Immobilization of exopolymeric substances from bacteria for metal removal: A study on characterization, optimization, reusability and toxicity.

This study investigated the immobilization of exopolymeric substances (EPS) from Bacillus cereus using sodium alginate to form EPS beads for metal removal. The EPS beads were characterized and their optimum biosorption conditions established (biosorbent dosage, initial metal concentration and pH of metal solutions). The EPS beads were also tested for reusability by using them continuously for five metal removal cycles with desorption process in between cycles. The toxicity of the treated metal solutions was tested by phytotoxicity tests. Results revealed that EPS beads demonstrated significantly higher metal removal efficiency (Pb: 99.26%, Cr: 50.73%, Cu: 48.94%, Zn: 29.81%, Cd: 20.29%) compared to plain alginate beads (without EPS) (Pb: 84.45%, Cu: 31%, Cr: 28.37%, Zn: 11.91%, Cd: 9.37%). SEM-EDX analysis detected Cu, Pb, Zn, Cd and Cr on the surface of EPS beads. Optimum conditions for Pb removal by EPS beads were from the use of 0.1 g of biosorbent at 100 mg/L initial metal concentration and pH 5. By contrast, Cu, Zn, Cd and Cr were optimally removed by 0.3 g of biosorbent at 25 mg/L initial metal concentration and pH 5. EPS beads can be reused up to five times while maintaining a high rate of metal removal efficiency (Pb- 99.52%, Cr- 89.23%, Cu- 89.17%, Zn-52.52%, Cd-39.12%). This was achieved through desorption with nitric acid that consistently recovered 76-93% of the metal adsorbed. FTIR analysis reveals that nitric acid is capable of restoring the functional groups present within EPS beads, allowing it to bind with metal ions in repeated cycles. Metal solutions treated with EPS beads were less toxic as seedling shoots (pre-treated: 0-10 cm, post-treated: 1.2-18.1 cm) and roots (pre-treated: 0-7.8 cm, post-treated: 0.8-15.1 cm) grew well, which suggested that reduced levels of metals led to reduced phytotoxicity. This study provides an insight into the use of EPS beads for metal removal, highlighting the benefits and reusability of the beads for future wastewater treatment.

Biodiscovery of Potential Antibacterial Diagnostic Metabolites from the Endolichenic Fungus Xylaria venustula Using LC-MS-Based Metabolomics.

Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC-MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for "dual" bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.

Mechanisms for metal removal established via electron microscopy and spectroscopy: a case study on metal tolerant fungi Penicillium simplicissimum.

Penicillium simplicissimum (isolate 10), a metal tolerant fungus, tolerated 1000 mg/L Cu and 500 mg/L Zn, but were inhibited by Cd (100 mg/L), evident by the Tolerance Index (TI) of 0.88, 0.83, and 0.08, respectively. Live cells of P. simplicissimum were more effective in removing Cr (88.6%), Pb (73.7%), Cu (63.8%), Cd (33.1%), and Zn (28.3%) than dead cells (5.3-61.7%). Microscopy approach via SEM-EDX and TEM-EDX suggested that metal removal involved biosorption and bioaccumulation, with metal precipitates detected on the cell wall, and in the cytoplasm and vacuoles. FTIR analysis revealed metals interacted with amino, carbonyl, hydroxyl, phosphoryl (except Cd) and nitro groups in the cell wall. Biosorption and bioaccumulation of metals by live cells reduced Cu and Pb toxicity, observed from good root and (4.00-4.28 cm) and shoot (8.07-8.36 cm) growth of Vigna radiata in the phytotoxicity assay.

The role of reactive oxygen species in the antimicrobial activity of pyochelin.

The increase in prevalence of antimicrobial-resistant bacteria (ARB) is currently a serious threat, thus there is a need for new antimicrobial compounds to combat infections caused by these ARB. An antimicrobial-producing bacterium, Burkholderia paludis was recently isolated and was able to produce a type of siderophore with antimicrobial properties, later identified as pyochelin. The chelating ability of pyochelin has been well-characterized but not for its antimicrobial characteristics. It was found that pyochelin had MIC values (MBC values) of 3.13 µg/mL (6.26 µg/mL) and 6.26 µg/mL (25.00 µg/mL) against three Enterococcus strains and four Staphylococcus strains. Pyochelin was able to inhibit E. faecalis ATCC 700802 (a vancomycin-resistant strain) in a time and dose dependent manner via killing kinetics assay. It was demonstrated that pyochelin enhanced the production of intracellular reactive oxygen species (ROS) over time, which subsequently caused a significant increase in malondialdehyde (MDA) production (a marker for lipid peroxidation) and ultimately led to cell death by disrupting the integrity of the bacterial membrane (validated via BacLight assay). This study has revealed the mechanism of action of pyochelin as an antimicrobial agent for the first time and has shown that pyochelin might be able to combat infections caused by E. faecalis in the future.

Intrinsic capabilities of Leptospermum javanicum in inducing apoptosis and suppressing the metastatic potential of human lung carcinoma cells.

The active isolate of LF1 in Leptospermum javanicum was further looked into its capabilities in provoking an apoptotic reaction and suppressing the metastasis process in treated non-small lung cancer cells. LF1 underwent isolation and purification to yield a white powder which was identified as Betulinic acid (BA) via NMR, LCMS and IR spectroscopy. The isolate, BA, which produced an encouraging cytotoxic effect against non-small lung cancer cells (A549 and NCI-H1299) through the MTT assay, was further assessed with TUNEL, Sub-G1 population quantification, acridine orange/ethidium bromide staining as well as activated caspase-3 detection. The results pointed towards the induction of apoptosis as a result of increasing doses of BA, regardless of the p53 status in both cell lines. Treatment with BA also prevented an effective attachment of the invasive A549 cells onto a new culture surface in addition to diminishing the migratory potential of treated cells across a porous membrane. Further investigation through the ELISA detection and gelatin zymography showed an adverse effect to production of matrix metalloproteinase-2 (MMP-2) while the levels of matrix metalloproteinase-9 (MMP-9) were not negatively affected. The findings from this study validate the potential of L. javanicum as a potential anti-cancer treatment as stated in our previous study. The isolate, BA not only showed a capacity in inducing apoptotic cell death in non-small lung cancer cells, but managed to distort the ability of the cancer cells in effectively undergoing the metastasis process.

A novel study based on adaptive metal tolerance behavior in fungi and SEM-EDX analysis.

Four fungal isolates: Simplicillium chinense (iso 9, accession no. KX425621), Penicillium simplicissimum (iso 10, KP713758), Trichoderma asperellum (iso 11, KP792512), and Coriolopsis sp. (1c3, KM403574) were subjected to a series of induced-tolerance training under high metal concentrations to determine if greater tolerance could be achieved from constant exposure to such conditions. Adaptive tolerance assay (Tolerance Index, TI) and Field-Emission Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX) characterized their metal tolerance. "Untrained" S. chinense, P. simplicissimum and T. asperellum showed tolerance towards 4000-4500ppm Al(III) (TI: 0.64-0.71), 1000ppm Cr(III) (0.52-0.83) and Pb(II) (0.32-0.88). With tolerance training, tolerance towards 2000-6000ppm Al(III), 500-3000ppm Pb(II) and 2000-3000ppm Cr(III) were achieved (TI: 0.01-0.82) compared to untrained cultures (0.00-0.59). In contrast, tolerance training for Coriolopsis sp. and P. simplicissimum was less successful, with TI values similar or lower than untrained cultures. SEM-EDX analysis proposed biosorption and bioaccumulation as mechanisms for metal removal. The latter was demonstrated with the removal of Cr(III) and Pb(II) by S. chinense (12.37 and 11.52mgg-1, respectively) and T. asperellum (10.44 and 7.50mgg-1). Induced-tolerance training may render benefit in the long run, but this delicate approach is suggestively species and metal dependent.

Newly Isolated Paenibacillus tyrfis sp. nov., from Malaysian Tropical Peat Swamp Soil with Broad Spectrum Antimicrobial Activity.

Emergence of antimicrobial resistance coupled with the slowdown in discovery of new antimicrobial compounds points to serious consequences for human health. Therefore, scientists are looking for new antimicrobial compounds from unique and understudied ecosystems such as tropical peat swamp forests. Over the course of isolating antimicrobial producing bacteria from North Selangor tropical peat swamp forest, Malaysia, a Gram variable, rod shaped, endospore forming, facultative anaerobic novel strain MSt1(T) that exerts potent and broad spectrum antimicrobial activity was isolated. Phylogenetic analysis using 16S rRNA gene sequences showed that strain MSt1(T) belonged to the genus Paenibacillus with the highest similarity to Paenibacillus elgii SD17(T) (99.5%). Whole genome comparison between strain MSt1(T) with its closely related species using average nucleotide identity (ANI) revealed that similarity between strain MSt1(T) with P. elgii B69 (93.45%) and Paenibacillus ehimensis A2 (90.42%) was below the recommended threshold of 95%. Further analysis using in silico pairwise DDH also showed that similarity between strain MSt1(T) with P. elgii B69 (55.4%) and P. ehimensis A2 (43.7%) was below the recommended threshold of 70%. Strain MSt1(T) contained meso-diaminopilemic acid in the cell wall and MK-7 as the major menaquinone. The major fatty acids of strain MSt1(T) were anteiso-C15:0 (48.2%) and C16:0 (29.0%) whereas the polar lipid profile consisted of phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, one unknown lipid, two unknown glycolipids, and one unknown phospholipid. Total DNA G+C content of strain MSt1(T) was 51.5 mol%. The extract from strain MSt1(T) exerted strong antimicrobial activity against Escherichia coli ATCC 25922 (MIC = 1.5 µg/mL), MRSA ATCC 700699 (MIC = 25 µg/mL) and Candida albicans IMR (MIC = 12.5 µg/mL). Partially purified active fraction exerted a strong effect against E. coli ATCC 25922 resulting in cell rupture when viewed with SEM. Based on distinctive taxonomic differences between strain MSt1(T) when compared to its closely related type species, we propose that strain MSt1(T) represents a novel species within the genus of Paenibacillus, for which the name Paenibacillus tyrfis sp. nov. (= DSM 100708(T) = MCCC 1K01247(T)) is proposed.

Burkholderia paludis sp. nov., an Antibiotic-Siderophore Producing Novel Burkholderia cepacia Complex Species, Isolated from Malaysian Tropical Peat Swamp Soil.

A novel Gram negative rod-shaped bacterium, designated strain MSh1T, was isolated from Southeast Pahang tropical peat swamp forest soil in Malaysia and characterized using a polyphasic taxonomy approach. The predominant cellular fatty acids (>10.0%) were C16:0 (31.7%), C17:0 cyclo (26.6%), and C19:0 cyclo ω8c (16.1%). The polar lipids detected were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The predominant ubiquinone was Q-8. This revealed that strain MSh1T belongs to the genus Burkholderia. The type strain MSh1T can be differentiated from other Burkholderia cepacia complex (Bcc) species by phylogenetic analysis of 16S rRNA gene sequence, multilocus sequence analysis (MLSA), average nucleotide identity (ANI) and biochemical tests. DNA-DNA relatedness values between strain MSh1T and closely related type strains were below the 70% threshold value. Based on this polyphasic study of MSh1T, it can be concluded that this strain represents a novel species within the Bcc, for which the name Burkholderia paludis sp. nov. is proposed. The type strain is MSh1T (= DSM 100703T = MCCC 1K01245T). The dichloromethane extract of MSh1T exhibited antimicrobial activity against four Gram positive bacteria (Enterococcus faecalis ATCC 29212, E. faecalis ATCC 700802, Staphylococcus aureus ATCC 29213, S. aureus ATCC 700699) and a Gram negative bacteria (Escherichia coli ATCC 25922). Further purification work has led to the isolation of Compound 1, pyochelin. Pyochelin demonstrated antimicrobial activity against four S. aureus strains and three E. faecalis strains with MIC-values of 3.13 µg/ml and 6.26 µg/ml, respectively. SEM analysis showed that the cellular morphology of E. faecalis ATCC 700802 was not affected by pyochelin; suggesting that it might target the intracellular components. Pyochelin, a siderophore with antimicrobial activity might be useful in treating bacterial infections caused by S. aureus and E. faecalis, however further work has to be done.