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Purpose Sewage sludge samples from a water treatment plant in Nigeria were subjected to an in-vessel composting (using sawdust as a bulking agent) and thermal sludge processing to improve its quality for agricultural applications. Methods Treated samples were analyzed for physicochemical and microbiological properties using standard analytical and aerobic culture protocols. Results Microbiological analysis of the initial fresh mixture (sewage sludge/sawdust) showed that the total heterotrophic bacteria was 1.17 × 106 CFU/g of fresh compost, coliforms 4.7 × 104 CFU/g, Salmonella sp., and Shigella sp. 7.3 × 104 CFU/g, yeasts and moulds 9.0 × 104 CFU/g. These values were significantly (p = 0.05) reduced after 40 days of in-vessel composting to 4.3 × 104 CFU/g for total heterotrophic bacteria, 7.4 × 102 CFU/g for coliforms, while yeasts and moulds, Salmonella and Shigella sp. were not detected in the final compost. The results of the physicochemistry revealed variation in pH, temperature, and nutrients status of treated sludge. Conclusion Salmonella sp., Staphylococcus aureus, and Shigella sp. were eliminated, while a 2-log reduction in coliform counts occurred after 40 days of composting. Composting had a better processing impact by increasing the ash as well as reducing the carbon/nitrogen ratio of treated sludge, while thermal processing improved the sulfate and phosphate components of treated sludge. The treated sludge (biosolids) met the permissible limits of microbiological and nutritional standards recommended by US EPA for land application of sludge and could, therefore, be used as a biofertilizer, soil conditioner and also for land reclamation.

The levels of some emerging organic pollutants (EOPs) including endocrine disrupting compounds, pharmaceuticals and personal care products were quantified in surface water of a freshwater ecosystem, the Ikpa River Basin, Nigeria using liquid chromatography/high resolution tandem mass spectrometry (LC-MS/MS). In addition, leachates and storm water samples collected from nearby dumpsites were also analysed to assess the effect on water quality. Seventeen compounds were detected at the nanogramme-per-litre levels and the ecological risks of selected compounds assessed based on predicted no-effect concentrations derived from comparison of toxicity data recorded for green algae, fish and invertebrate with the maximum measured environmental concentrations, to obtain risk quotients. Some of the compounds showed some level of widespread occurrence or persistence. Also, bisphenol A, chloramphenicol, erythromycin, triclocarban and triclosan were the most important EOPs detected in the study area that may pose detrimental effects to the aquatic organisms based on the outcome of the risk assessment.

The efficiency of removing 9 different pharmaceuticals, 5 carbamazepine metabolites, and 1 personal care product through wastewater treatment plants and constructed wetlands was investigated. The compound concentrations were measured using solid phase extraction followed by liquid chromatography quadrupole tandem mass spectrometry. For extraction confirmation and better accuracy, isotopic dilution and standards addition methods were employed. The reporting limits for the investigated compounds were less than 10 ng/L except TCEP (24 ng/L). The removal efficiencies were found to be inversely proportional to the octanol–water partition coefficients (log K ow ) after modification with ionizable functional groups (log D ow ); compounds with a higher hydrophilicity were more efficiently removed in the engineered processes through biological treatment mechanisms. Carbamazepine metabolites that were formed in the early stages of certain metabolic reactions exhibited enhanced removal efficiencies due to a decreased log D ow values. However, the removal efficiency of those formed in later stages did not increase, but rather fluctuated with large standard deviations. The removal behaviors of metabolites in biologically operating engineered systems need to be more extensively examined.

This review details recent progresses in the flavonoid biotransformation by bacterial non-heme dioxygenases, biphenyl dioxygenase (BDO), and naphthalene dioxygenase (NDO), which can initially activate biphenyl and naphthalene with insertion of dioxygen in stereospecfic and regiospecific manners. Flavone, isoflavone, flavanone, and isoflavanol were biotransformed by BDO from Pseudomonas pseudoalcaligenes KF707 and NDO from Pseudomonas sp. strain NCIB9816-4, respectively. In general, BDO showed wide range of substrate spectrum and produced the oxidized products, whereas NDO only metabolized flat two-dimensional substrates of flavone and isoflavone. Furthermore, biotransformation of B-ring skewed substrates, flavanone and isoflavanol, by BDO produced the epoxide products, instead of dihydrodiols. These results support the idea that substrate-driven reactivity alteration of the Fe-oxo active species may occur in the active site of non-heme dioxygenases. The study of flavonoid biotransformation by structurally-well defined BDO and NDO will provide the substrate structure and reactivity relationships and eventually establish the production of non-plant-originated flavonoids by means of microbial biotechnology.

Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC–MS and LC–NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2′ and C3′ on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme–substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 incorporated dioxygen at the C7 and C8 positions on the A-rings of flavone and isoflavone with different stereoselectivity, resulting in the formation of (7S,8S)-dihydroxy-2-phenyl-7,8-dihydro-4H-chromen-4-one (flavone-cis-(7S,8S)-dihydrodiol) and (7R,8R)-dihydroxy-3-phenyl-7,8-dihydro-4H-chromen-4-one (isoflavone-cis-(7R,8R)-dihydrodiol), respectively. In addition, NDO was shown to incorporate dioxygen at the C5 and C6 positions on the A-ring and the C2' and C3' positions on the B-ring of isoflavone, resulting in the production of (5S,6R)-dihydroxy-3-phenyl-5,6-dihydro-4H-chromen-4-one (isoflavone-cis-(5S,6R)-dihydrodiol) and 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one (isoflavone-cis-(2'R,3'S)-dihydrodiol), respectively. The metabolites were identified by LC/MS, (1)H, and (13)C NMR analyses and TD-SCF calculations combined with CD spectroscopy. In the case of flavone biotransformation, formation of flavone-(7S,8S)-dihydrodiol is likely to be the result of hydrogen bond interactions between the substrate and the active site of the dioxygenase. On the contrary, regioselective dioxygenation of isoflavone was found not to occur, and this may be due to the fact that the same hydrogen bonds that occur in the case of the flavone reaction cannot be established due to steric hindrance caused by the position of the B-ring. It is therefore proposed that the regioselectivity and stereoselectivity of NDO from strain NCIB 9816-4 are controlled by the position of the phenyl ring on flavone molecules.

The hypolipidemic agent gemfibrozil (GEM), which has been studied for its metabolism in humans and animals, was investigated to elucidate its primary metabolism by Cunninghamella elegans. The fungus produced ten metabolites (FM1-FM9 and FM6') from the biotransformation of GEM. Based on LC/MS/MS and NMR analyses, a major metabolite, FM7, was identified as 2'-hydroxymethyl GEM. FM6 was considered to be 5'-hydroxymethyl GEM, after comparison of results LC/MS, LC/MS/MS, and UV absorption spectra to FM7. The combined concentration of FM6 and FM7 was found to increase up to 0.83 mM by day 2, and then decreased gradually with incubation time, followed by a noticeable increase in the biotransformation product, FM1, up to 0.86 mM by day 15. NMR analyses confirmed that FM1 was 2',5'-dihydroxymethyl GEM. Further minor oxidations of the aromatic ring and carboxylic acid intermediates were also detected. Based upon these findings, the major fungal metabolic pathway for GEM is likely to occur via production of 2',5'-dihydroxymethyl GEM from 2'-hydroxymethyl GEM. These relatively rapid and diverse biotransformations of GEM by C. elegans suggest that depending upon conditions, it may also follow a similar biodegradation fate when released into the natural environment.

Purified atrazine chlorohydrolase (AtzA) was entrapped in the nanoparticles of biomimetically synthesized silica at the ambient condition within 20 min. Entrapped AtzA in biomimetic silica was less affected by pH change and showed higher thermostability than free enzymes. The entrapped AtzA was also more tolerant against proteolysis, with 80% of the initial activity remaining and retained 82% of the initial activity even after four cycles of usage. These results suggest that entrapment of AtzA in biomimetic silica could be utilized under diverse environmental conditions with the active catalytic performance sustained.

In this study we evaluated the persistence of diazinon, chlorpyrifos, profenofos, parathion, malathion and ethion in dead coral tissues of Indonesian coastal waters (Java, Bali, Sulawesi and Komodo). Comparison of the residue levels in coral tissues showed that the highest presence of organophosphate concentrations was detected in a coral sample collected from Java coastal waters. While medium amounts of a contaminant diazinon can still lead to detectable in Bali and Sulawesi coastal waters. Prominent contamination of organophosphate was not found in a sample collected from Komodo. Neither parathion nor malathion were detected in any of the samples. This result implies that the geographical variations of organophosphate compounds are determined by the possible usage of these chemicals around coastal waters at the present or in the past. There is need for further work to identify sources and fate of pesticide contaminants, as well as to improve monitoring of pesticide use.

The increasing use of herbicides in agriculture sectors have received great attention with respect to their potential toxic effect on reef-building corals of Indonesia. One chlorinated compound, 2,4-dichlorophenoxyacetat (2,4-D), has been widely used as a herbicide and has become a substantial environmental pollutant. Contamination of 2,4-D was assessed in stressed or dead coral tissues of Gal area fascicul arts from the coastal waters of Java Sea. Controlled tolerance experiment testing 2,4-D was performed on this coral. The effects of 2,4-D on coral mortality were investigated. Comparison of the residue levels in coral tissues showed that the highest presence of 2,4-D concentrations was detected in a sample collected from Jepara coastal waters. While small amounts of a contaminant 2,4-D can still lead to detectable in West Java and East Java coastal waters. Contamination of 2,4- D was not found in coral samples collected from Karimunjawa islands. The toxicity of 2,4-D on corals showed that short duration (48 h) laboratory test demonstrated dramatic effects on sloughing and death of coral. The LC50.96 was determined to be 18.82 mg L’ 2,4-D. These results demonstrated the possibility that 2,4-D herbicide could act and lead to coral mortality in the Java coastal waters

The fungus Mucor ramannianus was tested to elucidate the biological fate of a class of N-methyl carbamate pesticide carbofuran and its hydrolysed form carbofuran phenol. The elution profile obtained from analysis by high-pressure liquid chromatography equipped with a reverse-phase C-18 column showed that two peaks occurred after incubation of M. ramannianus with which 1 mM carbofuran was combined as a final concentration. In culture of M. ramannianus with 1 mM carbofuran phenol, it produced two other metabolites compared to metabolites of carbofuran. LC/MS analysis suggested that two of the metabolites produced from carbofuran phenol were most likely to be 2-hydoxy-3-(3-methylpropan-2-ol)phenol or 7a-(hydroxymethyl)-2,2-dimethylhexahydro-6H-furo[2,3-b]pyran-6-one and 3-hydroxycarbofuran-7-phenol.

In this study, the levels of 16 USEPA-prioritized polycyclic aromatic hydrocarbons (PAHs) were quantified in various environmental matrices in Ikpa River Basin and nearby dumpsites using gas chromatography-mass spectrometry (GC-MS). The levels of the contaminants were further subjected to models to estimate possible sources and potential risks. The results obtained revealed that nearby dumpsites could be the major source of contamination of the Ikpa River Basin. The total sum of PAHs in water and sediment samples gave 926.6 μg/l and 1099.7 μg/kg, respectively. Higher concentrations, 3025.8 μg/kg, 3645.7 μg/kg, and 2457.2 μg/l, were recorded for nearby municipal dumpsite soil, hospital dumpsite soil, and landfill leachates, respectively. Hierarchical cluster analysis (HCA) and PAH molecular diagnostic ratios (MDRs) suggest that PAH loading in the river basin were mostly of pyrogenic origin. The risk assessment indicated that exposure to PAHs through dermal contact with sediments was most significant than oral ingestion of water and children were the most vulnerable group. Non-cancer (toxic) risks due to exposure to PAHs by oral ingestion of water from Ikpa River were within acceptable limits as the calculated hazard quotients (HQs) and hazard indexes (HIs) were below unity, suggesting negligible or no toxic risk. However, toxic risks through dermal contact with sediments reached unacceptable limits as HI values exceeded unity for all sample stations. Estimated cancer risks due to oral ingestion of water reached the USEPA minimum risk level (3.14 × 10(-5)) requiring public notification while risks due to dermal adsorption of PAHs from the sediments reached levels (2.10 × 10(-1)) requiring remediation actions.

An anaerobic incubation mixture of two bacterial strains Eggerthella sp. Julong 732 and Lactobacillus sp. Niu-O16, which have been known to transform dihydrodaidzein to S-equol and daidzein to dihydrodaidzein respectively, produced S-equol from daidzein through dihydrodaidzein. The biotransformation kinetics of daidzein by the mixed cultures showed that the production of S-equol from daidzein was significantly enhanced, as compared to the production of S-equol from dihydrodaidzein by Eggerthella sp. Julong 732 alone. The substrate daidzein in the mixed culture was almost completely converted to S-equol in 24 h of anaerobic incubation. The increased production of S-equol from daidzein by the mixed culture is likely related to the increased bacterial numbers of Eggerthella sp. Julong 732. In the mixture cultures, the growth of Eggerthella sp. Julong 732 was significantly increased while the growth of Lactobacillus sp. Niu-O16 was suppressed as compared to either the single culture of Eggerthella sp. Julong 732 or Lactobacillus sp. Niu-O16. This is the first report in which two metabolic pathways to produce S-equol from daidzein by a mixed culture of bacteria isolated from human and bovine intestinal environments were successfully linked under anaerobic conditions.

A 3.0-kb DNA fragment containing an endo-inulinase gene was cloned from Arthrobacter sp. S37. It contained a single open reading frame of 2439 bp, encoding a polypeptide composed of signal peptide of 53 amino acids and mature protein of 759 amino acids. From the comparison with amino acids sequences of fructan hydrolases and invertase, five highly conserved regions including the β-fructosidase motif were found. The sequence of the endo-inulinase had the identity in the range of 13.3% to 16.0%.

Inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] secreted from Bacillus sp. snu-7 was purified 60.3-fold with a yield of 11.6% from a culture supernatant by ammonium sulfate precipitation, preparative isoelectrofocusing, anion exchange chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 62 kDa on SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was found to be Ala-Asp-Gly-Gln-Asp-Gly-Ala-Pro-Leu-Asn-Gln-Val-Asn-Thr-Tyr-Asp. The optimal pH and temperature for the enzyme reaction were 6.0 and 40°C, respectively. The enzyme was stable with a pH range of 4.0 to 7.0 and at up to 60°C. As the production of di-D-fructose 1,2':2,3' dianhydride increased in the course of enzyme reaction, the Km of the purified enzyme was estimated to be 5.4 mM. One mM each of Cu(2+), Fe(2+) and Hg(2+) inhibited the enzyme activity strongly. Exhaustive enzymatic digestion of inulin produced 1-kestose, 1-nystose, and 1-F-fructofuranosylnystose as well as di-D-fructose 1,2':2,3' dianhydride.

Escherichia coli cells containing the biphenyl dioxygenase genes bphA1A2A3A4 from Pseudomonas pseudoalcaligenes KF707 were found to biotransform isoflavone and produced a metabolite that was not found in a control experiment. Liquid chromatography/mass spectrometry (LC/MS) and (1)H and (13)C nuclear magnetic resonance (NMR) analyses indicated that biphenyl dioxygenase induced 2',3'-cis-dihydroxylation of the B-ring of isoflavone. In a previous report, the same enzyme showed dioxygenase activity toward flavone, producing flavone 2',3'-cis-dihydrodiol. Due to growing interest in flavone chemistry and the absolute configuration of natural products, time-dependent density functional theory (TD-DFT) calculations were combined with circular dichroism (CD) spectroscopy to determine the absolute configuration of the isoflavone dihydrodiol. By computational methods, the structure of the isoflavone metabolite was determined to be 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. This structure was confirmed further by the modified Mosher's method. The same protocol was applied to the flavone metabolite, and the absolute configuration was determined to be 2-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one. After determination of the absolute configurations of the biotransformation products, we suggest the binding mode of these substrate analogs to the enzyme active site.

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1-C3 and M1-M3). All six metabolites showed [M + H](+) at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography-mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k(cat)/K(m)) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k(cat), but not due to variations in K(m), consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK(a) of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK(a). Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.

KR-33028 ( N -[4-cyano-benzo[b]thiophene-2-carbonyl]guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. This study was performed to characterize the cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-33028. Hydroxylation (5-hydroxy- and 7-hydroxy-KR-33028) is major pathways for the metabolism of KR-33028 in human liver microsomes. Among the nine c-DNA expressed CYP isoforms tested, KR-33028 was 5-hydroxylated by CYP3A4 and 7-hydroxylated by CYP1A2, CYP3A4, and CYP2C19. These findings were supported by the combination of chemical inhibition studies in human liver microsomes and correlation analysis. Furafylline and ketoconazole potently inhibited hydroxylation of KR-33028 in human liver microsomes. Correlation analysis between the known CYP enzyme activities and the rates of the formation of 5-hydroxy- and 7-hydroxy-KR-33028 in the 16 human liver microsomes has showed significant correlations with CYP3A4-mediated midazolam 1′-hydroxylation and CYP1A2-mediated phenacetin O -deethylation, respectively. A 7-hydroxy-KR-33028 formation is also weakly correlated with CYP3A4-mediated midazolam 1′-hydroxylation. The kinetics of the major biotransformation of KR-33028 were studied: CYP3A4 mediated the formation of 5-hydroxy-KR-33028 from KR-33028 with Cl int = 0.22 μl/min/pmol CYP. The intrinsic clearance for 7-hydroxy-KR-33028 formation by CYP1A2, CYP2C19, and CYP3A4 were 0.26, 0.19, and 0.03 μl/min/pmol CYP, respectively. Taken together, these results provide evidence that CYP3A4 and CYP1A2 are the major isoforms responsible for the hydroxy metabolites formation from KR-33028.

A nuclear polyhedrosis virus ofHyphantriacunea replicated successfully in theTrichoplusiani cell line. Restriction endonuclease analysis of the viral DNA obtained from infected cell culture showed the same general homogeneity as that from virus isolated from diseased host larvae. Electron microscopic observations showed that the occluded virus from cell culture consists of rod-like nucleocapsids (31×320 nm) enveloped in aggregates and embedded in polyhedral inclusion bodies from 0.6 to 2.5 μm in diameter.