Synergy among extracellular adsorption, bio-precipitation and transmembrane transport of Penicillium oxalicum SL2 enhanced Pb stabilization.

As a potential bioremediation strain for Pb contamination, Penicillium oxalicum SL2 sometimes has secondary activation of Pb, so it is crucial to clarify its effect on Pb morphology and its intracellular response to Pb stress. We investigated the effect of P. oxalicum SL2 in medium on Pb2+ and Pb availability in eight minerals, and revealed the prioritization of Pb products. (i)Pb was stabilized within 30 days as Pb3(PO4)2 or Pb5(PO4)3Cl with sufficient phosphorus (P); (ii) under P deficiency but sulfur (S) sufficient, Pb was stabilized mainly in the form of PbSO4; (iii) under conditions of P and S deficiency, Pb was stabilized mainly in the form of PbC2O2. With the help of proteomic and metabolomics analysis, a total of 578 different proteins and 194 different metabolites were found to be matched in 52 pathways. Among them, the activation of chitin synthesis, oxalate production, sulfur metabolism and transporters improved the Pb tolerance of P. oxalicum SL2, and promoted the synergistic effect of extracellular adsorption, bio-precipitation and transmembrane transport on Pb stabilization. Our results fill the gap in the intracellular response of P. oxalicum SL2 to Pb and provide new insights into the development of bioremediation agent and technology for Pb contamination.

Simultaneous removal of multiple heavy metals from soil by washing with citric acid and ferric chloride.

The remediation of soil contaminated with multiple heavy metals is a matter of great concern due to its serious threat to the ecosystem and human health. Batch and slurry reactor soil washing experiments were conducted to explore the removal of Cd, Cr, Pb and Zn using 7 agents. Citric acid (CA) and ferric chloride (FeCl3) exhibited an obvious synergistic effect on the removal of heavy metals. Furthermore, the concentration of heavy metals in different soil particle size fractions was closely related to the soil element concentrations. Fine sand (0.05-0.25 mm) had a strong adsorption capacity for Cr and Pb because of the high Mn concentration. Notably, heavy metals in smaller-size soil particles could be efficiently removed by CA and FeCl3. After remediation, the bioavailability of heavy metals in soil decreased. The potential ecological risk of heavy metals in soil reduced from an extremely high level to a low level. Moreover, some elements (e.g. Al, Mn and Fe) and organic matter in soil were dissolved by CA and FeCl3, which accelerated the desorption of heavy metals from the soil. In a slurry reactor experiment, the removal efficiencies of Cd, Cr, Pb and Zn were 94.8%, 79.5%, 92.7% and 97.2%, respectively. The combination of CA and FeCl3 is a feasible practice to remediate soil contaminated by multiple heavy metals.

Ca2+ and SO42- accelerate the reduction of Cr(VI) by Penicillium oxalicum SL2.

Some ions in soils may affect the growth and metabolism of microorganisms and subsequently alter the remediation efficiency of Cr(VI). Here, the effects of different Ca2+ and SO42- levels on the reduction of Cr(VI) by Penicillium oxalicum SL2 were investigated. The results showed that Cr(VI) reduction by P. oxalicum SL2 in potato dextrose liquid (PDL) medium was accelerated by the presence of exogenous Ca2+ and SO42-. The Cr(VI) reduction rates were increased by 52.5% (200 mg L-1 Ca2+ treated) and 55.9% (2000 mg L-1 SO42- treated), respectively. High concentration of Ca2+ in medium resulted in the production of calcium oxalate crystals, which was contributed to the adsorption of chromium. In addition, X-ray absorption near-edge spectroscopy (XANES) analysis showed that P. oxalicum SL2 could reduce the toxicity of Cr(VI) by synthesizing cysteine (Cys) and reduced glutathione (GSH). The decrease of thiol compounds (Cys and GSH) in P. oxalicum SL2 mycelia treated with SO42- proved the alleviation of oxidative stress. In conclusion, exogenous Ca2+ could reduce the damage of Cr(VI) to P. oxalicum SL2 by maintaining the integrity of cell wall, and the addition of SO42- alleviated the Cr(VI) toxicity to P. oxalicum SL2, thus accelerating the reduction of Cr(VI).

Lead was mobilized in acid silty clay loam paddy soil with potassium dihydrogen phosphate (KDP) amendment.

The immobilization effectiveness between Pb and phosphorus in soil varies with soil types. To clarify the effect of phosphate on the availability of Pb in agricultural soil, a culture experiment with three types of paddy soil was performed with potassium dihydrogen phosphate (KDP) added. EDTA, DGT and in-situ solution extraction methods were used to represent different available Pb content. Results showed that the concentration of EDTA-Pb in HN soil was slightly elevated after exogenous KDP added. The supplement of 300 mg/kg KDP significantly increased the content of soluble Pb in both acid silty clay loam soil and neutral silty loam soil (increased by 104.65% and 65.12%, respectively). However, there was no significant influence of KDP on the concentration of DGT extracted Pb. XANES results showed that Pb(OH)2, PbHPO4, humic acid-Pb and GSH-Pb were the major speciation of Pb in soil colloids. The proportion of Pb(OH)2 and humic acid-bounded Pb in soil colloids were elevated after exogenous KDP added. Our results indicated that there was a mobilization effect of KDP on Pb by increasing the amount of colloidal Pb in soil solution, especially in acid silty clay loam paddy soil. Such colloid-facilitated transport might promote the uptake of Pb in rice and pose a potential threat to human health.

Investigation of lead bioimmobilization and transformation by Penicillium oxalicum SL2.

Fungi Penicillium oxalicum SL2 was applied for Pb2+ bioremediation in aqueous solution in this study. After 7 days of incubation at different initial concentrations of Pb2+ (0, 100, 500 and 2500 mg L-1), most of Pb2+ were removed (90, 98.3, and 86.2%), the maximum Pb content in mycelium reached about 155.6 mg g-1 dw. Meanwhile, the formation of extracellular secondary minerals and intracellular Pb-complex were observed and identified, the speciation of Pb in mycelium was also detected by X-ray absorption near-edge structure (XANES) spectroscopy, i.e., Pb-oxalate, Pb-citrate, Pb-hydrogen phosphate and Pb-glutathione analogues. In addition, content of glutathione and oxidized glutathione was increased under the exposure of Pb2+, which implied that glutathione might play a key role in Pb immobilization and detoxification in P. oxalicum SL2. This study elucidated partial mechanisms of Pb immobilization and speciation transformation of this strain, providing an alternative biomaterial in the bioremediation of Pb-contaminated wastewater.

Characterization of Penicillium oxalicum SL2 isolated from indoor air and its application to the removal of hexavalent chromium.

Removal of toxic Cr(VI) by microbial reduction is a promising approach to reducing its ecotoxicological impact. To develop bioremediation technologies, many studies have evaluated the application of microorganisms isolated from Cr(VI)-contaminated sites. Nonetheless, little attention has been given to microbes from the environments without a history of Cr(VI) contamination. In this study, we aimed to characterize the Cr(VI) tolerance and removal abilities of a filamentous fungus strain, SL2, isolated from indoor air. Based on phenotypic characterization and rDNA sequence analysis, SL2 was identified as Penicillium oxalicum, a species that has not been extensively studied regarding Cr(VI) tolerance and reduction abilities. SL2 showed high tolerance to Cr(VI) on solid and in liquid media, facilitating its application to Cr(VI)-contaminated environments. Growth curves of SL2 in the presence of 0, 100, 400, or 1000 mg/L Cr(VI) were well simulated by the modified Gompertz model. The relative maximal colony diameter and maximal growth rate decreased as Cr(VI) concentration increased, while the lag time increased. SL2 manifested remarkable efficacy of removing Cr(VI). Mass balance analysis indicated that SL2 removed Cr(VI) by reduction, and incorporated 0.79 mg of Cr per gram of dry biomass. In electroplating wastewater, the initial rate of Cr(VI) removal was affected by the initial contaminant concentration. In conclusion, P. oxalicum SL2 represents a promising new candidate for Cr(VI) removal. Our results significantly expand the knowledge on potential application of this microorganism.

Mitochondrial and endoplasmic reticulum pathways involved in microcystin-LR-induced apoptosis of the testes of male frog (Rana nigromaculata) in vivo.

Previous studies have shown that toxins produced by toxic cyanobacterial blooms are hazardous materials. In the present study, 1 µg/L microcystin-LR (MC-LR) was observed to induce apoptosis in the testes of male Rana nigromaculata via the mitochondrial and endoplasmic reticulum (ER) pathways at exposure times ranging from 7 d to 14 d. The results showed that reactive oxygen species production and malondialdehyde content were positively correlated with exposure time. Antioxidant enzyme contents, such as reduced glutathione and glutathione peroxidase rapidly decreased, implying that the defense system of the testes induces oxidative damage. MC-LR significantly stimulated the release of cytochrome c in the testes, thereby improving the protein expressions of Bax and caspases-3, 8, and 9 (p<0.01) and inhibiting the protein expression of Bcl-2 with prolonged exposure (p<0.01). Ultrastructural observations showed distention of the mitochondria and endoplasmic reticulum and deformation of the nucleolus. Moreover, prolonged exposure times strengthened and weakened the relative expression levels of C/EBP homologous protein and GRP78, respectively. These results indicate that MC-LR-induced apoptosis of the testes in male frogs in vivo may occur through the mitochondrial and ER pathways. It also further proves our previous findings that MC-LR can induce toxicity in the male reproductive system of R. nigromaculata in vitro. The findings show that MC-LR is highly hazardous to frogs and that the accepted drinking water limit of 1 µg/L MC-LR exerts significant toxicity to amphibians.

Regulation of nodularin-induced apoptosis by epigallocatechin-3-gallate on fish lymphocytes in vitro.

Nodularin is one of the most conspicuous and widespread pollutants that elicit water ecological hazards to fish, causing serious damage on the immune system and physiological functions. Nodularin can cause oxidative stress-induced apoptosis on fish lymphocytes. The regulatory effects of epigallocatechin-3-gallate (EGCG) at 10, 100, and 1000 µg/L levels on the antioxidant defense system and apoptosis of Carassius auratus lymphocytes exposed to a high dose of nodularin (100 µg/L) were quantified in vitro. EGCG reduced nodularin-induced oxidative damage on fish immune cells. This compound significantly increased the activities of superoxide dismutase and catalase and the level of glutathione but decreased the levels of intracellular reactive oxygen species and malondialdehyde. Flow cytometry results showed that the percentages of apoptotic cells after treatment with 10, 100, and 1000 µg/L EGCG for 12 h reached 27.9%, 19.1%, and 13.7%, respectively. By contrast, the nodularin alone-induced group showed a high percentage of apoptosis (44.2%). Western blot analysis showed the increased expression of bcl-2 and the decreased expression of bax and caspase-3 in EGCG-treated fish lymphocytes. EGCG also inhibited the potential collapse of the mitochondrial membrane. Overall, EGCG can inhibit nodularin-induced apoptosis and protect the normal immunity of fish by regulating bax/bcl-2 and blocking the downstream of mitochondrial apoptosis pathway with increased intracellular antioxidant enzyme activity.

Toxic effects of microcystin-LR on the reproductive system of male Rana nigromaculata in vitro.

This study aims to demonstrate that microcystin-LR (MC-LR) has toxic effects on the reproductive system of male Rana nigromaculata in vitro. R. nigromaculata were treated with 0, 0.1, 1, 10, and 100 nmol/L of MC-LR for 6 h. Results show that exposure to 1 nmol/L to 100 nmol/L of MC-LR decreased sperm motility and number of sperm cells and increased the sperm abnormality rate, whose values were significantly different from those of the control (P<0.01). Moreover, the same dosage of MC-LR increased reactive oxygen species production and malondialdehyde content. At the same time, antioxidant enzyme (catalase and glutathione S-transferase) activity and glutathione reduced content rapidly increased, whereas antioxidant enzyme superoxide dismutase activity significantly decreased. These results imply that the defense system of the testes quickly responds to oxidative stress. Ultrastructural observation shows distention of the mitochondria, endoplasmic reticulum, and Golgi apparatus and changes in the mitochondrial matrix color, cristae number, and morphology. Moreover, using real-time PCR, increased relative expressions of P450 aromatase and SF-1 genes were observed. The results demonstrate for the first time that MC-LR could induce toxicity in the male reproductive system of R. nigromaculata. The findings in this research will provide more insights into the relationships between aquatic microcystins and amphibians.

Apoptotic responses of Carassius auratus lymphocytes to nodularin exposure in vitro.

Nodularin, a metabolite of Nodularin spumigena, is widely detected in water blooms worldwide and causes serious negative effects on fish. The apoptosis-related cytotoxic effects and mechanisms of nodularin on Carassius auratus lymphocytes were investigated. Transmission electron microscopy results showed that nodularin-treated lymphocytes display a series of morphological changes, including condensed cytoplasm, nuclear chromatin agglutination and marginalization. DNA fragmentation was verified by the DNA-ladder and formation of sub-G1 DNA peaks. These cell characteristics confirmed the occurrence of apoptosis in lymphocytes. Flow cytometric results showed that the percentages of apoptotic cells incubated with 1, 5, 10, and 100 µg/L nodularin for 12 h reached 15.76%, 17.36%, 20.34% and 44.21%, respectively; controls showed low rates of apoptosis (2.4%). The mechanism of apoptosis induced by nodularin was determined, and results showed that nodularin exposure caused a significant increase in intracellular reactive oxygen species (ROS), loss of mitochondrial transmembrane potential in a dose-dependent manner, upregulation of intracellular Ca²âº, downregulation of Bcl-2 and upregulation of Bax expression at the mRNA and protein levels, and activation of caspase-3 and caspase-9 without caspase-8. In summary, all the results suggest that nodularin induces lymphocyte apoptosis via the mitochondrial apoptotic pathway and destroys the immune response of fish.