Production of Polyhydroxybutyrate by halotolerant Halomonas cerina YK44 using sugarcane molasses and soybean flour in tap water.

As environmental pollution intensifies, the interest in bioplastics is growing. The bioplastic polyhydroxyalkanoates (PHAs), which are produced and degraded by microorganisms, have received considerable attention. However, the production cost of PHA is still high, and several ways to increase economy of PHA production have been studied. Therefore, as one way of solution, Halomonas species were screened and evaluated with cheap substrates such as molasses and soybean flour. Among tested strains, Halomonas cerina YK44 was selected and used for polyhydroxybutyrate (PHB) production with molasses and soybean flour together, whose combination was not evaluated well before, in tap water. The medium composition optimization showed maximum PHB production at 4 % sugarcane molasses, 2 % NaCl, 0.05 % soybean flour, and pH 8 in tap water (9.2 g/L DCW, 7.3 g/L PHB, and 79.7 % PHB contents). However, cell growth of halotolerant H. cerina YK44 was disturbed by 0.2 % furfural, which existed in biomass based sugars as inhibitors. Physical and thermal analyses revealed that PHB film started from sugarcane molasses and soybean flour was no different from that initiated from simple sugars (Tm was 175.8 °C and 176.2 °C, PDI was 1.29, and 1.31, respectively).

Evaluation of simplified ester-linked fatty acid analysis (ELFA) for phospholipid fatty acid (PLFA) analysis of bacterial population.

Phospholipid fatty acid (PLFA) analysis is used for characterizing microbial communities based on their lipid profiles. This method avoids biases from PCR or culture, allowing data collection in a natural state. However, PLFA is labor-intensive due to lipid fractionation. Simplified ester-linked fatty acid analysis (ELFA), which skips lipid fractionation, offers an alternative. It utilizes base-catalyzed methylation to derivatize only lipids, not free fatty acids, and found glycolipid and neutral lipid fractions are scarcely present in most bacteria, allowing lipid fractionation to be skipped. ELFA method showed a high correlation to PLFA data (r = 0.99) and higher sensitivity than the PLFA method by 1.5-2.57-fold, mainly due to the higher recovery of lipids, which was 1.5-1.9 times higher than with PLFA. The theoretical limit of detection (LOD) and limit of quantification (LOQ) for the ELFA method indicated that 1.54-fold less sample was needed for analysis than with the PLFA method. Our analysis of three bacterial cultures and a simulated consortium revealed the effectiveness of the ELFA method by its simple procedure and enhanced sensitivity for detecting strain-specific markers, which were not detected in PLFA analysis. Overall, this method could be easily used for the population analysis of synthetic consortia.

A sustainable bioremediation of vanadium from marine environment and value-addition using potential thraustochytrids.

Rising concerns about global environmental degradation underscore the pressing need for effective solutions to combat heavy metal pollution. Industries such as semiconductor and steel production discharge vanadium into marine ecosystems, posing significant risks to both marine life and human health. The current study investigates efficacy of utilizing marine thraustochytrid for efficient vanadium removal outcompeting other microbial sources. By optimizing pH and temperature conditions during harvesting, achieved a remarkable 50.80 % enhancement in vanadium removal efficiency, from 19.31 to 29.12 mg/L. Furthermore, chelating agents EDTA and citric acid supplementation demonstrated promising enhancements, reaching up to 31.21 and 32.59 mg/L, respectively. Notably, vanadium-treated biomass supplemented with citric acid exhibited maximum enhancement in lipid content, from 58.47 to 75.34 %, indicating thraustochytrid's potential for biofuel production. This study presents a sustainable approach for industrial-scale vanadium bioremediation, aligning with Sustainable Development Goals focused on dual benefits of environmental protection and renewable energy.

A review on microbes mediated resource recovery and bioplastic (polyhydroxyalkanoates) production from wastewater.

BACKGROUND: Plastic is widely utilized in packaging, frameworks, and as coverings material. Its overconsumption and slow degradation, pose threats to ecosystems due to its toxic effects. While polyhydroxyalkanoates (PHA) offer a sustainable alternative to petroleum-based plastics, their production costs present significant obstacles to global adoption. On the other side, a multitude of household and industrial activities generate substantial volumes of wastewater containing both organic and inorganic contaminants. This not only poses a threat to ecosystems but also presents opportunities to get benefits from the circular economy. Production of bioplastics may be improved by using the nutrients and minerals in wastewater as a feedstock for microbial fermentation. Strategies like feast-famine culture, mixed-consortia culture, and integrated processes have been developed for PHA production from highly polluted wastewater with high organic loads. Various process parameters like organic loading rate, organic content (volatile fatty acids), dissolved oxygen, operating pH, and temperature also have critical roles in PHA accumulation in microbial biomass. Research advances are also going on in downstream and recovery of PHA utilizing a combination of physical and chemical (halogenated solvents, surfactants, green solvents) methods. This review highlights recent developments in upcycling wastewater resources into PHA, encompassing various production strategies, downstream processing methodologies, and techno-economic analyses. SHORT CONCLUSION: Organic carbon and nitrogen present in wastewater offer a promising, cost-effective source for producing bioplastic. Previous attempts have focused on enhancing productivity through optimizing culture systems and growth conditions. However, despite technological progress, significant challenges persist, such as low productivity, intricate downstream processing, scalability issues, and the properties of resulting PHA.

Dual Application of p-Nitrophenol Alkanoate-Based Assay for Soil Selection and Screening of Microbial Strains for Bioplastic Degradation.

With an increase in the commercialization of bioplastics, the importance of screening for plastic-degrading strains and microbes has emerged. Conventional methods for screening such strains are time-consuming and labor-intensive. Therefore, we suggest a method for quickly and effectively screening plastic-degrading microbial strains through dual esterase assays for soil and isolated strains, using p-nitrophenyl alkanoates as substrates. To select microbe-abundant soil, the total amount of phospholipid fatty acids (PLFAs) included in each soil sample was analyzed, and esterase assays were performed for each soil sample to compare the esterase activity of each soil. In addition, by analyzing the correlation coefficients and sensitivity between the amount of PLFAs and the degree of esterase activity according to the substrate, it was confirmed that substrate pNP-C2 is the most useful index for soil containing several microbes having esterase activity. In addition, esterase assays of the isolated strains allowed us to select the most active strain as the degrading strain, and 16S rRNA results confirmed that it was Bacillus sp. N04 showed the highest degradation activity for polybutylene succinate (PBS) as measured in liquid culture for 7 days, with a degradation yield of 99%. Furthermore, Bacillus sp. N04 showed degradation activity against various bioplastics. We propose the dual application of p-nitrophenyl alkanoates as an efficient method to first select the appropriate soil and then to screen for plastic-degrading strains in it, and conclude that pNP-C2 in particular, is a useful indicator.

Identification of Oil-Loving Cupriavidus necator BM3-1 for Polyhydroxyalkanoate Production and Assessing Contribution of Exopolysaccharide for Vegetable Oil Utilization.

Polyhydroxyalkanoates (PHA) have received attention owing to their biodegradability and biocompatibility, with studies exploring PHA-producing bacterial strains. As vegetable oil provides carbon and monomer precursors for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)), oil-utilizing strains may facilitate PHA production. Herein, Cupriavidus necator BM3-1, which produces 11.1 g/L of PHB with 5% vegetable oil, was selected among various novel Cupriavidus necator strains. This strain exhibited higher preference for vegetable oils over sugars, with soybean oil and tryptone determined to be optimal sources for PHA production. BM3-1 produced 33.9 g/L of exopolysaccharides (EPS), which was three-fold higher than the amount produced by H16 (10.1 g/L). EPS exhibited 59.7% of emulsification activity (EI24), higher than that of SDS and of EPS from H16 with soybean oil. To evaluate P(3HB-co-3HHx) production from soybean oil, BM3-1 was engineered with P(3HB-co-3HHx) biosynthetic genes (phaCRa, phaARe, and phaJPa). BM3-1/pPhaCJ produced 3.5 mol% of 3HHx and 37.1 g/L PHA. BM3-1/pCB81 (phaCAJ) produced 32.8 g/L PHA, including 5.9 mol% 3HHx. Physical and thermal analyses revealed that P(3HB-co-5.9 mol% 3HHx) was better than PHB. Collectively, we identified a novel strain with high vegetable oil utilization capacity for the production of EPS, with the option to engineer the strain for P(3HB-co-3HHx).

Recent advances in bio-based production of top platform chemical, succinic acid: an alternative to conventional chemistry.

Succinic acid (SA) is one of the top platform chemicals with huge applications in diverse sectors. The presence of two carboxylic acid groups on the terminal carbon atoms makes SA a highly functional molecule that can be derivatized into a wide range of products. The biological route for SA production is a cleaner, greener, and promising technological option with huge potential to sequester the potent greenhouse gas, carbon dioxide. The recycling of renewable carbon of biomass (an indirect form of CO2), along with fixing CO2 in the form of SA, offers a carbon-negative SA manufacturing route to reduce atmospheric CO2 load. These attractive attributes compel a paradigm shift from fossil-based to microbial SA manufacturing, as evidenced by several commercial-scale bio-SA production in the last decade. The current review article scrutinizes the existing knowledge and covers SA production by the most efficient SA producers, including several bacteria and yeast strains. The review starts with the biochemistry of the major pathways accumulating SA as an end product. It discusses the SA production from a variety of pure and crude renewable sources by native as well as engineered strains with details of pathway/metabolic, evolutionary, and process engineering approaches for enhancing TYP (titer, yield, and productivity) metrics. The review is then extended to recent progress on separation technologies to recover SA from fermentation broth. Thereafter, SA derivatization opportunities via chemo-catalysis are discussed for various high-value products, which are only a few steps away. The last two sections are devoted to the current scenario of industrial production of bio-SA and associated challenges, along with the author's perspective.

The green revolution of food waste upcycling to produce polyhydroxyalkanoates.

This review emphasizes the urgent need for food waste upcycling as a response to the mounting global food waste crisis. Focusing on polyhydroxyalkanoates (PHAs) as an alternative to traditional plastics, it examines the potential of various food wastes as feedstock for microbial fermentation and PHA production. The upcycling of food waste including cheese whey, waste cooking oil, coffee waste, and animal fat is an innovative practice for food waste management. This approach not only mitigates environmental impacts but also contributes to sustainable development and economic growth. Downstream processing techniques for PHAs are discussed, highlighting their role in obtaining high-quality materials. The study also addresses sustainability considerations, emphasizing biodegradability and recycling, while acknowledging the challenges associated with this path.

Controlled production of a polyhydroxyalkanoate (PHA) tetramer containing different mole fraction of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3 HV), 4 HV and 5 HV units by engineered Cupriavidus necator.

Polyhydroxyalkanoates (PHAs) are promising alternatives to existing petrochemical-based plastics because of their bio-degradable properties. However, the limited structural diversity of PHAs has hindered their application. In this study, high mole-fractions of Poly (39 mol% 3HB-co-17 mol% 3 HV-co-44 mol% 4 HV) and Poly (25 mol% 3HB-co-75 mol% 5 HV) were produced from 4- hydroxyvaleric acid and 5-hydroxyvaleric acid, using Cupriavidus necator PHB-4 harboring the gene phaCBP-M-CPF4 with modified sequences. In addition, the complex toxicity of precursor mixtures was tested, and it was confirmed that the engineered C. necator was capable of synthesizing Poly (32 mol% 3HB-co-11 mol% 3 HV-co-25 mol% 4 HV-co-32 mol% 5 HV) at low mixture concentrations. Correlation analyses of the precursor ratio and the monomeric mole fractions indicated that each mole fractions could be precisely controlled using the precursor proportion. Physical property analysis confirmed that Poly (3HB-co-3 HV-co-4 HV) is a rubber-like amorphous polymer and Poly (3HB-co-5 HV) has a high tensile strength and elongation at break. Poly (3HB-co-3 HV-co-4 HV-co-5 HV) had a much lower glass transition temperature than the co-, terpolymers containing 3 HV, 4 HV and 5 HV. This study expands the range of possible physical properties of PHAs and contributes to the realization of custom PHA production by suggesting a method for producing PHAs with various physical properties through mole-fraction control of 3 HV, 4 HV and 5 HV.

Reproducible Polybutylene Succinate (PBS)-Degrading Artificial Consortia by Introducing the Least Type of PBS-Degrading Strains.

Polybutylene succinate (PBS) stands out as a promising biodegradable polymer, drawing attention for its potential as an eco-friendly alternative to traditional plastics due to its biodegradability and reduced environmental impact. In this study, we aimed to enhance PBS degradation by examining artificial consortia composed of bacterial strains. Specifically, Terribacillus sp. JY49, Bacillus sp. JY35, and Bacillus sp. NR4 were assessed for their capabilities and synergistic effects in PBS degradation. When only two types of strains, Bacillus sp. JY35 and Bacillus sp. NR4, were co-cultured as a consortium, a notable increase in degradation activity toward PBS was observed compared to their activities alone. The consortium of Bacillus sp. JY35 and Bacillus sp. NR4 demonstrated a remarkable degradation yield of 76.5% in PBS after 10 days. The degradation of PBS by the consortium was validated and our findings underscore the potential for enhancing PBS degradation and the possibility of fast degradation by forming artificial consortia, leveraging the synergy between strains with limited PBS degradation activity. Furthermore, this study demonstrated that utilizing only two types of strains in the consortium facilitates easy control and provides reproducible results. This approach mitigates the risk of losing activity and reproducibility issues often associated with natural consortia.

Development in health-promoting essential polyunsaturated fatty acids production by microalgae: a review.

Polyunsaturated fatty acids (PUFAs) found in microalgae, primarily omega-3 (ω-3) and omega-6 (ω-6) are essential nutrients with positive effects on diseases such as hyperlipidemia, atherosclerosis, and coronary risk. Researchers still seek improvement in PUFA yield at a large scale for better commercial prospects. This review summarizes advancements in microalgae PUFA research for their cost-effective production and potential applications. Moreover, it discusses the most promising cultivation modes using organic and inorganic sources. It also discusses biomass hydrolysates to increase PUFA production as an alternative and sustainable organic source. For cost-effective PUFA production, heterotrophic, mixotrophic, and photoheterotrophic cultivation modes are assessed with traditional photoautotrophic production modes. Also, mixotrophic cultivation has fascinating sustainable attributes over other trophic modes. Furthermore, it provides insight into growth phase (stage I) improvement strategies to accumulate biomass and the complementing effects of other stress-inducing strategies during the production phase (stage II) on PUFA enhancement under these cultivation modes. The role of an excessive or limiting range of salinity, nutrients, carbon source, and light intensity were the most effective parameter in stage II for accumulating higher PUFAs such as ω-3 and ω-6. This article outlines the commercial potential of microalgae for omega PUFA production. They reduce the risk of diabetes, cardiovascular diseases (CVDs), cancer, and hypertension and play an important role in their emerging role in healthy lifestyle management.

Establishment of efficient 5-hydroxyvaleric acid production system by regenerating alpha-ketoglutaric acid and its application in poly(5-hydroxyvaleric acid) production.

5-hydroxyvaleric acid (5-HV) is a versatile C5 intermediate of bio-based high-value chemical synthesis pathways. However, 5-HV production faces a few shortcomings involving the supply of cofactors, especially α-ketoglutaric acid (α-KG). Herein, we established a two-cell biotransformation system by introducing L-glutamate oxidase (GOX) to regenerate α-KG. Additionally, the catalase KatE was adapted to inhibit α-KG degradation by the H2O2 produced during GOX reaction. We searched for the best combination of genes and vectors and optimized the biotransformation conditions to maximize GOX effectiveness. Under the optimized conditions, 5-HV pathway with GOX showed 1.60-fold higher productivity than that of without GOX, showing 11.3 g/L titer. Further, the two-cell system with GOX and KatE was expanded to produce poly(5-hydroxyvaleric acid) (P(5HV)), and it reached at 412 mg/L of P(5HV) production and 20.5% PHA contents when using the biotransformation supernatant. Thus, the two-cell biotransformation system with GOX can potentially give the practical and economic alternative of 5-HV production using bio-based methods. We also propose direct utilization of 5-HV from bioconversion for P(5HV) production.

Indigo production goes green: a review on opportunities and challenges of fermentative production.

Indigo is a widely used dye in various industries, such as textile, cosmetics, and food. However, traditional methods of indigo extraction and processing are associated with environmental and economic challenges. Fermentative production of indigo using microbial strains has emerged as a promising alternative that offers sustainability and cost-effectiveness. This review article provides a critical overview of microbial diversity, metabolic pathways, fermentation strategies, and genetic engineering approaches for fermentative indigo production. The advantages and limitations of different indigo production systems and a critique of the current understanding of indigo biosynthesis are discussed. Finally, the potential application of indigo in other sectors is also discussed. Overall, fermentative production of indigo offers a sustainable and bio-based alternative to synthetic methods and has the potential to contribute to the development of sustainable and circular biomanufacturing.

Understanding microplastic pollution: Tracing the footprints and eco-friendly solutions.

Microplastics (MPs) pollution has emerged as a critical environmental issue with far-reaching consequences for ecosystems and human health. These are plastic particles measuring <5 mm and are categorized as primary and secondary based on their origin. Primary MPs are used in various products like cosmetics, scrubs, body wash, and toothpaste, while secondary MPs are generated through the degradation of plastic products. These have been detected in seas, rivers, snow, indoor air, and seafood, posing potential risks to human health through the food chain. Detecting and quantifying MPs are essential to understand their distribution and abundance in the environment. Various microscopic (fluorescence microscopy, scanning electron microscopy) and spectroscopy techniques (FTIR, Raman spectroscopy, X-ray photoelectron spectroscopy) have been reported to analyse MPs. Despite the challenges in scalable removal methods, biological systems have emerged as promising options for eco-friendly MPs remediation. Algae, bacteria, and fungi have shown the potential to adsorb and degrade MPs in wastewater treatment plants (WWTPs) offering hope for mitigating this global crisis. This review examines the sources, impacts, detection, and biological removal of MPs, highlighting future directions in this crucial field of environmental conservation. By fostering global collaboration and innovative research a path towards a cleaner and healthier planet for future generations can be promised.

Current paradigms in employing self-assembled structures: Drug delivery implications with improved therapeutic potential.

Recent efforts have focused on developing improved drug delivery systems with enhanced therapeutic efficacy and minimal side effects. Micelles, self-assembled from amphiphilic block copolymers in aqueous solutions, have gained considerable attention for drug delivery. However, there is a need to further enhance their efficiency. These micelles offer benefits like biodegradability, biocompatibility, sustained drug release, and improved patient compliance. Yet, researchers must address stability issues and reduce toxicity. Nanoscale self-assembled structures have shown promise as efficient drug carriers, offering an alternative to conventional methods. Fine-tuning at the monomeric and molecular levels, along with structural modifications, is crucial for optimal drug release profiles. Various strategies, such as entrapping hydrophobic drugs and using polyethylene oxide diblock copolymer micelles to resist protein adsorption and cellular adhesion, protect the hydrophobic core from degradation. The polyethylene oxide corona also provides stealth properties, prolonging blood circulation for extended drug administration. Amphiphilic copolymers are attractive for drug delivery due to their adjustable properties, allowing control over micelle size and morphology. Emerging tools promise complex and multifunctional platforms. This article summarizes about the challenges as far as the use of micelles is concerned, including optimizing performance, rigorous pre-clinical and clinical research, and suggests further improvement for drug delivery efficacy.

Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase.

L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.

Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824.

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

Microbial alchemy: upcycling of brewery spent grains into high-value products through fermentation.

Spent grains are one of the lignocellulosic biomasses available in abundance, discarded by breweries as waste. The brewing process generates around 25-30% of waste in different forms and spent grains alone account for 80-85% of that waste, resulting in a significant global waste volume. Despite containing essential nutrients, i.e., carbohydrates, fibers, proteins, fatty acids, lipids, minerals, and vitamins, efficient and economically viable valorization of these grains is lacking. Microbial fermentation enables the valorization of spent grain biomass into numerous commercially valuable products used in energy, food, healthcare, and biomaterials. However, the process still needs more investigation to overcome challenges, such as transportation, cost-effective pretreatment, and fermentation strategy. to lower the product cost and to achieve market feasibility and customer affordability. This review summarizes the potential of spent grains valorization via microbial fermentation and associated challenges.

Tryptophan-Based Hyperproduction of Bioindigo by Combinatorial Overexpression of Two Different Tryptophan Transporters.

Indigo is a valuable, natural blue dye that has been used for centuries in the textile industry. The large-scale commercial production of indigo relies on its extraction from plants and chemical synthesis. Studies are being conducted to develop methods for environment-friendly and sustainable production of indigo using genetically engineered microbes. Here, to enhance the yield of bioindigo from an E. coli whole-cell system containing tryptophanase (TnaA) and flavin-containing monooxygenase (FMO), we evaluated tryptophan transporters to improve the transport of aromatic compounds, such as indole and tryptophan, which are not easily soluble and passable through cell walls. Among the three transporters, Mtr, AroP, and TnaB, AroP enhanced indigo production the most. The combination of each transporter with AroP was also evaluated, and the combination of AroP and TnaB showed the best performance compared to the single transporters and two transporters. Bioindigo production was then optimized by examining the culture medium, temperature, isopropyl ß-D-1-thiogalactopyranoside concentration, shaking speed (rpm), and pH. The novel strain containing aroP and tnaB plasmid with tnaA and FMO produced 8.77 mM (2.3 g/l) of bioindigo after 66 h of culture. The produced bioindigo was further recovered using a simple method and used as a watercolor dye, showing good mixing with other colors and color retention for a relatively long time. This study presents an effective strategy for enhancing indigo production using a combination of transporters.