Preliminary forensic assessment of the visualised fingerprints on nonporous substrates immersed in water using the green and optimised novel nanobio-based reagent.

The discovery of forensic evidence (e.g. weapons) during forensic underwater investigations has seen an increasing trend. To date, small particle reagent (SPR) has been one of the routinely used methods for visualising fingerprints on wet, non-porous substrates. However, the long term use of SPR is detrimental to humans and environment due to the use of toxic chemicals. Although previously we have successfully developed and optimised a greener nanobio-based reagent (NBR), its suitable practical use in a more realistic scene (e.g. outdoor pond) was not evaluated. Therefore, this present research is aimed at (1) investigating the performance of NBR against the benchmark SPR in visualising fingerprints immersed in a natural outdoor pond and (2) evaluating the greenness of NBR against the analytical Eco-Scale. Results showed that the performance of the optimised NBR was mostly comparable (University of Canberra (UC) comparative scale: 0) with SPR at visualising fingerprints on three different non-porous substrates immersed in a natural outdoor pond. Observably, the NBR had higher preference towards aged fingerprints (up to 4 weeks of immersion). In addition, its greenness assessment revealed 76 points, indicating 'excellent green analysis'. The findings gathered here further supported the practical use of the NBR in forensic investigations.

Hepatoprotective effects of flexirubin, a novel pigment from Chryseobacterium artocarpi, against carbon tetrachloride-induced liver injury: An in vivo study and molecular modeling.

Liver injuries caused by various industrial chemicals represent a serious health concern worldwide. Flexirubins are a novel class of naturally occurring bacterial pigments whose bioactivity remains largely unexplored. The present study evaluated the hepatoprotective effects of flexirubin pigment extracted from the bacterium Chryseobacterium artocarpi against CCl4-induced acute liver injury in mice. Flexirubin was applied at three different oral doses, 125, 250 and 500 mg/kg bw/d for seven consecutive days. Treatment of animals with flexirubin before exposure to CCl4 (10 mL/kg bw dissolved in olive oil, 1:1 v/v) significantly decreased the elevated serum levels of ALT, AST, ALP, LDH and TBL. Flexirubin pretreatment showed a great capability for attenuating the CCl4-induced oxidative stress by decreasing the level of liver MDA, and increasing the antioxidant enzyme activities of liver SOD and CAT, and the levels of GSH and TAC. Flexirubin also alleviated the histopathological alterations in liver by prohibiting steatosis, ballooning degeneration, leukocytic infiltration and necrosis. Immunohistochemical analysis demonstrated that flexirubin has a significant anti-apoptotic activity against CCl4 via upregulation of Bcl-2, and downregulation of Bax, Caspase-3 and TGF-ß1. Flexirubin also exhibited a remarkable anti-inflammatory activity against CCl4 through its suppressive action on TNF-α, COX-2 and CD-45. Flexirubin could trigger upregulation of the Nrf2/HO-1 signaling pathway mediating protection against CCl4. In silico molecular docking revealed flexirubin as a potential inhibitor against two target proteins, TGF-ß1 and TACE. The results proved the effectiveness of flexirubin as a significant source of natural compounds for its use in drug formulation strategies to offer protection against hepatotoxins.

Antioxidant Activity Evaluation of FlexirubinType Pigment from Chryseobacterium artocarpi CECT 8497 and Related Docking Study.

The current research is focused on studying the biological efficacy of flexirubin, a pigment extracted from Chryseobacterium artocarpi CECT 8497.Different methods such as DPPH, H2O2, NO•, O2•-, •OH, lipid peroxidation inhibition by FTC and TBA, ferric reducing and ferrous chelating activity were carried out to evaluate the antioxidant activity of flexirubin. Molecular docking was also carried out, seeking the molecular interactions of flexirubin and a standard antioxidant compound with SOD enzyme to figure out the possible flexirubin activity mechanism. The new findings revealed that the highest level of flexirubin exhibited similar antioxidant activity as that of the standard compound according to the H2O2, •OH, O2•-, FTC and TBA methods. On the other hand, flexirubin at the highest level has shown lower antioxidant activity than the positive control according to the DPPH and NO• and even much lower when measured by the FRAP method. Molecular docking showed that the interaction of flexirubin was in the binding cavity of the SOD enzyme and did not affect its metal-binding site. These results revealed that flexirubin has antioxidant properties and can be a useful therapeutic compound in preventing or treating free radical-related diseases.

Box-Behnken design optimisation of a green novel nanobio-based reagent for rapid visualisation of latent fingerprints on wet, non-porous substrates.

OBJECTIVE: Optimisation of the green novel nanobio-based reagent (NBR) for rapid visualisation of groomed fingerprints on wet non-porous substrates using response surface methodology and assessment of its stability and sensitivity were attempted for forensic applications. RESULTS: Scanning electron microscopy images demonstrated successful attachments of NBR onto the constituents of fingerprints on the substrates. The highest average quality of visualised fingerprints was attained at the optimum condition (100 mg of CRL; 75 mg of acid-functionalised multi-walled carbon nanotubes; 5 h of immobilisation). The NBR produced comparable average quality of fingerprints with the commercially available small particle reagent, even after 4 weeks of storage (without any preservatives) in both chilled and sultry conditions. The NBR was sensitive enough to visualise the increasingly weaker fingerprints, particularly on glass slides. CONCLUSION: The optimised novel NBR could be the relatively greener option for visualising latent fingerprints on wet, non-porous substrates for forensic applications.

Antibacterial mode of action of violacein from Chromobacterium violaceum UTM5 against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA).

Violacein, violet pigment produced by Chromobacterium violaceum, has attracted much attention recently due to its pharmacological properties including antibacterial activity. The present study investigated possible antibacterial mode of action of violacein from C. violaceum UTM5 against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) strains. Violet fraction was obtained by cultivating C. violaceum UTM5 in liquid pineapple waste medium, extracted, and fractionated using ethyl acetate and vacuum liquid chromatography technique. Violacein was quantified as major compound in violet fraction using HPLC analysis. Violet fraction displayed bacteriostatic activity against S. aureus ATCC 29213 and methicillin-resistant S. aureus ATCC 43300 with minimum inhibitory concentration (MIC) of 3.9 µg/mL. Fluorescence dyes for membrane damage and scanning electron microscopic analysis confirmed the inhibitory effect by disruption on membrane integrity, morphological alternations, and rupture of the cell membranes of both strains. Transmission electron microscopic analysis showed membrane damage, mesosome formation, and leakage of intracellular constituents of both bacterial strains. Mode of action of violet fraction on the cell membrane integrity of both strains was shown by release of protein, K+, and extracellular adenosine 5'-triphosphate (ATP) with 110.5 µg/mL, 2.34 µg/mL, and 87.24 ng/µL, respectively, at 48 h of incubation. Violet fraction was toxic to human embryonic kidney (HEK293) and human fetal lung fibroblast (IMR90) cell lines with LC50 value of 0.998 ± 0.058 and 0.387 ± 0.002 µg/mL, respectively. Thus, violet fraction showed a strong antibacterial property by disrupting the membrane integrity of S. aureus and MRSA strains. This is the first report on the possible mode of antibacterial action of violet fraction from C. violaceum UTM5 on S. aureus and MRSA strains.

Synthesis of flexirubin-mediated silver nanoparticles using Chryseobacterium artocarpi CECT 8497 and investigation of its anticancer activity.

In this work, the synthesis of silver nanoparticles from a pigment produced by a recently-discovered bacterium, Chryseobacterium artocarpi CECT 8497, was achieved, followed by an investigation of its anticancer properties. The bacterial pigment was identified as flexirubin following NMR ((1)H NMR and (13)C NMR), UV-Vis, and LC-MS analysis. An aqueous silver nitrate solution was treated with isolated flexirubin to produce silver nanoparticles. The synthesised silver nanoparticles were subsequently characterised by UV-Vis spectroscopy, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDX), X-Ray Diffraction (XRD), and Fourier Transform Infrared (FTIR) Spectroscopy methodologies. Furthermore, the anticancer effects of synthesised silver nanoparticles in a human breast cancer cell line (MCF-7) were evaluated. The tests showed significant cytotoxicity activity of the silver nanoparticles in the cultured cells, with an IC50 value of 36µgmL(-1). This study demonstrates that silver nanoparticles, synthesised from flexirubin from C. artocarpi CECT 8497, may have potential as a novel chemotherapeutic agent.

Optimization of culture conditions for flexirubin production by Chryseobacterium artocarpi CECT 8497 using response surface methodology.

Flexirubins are the unique type of bacterial pigments produced by the bacteria from the genus Chryseobacterium, which are used in the treatment of chronic skin disease, eczema etc. and may serve as a chemotaxonomic marker. Chryseobacterium artocarpi CECT 8497, an yellowish-orange pigment producing strain was investigated for maximum production of pigment by optimizing medium composition employing response surface methodology (RSM). Culture conditions affecting pigment production were optimized statistically in shake flask experiments. Lactose, l-tryptophan and KH2PO4 were the most significant variables affecting pigment production. Box Behnken design (BBD) and RSM analysis were adopted to investigate the interactions between variables and determine the optimal values for maximum pigment production. Evaluation of the experimental results signified that the optimum conditions for maximum production of pigment (521.64 mg/L) in 50 L bioreactor were lactose 11.25 g/L, l-tryptophan 6 g/L and KH2PO4 650 ppm. Production under optimized conditions increased to 7.23 fold comparing to its production prior to optimization. Results of this study showed that statistical optimization of medium composition and their interaction effects enable short listing of the significant factors influencing maximum pigment production from Chryseobacterium artocarpi CECT 8497. In addition, this is the first report optimizing the process parameters for flexirubin type pigment production from Chryseobacterium artocarpi CECT 8497.

Chryseobacterium artocarpi sp. nov., isolated from the rhizosphere soil of Artocarpus integer.

A bacterial strain, designated UTM-3(T), isolated from the rhizosphere soil of Artocarpus integer (cempedak) in Malaysia was studied to determine its taxonomic position. Cells were Gram-stain-negative, non-spore-forming rods, devoid of flagella and gliding motility, that formed yellow-pigmented colonies on nutrient agar and contained MK-6 as the predominant menaquinone. Comparative analysis of the 16S rRNA gene sequence of strain UTM-3(T) with those of the most closely related species showed that the strain constituted a distinct phyletic line within the genus Chryseobacterium with the highest sequence similarities to Chryseobacterium lactis NCTC 11390(T), Chryseobacterium viscerum 687B-08(T), Chryseobacterium tructae 1084-08(T), Chryseobacterium arthrosphaerae CC-VM-7(T), Chryseobacterium oncorhynchi 701B-08(T), Chryseobacterium vietnamense GIMN1.005(T), Chryseobacterium bernardetii NCTC 13530(T), Chryseobacterium nakagawai NCTC 13529(T), Chryseobacterium gallinarum LMG 27808(T), Chryseobacterium culicis R4-1A(T), Chryseobacterium flavum CW-E2(T), Chryseobacterium aquifrigidense CW9(T), Chryseobacterium ureilyticum CCUG 52546(T), Chryseobacterium indologenes NBRC 14944(T), Chryseobacterium gleum CCUG 14555(T), Chryseobacterium jejuense JS17-8(T), Chryseobacterium oranimense H8(T) and Chryseobacterium joostei LMG 18212(T). The major whole-cell fatty acids were iso-C15 : 0 and iso-C17 : 1ω9c, followed by summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1ω7t) and iso-C17 : 0 3-OH, and the polar lipid profile consisted of phosphatidylethanolamine and several unknown lipids. The DNA G+C content strain UTM-3(T) was 34.8 mol%. On the basis of the phenotypic and phylogenetic evidence, it is concluded that the isolate represents a novel species of the genus Chryseobacterium, for which the name Chryseobacterium artocarpi sp. nov. is proposed. The type strain is UTM-3(T) ( = CECT 8497(T) = KCTC 32509(T)).

Isotherm kinetics of Cr(III) removal by non-viable cells of Acinetobacter haemolyticus.

The potential use of non-viable biomass of a Gram negative bacterium i.e. Acinetobacter haemolyticus to remove Cr(III) species from aqueous environment was investigated. Highest Cr(III) removal of 198.80 mg g(-1) was obtained at pH 5, biomass dosage of 15 mg cell dry weight, initial Cr(III) of 100 mg L(-1) and 30 min of contact time. The Langmuir and Freundlich models fit the experimental data (R(2)>0.95) while the kinetic data was best described using the pseudo second-order kinetic model (R(2)>0.99). Cr(III) was successfully recovered from the bacterial biomass using either 1M of CH(3)COOH, HNO(3) or H(2)SO(4) with 90% recovery. TEM and FTIR suggested the involvement of amine, carboxyl, hydroxyl and phosphate groups during the biosorption of Cr(III) onto the cell surface of A. haemolyticus. A. haemolyticus was also capable to remove 79.87 mg g(-1) Cr(III) (around 22.75%) from raw leather tanning wastewater. This study demonstrates the potential of using A. haemolyticus as biosorbent to remove Cr(III) from both synthetic and industrial wastewater.

Bacterial reduction of Cr(VI) at technical scale--the Malaysian experience.

The bacterial reduction of Cr(VI) from industrial wastewater was evaluated using a 2.0-m(3) bioreactor. Liquid pineapple waste was used as a nutrient for the biofilm community formed inside the bioreactor. The use of rubber wood sawdust as packing material was able to immobilize more than 10(6) CFU mL(-1) of Acinetobacter haemolyticus cells after 3 days of contact time. Complete reduction of 15-240 mg L(-1) of Cr(VI) was achieved even after 3 months of bioreactor operation. Cr(VI) was not detected in the final effluent fraction indicating complete removal of Cr from solution from the flocculation/coagulation step and the unlikely re-oxidation of Cr(III) into Cr(VI). Impatiens balsamina L. and Gomphrena globosa L. showed better growth in the presence of soil-sludge mixture compared to Coleus scutellarioides (L.) Benth. Significant amounts of Cr accumulated at different sections of the plants indicate its potential application in Cr phytoremediation effort. The bacterial-based system was also determined not to be detrimental to human health based on the low levels of Cr detected in the hair and nail samples of the plant operators. Thus, it can be said that bacterial-based Cr(VI) treatment system is a feasible alternative to the conventional system especially for lower Cr(VI) concentrations, where sludge generated can be used as growth supplement for ornamental plant as well as not detrimental to the health of the workers.

Production and characterization of violacein by locally isolated Chromobacterium violaceum grown in agricultural wastes.

The present work highlighted the production of violacein by the locally isolated Chromobacterium violaceum (GenBank accession no. HM132057) in various agricultural waste materials (sugarcane bagasse, solid pineapple waste, molasses, brown sugar), as an alternative to the conventional rich medium. The highest yield for pigment production (0.82 g L⁻¹) was obtained using free cells when grown in 3 g of sugarcane bagasse supplemented with 10% (v/v) of L-tryptophan. A much lower yield (0.15 g L⁻¹) was obtained when the cells were grown either in rich medium (nutrient broth) or immobilized onto sugarcane bagasse. Violacein showed similar chemical properties as other natural pigments based on the UV-Vis, Fourier transform infrared spectroscopy, thin-layer chromatography, nuclear magnetic resonance, and mass spectrometry analysis. The pigment is highly soluble in acetone and methanol, insoluble in water or non-polar organic solvents, and showed good stability between pH 5-9, 25-100 °C, in the presence of light metal ions and oxidant such as H2O2. However, violacein would be slowly degraded upon exposure to light. This is the first report on the use of cheap and easily available agricultural wastes as growth medium for violacein-producing C. violaceum.

Pilot-scale removal of chromium from industrial wastewater using the ChromeBac system.

The enzymatic reduction of Cr(VI) to Cr(III) by Cr(VI) resistant bacteria followed by chemical precipitation constitutes the ChromeBac system. Acinetobacter haemolyticus was immobilized onto carrier material inside a 0.2m(3) bioreactor. Neutralized electroplating wastewater with Cr(VI) concentration of 17-81 mg L(-1) was fed into the bioreactor (0.11-0.33 m(3)h(-1)). Complete Cr(VI) reduction to Cr(III) was obtained immediately after the start of bioreactor operation. Together with the flocculation, coagulation and filtration, outflow concentration of less than 0.02 mg Cr(VI)L(-1) and 1mg total CrL(-1) were always obtained. Performance of the bioreactor was not affected by fluctuations in pH (6.2-8.4), Cr(VI) (17-81 mg L(-1)), nutrient (liquid pineapple waste, 1-20%v/v) and temperature (30-38 degrees C). Standby periods of up to 10 days can be tolerated without loss in activity. A robust yet effective biotechnology to remove chromium from wastewater is thus demonstrated.

Biological detoxification of Cr(VI) using wood-husk immobilized Acinetobacter haemolyticus.

Acinetobacter haemolyticus, a Gram-negative aerobic locally isolated bacterium, immobilized on wood-husk showed the ability to detoxify Cr(VI) to Cr(III). Wood-husk, a natural cellulose-based support material, packed in an upward-flow column was used as support material for bacterial attachment. Around 97% of the Cr(VI) in wastewater containing 15 mg L(-1) of Cr(VI) was reduced at a flow rate of 8.0 mL min(-1). The wastewater containing Cr(VI) was added with liquid pineapple wastewater as nutrient source for the bacteria. Electron microscopic examinations of the wood-husk after 42 days of column operation showed gradual colonization of the wood-husk by bacterial biofilm. The use of 0.1% (v/v) formaldehyde as a disinfecting agent inhibited growth of bacteria present in the final wastewater discharge. This finding is important in view of the ethical code regarding possible introduction of exogenous bacterial species into the environment.

Hexavalent chromium reduction by Acinetobacter haemolyticus isolated from heavy-metal contaminated wastewater.

Possible application of a locally isolated environmental isolate, Acinetobacter haemolyticus to remediate Cr(VI) contamination in water system was demonstrated. Cr(VI) reduction by A. haemolyticus seems to favour the lower concentrations (10-30 mg/L). However, incomplete Cr(VI) reduction occurred at 70-100 mg/L Cr(VI). Initial specific reduction rate increased with Cr(VI) concentrations. Cr(VI) reduction was not affected by 1 or 10 mM sodium azide (metabolic inhibitor), 10 mM of PO(4)3-, SO4(2-), SO(3)2-, NO3- or 30 mg/L of Pb(II), Zn(II), Cd(II) ions. However, heat treatment caused significant dropped in Cr(VI) reduction to less than 20% only. A. haemolyticus cells loses its shape and size after exposure to 10 and 50 mg Cr(VI)/L as revealed from TEM examination. The presence of electron-dense particles in the cytoplasmic region of the bacteria suggested deposition of chromium in the cells.