Carlos G. Dosoretz

Irpex lacteus, a white rot fungus applicable to water and soil bioremediation

Abstract Growth parameters, ligninolytic enzyme activities and ability to degrade polycyclic aromatic hydrocarbons by the fungus Irpex lacteus were characterized and compared with those of other white rot fungi capable of rapid decolorization of poly R-478 and Remazol Brilliant Blue R dyes. I. lacteus was able to grow on mineral and complex media and efficiently colonized sterile and non-sterile soil by exploratory mycelium growing from a wheat straw inoculum. In shallow stationary cultures growing on high nitrogen mineral medium containing 45 mM ammonium as nitrogen source, the fungus produced lignin peroxidase (LIP), Mn-dependent peroxidase (MnP) and laccase simultaneously, the respective maximal activities of 70, 970 and 36 U/l being attained around day 18. Growing in nitrogen-limited medium (2.4 mM ammonium), no LIP was formed and levels of MnP and laccase decreased significantly. During growth in sterile soil, the fungus synthesized LIP and laccase but not MnP. I. lacteus efficiently removed three- and four-ringed PAHs from liquid media and artificially spiked soil. The variety of ligninolytic enzymes, robust growth, capability of soil colonization and resistance to inhibitory action of soil bacteria make I. lacteus a suitable fungal organism for use in bioremediation.

Potential Use of Cutinase in Enzymatic Scouring of Cotton Fiber Cuticle

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 reversephase 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.

1-Octen-3-ol and 13-hydroperoxylinoleate are products of distinct pathways in the oxidative breakdown of linoleic acid by Pleurotus pulmonarius

Abstract The oxidative breakdown of linoleic acid leading to the formation of the mushroom flavor alcohol 1-octen-3-ol by mycelial homogenate of the edible mushroom Pleurotus pulmonarius grown in submerged culture was studied. The compounds 13-hydroperoxy- cis -9, trans -11-octadecadienoic acid (13-HPOD) and 10-oxo- trans -8-decenoic acid (10-oxo-acid) were found to be the major nonvolatile metabolites associated with the enzymatic cleavage of linoleic acid to 1-octen-3-ol. Whereas 1-octen-3-ol and 10-oxo-acid were produced at all linoleic acid concentrations studied (up to 8 mM), 13-HPOD was absent at linoleic acid concentrations below 1 mM but became the major nonvolatile product of the enzymatic oxidation at concentrations above 1 mM. Despite its accumulation, 13-HPOD was found not to be the precursor of 1-octen-3-ol when it was supplied as the substrate instead of linoleic acid. Periodic addition of linoleic acid during the course of the reaction maintained 1-octen-3-ol formation at a constant rate while 13-HPOD accumulated, suggesting that 1-octen-3-ol formation may be limited by either competitive product inhibition or substrate availability. These results suggest the involvement of two different lipoxygenases in 1-octen-3-ol and 13-HPOD formation. Mild heat treatment of the pellets before homogenization and incubation completely inhibited 1-octen-3-ol formation and resulted in the accumulation of an additional HPOD, possibly a 1-octen-3-ol precursor. It would appear, therefore, that 13-HPOD accumulation takes place in parallel with 1-octen-3-ol and 10-oxo-acid biosynthesis, suggesting that these are two distinct biosynthetic pathways catalyzed by two different lipoxygenases.

Reactive Oxygen Species and Induction of Lignin Peroxidase in Phanerochaete chrysosporium

ABSTRACT We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O2) 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 O2 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 O2 (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 O2 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 O2 is at least partially mediated by the intracellular ROS.

Microbial degradation of aromatic and polyaromatic toxic compounds adsorbed on powdered activated carbon

Abstract The microbial degradation of phenol (300–1300 mg l −1 ), p -nitrophenol (50–500 mg l −1 ), and phenanthrene (50–300 mg l −1 ) adsorbed on powdered activated carbon (PAC) was studied. The combination of physical sorption and biological degradation on PAC, which serves as adsorbent carrier, was compared with the degradation performance of suspended cells and cultures attached to sand, which serves as an example of an inert support. Almost similar profiles of degradation were obtained in the cultures with PAC for the three compounds studied, over the entire concentration range. However, at the higher concentrations, degradation was dramatically inhibited, or even ceased completely, in suspended or sand-attached cultures, even though specialized mixed bacterial cultures adapted to high toxicant concentrations were used in each case. Degradation with PAC assumes an intrinsic relationship between the activated carbon and bacteria in the same environment, thus regenerating the surface of the adsorbent/ carrier as diffusion and degradation proceed. The adsorbed material desorbs, diffuses out of the carbon and can then be metabolized. Our results highlight the applicability of activated carbon, in either powdered or granular form, to achieve a steady-state operation in continuous process, for the treatment of toxic chemicals, even with specialized bacteria.

Gene silencing by RNA Interference in the white rot fungus Phanerochaete chrysosporium.

ABSTRACT 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.

Atrazine degradation under denitrifying conditions in continuous culture of Pseudomonas ADP.

The simultaneous removal of atrazine and nitrate in continuous culture under denitrifying conditions using Pseudomonas sp. strain ADP was investigated. Under all operational conditions the nitrate removal efficiency was always higher than 90%, while atrazine degradation deteriorated with time due to contamination by foreign denitrifying bacteria, lacking the ability to degrade atrazine. Recovery of atrazine degradation ability was achieved by applying aerobic conditions with atrazine as the sole nitrogen source.

Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases

Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29 degrees were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k(i) = 2.03 x 10(9) M(-1) min(-1)) > mouse AChE (MoAChE) approximately equal to fetal bovine serum AChE (FBS-AChE) approximately equal to equine BChE (EqBChE); for DEP-4PAM, HuBChE (k(i) = 0.71 x 10(9) M(-1) min(-1)) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k(cat)/Km = 3.56 x 10(7) M(-1) min(-1) and Km = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.

Integrated experimental investigation and mathematical modeling of brackish water desalination and wastewater treatment in microbial desalination cells.

Desalination of brackish water can provide freshwater for potable use or non potable applications such as agricultural irrigation. Brackish water desalination is especially attractive to microbial desalination cells (MDCs) because of its low salinity, but this has not been well studied before. Herein, three brackish waters prepared according to the compositions of actual brackish water in three locations in Israel were examined with domestic wastewater as an electron source in a bench-scale MDC. All three brackish waters could be effectively desalinated with simultaneous wastewater treatment. The MDC achieved the highest salt removal rate of 1.2 g L(-1) d(-1) with an initial salinity of 5.9 g L(-1) and a hydraulic retention time (HRT) of 0.8 d. The desalinated brackish water could meet the irrigation standard of both salinity (450 mg L(-1) TDS) and the concentrations of major ionic species, given a sufficient HRT. The MDC also accomplished nearly 70% removal of organic compounds in wastewater with Coulombic efficiency varied between 5 and 10%. A previously developed MDC model was improved for brackish water desalination, and could well predict salinity variation and the concentrations of individual ions. The model also simulated a staged operation mode with improved desalination performance. This integrated experiment and mathematical modeling approach provides an effective method to understand the key factors in brackish water desalination by MDCs towards further system development.

Characterization of atrazine degradation and nitrate reduction by Pseudomonas sp. strain ADP

Concomitant atrazine degradation and nitrate reduction by a pure culture of Pseudomonas sp. strain ADP were studied. Under anoxic conditions, Ps ADP grew well and degraded atrazine efficiently in the presence of nitrate. Similar atrazine degradation rates were observed under both anoxic and aerobic conditions: 30.7±2.83 and 36.2±0.44 mg atrazine g−1 cell h−1, respectively. A high denitrification rate of 90.8±8.22 mg NO3−-N g−1 cell h−1 was also observed using Ps ADP with citrate as the electron donor. The required citrate to nitrate ratio for complete denitrification was 5.11±0.15 g citrate g−1 NO3−-N.