Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer ß-sheets, and ß-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 µM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5'-ACCCCAATTACGAAAATTTTT-3'), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

Enhancing column bioleaching of chalcocite by isolated iron metabolism partners Leptospirillum ferriphilum/Acidiphilium sp. coupling with systematically utilizing cellulosic waste.

The iron metabolism partners Leptospirillum ferriphilum and Acidiphilium sp. were screened from industrial bioheap site. An integrated multi-stage strategy was proposed to improve chalcolite column bioleaching coupling with synergistical utilization of cellulosic waste such as acid hydrolysate of aquatic plants. L. ferriphilum was used to accelerate the initial iron metabolism, and Acidithiobacillus caldus maintained a lower pH in the middle stage, while Acidiphilium sp. greatly inhibited jarosite passivation in the later stage. Meanwhile, L. ferriphilum (38.3 %) and Acidiphilium sp. (37.0 %) dominated the middle stage, while the abundance of Acidiphilium sp. reached 63.5 % in the later stage. The ferrous, sulfate ion and biomass were improved and the transcriptional levels of some biofilm and morphology related genes were significantly up-regulated. The final Cu2+ concentration reached 325.5 mg·L-1, improved by 43.8 %. Moreover, Canonical Correlation Analysis (CCA) analysis between bioleaching performance, iron/sulfur metabolism and community verified the important role of iron metabolism partners.

The digestive behavior of pectin in human gastrointestinal tract: a review on fermentation characteristics and degradation mechanism.

Pectin is widely spread in nature and it develops an extremely complex structure in terms of monosaccharide composition, glycosidic linkage types, and non-glycosidic substituents. As a non-digestible polysaccharide, pectin exhibits resistance to human digestive enzymes, however, it is easily utilized by gut microbiota in the large intestine. Currently, pectin has been exploited as a novel functional component with numerous physiological benefits, and it shows a promising prospect in promoting human health. In this review, we introduce the regulatory effects of pectin on intestinal inflammation and metabolic syndromes. Subsequently, the digestive behavior of pectin in the upper gastrointestinal tract is summarized, and then it will be focused on pectin's fermentation characteristics in the large intestine. The fermentation selectivity of pectin by gut bacteria and the effects of pectin structure on intestinal microecology were discussed to highlight the interaction between pectin and bacterial community. Meanwhile, we also offer information on how gut bacteria orchestrate enzymes to degrade pectin. All of these findings provide insights into pectin digestion and advance the application of pectin in human health.

Enhancement of acid tolerance of Escherichia coli by introduction of molecule chaperone CbpA from extremophile.

Molecular chaperone CbpA from extreme acidophile Acidithiobacillus caldus was applied to improve acid tolerance of Escherichia coli via CRISPR/Cas9. Cell growth and viability of plasmid complementary strain indicated the importance of cbpAAc for bacteria acid tolerance. With in situ gene replacement by CRISPR/Cas9 system, colony formation unit (CFU) of genome recombinant strain BL21-ΔcbpA/AccbpA showed 7.7 times higher cell viability than deficient strain BL21-ΔcbpA and 2.3 times higher than wild type. Cell morphology observation using Field Emission Scanning Electron Microscopy (FESEM) revealed cell breakage of BL21-ΔcbpA and significant recovery of BL21-ΔcbpA/AccbpA. The intracellular ATP level of all strains gradually decreased along with the increased stress time. Particularly, the value of recombinant strain was 56.0% lower than that of deficient strain after 5 h, indicating that the recombinant strain consumed a lot of energy to resist acid stress. The arginine concentration in BL21-ΔcbpA/AccbpA was double that of BL21-ΔcbpA, while the aspartate and glutamate contents were 14.8% and 6.2% higher, respectively, compared to that of wild type. Moreover, RNA-Seq analysis examined 93 genes down-regulated in BL21-ΔcbpA compared to wild type strain, while 123 genes were up-regulated in BL21-ΔcbpA/AccbpA compared to BL21-ΔcbpA, with an emphasis on energy metabolism, transport, and cell components. Finally, the working model in response to acid stress of cbpA from A. caldus was developed. This study constructed a recombinant strain resistant to acid stress and also provided a reference for enhancing microorganisms' robustness to various conditions.

Improving acid resistance of Escherichia coli base on the CfaS-mediated membrane engineering strategy derived from extreme acidophile.

Industrial microorganisms used for the production of organic acids often face challenges such as inhibited cell growth and reduced production efficiency due to the accumulation of acidic metabolites. One promising way for improving the acid resistance of microbial cells is to reconstruct their membranes. Herein, the overexpression of cfa2 from extreme acidophile endowed E. coli with high-performance on resistance to the acid stress. The engineered strain M1-93-Accfa2, constructed by CRISPR/Cas9-mediated chromosome integration, also exhibited a significantly higher resistance to severe acid stress. The analysis of fatty acid profiles indicated that the proportion of Cy-19:0 in the cell membrane of M1-93-Accfa2 increased by 5.26 times compared with the control, while the proportion of C18:1w9c decreased by 5.81 times. Correspondingly, the permeability and fluidity of the membrane decreased significantly. HPLC analysis demonstrated that the contents of intracellular glutamic acid, arginine, methionine and aspartic acid of M1-93-Accfa2 were 2.59, 2.04, 22.07 and 2.65 times that of the control after environmental acidification, respectively. Meanwhile, transmission electron microscopy observation indicated that M1-93-Accfa2 could maintain a plumper cell morphology after acid stimulation. M1-93-Accfa2 also exhibited higher-performance on the resistance to organic acids, especially succinic acid stress. These results together demonstrated the great potential of M1-93-Accfa2 constructed here in the production of organic acids.

Functional bacterial cellulose nanofibrils with silver nanoparticles and its antibacterial application.

Bacterial cellulose (BC) with good biocompatibility and superior mechanical properties has broad applications. BC functionalized with silver nanoparticles (AgNPs) has been assessed as an antimicrobial membrane for wound-healing treatment. During the AgNPs synthesis, avoiding the use of toxic chemicals is very necessary for the development of environmentally friendly procedures. Herein, a Komagataeibacter xylinus-based direct biosynthetic method to fabricate D-Saccharic acid potassium salt (SA)-grafted BC (SABC) through in situ bacterial metabolism was firstly explored. Subsequently, the SABC pellicles were immersed in AgNO3 solution for ion-exchanged process, and the silver nanoparticles (AgNPs) with diameter of ∼25.2 nm were in situ synthesized on SABC nanofiber surfaces by thermal reduction instead of using a reducing agent. The morphology and microstructure of SABC/AgNPs pellicles were analyzed by field-emission scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, and X-ray photoelectron spectra. Moreover, antibacterial activity measurement performed against the Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) by disk diffusion and plate count methods, showed high-efficiency bacteria-killing performance of SABC/AgNPs pellicles. This work proposed a new method by using microbial metabolism to prepare BC pellicles with functional groups, and antimicrobial films containing AgNPs was prepared by thermal reduction, exhibiting valuable prospects in wound healing treatment.

Molecular Insights into a Novel Cu(I)-Sensitive ArsR/SmtB Family Repressor in Extremophile Acidithiobacillus caldus.

Acidithiobacillus caldus is a common bioleaching bacterium that is inevitably exposed to extreme copper stress in leachates. The ArsR/SmtB family of metalloregulatory repressors regulates homeostasis and resistance in bacteria by specifically responding to metals. Here, we characterized A. caldus Cu(I)-sensitive repressor (AcsR) and gained molecular insights into this new member of the ArsR/SmtB family. Transcriptional analysis indicated that the promoter (PIII) of acsR was highly active in Escherichia coli but inhibited upon AcsR binding to the PIII-acsR region. Size exclusion chromatography and circular dichroism spectra revealed that CuI-AcsR shared an identical assembly state with apo-AcsR, as a dimer with fewer α helices, more extended strands, and more ß turns. Mutation of the cysteine site in AcsR did not affect its assembly state. Copper(I) titrations revealed that apo-AcsR bound two Cu(I) molecules per monomer in vitro with an average dissociation constant (KD) for bicinchoninic acid competition of 2.55 × 10-9 M. Site-directed mutation of putative Cu(I)-binding ligands in AcsR showed that replacing Cys64 with Ala reduces copper binding ability from two Cu(I) molecules per monomer to one, with an average KD of 6.05 × 10-9 M. Electrophoretic mobility shift assays revealed that apo-AcsR has high affinity for the 12-2-12 imperfect inverted repeats P2245 and P2270 in the acsR gene cluster and that Cu-loaded AcsR had lower affinity for DNA fragments than apo-AcsR. We developed a hypothetical working model of AcsR to better understand Cu resistance mechanisms in A. caldus. IMPORTANCE Copper (Cu) resistance among various microorganisms is attracting interest. The chemolithoautotrophic bacterium A. caldus, which can tolerate extreme copper stress (≥10 g/L Cu ions), is typically used to bioleach chalcopyrite (CuFeS2). Understanding of Cu resistance in A. caldus is limited due to scant investigation and the absence of efficient gene manipulation tools. Here, we characterized a new member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional proteins that repress operons linked to stress-inducing concentrations of heavy metal ions. This protein can bind two Cu(I) molecules per monomer and negatively regulate its gene cluster. Members of the ArsR/SmtB family have not been investigated in A. caldus until now. The discovery of this novel protein enriches understanding of Cu homeostasis in A. caldus.

Pulsed electric field as a promising technology for solid foods processing: A review.

A pulsed electric field (PEF) induces cell electroporation for solid foods, thereby allowing PEF to be used as a pretreatment method for extraction, drying, peeling, freeze-thawing, and cooking by increasing mass transfer. Currently, popular research mainly focuses on the process and results of the application of PEF to solid food processing. Therefore, this review summarizes the impact of PEF on the quality of solid food, the evaluation techniques of PEF-treated tissues and the characteristics of PEF treatment chambers. Furthermore, other pretreatments, including freezing and peeling, typically used in the meat and vegetable sectors, are also discussed alongside PEF to evaluate the impact on its effects. Finally, this article examined the main obstacles and prospects of PEF in solid food processing. This evaluation is anticipated to expand future PEF research paths in the solid food industry.

Orally Administered Xylo-Oligosaccharides (XOS) Ameliorates Diarrhea Symptoms in Mice via Intestinal Barrier Improvement and Gut Microbiota Modulation.

Xylo-oligosaccharides (XOS) has a bidirectional regulatory effect, and can be fermented by gut microbiota. This study aims to investigate how XOS alleviates Folium Sennae extracts induced diarrhea in mice. KM mice are given different concentration XOS for 3 weeks, and then Folium Sennae (Senna alexandrina Mill) extracts solutions (0.8 g mL-1 ) are given to establish diarrhea model. The results show that doses of XOS at 0.25 mg g-1 BW day-1 and higher could alleviate diarrhea symptoms by decreasing the disease activity indexes, down-regulating pro-inflammatory factors, up-regulating anti-inflammatory factor, and suppressing the intestinal permeability in colon tissues. XOS also regulates the composition of gut microbiota via increasing the abundance Prevotellaceae, Ruminococcus, and Bifidobacterium. The results suggest that the dietary supplementation XOS could be a more effective choice to prevent and alleviate diarrhea.

EpsRAc is a copper-sensing MarR family transcriptional repressor from Acidithiobacillus caldus.

The MarR family, as multiple antibiotic resistance regulators, is associated with the resistance of organisms to unfavorable conditions. MarR family extracellular polymeric substances (EPS)-associated transcriptional regulator (EpsRAc) was closely associated with copper resistance in Acidithiobacillus caldus (A. caldus). Transcriptional analysis showed high activity of the epsR promoter (PI) in Escherichia coli and differential response to metal ions. The copper content and UV absorption spectrum of the co-purified protein did not increase, but a stoichiometry of 0.667 mol Cu(I) per EpsRAc monomer was observed in vitro in copper titration experiments, suggesting that Cu(II) acts with low affinity in binding to the EpsRAc protein. Electrophoretic mobility shift assays (EMSA) demonstrated that EpsRAc could bind to its own promoter in vitro, and the binding region was the palindrome sequence TGTTCATCGTGTGTGAGCACACA. EpsRAc negatively regulated its own gene expression, whereas Cu(II) mitigates this negative effect. EpsRAc did not bind to other neighboring gene promoters. Finally, we developed a working model to illustrate the regulatory mechanism of A. caldus in response to extreme copper stress. KEY POINTS: • Identification of a MarR family EPS-associated transcriptional regulator, named EpsRAc. • Cu(I) can bind to the EpsRAc protein with low affinity. • EpsRAc negatively regulates the expression of epsR, and Cu(II) can alleviate this negative regulation.

Simultaneously pollutant removal and S0 recovery from composite wastewater containing Cr(VI)-S2- based on biofilm enhancement.

Bioaugmentation of extracellular polymeric substances-producing bacteria was applied in pollutant removal and S0 recovery from composite wastewater in a mixotrophic denitrification system. In the presence of 200 mg·L-1 S2- and 50 mg·L-1 Cr(VI), the removal efficiencies of chemical oxygen demand, NO3-, S2- and Cr(VI) were 86.38%, 91.82%, 95.75%, and 100.00% respectively, while S0 recovery efficiency reached 79.17%. Increased contents of protein and polysaccharide, especially the high ratio of protein/polysaccharide verified the structural stability of biofilm was promoted by biofilm enhancement. The widespread distribution of bacteria/extracellular polymeric substance (EPS) revealed the more obvious biofilms formation in biofilm-enhanced group. High-throughput sequencing analysis showed that EPS-producing bacteria (Flavobacterium, Thauera, Thiobacillus and Simplicispira) were dominant bacteria in the biofilm-enhanced group. Moreover, by comprehensive considering of redundancy analysis, the colonization of selected bacteria improved the robustness of the reactor and treatment performance to wastewater contained toxic pollutions.

Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity.

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis-fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126-fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.

Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

Simultaneous denitrification and desulfurization-S0 recovery of wastewater in trickling filters by bioaugmentation intervention based on avoiding collapse critical points.

In order to better achieve efficiently simultaneous desulfurization and denitrification/S0 recovery of wastewater, the intervention of sulfur oxidizing bacteria (SOB) and denitrifying bacteria (DNB) was employed to avoid the collapse critical points (the dramatically decrease of S/N removal efficiency) under the fluctuated load. With the assistance of DNB and SOB, collapse critical point of trickling filter (TF) was delayed from the P8 (105-114 d) to P10 stage (129-138 d). The treatment efficiency of nitrogen and sulfur was the highest with the S/N ratio of 3:1. The bioaugmentation of DNB and SOB at collapse critical point could effectively regulated collapse situation, which further increased the maximum system utilization/elimination capacity to 4.50 kg S m-3·h-1 and 0.90 kg N m-3·h-1 (increased by 56.89% and 65.56% in comparison to control). High-throughput sequencing analysis indicated that Proteobacteria (average 78.59%) and Bacteroidetes (average 9.30%) were dominant bacteria in the reactor at all stages. As the reaction proceeds, the microbial community was gradually dominated by some functional genera such as Chryseobacterium (average 2.97%), Halothiobacillus (average 22.71%), Rhodanobacter (average 14.02%), Thiobacillus (average 9.01%), Thiomonas (average 16.70%) and Metallibacterium (average 21.63%), which could remove nitrate or sulfide. Both of Principal Component Analysis (PCA) and Canonical Correlation Analysis (CCA) demonstrated the important role of DNB/SOB during the long-term run in the trickling filters (TFs).

The adaptation mechanisms of Acidithiobacillus caldus CCTCC M 2018054 to extreme acid stress: Bioleaching performance, physiology, and transcriptomics.

To understand the acid-resistant mechanism of bioleaching microorganism Acidithiobacillus caldus CCTCC M 2018054, its physiology and metabolic changes at the transcriptional level under extreme acid stress were systemically studied. Scanning electron microscopy (SEM), Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) showed that with an increase in acidity, the absorption peak of sulfur oxidation-related functional groups such as S-O decreased significantly, and a dense sulfur passivation film appeared on the surface of the ore. Confocal laser scanning microscopy (CLSM) revealed that coverage scale of extracellular polymeric substance (EPS) and biofilm fluctuated accordingly along with the increasing acid stress (pH-stat 1.5, 1.2 0.9 and 0.6) during the bioleaching process. In response to acid stress, the increased levels of intracellular glutamic acid, alanine, cysteine, and proline contributed to the maintenance of intracellular pH homeostasis via decarboxylation and alkaline neutralization. Higher unsaturated fatty acid content was closely related to membrane fluidity. Up to 490 and 447 differentially expressed genes (DEGs) were identified at pH 1.5 vs pH 1.2 and pH 1.2 vs pH 0.9, respectively, and 177 common DEGs were associated with two-component system (TCS) regulation, transporter regulation, energy metabolism, and stress response. The upregulation of kdpB helped cells defend against proton invasion, whereas the downregulation of cysB and cbl implied stronger oxidation of sulfur compounds. The transcriptional level of sqr, sor, and soxA was significantly increased and consolidated the energy supply needed for resisting acid stress. Furthermore, eight of the identified DEGs (sor, cbl, ompA, atpF, nuoH, nuoC, sqr, grxB) were verified as being related to the acid stress response process. This study contributes toward expanding the application of these acidophiles in industrial bioleaching.

Enhancement of the Isomerization Activity and Thermostability of Cellobiose 2-Epimerase from Caldicellulosiruptor saccharolyticus by Exchange of a Flexible Loop.

Cellobiose 2-epimerase (CE) offers a promising enzymatic approach to produce lactulose. However, its application is limited by the unsatisfactory isomerization activity and thermostability. Our study attempted to optimize the catalytic performances of CEs by flexible loop exchange, for which four mutants were constructed using CsCE (CE from Caldicellulosiruptor saccharolyticus) as a template. As a result, all mutants maintained the same catalytic directions as the templates. Mutant RmC displayed a 2.2- and 1.34-fold increase in the isomerization activity and catalytic efficiency, respectively. According to the results of molecular dynamics (MD) simulations, it was revealed that the loop exchange in RmC enlarged the entrance of the active site for substrate binding and benefited proton transfer involved in the isomerization process. Besides, the t1/2 of mutant StC at 70 °C was increased from 29.07 to 38.29 h, owing to the abundance of rigid residues (proline) within the flexible loop of StC. Our work demonstrated that the isomerization activity and thermostability of CEs were closely related to the flexible loop surrounding the active site, which provides a new perspective to engineer CEs for higher lactulose production.

Simultaneously enhance iron/sulfur metabolism in column bioleaching of chalcocite by pyrite and sulfur oxidizers based on joint utilization of waste resource.

In chalcocite (Cu2S) bioleaching, the lack of iron metabolism is a key restricting factor. As the most common sulfide mineral, pyrite (FeS2) can release Fe(Ⅱ) and compensate for the iron metabolism deficiency in chalcocite bioleaching. The bioleaching of chalcocite in an imitated industrial system was improved by enhancing the iron-sulfur metabolism simultaneously using pyrite and sulfur oxidizers based on the joint utilization of waste resources, while the bioleaching performance and community structure in the leachate were systematically investigated. Due to the active sulfur/iron metabolism, the pH reached 1.2, and Fe3+ was increased by 77.78%, while the biomass of planktonic cells was improved to 2.19 × 107 cells/mL. Fourier transform infrared reflection (FTIR) and X-ray diffraction (XRD) analysis results showed that more iron-sulfur crystals were produced due to more active iron-sulfur metabolism. Scanning electron microscopy (SEM) revealed that many derivative particles and corrosion marks appeared on the surface of the ore, implying that the mineral-microbe interaction was strengthened. Confocal laser scanning microscopy (CLSM) showed the accumulation of cells and extracellular polymeric substances (EPS) on the ore surface, indicating a stronger contact leaching mechanism. Furthermore, the community structure and canonical correspondence analysis (CCA) demonstrated that the introduction of sulfur-oxidizing bacteria and pyrite could maintain the diversity of dominant leaching microorganisms at a high level. Sulfobacillus (27.75%) and Leptospirllillum (20.26%) were the dominant sulfur-oxidizing and iron-oxidizing bacteria during the bioleaching process. With the accumulation of multiple positive effects, the copper ion leaching rate was improved by 44.8%. In general, this new type of multiple intervention strategy can provide an important guide for the bioleaching of low-grade ores.

Bioaugmentation potential evaluation of a bacterial consortium composed of isolated Pseudomonas and Rhodococcus for degrading benzene, toluene and styrene in sludge and sewage.

Bioaugmentation was conducted using a bacterial consortium of Pseudomonas putida SW-3 and Rhodococcus ruber SS-4, to test their ability to degrade benzene, toluene, and styrene (BTS). SW-3 and SS-4 were isolated from domestic sludge and sewage samples to establish a synthetic consortium with an optimized ratio of 2:1 to reach a degradation efficiency of 82.5-89.8% of BTS. The bacterial consortium was inoculated with sludge and sewage samples at a ratio of 2:1, resulting in a degradation efficiency of 97.9% and 92.7%, respectively, at a BTS concentration of 1800 mg·L-1. Analysis of bacterial community structure following bioaugmentation indicated an increase in abundance of BTS-degrading bacteria, particularly Acinetobacter and Pseudoxanthomonas in sludge and Pseudomonas in sewage, enhancing the collective BTS degradation ability of the bacterial community. Principal component analysis demonstrated that a more balanced bacterial community structure was established following intervention. This indicated that the selected bacteria are excellent candidates for bioaugmentation.

Chitosan/casein based microparticles with a bilayer shell-core structure for oral delivery of nattokinase.

Nattokinase is a thrombolytic enzyme obtained from Japanese traditional food natto for prevention and cure of thrombosis-related cardiovascular diseases. However, the effectiveness of nattokinase through oral intake is limited, due to the loss of thrombolytic activity in the acidic gastric juice. In this study, we develop a functional oral delivery system of nattokinase, in which chitosan microparticles were used as the carrier core to load nattokinase via genipin crosslinking and then covered by a casein-based protective shell via transglutaminase (TG) crosslinking. The results of in vitro and in vivo assays, in the aspects of bioactivity, release dynamics, and therapeutic effects, indicated that the bilayer shell-core structure could protect loaded nattokinase from destruction in the gastric juice and achieve its controlled-release in the intestine. This work demonstrates the availability of bilayer shell-core structure design in oral delivery of nattokinase and shows its high potential for application as an anti-thrombosis functional food additive.

Adaptive defensive mechanism of bioleaching microorganisms under extremely environmental acid stress: Advances and perspectives.

Bioleaching microorganisms inhabit extremely acidic environments (pH < 3.0) and can be used in the biohydrometallurgical industry. They employ several strategies, such as biofilms formation, mechanical defense, membrane reversal potential, proton efflux, intracellular buffering, and protection, or repair mechanism of biomacromolecules, to maintain their intracellular pH within a narrow range, which is close to neutral, for conducting normal physiological activity. In this review, we describe the effects of these strategies on the homeostasis of intracellular pH of bioleaching microorganisms and the relevant energy metabolism. The potential significance of horizontal gene transfer and gene loss in their adaptation to the environment, and the prospect of new technologies, such as cryo EM technology, in revealing the potential acid-resistant components have also been discussed.