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Abstract 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. Main body of abstract 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. Graphical abstract

Microbial exopolysaccharides (EPS) are high molecular weight carbohydrate-based materials produced and secreted in response to external environmental stress to protect microbial cells. These molecules also contain non-carbohydrate moieties including acetate, pyruvate, phosphate, and succinate along with sugar molecules. EPS has been incorporated into industrial operations as a gelling, emulsifying, and stabilizing agent to increase viscosity. Besides, these molecules have also shown high biocompatibility, immunomodulatory, antitumor, anti-microbial, and anti-viral activity which suggest their possible applications in healthcare. However, these molecules are facing some commercial shortcomings due to low stability, bioactivity, and poor mechanical and tensile strength in comparison to the required standard. Hence use of composites of EPS with other polymers and moieties has been suggested for higher performance. Especially in the context of inorganic moieties like silver, gold nanomaterials, clay, salts, and minerals will add bioactivity and functional sites for the attachment of drug molecules and other compounds. The current review summarizes the higher physical and chemical characteristics of EPS composites with inorganic materials and their immense potential in the biomedical sector.

Although efforts have been undertaken to produce polyhydroxyalkanoates (PHA) with various monomers, the low yield of PHAs because of complex metabolic pathways and inhibitory substrates remains a major hurdle in their analyses and applications. Therefore, we investigated the feasibility of mass production of PHAs containing 5-hydroxyvalerate (5HV) using δ-valerolactone (DVL) without any pretreatment along with the addition of plant oil to achieve enough biomass. We identified that PhaCBP-M-CPF4, a PHA synthase, was capable of incorporating 5HV monomers and that C. necator PHB−4 harboring phaCBP-M-CPF4 synthesized poly(3HB-co-3HHx-co-5HV) in the presence of bean oil and DVL. In fed-batch fermentation, the supply of bean oil resulted in the synthesis of 49 g/L of poly(3HB-co-3.7 mol% 3HHx-co-5.3 mol%5HV) from 66 g/L of biomass. Thermophysical studies showed that 3HHx was effective in increasing the elongation, whereas 5HV was effective in decreasing the melting point. The contact angles of poly(3HB-co-3HHx-co-5HV) and poly(3HB-co-3HHx) were 109 and 98°, respectively. In addition, the analysis of microbial degradation confirmed that poly(3HB-co-3HHx-co-5HV) degraded more slowly (82% over 7 days) compared to poly(3HB-co-3HHx) (100% over 5 days). Overall, the oil-based fermentation strategy helped produce more PHA, and the mass production of novel PHAs could provide more opportunities to study polymer properties.

Abstract Background Bioplastics are attracting considerable attention, owing to the increase in non-degradable waste. Using microorganisms to degrade bioplastics is a promising strategy for reducing non-degradable plastic waste. However, maintaining bacterial viability and activity during culture and storage remains challenging. With the use of conventional methods, cell viability and activity was lost; therefore, these conditions need to be optimized for the practical application of microorganisms in bioplastic degradation. Therefore, we aimed to optimize the feasibility of the lyophilization method for convenient storage and direct use. In addition, we incoporated protective reagents to increase the viability and activity of lyophilized microorganisms. By selecting and applying the best protective reagents for the lyophilization process and the effects of additives on the growth and PHB-degrading activity of strains were analyzed after lyophilization. For developing the lyophilization method for protecting degradation activity, it may promote practical applications of bioplastic-degrading bacteria. Results In this study, the polyhydroxybutyrate (PHB)-degrading strain, Bacillus sp. JY14 was lyophilized with the use of various sugars as protective reagents. Among the carbon sources tested, raffinose was associated with the highest cell survival rate (12.1%). Moreover, 7% of raffionose showed the highest PHB degradation yield (92.1%). Therefore, raffinose was selected as the most effective protective reagent. Also, bacterial activity was successfully maintained, with raffinose, under different storage temperatures and period. Conclusions This study highlights lyophilization as an efficient microorganism storage method to enhance the applicability of bioplastic-degrading bacterial strains. The approach developed herein can be further studied and used to promote the application of microorganisms in bioplastic degradation.

Abstract Polyhydroxyalkanoate (PHA) is one of the most promising materials for replacing petroleum-based plastics, and it can be produced from various renewable biomass sources. In this study, PHA production was conducted using Halomonas sp. YLGW01 utilizing mixed volatile fatty acids (VFAs) as carbon sources. The ratio and concentration of carbon and nitrogen sources were optimized through mixture analysis and organic nitrogen source screening, respectively. It was found that the highest cell dry weight (CDW) of 3.15 g/L and PHA production of 1.63 g/L were achieved when the ratio of acetate to lactate in the mixed VFAs was 0.45:0.55. Furthermore, supplementation of organic nitrogen sources such as soytone resulted in a ninefold increase in CDW (reaching 2.32 g/L) and a 22-fold increase in PHA production (reaching 1.60 g/L) compared to using inorganic nitrogen sources. Subsequently, DO-stat, VFAs consumption rate stat, and pH–stat fed-batch methods were applied to investigate and evaluate PHA productivity. The results showed that when pH–stat-based VFAs feeding was employed, a CDW of 7 g/L and PHA production of 5.1 g/L were achieved within 68 h, with a PHA content of 73%. Overall, the pH–stat fed-batch strategy proved to be effective in enhancing PHA production by Halomonas sp. YLGW01 utilizing VFAs.

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).

Antibiotic-induced gut microbiota disruption constitutes a major risk factor for Clostridioides difficile infection (CDI). Further, antibiotic therapy, which is the standard treatment option for CDI, exacerbates gut microbiota imbalance, thereby causing high recurrent CDI incidence. Consequently, probiotic-based CDI treatment has emerged as a long-term management and preventive option. However, the mechanisms underlying the therapeutic effects of probiotics for CDI remain uninvestigated, thereby creating a knowledge gap that needs to be addressed. To fill this gap, we used a multiomics approach to holistically investigate the mechanisms underlying the therapeutic effects of probiotics for CDI at a molecular level. We first screened Bifidobacterium longum owing to its inhibitory effect on C. difficile growth, then observed the physiological changes associated with the inhibition of C. difficile growth and toxin production via a multiomics approach. Regarding the mechanism underlying C. difficile growth inhibition, we detected a decrease in intracellular adenosine triphosphate (ATP) synthesis due to B. longum–produced lactate and a subsequent decrease in (deoxy)ribonucleoside triphosphate synthesis. Via the differential regulation of proteins involved in translation and protein quality control, we identified B. longum–induced proteinaceous stress. Finally, we found that B. longum suppressed the toxin production of C. difficile by replenishing proline consumed by it. Overall, the findings of the present study expand our understanding of the mechanisms by which probiotics inhibit C. difficile growth and contribute to the development of live biotherapeutic products based on molecular mechanisms for treating CDI.

Hibiscus syriacus L. extract (HSE) traditionally has been recognized as promising natural resources for the treatment of diseases related to hyper-inflammation. This study aimed to investigate the anti-inflammatory effect of HSE in LPS-stimulated Raw 264.7 cells. For this purpose, we determined the optimal extraction method and found that the ultrasonic extraction showed the most effective activity for reduction of free radical species by DPPH assay. Then, we performed PGE2 concentration analysis, COX-2 activity assay, proliferation assay, apoptosis detection, and western blotting analysis. HSE decreased PGE2 concentration and COX-2 activity and reduced the expression of COX-2 and IL6 in LPS stimulated Raw 264.7 cells. In addition, we found that HSE inhibited the proliferation and induced apoptosis with increase in the expression of pro-apoptotic proteins such as caspase-3, -7, -9, Bax, Bim, FOXO3, and p53 in LPS-stimulated Raw 264.7 cells. Taken together, we found that HSE effectively suppressed LPS-stimulated Raw 264.7 cells suggesting that HSE has the potential to treat inflammation via regulation of proinflammatory COX-2, IL6, and PGE2 and induction of apoptosis.

Bilobetin is a biflavonoid isolated from the leaves of Ginkgo biloba. Bilobetin displays several biological effects, however, the activities of bilobetin against cancer have been solely demonstrated. Thus, the aim of this study is investigating the apoptotic effects of and anticancer mechanism of bilobetin in Huh7 and HepG2 cells, both of human hepatocellular carcinoma (HCC) cell lines. MTT, cell counting, and colony formation assay showed that anti-proliferation effect of bilobetin. Cell cycle analysis revealed that bilobetin induces increase of population of the sub G1 phase. Annexin V/PI staining and TUNEL assay showed that bilobetin treatment promotes apoptotic cell death and DNA fragmentation. Bilobetin also induces ROS elevation and DNA damage in HCC cells. Western blot analysis elucidated that bilobetin displays apoptotic signaling pathway in HCC cells via upregulating cleaved PARP, cleaved caspase3 and Bax and downregulating CYP2J2. Bilobetin inhibited CYP2J2-catalyzed terfenadine and ebastine hydroxylase activities with IC50 values of 0.81 and 2.21 μM, respectively. Transfection of CYP2J2 siRNA increased the anticancer effect of bilobetin in HCC cells through the inhibition of CYP2J2 expression. Taken together, this study suggests that bilobetin induces apoptosis against HCC cells via ROS level elevation and inhibition of CYP2J2.

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.

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.

Clostridioides difficile is a gram-positive anaerobic bacterium that causes antibiotic-associated infections in the gut. C. difficile infection develops in the intestine of a host with an imbalance of the intestinal microbiota and, in severe cases, can lead to toxic megacolon, intestinal perforation, and even death. Despite its severity and importance, however, the lack of a model to understand host-pathogen interactions and the lack of research results on host cell effects and response mechanisms under C. difficile infection remain limited. Here, we developed an in vitro anaerobic-aerobic C. difficile infection model that enables direct interaction between human gut epithelial cells and C. difficile through the Mimetic Intestinal Host–Microbe Interaction Coculture System. Additionally, an integrative multiomics approach was applied to investigate the biological changes and response mechanisms of host cells caused by C. difficile in the early stage of infection. The C. difficile infection model was validated through the induction of disaggregation of the actin filaments and disruption of the intestinal epithelial barrier as the toxin-mediated phenotypes following infection progression. In addition, an upregulation of stress-induced chaperones and an increase in the ubiquitin proteasomal pathway were identified in response to protein stress that occurred in the early stage of infection, and downregulation of proteins contained in the electron transfer chain and ATP synthase was observed. It has been demonstrated that host cell energy metabolism is inhibited through the glycolysis of Caco-2 cells and the reduction of metabolites belonging to the TCA cycle. Taken together, our C. difficile infection model suggests a new biological response pathway in the host cell induced by C. difficile during the early stage of infection at the molecular level under anaerobic-aerobic conditions. Therefore, this study has the potential to be applied to the development of future therapeutics through basic metabolic studies of C. difficile infection.

Microbial exopolysaccharides (EPSs), e.g., xanthan, dextran, gellan, curdlan, etc., have significant applications in several industries (pharma, food, textiles, petroleum, etc.) due to their biocompatibility, nontoxicity, and functional characteristics. However, biodegradability, poor cell adhesion, mineralization, and lower enzyme activity are some other factors that might hinder commercial applications in healthcare practices. Some EPSs lack biological activities that make them prone to degradation in ex vivo, as well as in vivo environments. The blending of EPSs with other natural and synthetic polymers can improve the structural, functional, and physiological characteristics, and make the composites suitable for a diverse range of applications. In comparison to EPS, composites have more mechanical strength, porosity, and stress-bearing capacity, along with a higher cell adhesion rate, and mineralization that is required for tissue engineering. Composites have a better possibility for biomedical and healthcare applications and are used for 2D and 3D scaffold fabrication, drug carrying and delivery, wound healing, tissue regeneration, and engineering. However, the commercialization of these products still needs in-depth research, considering commercial aspects such as stability within ex vivo and in vivo environments, the presence of biological fluids and enzymes, degradation profile, and interaction within living systems. The opportunities and potential applications are diverse, but more elaborative research is needed to address the challenges. In the current article, efforts have been made to summarize the recent advancements in applications of exopolysaccharide composites with natural and synthetic components, with special consideration of pharma and healthcare applications.

Abstract Methicillin-resistant Staphylococcus aureus (MRSA) strains are distinct from general Staphylococcus strains with respect to the composition of the membrane, ability to form a thicker biofilm, and, importantly, ability to modify the target of antibiotics to evade their activity. The agr gene is an accessory global regulator of gram-positive bacteria that governs virulence or resistant mechanisms and therefore an important target for the control of resistant strains. However, the mechanism by which agr impacts resistance to β-lactam antibiotics remains unclear. In the present study, we found the Δagr mutant strain having higher resistance to high concentrations of β-lactam antibiotics such as oxacillin and ampicillin. To determine the influence of variation in the microenvironment of cells between the parental and mutant strains, fatty acid analysis of the supernatant, total lipids, and phospholipid fatty acids were compared. The Δagr mutant strain tended to produce fewer fatty acids and retained lower amounts of C16, C18 fatty acids in the supernatant. Phospholipid analysis showed a dramatic increase in the hydrophobic longer-chain fatty acids in the membrane. To target membrane, we applied several surfactants and found that sorbitan monolaurate (Span20) had a synergistic effect with oxacillin by decreasing biofilm formation and growth. These findings indicate that agr deletion allows for MRSA to resist antibiotics via several changes including constant expression of mecA, fatty acid metabolism, and biofilm thickening.

Faecalibacterium prausnitzii, a major commensal bacterium in the human gut, is well known for its anti-inflammatory effects, which improve host intestinal health. Although several studies have reported that inulin, a well-known prebiotic, increases the abundance of F. prausnitzii in the intestine, the mechanism underlying this effect remains unclear. In this study, we applied liquid chromatography tandem mass spectrometry (LC-MS/MS)-based multiomics approaches to identify biological and enzymatic mechanisms of F. prausnitzii involved in the selective digestion of inulin. First, to determine the preference for dietary carbohydrates, we compared the growth of F. prausnitzii in several carbon sources and observed selective growth in inulin. In addition, an LC-MS/MS-based intracellular proteomic and metabolic profiling was performed to determine the quantitative changes in specific proteins and metabolites of F. prausnitzii when grown on inulin. Interestingly, proteomic analysis revealed that the putative proteins involved in inulin-type fructan utilization by F. prausnitzii, particularly β-fructosidase and amylosucrase were upregulated in the presence of inulin. To investigate the function of these proteins, we overexpressed bfrA and ams, genes encoding β-fructosidase and amylosucrase, respectively, in Escherichia coli, and observed their ability to degrade fructan. In addition, the enzyme activity assay demonstrated that intracellular fructan hydrolases degrade the inulin-type fructans taken up by fructan ATP-binding cassette transporters. Furthermore, we showed that the fructose uptake activity of F. prausnitzii was enhanced by the fructose phosphotransferase system transporter when inulin was used as a carbon source. Intracellular metabolomic analysis indicated that F. prausnitzii could use fructose, the product of inulin-type fructan degradation, as an energy source for inulin utilization. Taken together, this study provided molecular insights regarding the metabolism of F. prauznitzii for inulin, which stimulates the growth and activity of the beneficial bacterium in the intestine.

Coffee waste is an abundant biomass that can be converted into high value chemical products, and is used in various renewable biological processes. In this study, oil was extracted from spent coffee grounds (SCGs) and used for polyhydroxyalkanoate (PHA) production through Pseudomonas resinovorans. The oil–extracted SCGs (OESCGs) were hydrolyzed and used for biohydrogen production through Clostridium butyricum DSM10702. The oil extraction yield through n–hexane was 14.4%, which accounted for 97% of the oil present in the SCGs. OESCG hydrolysate (OESCGH) had a sugar concentration of 32.26 g/L, which was 15.4% higher than that of the SCG hydrolysate (SCGH) (27.96 g/L). Hydrogen production using these substrates was 181.19 mL and 136.58 mL in OESCGH and SCGH media, respectively. The consumed sugar concentration was 6.77 g/L in OESCGH and 5.09 g/L in SCGH media. VFA production with OESCGH (3.58 g/L) increased by 40.9% compared with SCGH (2.54 g/L). In addition, in a fed–batch culture using the extracted oil, cell dry weight was 5.4 g/L, PHA was 1.6 g/L, and PHA contents were 29.5% at 24 h.

Polyhydroxybutyrate (PHB) is a biodegradable bioplastic with potential applications as an alternative to petroleum-based plastics. However, efficient PHB production remains difficult. The main cost of PHB production is attributed to carbon sources; hence, finding inexpensive sources is important. Galactose is a possible substrate for polyhydroxyalkanoate production as it is abundant in marine environments. Marine bacteria that produce PHB from galactose could be an effective resource that can be used for efficient PHB production. In this study, to identify a galactose utilizing PHB producer, we examined 16 Halomonas strains. We demonstrated that Halomonas cerina (Halomonas sp. YK44) has the highest growth and PHB production using a culture media containing 2% galactose, final 4% NaCl, and 0.1% yeast extract. These culture conditions yielded 8.98 g/L PHB (78.1% PHB content (w/w)). When galactose-containing red algae (Eucheuma spinosum) hydrolysates were used as a carbon source, 5.2 g/L PHB was produced with 1.425% galactose after treatment with activated carbon. Since high salt conditions can be used to avoid sterilization, we examined whether Halomonas sp. YK44 could produce PHB in non-sterilized conditions. Culture media in these conditions yielded 72.41% PHB content. Thus, Halomonas sp. YK44 is robust against contamination, allowing for long-term culture and economical PHB production.

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most well-known human pathogens, houses many virulence factors and regulatory proteins that confer resistance to diverse antibiotics. Although they have been investigated intensively, the correlations among virulence factors, regulatory proteins and antibiotic resistance are still elusive. We aimed to identify the most significant global MRSA regulator by concurrently analyzing protein-binding and several promoters under same conditions and at the same time point. DNA affinity capture assay (DACA) was performed with the promoters of mecA, sarA, and sarR, all of which significantly impact survival of MRSA. Here, we show that SarA protein binds to all three promoters. Consistent with the previous reports, ΔsarA mutant exhibited weakened antibiotic resistance to oxacillin and reduced biofilm formation. Additionally, production and activity of many virulence factors such as phenol-soluble modulins (PSM), α-hemolysin, motility, staphyloxanthin, and other related proteins were decreased. Comparing the sequence of SarA with that of clinical strains of various lineages showed that all sequences were highly conserved, in contrast to that observed for AgrA, another major regulator of virulence and resistance in MRSA. We have demonstrated that SarA regulates antibiotic resistance and the expression of various virulence factors. Our results warrant that SarA could be a leading target for developing therapeutic agents against MRSA infections.

Polybutylene succinate (PBS) is a bioplastic substitute for synthetic plastics that are made from petroleum-based products such as polyethylene and polypropylene. However, the biodegradation rate of PBS is still low and similar to that of polylactic acid (PLA). Moreover, our knowledge about degrader species is limited to a few fungi and mixed consortia. Here, to identify a bacterial degrader to accelerate PBS degradation, we screened and isolated Terribacillus sp. JY49, which showed significant degradability. In order to optimize solid and liquid culture conditions, the effect of factors such as temperature, additional carbon sources, and salt concentrations on degradation was confirmed. We observed a degradation yield of 22.3% after 7 days when adding 1% of glucose. Additionally, NaCl was added to liquid media, and degradation yield was decreased but PBS films were broken into pieces. Comparing the degree of PBS degradation during 10 days, the degradation yield was 31.4% after 10 days at 30 °C. Alteration of physical properties of films was analyzed by using scanning electron microscopy (SEM), gel permeation chromatography (GPC), and Fourier transform infrared (FT-IR). In addition, Terribacillus sp. JY49 showed clear zones on poly(butylene adipate-co-terephthalate) (PBAT), polycaprolactone (PCL), and copolymers such as P(3HB-co-3HV) and P(3HV-co-4HB), exhibiting a broad spectrum of degradation activities on bioplastics. However, there was no significant difference in absorbance when esterase activity was examined for different types of bioplastics. Overall, Terribacillus sp. JY49 is a potential bacterial strain that can degrade PBS and other bioplastics, and this is the first report of Terribacillus sp. as a bioplastic degrader.

As a biodegradable plastic, polyhydroxybutyrate (PHB) has relatively poor mechanical properties, preventing its wider use. Various plasticizers have been studied to improve the mechanical properties of PHB; however, due to the slow degradation speed in the soil environment and lack of evaluation methods, studies on the degradation of PHB with plasticizers are rarely reported. In this study, by applying Microbulbifer sp. SOL66, which is able to degrade PHB very quickly, a benign plasticizer was evaluated with good properties and good degradability, not inhibiting microbial activities. Eight different plasticizers were applied with PHB and Microbulbifer sp. SOL66, PHB film containing 10% and 20% tributyl citrate showed significant biodegradability of PHB. It was confirmed that tributyl citrate could increase the speed of PHB degradation by Microbulbifer sp. SOL66 by 88% at 1 day, although the degree of degradation was similar after 3 days with and without tributyl citrate. By the analysis of microbial degradation, physical, chemical, and mechanical properties, tributyl citrate was shown not only to improve physical, chemical, and mechanical properties but also the speed of microbial degradation.

Bacteria can evade antibiotics by acquiring resistance genes, as well as switching to a non-growing dormant state without accompanying genetic modification. Bacteria in this quiescent state are called persisters, and this non-inheritable ability to withstand multiple antibiotics is referred to as antibiotic tolerance. Although all bacteria are considered to be able to form antibiotic-tolerant persisters, the antibiotic tolerance of extremophilic bacteria is poorly understood. Previously, we identified the psychrotolerant bacterium Pseudomonas sp. B14-6 from the glacier foreland of Midtre Lovénbreen in High Arctic Svalbard. Herein, we investigated the resistance and tolerance of Pseudomonas sp. B14-6 against aminoglycosides at various temperatures. This bacterium was resistant to streptomycin and susceptible to apramycin, gentamicin, kanamycin, and tobramycin. The two putative aminoglycoside phosphotransferase genes aph1 and aph2 were the most likely contributors to streptomycin resistance. Notably, unlike the mesophilic Pseudomonas aeruginosa PA14, this cold-adapted bacterium demonstrated reduced susceptibility to all tested aminoglycosides in a temperature-dependent manner. Pseudomonas sp. B14-6 at a lower temperature formed the persister cells that shows tolerance to the 100-fold minimum inhibitory concentration (MIC) of gentamicin, as well as the partially tolerant cells that withstand 25-fold MIC gentamicin. The temperature-dependent gentamicin tolerance appears to result from reduced metabolic activity. Lastly, the partially tolerant Pseudomonas sp. B14-6 cells could slowly proliferate under the bactericidal concentrations of aminoglycosides. Our results demonstrate that Pseudomonas sp. B14-6 has a characteristic ability to form cells with a range of tolerance, which appears to be inversely proportional to its growth rate.

Polyhydroxyalkanoate (PHA) is a biodegradable plastic that can be used to replace petroleum-based plastic. In addition, as a medium-chain-length PHA (mcl-PHA), it can be used to provide elastomeric properties in specific applications. Because of these characteristics, recently, there has been much research on mcl-PHA production using inexpensive biomass materials as substrates. In this study, mcl-PHA producers were screened using alkanes (n-octane, n-decane, and n-dodecane) as sources of carbon. The amount of PHA produced by Pseudomonas resinovorans using sole n-octane, n-decane, or n-dodecane was 0.48 g/L, 0.27 g/L, or 0.07 g/L, respectively, while that produced using mixed alkane was 0.74 g/L. As a larger amount of PHA was produced using mixed alkane compared with sole alkane, a statistical mixture analysis was used to determine the optimal ratio of alkanes in the mixture. The optimal ratio predicted by the analysis was a medium with 9.15% n-octane, 6.44% n-decane, and 4.29% n-dodecane. In addition, through several concentration-specific experiments, the optimum concentrations of nitrogen and phosphorus for cell growth and maximum PHA production were determined as 0.05% and 1.0%, respectively. Finally, under the determined optimal conditions, 2.1 g/L of mcl-PHA and 60% PHA content were obtained using P. resinovorans in a 7 L fermenter.

Continuous urbanization and modernization have increased the burning of fossil fuels to meet energy needs across the globe, emanating environmental pollution and depleting fossil fuels. Therefore, a shift towards sustainable and renewable energy is necessary. Several techniques to exploit biomass to yield energy are trending, with pyrolysis one of them. Usually, a single feedstock is employed in pyrolysis for anoxygenic generation of biochar together with bio-oil at elevated temperatures (350–600 °C). Bio-oil produced through pyrolysis can be upgraded to crude oil after some modification. However, these modifications of bio-oil are one of the major drawbacks for its large-scale adoption, as upgradation increases the overall cost. Therefore, in recent years the scientific community has been researching co-pyrolysis technology that involves the pyrolysis of lignocellulosic biomass waste with non-biodegradable waste. Co-pyrolysis reduces the need for post-modification of bio-oil, unlike pyrolysis of a single feedstock. This review article discusses the recent advancements and technological challenges in waste biomass co-pyrolysis, the mechanism of co-pyrolysis, and factors that affect co-pyrolysis. The current study critically analyzes different recent research articles presented in databases such as PubMed, MDPI, ScienceDirect, Springer, etc. Hence, this review is one-of-a-kind in that it attempts to explain each and every aspect of the co-pyrolysis process and its current progress in the scientific field. Consequently, this review also compiles the remarkable achievements in co-pyrolysis and recommendations for the future.

Wastewater is not a liability, instead considered as a resource for microbial fermentation and value-added products. Most of the wastewater contains various nutrients like nitrates and phosphates apart from the organic constituents that favor microbial growth. Microalgae are unicellular aquatic organisms and are widely used for wastewater treatment. Various cultivation methods such as open, closed, and integrated have been reported for microalgal cultivation to treat wastewater and resource recovery simultaneously. Microalgal growth is affected by various factors such as sunlight, temperature, pH, and nutrients that affect the growth rate of microalgae. Microalgae can consume urea, phosphates, and metals such as magnesium, zinc, lead, cadmium, arsenic, etc. for their growth and reduces the biochemical oxygen demand (BOD). The microalgal biomass produced during the wastewater treatment can be further used to produce carbon-neutral products such as biofuel, feed, bio-fertilizer, bioplastic, and exopolysaccharides. Integration of wastewater treatment with microalgal bio-refinery not only solves the wastewater treatment problem but also generates revenue and supports a sustainable and circular bio-economy. The present review will highlight the current and advanced methods used to integrate microalgae for the complete reclamation of nutrients from industrial wastewater sources and their utilization for value-added compound production. Furthermore, pertaining challenges are briefly discussed along with the techno-economic analysis of current pilot-scale projects worldwide.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes severe diseases in humans. For decades, MRSA has acquired substantial resistance against conventional antibiotics through regulatory adaptation, thereby posing a challenge for treating MRSA infection. One of the emerging strategies to combat MRSA is the combinatory use of antibacterial agents. Based on the dramatic change in phospholipid fatty acid (PLFA) composition of MRSA in previous results, this study investigated branched-chain amino acid derivatives (precursors of fatty acid synthesis of cell membrane) and discovered the antimicrobial potency of D-norvaline. The compound, which can act synergistically with oxacillin, is among the three leucine-tRNA synthetase inhibitors with high potency to inhibit MRSA cell growth and biofilm formation. PLFA analysis and membrane properties revealed that D-norvaline decreased the overall amount of PLFA, increasing the fluidity and decreasing the hydrophobicity of the bacterial cell membrane. Additionally, we observed genetic differences to explore the response to D-norvaline. Furthermore, deletion mutants and clinically isolated MRSA strains were treated with D-norvaline. The study revealed that D-norvaline, with low concentrations of oxacillin, was effective in killing several MRSA strains. In summary, our findings provide a new combination of aminoacyl-tRNA synthetase inhibitor D-norvaline and oxacillin, which is effective against MRSA.

Having the advantage of eco-friendly decomposition, bioplastics could be used to replace petroleum-based plastics. In particular, poly(3-hydroxybutyrate) (PHB) is one of the most commercialized bioplastics, however, necessitating the introduction of PHB-degrading bacteria for its effective disposal. In this study, Microbulbifer sp. SOL66 (94.18% 16S rRNA with similarity to Microbulbifer hydrolyticus) demonstrated the highest degradation activity among five newly screened Microbulbifer genus strains. Microbulbifer sp. SOL66 showed a rapid degradation yield, reaching 98% in 4 days, as monitored by laboratory scale, gas chromatography-mass spectrometry, scanning electron microscopy, gel permeation chromatography, and Fourier transform infrared spectroscopy. The PHB film was completely degraded within 7 days at 37 °C in the presence of 3% NaCl. When 1% xylose and 0.4% ammonium sulfate were added, the degradation activity increased by 17% and 24%, respectively. In addition, this strain showed biodegradability on pellets of poly(3-hydroxybutyrate-co-4-hydroxybutyrate), as confirmed by weight loss and physical property changes. We confirmed that Microbulbifer sp. SOL66 has a great ability to degrade PHB, and has rarely been reported to date.

Crude oil is a major energy source that is exploited globally to achieve economic growth. To meet the growing demands for oil, in an environment of stringent environmental regulations and economic and technical pressure, industries have been required to develop novel oil salvaging techniques. The remaining ~70% of the world’s conventional oil (one-third of the available total petroleum) is trapped in depleted and marginal reservoirs, and could thus be potentially recovered and used. The only means of extracting this oil is via microbial enhanced oil recovery (MEOR). This tertiary oil recovery method employs indigenous microorganisms and their metabolic products to enhance oil mobilization. Although a significant amount of research has been undertaken on MEOR, the absence of convincing evidence has contributed to the petroleum industry’s low interest, as evidenced by the issuance of 400+ patents on MEOR that have not been accepted by this sector. The majority of the world’s MEOR field trials are briefly described in this review. However, the presented research fails to provide valid verification that the microbial system has the potential to address the identified constraints. Rather than promising certainty, MEOR will persist as an unverified concept unless further research and investigations are carried out.

Abstract Climate change causes permafrost thawing, and we are confronted with the unpredictable risk of newly discovered permafrost microbes that have disease-causing capabilities. Here, we first characterized the detailed chemical structure of the lipid A moiety from a Pseudomonas species that was isolated from thawing arctic permafrost using MALDI-based mass spectrometric approaches (i.e., MALDI-TOF MS and MALDI-QIT-TOF MSn). The MALDI multi-stage mass spectrometry (MS) analysis of lipid A extracted from the Pseudomonas sp. strain PAMC 28618 demonstrated that the hexaacyl lipid A ([M−H]− at m/z 1616.5) contains a glucosamine (GlcN) disaccharide backbone, two phosphates, four main acyl chains and two branched acyl chains. Moreover, the lipid A molecule–based structural activity relationship with other terrestrial Gram-negative bacteria indicated that strain PAMC 28618 has an identical lipid A structure with the mesophilic Pseudomonas cichorii which can cause rot disease in endive (Cichorium endivia) and that their bacterial toxicities were equivalent. Therefore, the overall lipid A validation process provides a general strategy for characterizing bacteria that have been isolated from arctic permafrost and analyzing their respective pathogenicities.

Crude oil degumming by phospholipid removal is crucial to guarantee oil quality. Phospholipase degumming could produce green vegetable oil by reducing energy consumption and protecting the environment. To develop a novel phospholipase for oil degumming, we cloned the Serratia marcescens outer membrane phospholipase A gene (OM-PLA1) and expressed its 33 KDa protein in engineered Escherichia coli BL21(DE3). OM-PLA1 activity reached 18.9 U mL−1 with the induction of 0.6 mM isopropyl β-D-1-thiogalactopyranoside for 4 h. The optimum temperature and pH were 50°C and 7.5, respectively. Mg2+, Ca2+, Co2+, and Mn2+ at 0.1 mM L−1 significantly increased OM-PLA1 activity. The kinetic equations of OM-PLA1 and Lecitase Ultra were y = 13.7x+0.74 (Km = 18.53 mM, Vmax = 1.35 mM min−1) and y = 24.42x+0.58 (Km = 42.1 mM, Vmax = 1.72 mM min−1), respectively. The phosphorus content decreased from 22.6 to 9.3 mg kg−1 with the addition of 15 units of free recombinant OM-PLA1 into 150 g of crude rapeseed oil. OM-PLA1 has the close degumming efficiency with Lecitase Ultra. The S. marcescens outer membrane phospholipase gene (OM-PLA1) possessed higher substrate affinity and catalytic efficiency than Lecitase Ultra. This study provides an alternative approach to achieve crude vegetable oil degumming with enzymatic technology.

Arctic bacteria employ various mechanisms to survive harsh conditions, one of which is to accumulate carbon and energy inside the cell in the form of polyhydroxyalkanoate (PHA). Whole-genome sequencing of a new Arctic soil bacterium Pseudomonas sp. B14-6 revealed two PHA-production-related gene clusters containing four PHA synthase genes (phaC). Pseudomonas sp. B14-6 produced poly(6% 3-hydroxybutyrate-co-94% 3-hydroxyalkanoate) from various carbon sources, containing short-chain-length PHA (scl-PHA) and medium-chain-length PHA (mcl-PHA) composed of various monomers analyzed by GC-MS, such as 3-hydroxybutyrate, 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, 3-hydroxydodecenoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecanoic acid. By optimizing the PHA production media, we achieved 34.6% PHA content using 5% fructose, and 23.7% PHA content using 5% fructose syrup. Differential scanning calorimetry of the scl-co-mcl PHA determined a glass transition temperature (Tg) of 15.3 °C, melting temperature of 112.8 °C, crystallization temperature of 86.8 °C, and 3.82% crystallinity. In addition, gel permeation chromatography revealed a number average molecular weight of 3.6 × 104, weight average molecular weight of 9.1 × 104, and polydispersity index value of 2.5. Overall, the novel Pseudomonas sp. B14-6 produced a polymer with high medium-chain-length content, low Tg, and low crystallinity, indicating its potential use in medical applications.

Polyhydroxyalkanoates (PHAs) are attractive new bioplastics for the replacement of plastics derived from fossil fuels. With their biodegradable properties, they have also recently been applied to the medical field. As poly(3-hydroxybutyrate) produced by wild-type Ralstonia eutropha has limitations with regard to its physical properties, it is advantageous to synthesize co- or terpolymers with medium-chain-length monomers. In this study, tung oil, which has antioxidant activity due to its 80% α-eleostearic acid content, was used as a carbon source and terpolymer P(53 mol% 3-hydroxybytyrate-co-2 mol% 3-hydroxyvalerate-co-45 mol% 3-hydroxyhexanoate) with a high proportion of 3-hydroxyhexanoate was produced in R. eutropha Re2133/pCB81. To avail the benefits of α-eleostearic acid in the tung oil-based medium, we performed partial harvesting of PHA by using a mild water wash to recover PHA and residual tung oil on the PHA film. This resulted in a film coated with residual tung oil, showing antioxidant activity. Here, we report the first application of tung oil as a substrate for PHA production, introducing a high proportion of hydroxyhexanoate monomer into the terpolymer. Additionally, the residual tung oil was used as an antioxidant coating, resulting in the production of bioactive PHA, expanding the applicability to the medical field.

The overuse of antibiotics has led to the emergence of multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). MRSA is difficult to kill with a single antibiotic because it has evolved to be resistant to various antibiotics by increasing the PBP2a (mecA) expression level, building up biofilm, introducing SCCmec for multidrug resistance, and changing its membrane properties. Therefore, to overcome antibiotic resistance and decrease possible genetic mutations that can lead to the acquisition of higher antibiotic resistance, drug combination therapy was applied based on previous results indicating that MRSA shows increased susceptibility to free fatty acids and surfactants. The optimal ratio of three components and the synergistic effects of possible combinations were investigated. The combinations were directly applied to clinically isolated strains, and the combination containing 15 μg/mL of oxacillin was able to control SCCmec type III and IV isolates having an oxacillin minimum inhibitory concentration (MIC) up to 1024 μg/mL; moreover, the combination with a slightly increased oxacillin concentration was able to kill SCCmec type II. Phospholipid analysis revealed that clinical strains with higher resistance contained a high portion of 12-methyltetradecanoic acid (anteiso-C15:0) and 14-methylhexadecanoic acid (anteiso-C17:0), although individual strains showed different patterns. In summary, we showed that combinatorial therapy with a low concentration of oxacillin controlled different laboratory and highly diversified clinical MRSA strains.

Rapid development in the printing and dying industry produces large amounts of wastewater, and its discharge in the environment causes pollution. Keeping in view the carcinogenic and mutagenic properties of various dyes, it is important to treat dyed wastewater. Maple leaf biochars were produced at different pyrolysis temperatures, i.e., 350 °C, 550 °C, and 750 °C, characterized for physicochemical properties and used for the removal of cationic (methylene blue (MB)) and anionic dye (congo red (CR)). Response surface methodology (RSM) using three variables, i.e., pH (4, 7, and 10), pyrolysis temperature (350 °C, 550 °C, and 750 °C), and adsorption temperature (20 °C, 30 °C, and 40 °C), was designed to find the optimum condition for dyes removal. X-ray diffraction (XRD) analysis showed an increase in CaCO3 crystallinity and a decrease in MgCO3 crystallinity with the increase of pyrolysis temperature. RSM design results showed that maple biochar showed maximum adsorption capacity for cationic dye at higher pH (9–10) and for anionic dye at pH 4-6, respectively. Under the selected condition of pH 7 and an adsorption temperature of 30 °C, biochar MB550 was able to remove MB and CR by 68% and 74%, respectively, from dye mixtures. Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses showed that MB550 was able to remove both dyes simultaneously from the aqueous mixtures.

Phenol-soluble modulins (PSMs) are major determinants of Staphylococcus aureus virulence and their increased production in community-associated methicillin-resistant S. aureus (CA-MRSA) likely contributes to the enhanced virulence of MRSA strains. Here, we analyzed the differences in bacterial cell aggregation according to PSM presence in the specific human cerebrospinal fluid (CSF) environment. CSF samples from the intraventricular or lumbar intrathecal area of each patient and tryptic soy broth media were mixed at a 1:1 ratio, inoculated with WT and PSM-deleted mutants (Δpsm) of the CA-MRSA strain, USA300 LAC, and incubated overnight. Cell aggregation images were acquired after culture and image analysis was performed. The cell aggregation ratio in WT samples differed significantly between the two sampling sites (intraventricular: 0.2% vs. lumbar intrathecal: 6.7%, p < 0.001). The cell aggregation ratio in Δpsm samples also differed significantly between the two sampling sites (intraventricular: 0.0% vs. lumbar intrathecal: 1.2%, p < 0.001). Division of the study cases into two groups according to the aggregated area ratio (WT/Δpsm; group A: ratio of ≥ 2, group B: ratio of < 2) showed that the median aggregation ratio value differed significantly between groups A and B (5.5 and 0, respectively, p < 0.001). The differences in CSF distribution and PSM presence within the specific CSF environment are significant factors affecting bacterial cell aggregation.

Polyhydroxyalkanoate (PHA) is a potential substitute for petroleum-based plastics and can be produced by many microorganisms, including recombinant Escherichia coli. For efficient conversion of substrates and maximum PHA production, we performed multiple engineering of branched pathways in E. coli. We deleted four genes (pflb, ldhA, adhE, and fnr), which contributed to the formation of byproducts, using the CRISPR/Cas9 system and overexpressed pntAB, which catalyzes the interconversion of NADH and NADPH. The constructed strain, HR002, showed accumulation of acetyl-CoA and decreased levels of byproducts, resulting in dramatic increases in cell growth and PHA content. Thus, we demonstrated the effects of multiple engineering for redirecting carbon flux into PHA production without any concerns regarding simultaneous deletion.