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The present study explored bacterial aerobic biodegradation of reduced carbon-contaminants (RCC) in a pilot system mimicking remediation of a saturated aquifer in a permeable reactive biobarrier (PRBB). Bioaugmentation was performed with a pure culture of Pseudomonas putida macro-encapsulated in a cellulose-acetate porous envelope and integrated transversely to the flow trajectory of the fluid in the biobarrier and compared with controls without capsules. The macro-encapsulation technique applied allowed the incorporation of a built-in nutrient core for the slow release of macronutrients, i.e. N, P, instead of exogenous nutrients supply. 3-Chlorophenol (3CP) at a concentration range of 350-500 mg/L was chosen as an RCC model compound. The findings indicate efficient 3CP biodegradation during the PRBB operation with a similar degree of transformation (76 ± 2% and 72 ± 2%) and mineralization (55 ± 4% vs. 49 ± 3%) for exogenous and built-in nutrients supply, respectively. The extent of dechlorination in both cases (54 ± 10% vs. 40 ± 2%, respectively) followed mineralization rather than transformation, suggesting that Cl - release took place in late transformation stages. Negligible decontamination was observed in the control system without bioaugmentation. Concluding, tailored PRBB with macro-capsules incorporating a built-in nutrient core to support bacterial growth presents a significant environmental advantage controlling excess nutrients release required in bioremediation of oligotrophic systems.

This study explored the stability of partial ammonium oxidation at low feed concentration (50 g N/m 3 ), suitable for anammox process, in continuous fixed bed up-flow biofilm reactors with external recirculation-aeration. The reactors, filled with crushed basalt, were fed with synthetic medium at 20-25 °C at constant flow-rate with limiting dissolved oxygen concentration controlled by the recirculation ratio (R). Successful nitritation was achieved at R ≅ 4-6 with approx. 50% of NH 4 + oxidized to NO 2 - with <5% NO 3 - accumulation. q-PCR analysis along the reactor showed ammonia oxidizing bacteria being the prevalent nitrifiers over the three-fourths of the bed in the flow direction, negligible denitrifiers and absent ammonium oxidizing archaea. A numerical model for predicting the concentration of the nitrogen species and DO was formulated. The model successfully predicted the experimental results and displayed good sensitivity to intrinsic oxygen uptake parameters. The proposed numerical model can serve both as an operational and design tool., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study aimed at evaluating the contribution of low voltage electric field, both alternating (AC) and direct (DC) currents, on the prevention of bacterial attachment and cell inactivation to highly electrically conductive self-supporting carbon nanotubes (CNT) membranes at conditions which encourage biofilm formation. A mutant strain of Pseudomonas putida S12 was used a model bacterium and either capacitive or resistive electrical circuits and two flow regimes, flow-through and cross-flow filtration, were studied. Major emphasis was placed on AC due to its ability of repulsing and inactivating bacteria. AC voltage at 1.5 V, 1 kHz frequency and wave pulse above offset (+0.45) with 100Ω external resistance on the ground side prevented almost completely attachment of bacteria (>98.5%) with concomitant high inactivation (95.3 ± 2.5%) in flow-through regime. AC resulted more effective than DC, both in terms of biofouling reduction compared to cathodic DC and in terms of cell inactivation compared to anodic DC. Although similar trends were observed, a net reduced extent of prevention of bacterial attachment and inactivation was observed in filtration as compared to flow-through regime, which is mainly attributed to the permeate drag force, also supported by theoretical calculations in DC in capacitive mode. Electrochemical impedance spectroscopy analysis suggests a pure resistor behavior in resistance mode compared to involvement of redox reactions in capacitance mode, as source for bacteria detachment and inactivation. Although further optimization is required, electrically polarized CNT membranes offer a viable antibiofouling strategy to hinder biofouling and simplify membrane care during filtration., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Commercial activated carbon (CAC) and biochar are useful adsorbents for removing heavy metals (HM) from water, but their production is costly. Biochar production from olive solid waste from two olive cultivars (Picual and Souri) and two oil production process (two- or three-phase) and two temperatures (350 and 450°C) was tested. The biochar yield was 24-35% of the biomass, with a surface area of 1.65-8.12m 2 g -1 , as compared to 1100m 2 g -1 for CAC. Picual residue from the two-phase milling technique, pyrolysed at 350°C, had the best cumulative removal capacity for Cu +2 , Pb +2 , Cd +2 , Ni +2 and Zn +2 with more than 85% compared to other biochar types and CAC. These results suggest that surface area cannot be used as a sole predictor of HM removal capacity. FTIR analysis revealed the presence of different functional groups in the different biochar types, which may be related to the differences in absorbing capacities., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A juxtaposition of regional cuisines and recent prospective studies of fish consumption in China and Japan points to fresh horseradish and/or radish (HRR) as possible contributors to delaying age-related dementia. The hypothesis is that the inverse association found sometimes between fish intake and cognitive decline is partially due to exposure of the oral cavity to active peroxidases from HRR served in conjunction with fish. This hypothesis can be tested by specifically looking at whether HRR is consumed with fish and whether such HRR is prepared in a way that preserves activity of HRR peroxidases. It is possible that by putting active HRR peroxidases in their mouths, elderly people supplement their age-diminished salivary antioxidant capacity and break down additional hydrogen peroxide (H 2 O 2 ) in the oral cavity before it can migrate into the brain, thus decreasing the incidence of brain cell death induction by chronically-elevated H 2 O 2 . Intentional exposure of the oral cavity to active HRR peroxidases could be a prophylactic for delaying dementia. Because vegetable peroxidases are inactivated by gastric juices, it will be difficult to obtain benefit from HRR peroxidases' antioxidant effect via ingestion in encapsulated dietary supplements., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Removal of micropollutants from reverse osmosis (RO) brines of wastewater desalination by oxidation processes is influenced by the scavenging capacity of brines components, resulting in the accumulation of transformation products (TPs) rather than complete mineralization. In this work the iodinated contrast media diatrizoate (DTZ) was used as model compound due to its relative resistance to oxidation. Identification of TPs was performed in ultrapure water (UPW) and RO brines applying nonthermal plasma (NTP) and UVA-TiO2 as oxidation techniques. The influence of main RO brines components in the formation and accumulation of TPs, such as chloride, bicarbonate alkalinity and humic acid, was also studied during UVA-TiO2. DTZ oxidation pattern in UPW resulted similar in both UVA-TiO2 and NTP achieving 66 and 61% transformation, respectively. However, DTZ transformation in RO brines was markedly lower in UVA-TiO2 (9%) than in NTP (27%). These differences can be attributed to the synergic effect of RO brines components during NTP. Moreover, reactive species other than hydroxyl radical contributed to DTZ transformation, i.e., direct photolysis in UVA-TiO2 and direct photolysis + O3 in NTP accounted for 16 and 23%, respectively. DTZ transformation led to iodide formation in both oxidation techniques but it further oxidized to iodate by ozone in NTP. In total 14 transformation products were identified in UPW of which 3 were present only in UVA-TiO2 and 2 were present exclusively in NTP; 5 of the 14 TPs were absent in RO brines. Five of them were new and were denoted as TP-474A/B, TP-522, TP-586, TP-602, TP-628. TP-522 (mono-chlorinated) was elucidated only in presence of high chloride titer-synthetic water matrix in NTP, most probably formed by active chlorine species generated in situ. TPs accumulation in RO brines was markedly different in comparison to UPW. This denotes the influence of RO brines components in the formation of reactive species that could further attack DTZ/TPs and/or scavenging performed by these brine components that could limit further TPs degradation. Five plausible degradation pathways are proposed for DTZ transformation in UPW., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Kinetic studies on the stability of the pain killer paracetamol in Al-Quds activated sludge demonstrated that paracetamol underwent biodegradation within less than one month to furnish p-aminophenol in high yields. Characterizations of bacteria contained in Al-Quds sludge were accomplished. It was found that Pseudomonas aeruginosa is the bacterium most responsible for the biodegradation of paracetamol to p-aminophenol and hydroquinone. Batch adsorptions of paracetamol and its biodegradation product (p-aminophenol) by activated charcoal and a composite micelle (octadecyltrimethylammonium)-clay (montmorillonite) were determined at 25°C. Adsorption was adequately described by a Langmuir isotherm, and indicated better efficiency of removal by the micelle-clay complex. The ability of bench top reverse osmosis (RO) plant as well as advanced membrane pilot plant to remove paracetamol was also studied at different water matrixes to test the effect of organic matter composition. The results showed that at least 90% rejection was obtained by both plants. In addition, removal of paracetamol from RO brine was investigated by using photocatalytic processes; optimal conditions were found to be acidic or basic pH, in which paracetamol degraded in less than 5 min. Toxicity studies indicated that the effluent and brine were not toxic except for using extra low energy membrane which displayed a half maximal inhibitory concentration (IC-50) value of 80%.

Microbial desalination cells (MDCs) taking advantage of energy in wastewater to drive desalination represent a promising approach for energy-efficient desalination, but concerns arise whether contaminants in wastewater could enter the desalinated stream across ion exchange membranes. Such back diffusion of contaminants from the anolyte into the desalinated stream could be controlled by two mechanisms, Donnan effect and molecule transport. This study attempted to understand those mechanisms for inorganic and organic compounds in MDCs through two independently conducted experiments. Donnan effect was found to be the dominant mechanism under the condition without current generation. Under open circuit condition, the MDC fed with 5 g L(-1) salt solution exhibited 1.9 ± 0.7%, 10.3 ± 1.3%, and 1.8 ± 1.2% back diffusion of acetic, phosphate, and sulfate ions, respectively. Current generation effectively suppressed Donnan effect from 68.2% to 7.2%, and then molecule transport became more responsible for back diffusion. A higher initial salt concentration (35 g L(-1)) and a shorter HRT (1.0 d) led to the highest concentration gradient, resulting in the most back diffusion of 7.1 ± 1.2% and 6.8 ± 3.1% of phosphate and sulfate ions, respectively. Three representative organic compounds were selected for test, and it was found that organic back diffusion was intensified with a higher salt concentration gradient and molecular weight played an important role in compound movement. Principal component analysis confirmed the negative correlation between Donnan effect and current, and the positive correlation between molecule transport and concentration gradient related conditions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The hydrothermal pretreatment of olive mill solid waste amended with 0.6M organic acids was studied at temperatures between 100 and 170°C. Acetic and formic acids which are endogenous intermediates of hemiacetyl splitting at subcritical conditions were tested. Formic acid, with smaller molecular size and lower pKa, was found to be more effective than acetic in the entire range of temperatures tested. Yield of enzymatic hydrolysis was significantly enhanced (>2 folds) at temperatures above 140°C. Concentration of aldehyde byproducts in the medium increased with temperature and pressure and addition of organic acids, however, the highest concentration detected (ca 1g/L) did not surpass values reported as inhibitory of sugars fermentation to ethanol by either yeast or bacteria. Aldehyde production was more affected by temperature than by acid addition. Concluding, addition of formic acid to hydrothermal pretreatment at relatively mild temperatures (140-170°C) and pressure (10-13 atm) improved saccharification yield while saving energy., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Among the main restrictions for the implementation of advanced oxidation processes (AOPs) for removal of micropollutants present in reverse osmosis (RO) brines of secondary effluents account the quenching performed by background organic and inorganic constituents. Natural organic matter (NOM) and soluble microbial products (SMP) are the main effluent organic matter constituents. The inorganic fraction is largely constituted by chlorides and bicarbonate alkalinity with sodium and calcium as main counterions. The quenching influence of these components, separately and their mixture, in the transformation of model compounds by UVA/TiO2 was studied applying synthetic brines solutions mimicking 2-fold concentrated RO secondary effluents brines. The results were validated using fresh RO brines. Diatrizoate (DTZ) and iopromide (IOPr) were used as model compound. They have been found to exhibit relative high resistance to oxidation process and therefore represent good markers for AOPs techniques. Under the conditions applied, oxidization of DTZ in the background of RO brines was strongly affected by quenching effects. The major contribution to quenching resulted from organic matter (≈70%) followed by bicarbonate alkalinity (≈30%). NOM displayed higher quenching than SMP in spite of its relative lower concentration. Multivalent cations, i.e., Ca(+2), were found to decrease effectiveness of the technique due to agglomeration of the catalyst. However this influence was lowered in presence of NOM. Different patterns of transformation were found for each model compound in which a delayed deiodination was observed for iopromide whereas diatrizoate oxidation paralleled deiodination., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The biotransformation of five selected pharmaceuticals ibuprofen (IBP), ketoprofen (KTP), carbamazepine (CBZ), dexamethasone (DXM) and iopromide (IOP) by a stable nitrifying enrichment culture was investigated at concentrations ranging between 25 μg/L and 2mg/L. Complete biotransformation was observed only for IBP and KTP, although, an inverse correlation between transformation rate and concentration was found. The transformation pattern observed is consistent with ammonia monooxygenase (AMO) activity. The metabolic succession of the compounds according to the biotransformation rates was: IBP>KTP>DXM>CBZ>IOP. A linear correlation between the calculated diffusive flux of the model compounds across a bilayer membrane and their biotransformation rates was found. Our results support the concept that augmentation with nitrifying activity can enhance the removal of trace organic pollutants during effluent treatment. Furthermore, ammonia-oxidizing activity appears as a good indicator for estimation of potential of biodegradability of pharmaceuticals, especially at low concentrations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The concept of suppressing biofouling formation using an antibacterial feed spacer was investigated in a bench scale-cross flow system mimicking a spiral wound membrane configuration. An antibacterial composite spacer containing zinc oxide-nanoparticles was constructed by modification of a commercial polypropylene feed spacer using sonochemical deposition. The ability of the modified spacers to repress biofilm development on membranes was evaluated in flow-through cells simulating the flow conditions in commercial spiral wound modules. The experiments were performed at laminar flow (Re = 300) with a 200 kDa molecular weight cut off polysulfone ultrafiltration membrane using Pseudomonas putida S-12 as model biofilm bacteria. The modified spacers reduced permeate flux decrease at least by 50% compared to the unmodified spacers (control). The physical properties of the modified spacer and biofilm development were evaluated using high resolution/energy dispersive spectrometry-scanning electron microscopy, atomic force microscopy and confocal laser scanning microscopy imaging (HRSEM, EDS, AFM and CLSM). HRSEM images depicted significantly less bacteria attached to the membranes exposed to the modified spacer, mainly scattered and in a sporadic monolayer structure. AFM analysis indicated the influence of the modification on the spacer surface including a phase change on the upper surface. Dead-live staining assay by CLSM indicated that most of the bacterial cells attached on the membranes exposed to the modified spacer were dead in contrast to a developed biofilm which was predominant in the control samples., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The oxidation of trace pharmaceutical compounds in wastewater desalination streams by nonthermal plasma (NTP) was evaluated. Brines from a two stage-RO pilot plant process as well as two sources of tertiary effluents, ultrafiltrated secondary effluents and membrane biological reactor effluents, were comparatively tested with ultra-pure water. The non-ionic and ionic iodinated contrast media (ICM) compounds, iopromide (IOPr) and diatrizoate (DTZ), respectively, were used as model compounds. The neurostabilizer drug carbamazepine (CBZ) was used for reference purposes. Based on deiodination profiles, two distinct patterns of initial oxidation could be established for the ICM. The time profile of deiodination and transformation paralleled for DTZ, indicating that transformation of the aromatic ring is the main initial pattern of transformation. For IOPr, a considerable lag phase of deiodination was observed, suggesting that oxidation of the alkyl chains rather than ring oxidation is the main pattern of initial transformation. Although transformation rate of IOPr was higher compared to DTZ, the rate and degree of deiodination was higher for DTZ than IOPr. Both ICM displayed a markedly lower susceptibility to NTP oxidation compared to CBZ. However, the kinetics of IOPr transformation seems to be less affected by the water matrixes, compared to DTZ and CBZ. Whereas NTP mediated oxidation of ICM followed first-order kinetics, a better fit to Harris model was found for CBZ. As a result of the NTP oxidation, treated brines and effluents displayed a substantial increase in biodegradability (measured as BOD). To conclude, NTP displayed a high potential for treating reluctant pharmaceuticals active compounds such as ICM, even at the background of relatively high DOC concentrations, as can be found in treated effluents and desalination brines, and with no need for chemical additives., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The use of lignin peroxidase (LIP) as an alternative method for the removal of four catechols (1,2-dihydroxybenzene): catechol (CAT), 4-chlorocatechol (4-CC), 4,5-dichlorocatechol (4,5-DCC) and 4-methylcatechol (4-MC) typical pollutants in wastewater derived from oil and paper industries, was evaluated. The removal of 2mM catecholic substrates by 1 microM LIP after 1h was in the following order: 4,5-DCC (95%)>4-CC(90%)>CAT(55%)>4-MC(43%). Except for 4-MC, all reactions were accompanied by the formation of insoluble products, leading to LIP precipitation. LIP was exposed to soluble or insoluble product-dependent inactivation, depending on the substrates tested, immediately at the start of the reactions. Despite immediate enzyme inactivation, removal of catecholic substrates continued, resulting in oligomeric product formation. Major oxidation products analyzed were compatible with dimeric, trimeric and tetrameric structures. Ether linkages and a benzoquinone structure were detected in two purified oligochlorocatechols. Catechol derivatives removal initiated by LIP, seems to be different for each catecholic substrate in terms of substrate consumption and transformation, and of enzyme activity.

The effectiveness of RNA interference (RNAi) is demonstrated in the lignin-degrading fungus Phanerochaete chrysosporium. The manganese-containing superoxide dismutase gene (MnSOD1) was used as the target for RNAi. The plasmid constructed for gene silencing contained a transcriptional unit for hairpin RNA expression. Significantly lower MnSOD expression at both the mRNA and protein activity levels was detected in RNAi transformants. Furthermore, even though P. chrysosporium possesses three copies of the MnSOD gene, this RNAi construct was sufficient to decrease the enzymatic activity by as much as 70% relative to control levels. Implementation of the RNAi technique in P. chrysosporium provides an alternative genetic tool for studies of gene function, particularly of essential genes or gene families.

The adsorption of dissolved organic matter (DOM) on granular-activated carbon (GAC) as a pretreatment to reverse osmosis (RO) desalination of membrane bioreactor (MBR) effluents was studied in lab- and pilot-scale columns. The pattern and efficiency of DOM adsorption and fate of the hydrophobic (HPO), transphilic (TPI) and hydrophilic (HPI) fractions were characterized, as well as their impact on organic fouling of the RO membranes. Relatively low DOM adsorption capacity and low intensity of adsorption were observed in batch studies. Continuous adsorption experiments performed within a range of hydraulic velocities of 0.9-12m/h depicted permissible values within the mass transfer zone up to 1.6m/h. The breakthrough curves within this range displayed a non-adsorbable fraction of 24+/-6% and a biodegradable fraction of 49+/-12%. Interestingly, the adsorbable fraction remained almost constant ( approximately 30%) in the entire hydraulic range studied. Comparative analysis by HPO interaction chromatography showed a steady removal (63-66%) of the HPO fraction. SUVA index and Fourier Transform Infrared (FTIR) spectra indicated that DOM changes during the adsorption phase were mainly due to elution of the more HPI components. GAC pretreatment in pilot-scale columns resulted in 80-90% DOM removal from MBR effluents, which in turn stabilized membrane permeability and increased permeate quality. FTIR analysis indicated that the residual DOM present in the RO permeate, regardless of the pretreatment, was mainly of HPI character (e.g., low-molecular-weight humics linked to polysaccharides and proteins). The DOM removed by GAC pretreatment is composed mainly of HPO and biodegradable components, which constitutes the fraction primarily causing organic fouling.

The structure and microbial communities of biofilms developing on cross-flow nanofiltration (NF) membranes at different temperatures (20, 25 or 34 degrees C) and operation lengths (8h-24days) were studied. Feedwater comprised tertiary quality wastewater effluent or synthetic media mimicking effluents of intermediate quality. After each run, the membranes were autopsied for bacterial enumeration, bacterial community composition and microscopy visualization (SEM, CLSM and AFM/NSOM). Community composition was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) coupled with sequence analysis of 16S rRNA gene fragments from dominant bands. Deposition of polysaccharides and initial bacterial colonization were observed within 8h, whereas developed biofilms markedly affecting membrane permeability were evident from days 2-3 onwards. Regardless of applied conditions, the heterotrophic plate counts in the biofilm were 3-4x10(6)CFU/cm(2) and the thickness of the biofouling layer was 20-30microm. From a total of 22 sequences obtained from 14 independent experiments, most species identified were Gram negative (19 of 22 sequences). Proteobacteria were found to be a prevalent group in all cases (16 of 22 sequences) and among it, the beta-subclass was the most predominant (8 sequences), followed by the gamma-subclass (5 sequences). Pseudomonas/Burkholderia, Ralstonia, Bacteroidetes and Sphingomonas were the dominant groups found in most cases. Even though the microbial population might be important with respect to biofouling patterns, membrane permeability decline seems to be more substantially influenced by the formation and accumulation of exopolymeric substances (EPS).

A favorable microenvironment for biofilm growth on GAC particles was shown using green fluorescent protein (GFP) as a marker for a phenol degrading bacterium, Pseudomonas putida F1. The dispersion of P. putida F1 in a biofilm covering granulated activated carbon (GAC) particles was monitored and compared to a biofilm on non-activated granular carbon particles. Laser scanning confocal microscopy (LSCM) micrographs of the biofilms taken from two fluidized bed reactors operating under identical conditions, showed higher fluorescent green areas in the GAC biofilm, especially close to the GAC surface. Quantitative analysis of the biofilm by COMSTAT, a three-dimensional biofilm structure analysis program, showed higher biomass concentration and higher viability in the GAC covered biofilm vs. the non-activated carbon biofilm. In addition, better effluent quality was measured for the BGAC reactor, which strongly suggests a significantly larger biofilm surface area available to the substrate, as opposed to that of the non-activated carbon carrier reactor.

Phytopathogenic fungi penetrate plants by breaking down the cuticular barrier with cutinase. Cutinases are extracellular hydrolytic enzymes that degrade cutin, a polyester composed of hydroxy and epoxy fatty acids. Until now, cutinase has been recognized by its ability to release labeled cutin monomers or by a non-specific esterase assay based on the hydrolysis of p-nitrophenyl esters of short fatty acids. In this work, an insoluble p-nitrophenyl derivative was synthesized and purified, and its structure was determined to be 4-nitrophenyl (16-methyl sulfone ester) hexadecanoate (pNMSEH) by nuclear magnetic resonance (H+ NMR) analysis. pNMSEH was tested as a new cutinase substrate with Pseudomonas mandocino cutinase and porcine liver esterase. While a linear release over time of p-nitrophenol (pNP) was recorded in the presence of cutinase, no response was obtained with the esterase. The calculated kinetic parameters of pNMSEH hydrolysis by cutinase revealed a high specificity (Km=1.8mM), albeit a low catalytic rate (Vmax=10.5 micromol min(-l)l(-1)). This new synthetic substrate may be helpful for detecting and assaying cutinase activity in mixed solutions, such as crude fungal extracellular extracts.

A process is described in which toxic aromatic compounds are converted by toluene dioxygenase and in turn toluene cis-dihydrodiol dehydrogenase to catechols which are further polymerized by peroxidase-catalyzed oxidation producing polycatechols. Three approaches for obtaining catechols were employed: (1) addition of halogenated aromatics to P. putida F1, resulting in the accumulation of halogenated catechols; (2) inhibition of catechol 2,3-dioxygenase of P. putida F1 by known aromatic and aliphatic inhibitors; and (3) overexpression of toluene dioxygenase and toluene cis-dihydrodiol dehydrogenase genes in E. coli JM109. The process is suitable for producing novel catechols that upon oxidation may yield polymers with unique properties, presenting a tool for producing tailor-made biopolymers. Formation of 3-chlorocatechol from chlorobenzene, 3,4-dichlorocatechol from 1,2-dichlorobenzene, and catechol from benzene and their subsequent oxidation and polymerization was demonstrated. Oxidation of catechol yielded polymers with molecular weights of up to 4000 Daltons. Their apparently high water solubility eliminates the need for water-miscible solvents. In aqueous solution oxidation of catechols was rapid, yet the presence of 20%, 30%, and 40% ethanol, resulted in a rate decrease of 31%, 95%, and 93%, respectively. The advantage is that significantly less peroxidase is required for performing the reactions if miscible solvents are not employed. Furthermore, water-soluble polymers may be desirable for many applications.

Cellulose-binding domains (CBDs) are characterized by their ability to strongly bind to different forms of cellulose. This study examined the use of a recombinant CBD fused to the reporter enzyme beta-glucuronidase (CBD-GUS) to determine the extent of removal of the water-repellent waxy component of cotton fiber cuticles following hydrolytic treatment, i.e., scouring. The CBD-GUS test displayed higher sensitivity and repeatability than conventional water absorb techniques applied in the textile industry. Increases in the levels of CBD-GUS bound to the exposed cellulose correlated to increases in the fabric's hydrophilicity as a function of the severity of the scouring treatment applied, clearly indicating that the amount of bound enzyme increases proportionally with the amount of available binding sites. The binding of CBD-GUS also gave measurable and repeatable results when used on untreated or raw fabrics in comparison with conventional water drop techniques. The quantitative response of the reaction as bound enzyme activity was optimized for fully wettable fabrics. A minimal free enzyme concentration-to-swatch weight ratio of 75:1 was found to be necessary to ensure enzyme saturation (i.e., a linear response), corresponding to a free enzyme-to-bound enzyme ratio of at least 3:5.

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O(2)) of Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concentration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed when the fungus was exposed to pure O(2) gas. The specific activities of manganese superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher in cultures exposed to pure O(2) (oxygenated cultures) than in cultures grown with atmospheric air. Significantly higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP) was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP levels were obtained at high O(2) concentrations and in cultures grown with atmospheric air. These results suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O(2) is at least partially mediated by the intracellular ROS.

The steady state kinetic parameters Km and kcat for the oxidation of phenolic substrates by lignin peroxidase correlated with the presteady state kinetic parameters Kd and k for the reaction of the enzyme intermediate compound II with the substrates, indicating that the latter is the rate-limiting step in the catalytic cycle. ln Km and ln Kd values for phenolic substrates correlated with redox properties, unlike ln kcat and ln k. This finding suggests that in contrast to horseradish peroxidase, electron transfer is not the rate-limiting step during oxidation by lignin peroxidase compound II. A mechanism is proposed for lignin peroxidase compound II reactions consisting of an equilibrium electron transfer step followed by a subsequent rate-limiting step. Analysis of the correlation coefficients for linear relationships between ln Kd and ln Km and different calculated redox parameters supports a mechanism in which the acidic forms of phenols are oxidized by lignin peroxidase and electron transfer is coupled with proton transfer. 1,2-Dimethoxyarenes did not comply with the trend for phenolic substrates, which may be a result of more than one substrate binding site on lignin peroxidase and/or alternative binding modes. This behavior was supported by analogue studies with the 1,2-dimethoxyarenes veratric acid and veratryl aldehyde, both of which are not oxidized by lignin peroxidase. Inclusion of either had little effect on the rate of oxidation of phenolic substrates yet resulted in a decrease in the oxidation rate of 1,2-dimethoxyarene substrates, which was considerable for veratryl alcohol and less pronounced for 3,4-dimethoxyphenethylalcohol and 3,4-dimethoxycinnamic acid, in particular in the presence of veratric acid.

An adsorbing biofilm carrier, like granular activated carbon (GAC), can be the source of an extra flux of pollutant to the biofilm in addition to the bulk liquid. This double flux can improve the performance of a biological GAC (BGAC) reactor as compared to a nonabsorbing carrier reactor but only under conditions of pollutant partial penetration in the biofilm. Pollutant partial penetration in a biofilm often occurs in treatment processes where very low effluent concentrations are required. However, under these conditions, adsorption in BGAC reactors is questionable and requires the existence of biofilm free areas on the GAC carrier. The purpose of this investigation is to prove that under normal BGAC fluidized bed reactor operational conditions patchy biofilm coverage with exposed areas of GAC develops. Adsorption and desorption through these exposed areas can explain the widely debated advantage of BGAC reactors regarding higher biofilm activity. The patchy-like nature of the biofilm coverage on the GAC particles was verified using experimental and modeling tools. Comparison between a nonadsorbing granular carbon carrier and a GAC carrier with an atrazine degrading biofilm (Pseudomonas ADP) under conditions of atrazine partial penetration in the biofilm showed higher biodegradation and lower effluent atrazine concentrations in the BGAC reactor.

A fused protein consisting of cellulose-binding domain (CBD) and horseradish peroxidase (HRP) was constructed and expressed in Escherichia coli. Refolded recombinant CBD-HRP (95% recovery yield) was bound to microcrystalline cellulose and applied for the oxidation of a model toxic phenol, 4-bromophenol (BP). Oxidation of BP by CBD-HRP resulted in the formation of dimers to pentamers as evidenced by mass spectrometry analysis. When immobilized, the vast majority of the oxidation products adsorbed to the cellulose matrix. CBD-HRP (0.75 pyrogallol units) bound to 0.1 g cellulose was packed in a column, connected to an HPLC pump and monitoring system, and column performance and capacity were studied under various operating conditions. When performance was studied as a function of BP loading rate at a constant H(2)O(2) loading rate of 1500 nmol/min, V(app) (max) and K(m) (app) were calculated to be 5.29 +/- 0.46 micromol mL min and 644.9 +/- 114.3 microM, respectively. Immobilized CBD-HRP exhibited enhanced stability to H(2)O(2) and oxidized considerably more BP than free CBD-HRP. Inclusion of gelatin, which suppresses product-dependent inactivation, further increased the amount of BP oxidation. These findings may have potential impact in terms of enzyme supply in high-rate treatment of wastewater contaminated with toxic phenols, since the susceptibility of peroxidases to both H(2)O(2) - and product-dependent inactivation demands continuous supply of fresh enzyme., (Copyright 2003 Wiley Periodicals, Inc.)

The present study characterized the ability of a bacterial cutinase to improve the wettability of raw cotton fabrics by specific hydrolysis of the cutin structure of the cuticle. The effect of cutinase was studied alone and in coreaction with pectin lyase. The changes in both the fabric and the reaction fluid were measured and compared to enzymatic hydrolysis with polygalacturonase, and to chemical hydrolysis with boiling NaOH. Water absorbancy, specific staining, fabric weight loss, and evaporative light-scattering reverse-phase high-performance liquid chromatography analysis of chloroform extract of the reaction fluid were measured to assess the enzymatic hydrolysis of the cuticle waxy layer. The pattern and extent of hydrolysis of the major cuticle constituents depended on the enzyme type and titers employed and paralleled the degree of wettability obtained. The combination of cutinase and pectin lyase resulted in a synergistic effect. The use of detergents improved enzymatic scouring. The major products released to the reaction medium by the cutinase treatment were identified by gas chromatography/mass spectrometry analysis as C:16 and C:18 saturated fatty acid chains.