyl donor. An optimal reaction condition was determined by evaluating the effect of various variables (enzyme type, enzyme concentration, acyl donor, molar ratio of reactants, reaction temperature, and solvent) on the synthesis of naringin acetate. The optimal condition was as follows: 3 g/L of Lipozyme TL IM, molar ratio of 1:5 (naringin:acyl donor), reaction temperature of 40 °C, and acetonitrile as the reaction solvent. Under this condition, the maximum conversion to naringin acetate from acetic anhydride and vinyl acetate was achieved at approximately 98.5% (8 h) and 97.5% (24 h), respectively. Compared to the previously reported values, a high conversion was achieved within a short time, confirming the commercial potential of the process.

Consolidated microbial production of four-, five-, and six-carbon organic acids from crop residues: Current status and perspectives.

The production of platform organic acids has been heavily dependent on petroleum-based industries. However, petrochemical-based industries that cannot guarantee a virtuous cycle of carbons released during various processes are now facing obsolescence because of the depletion of finite fossil fuel reserves and associated environmental pollutions. Thus, the transition into a circular economy in terms of the carbon footprint has been evaluated with the development of efficient microbial cell factories using renewable feedstocks. Herein, the recent progress on bio-based production of organic acids with four-, five-, and six-carbon backbones, including butyric acid and 3-hydroxybutyric acid (C4), 5-aminolevulinic acid and citramalic acid (C5), and hexanoic acid (C6), is discussed. Then, the current research on the production of C4-C6 organic acids is illustrated to suggest future directions for developing crop-residue based consolidated bioprocessing of C4-C6 organic acids using host strains with tailor-made capabilities.

Improved Productivity of Naringin Oleate with Flavonoid and Fatty Acid by Efficient Enzymatic Esterification.

Naringin is a flavonoid found in citrus fruits. It exhibits biological activities, such as anticancer and antioxidant effects, but it suffers from low solubility and low stability in lipophilic systems. These drawbacks lead to difficulties in the commercial application of naringin, but they can be overcome through esterification. In this study, naringin oleate was synthesized by enzymatic esterification and optimal conditions for the reaction were investigated. Experiments were conducted focusing on the following parameters: enzyme type, enzyme concentration, molar ratio of naringin to oleic acid, reaction temperature, and reaction solvent. We further confirmed the degree of esterification based on the difference in the initial and the final naringin concentrations. A conversion of 93.10% was obtained under optimized conditions (Lipozyme TL IM 10 g/L, molar ratio 1:20, reaction temperature 40 °C, acetonitrile as solvent, and 48 h reaction time). Thus, naringin oleate, a high value-added material that overcomes the low hydrophobicity of naringin and enhances its performance, was obtained through esterification of naringin using oleic acid. This study presented a method for the efficient enzymatic synthesis that could ensure high conversion within a shorter reaction time compared with that required in previously reported methods.

Microbial cell factories for the production of three-carbon backbone organic acids from agro-industrial wastes.

At present, mass production of basic and valuable commodities is dependent on linear petroleum-based industries, which ultimately makes the depletion of finite natural reserves and accumulation of non-biodegradable and hazardous wastes. Therefore, an ecofriendly and sustainable solution should be established for a circular economy where infinite resources, such as agro-industrial wastes, are fully utilized as substrates in the production of target value-added chemicals. Hereby, recent advances in metabolic engineering strategies and techniques used in the development of microbial cell factories for enhanced production of three-carbon platform chemicals such as lactic acid, propionic acid, and 3-hydroxypropionic acid are discussed. Further developments and future perspectives in the production of these organic acids from agro-industrial wastes from the dairy, sugar, and biodiesel industries are also highlighted to demonstrate the importance of waste-based biorefineries for organic acid production.

Recent advances in the microbial production of C4 alcohols by metabolically engineered microorganisms.

BACKGROUND: The heavy global dependence on petroleum-based industries has led to serious environmental problems, including climate change and global warming. As a result, there have been calls for a paradigm shift towards the use of biorefineries, which employ natural and engineered microorganisms that can utilize various carbon sources from renewable resources as host strains for the carbon-neutral production of target products. PURPOSE AND SCOPE: C4 alcohols are versatile chemicals that can be used directly as biofuels and bulk chemicals and in the production of value-added materials such as plastics, cosmetics, and pharmaceuticals. C4 alcohols can be effectively produced by microorganisms using DCEO biotechnology (tools to design, construct, evaluate, and optimize) and metabolic engineering strategies. SUMMARY OF NEW SYNTHESIS AND CONCLUSIONS: In this review, we summarize the production strategies and various synthetic tools available for the production of C4 alcohols and discuss the potential development of microbial cell factories, including the optimization of fermentation processes, that offer cost competitiveness and potential industrial commercialization.

A shortcut to carbon-neutral bioplastic production: Recent advances in microbial production of polyhydroxyalkanoates from C1 resources.

Since the 20th century, plastics that are widely being used in general life and industries are causing enormous plastic waste problems since improperly discarded plastics barely degrade and decompose. Thus, the demand for polyhydroxyalkanoates (PHAs), biodegradable polymers with material properties similar to conventional petroleum-based plastics, has been increased so far. The microbial production of PHAs is an environment-friendly solution for the current plastic crisis, however, the carbon sources for the microbial PHA production is a crucial factor to be considered in terms of carbon-neutrality. One­carbon (C1) resources, such as methane, carbon monoxide, and carbon dioxide, are greenhouse gases and are abundantly found in nature and industry. C1 resources as the carbon sources for PHA production have a completely closed carbon loop with much advances; i) fast carbon circulation with direct bioconversion process and ii) simple fermentation procedure without sterilization as non-preferable nutrients. This review discusses the biosynthesis of PHAs based on C1 resource utilization by wild-type and metabolically engineered microbial host strains via biorefinery processes.

Chemo-Biological Upcycling of Poly(ethylene terephthalate) to Multifunctional Coating Materials.

Chemo-biological upcycling of poly(ethylene terephthalate) (PET) developed in this study includes the following key steps: chemo-enzymatic PET depolymerization, biotransformation of terephthalic acid (TPA) into catechol, and its application as a coating agent. Monomeric units were first produced through PET glycolysis into bis(2-hydroxyethyl) terephthalate (BHET), mono(2-hydroxyethyl) terephthalate (MHET), and PET oligomers, and enzymatic hydrolysis of these glycolyzed products using Bacillus subtilis esterase (Bs2Est). Bs2Est efficiently hydrolyzed glycolyzed products into TPA as a key enzyme for chemo-enzymatic depolymerization. Furthermore, catechol solution produced from TPA via a whole-cell biotransformation (Escherichia coli) could be directly used for functional coating on various substrates after simple cell removal from the culture medium without further purification and water-evaporation. This work demonstrates a proof-of-concept of a PET upcycling strategy via a combination of chemo-biological conversion of PET waste into multifunctional coating materials.

Chemoautotroph Cupriavidus necator as a potential game-changer for global warming and plastic waste problem: A review.

Cupriavidus necator, a versatile microorganism found in both soil and water, can have both heterotrophic and lithoautotrophic metabolisms depending on environmental conditions. C. necator has been extensively examined for producing Polyhydroxyalkanoates (PHAs), the promising polyester alternatives to petroleum-based synthetic polymers because it has a superior ability for accumulating a considerable amount of PHAs from renewable resources. The development of metabolically engineered C. necator strains has led to their application for synthesizing biopolymers, biofuels and biochemicals such as ethanol, isobutanol and higher alcohols. Bio-based processes of recombinant C. necator have made much progress in production of these high-value products from biomass wastes, plastic wastes and even waste gases. In this review, we discuss the potential of C. necator as promising platform host strains that provide a great opportunity for developing a waste-based circular bioeconomy.

Improving the organic solvent resistance of lipase a from Bacillus subtilis in water-ethanol solvent through rational surface engineering.

Given that lipase is an enzyme applicable in various industrial fields and water-miscible organic solvents are important reaction media for developing industrial-scale biocatalysis, a structure-based strategy was explored to stabilize lipase A from Bacillus subtilis in a water-ethanol cosolvent. Site-directed mutagenesis of ethanol-interacting sites resulted in 4 mutants, i.e., Ser16Gly, Ala38Gly, Ala38Thr, and Leu108Asn, which were stable in 50% ethanol and had up to 1.8-fold higher stability than the wild-type. In addition, Leu108Asn was more thermostable at 45 °C than the wild type. The results discussed in this study not only provide insights into strategies for enzyme engineering to improve organic solvent resistance but also suggest perspectives on pioneering routes for constructing enzyme-based biorefineries to produce value-added fuels and chemicals.

Rapid analysis of polyhydroxyalkanoate contents and its monomer compositions by pyrolysis-gas chromatography combined with mass spectrometry (Py-GC/MS).

Here, we report an analysis method for determining PHA (polyhydroxyalkanoates) contents and their monomer composition in microbial cells based on pyrolysis gas chromatography combined with mass spectrometry (Py-GC/MS). Various kinds of microbial cells accumulating different PHA contents and monomer compositions were prepared through the cultivation of Ralstonia eutropha and recombinant Escherichia coli. Py-GC/MS could analyse these samples in a short time without complicated pretreatment steps. Characteristic peaks such as 2-butenoic acid, 2-pentenoic acid, and hexadecanoic acid regarding PHA compositions and cell components were identified. Considering constituents of cells and ratios of peak areas of dehydrated monomers to hexadecanoic acid, a simple equation for estimation of PHA contents in microbial cells was derived. Also, monomer compositions of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in R. eutropha could be successfully determined based on peak area of 2-butenoic acid and 2-pentenoic acid of Py-GC/MS, which are the corresponding species of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) in PHBV. Correlation of results between GC-FID and Py-GC/MS could be fitted very well. This method shows similar results for the samples obtained from same experimental conditions, allowing rapid and reliable analysis. Py-GC/MS can be a promising tool to rapidly screen PHA-positive strains based on polymer contents along with monomer compositions.