Responses of oil degrader enzyme activities, metabolism and degradation kinetics to bean root exudates during rhizoremediation of crude oil contaminated soil.

During rhizoremediation process, plant roots secrete the specific exudates which enhance or stimulate growth and activity of microbial community in the rhizosphere resulting in effective degradation of pollutants. The present study characterized cowpea (CP) and mung bean (MB) root exudates and examined their influences on the degradation of total petroleum hydrocarbons (TPHs) and polycyclic aromatic hydrocarbons (PAHs) by the two oil degraders Micrococcus luteus WN01 and Bacillus cereus W2301. The effects of root exudates on soil microbial population dynamic and their enzymes dehydrogenase (DHA), and catechol 2,3 dioxygenase (C23O) activities were assessed. Both root exudates enhanced the degradation by both oil degraders. Cowpea root exudates maximized the removal of TPHs and PAHs by M. luteus WN01. Both bacterial population and DHA increased significantly in the presence of both root exudates. However, the C23O activities were significantly higher in WN01 treated. No significant influence of root exudates was observed on the C23O activities of W2301 treated. By using gas chromatography -mass spectroscopy, the dominant compounds found in cowpea and mung bean root exudates were 4-methoxy-cinnamic acid and terephthalic acid. Found in lower amount were propionic, malonic acid, and citric acid which were associated with enhanced PAHs desorption from soil and subsequent degradation. Novelty statement This is the first study to characterize the low molecular weight organic acids from root exudates of cowpea and mung bean and their influences on hydrocarbon desorption and hence enhancing the biodegradation process. The findings of the present study will greatly contribute to a better understanding of plant-microbe interaction in total petroleum hydrocarbons contaminated soil.

Examination of the Metallothionein Gene Family in Greater Duckweed Spirodela polyrhiza.

Duckweeds are aquatic plants that proliferate rapidly in a wide range of freshwaters, and they are regarded as a potential source of sustainable biomass for various applications and the cost-effective bioremediation of heavy metal pollutants. To understand the cellular and molecular basis that underlies the high metal tolerance and accumulation capacity of duckweeds, we examined the forms and transcript profiles of the metallothionein (MT) gene family in the model duckweed Spirodela polyrhiza, whose genome has been completely sequenced. Four S. polyrhiza MT-like genes were identified and annotated as SpMT2a, SpMT2b, SpMT3, and SpMT4. All except SpMT2b showed high sequence homology including the conserved cysteine residues with the previously described MTs from flowering plants. The S. polyrhiza genome appears to lack the root-specific Type 1 MT. The transcripts of SpMT2a, SpMT2b, and SpMT3 could be detected in the vegetative whole-plant tissues. The transcript abundance of SpMT2a was upregulated several-fold in response to cadmium stress, and the heterologous expression of SpMT2a conferred copper and cadmium tolerance to the metal-sensitive ∆cup1 strain of Saccharomyces cerevisiae. Based on these results, we proposed that SpMT2a may play an important role in the metal detoxification mechanism of duckweed.

Phytoremediation of soil co-contaminated with zinc and crude oil using Ocimum gratissimum (L.) in association with Pseudomonas putida MU02.

Soil adulteration by organic and inorganic contaminants chiefly in industrial and agricultural area is one of the major problems faced by the world today. Phytoremediation using aromatic plant such as Ocimum is a sound, economically reasonable and an eco-friendly approach. Besides, microbial assisted phytoremediation can bring about plant-promoted microbial degradation, plant uptake and phytodegradation of soil contaminants. In this study, the ability of Ocimum gratissimum in association with Pseudomonas putida MU02 to remediate zinc and crude oil contaminated soil (2,000 mg/kg, 1% w w-1 crude oil) was investigated in a 60-day pot experiment. Four different treatments and a control (clean soil + plant) were employed for the study: co-contaminated soil only; co-contaminated soil with O. gratissimum; co-contaminated with P. putida inoculum; co-contaminated soil with O. gratissimum and P. putida. Comparatively, the highest TPH deduction efficiency (75.87%) was shown by the treatment comprising of O. gratissimum and P. putida while highest metal uptake was shown by the treatment with plant only. Better TPH removal associated with higher number of bacteria in the vegetated and inoculated pots could be attributed to the rhizopheric effect of the plants. Altogether, the association of O. gratissimum with P. putida can be a potential candidate for the remediation of co-contaminated soil.

Bacteria-assisted phytoremediation of fuel oil and lead co-contaminated soil in the salt-stressed condition by chromolaena odorata and Micrococcus luteus.

Bioremediation of lead-petroleum co-contaminated soil under salt-stressed condition has been investigated. In this study, the co-contaminated soil (780 mg kg-1 Pb and 27,000 mg kg-1 TPHs) under the high salinity (EC 7.79 ds m-1) was used as a model soil to be remediated by Chromolaena odorata inoculated with Micrococcus luteus. The results showed that salt stress caused a marked reduction in dry biomass and stem height, and high accumulation of proline. The presence of salt did not affect the total amount of chlorophyll in plant tissues. No toxicity symptoms were evident from plant morphology after three months of exposure. Drastic differences in the accumulation patterns of Pb in C. odorata grown on saline and non-saline soils were observed and indicated that salinity negatively affected Pb uptake and accumulation. A high rate of degradation of TPHs was observed in non-saline soils with or without bacterial inoculation. Salinity stress showed no significant different in the proportion of TPH degradation with added or non-added M. luteus. The tolerance of C. odorata and M. luteus to moderate concentrations of Pb and fuel oil made them very good candidates for the use in bacteria-assisted phytoremediation of lead-fuel oil co-contaminated soils under the mild saline soils.

Phytoremediation of fuel oil and lead co-contaminated soil by Chromolaena odorata in association with Micrococcus luteus.

Phytoremediation is widely promoted as a cost-effective technology for treating heavy metal and total petroleum hydrocarbon (TPH) co-contaminated soil. This study investigated the concurrent removal of TPHs and Pb in co-contaminated soil (27,000 mg kg(-1) TPHs, 780 mg kg(-1) Pb) by growing Siam weed (Chromolaena odorata) in a pot experiment for 90 days. There were four treatments: co-contaminated soil; co-contaminated soil with C. odorata only; co-contaminated soil with C. odorata and Micrococcus luteus inoculum; and co-contaminated soil with M. luteus only. C. odorata survived and grew well in the co-contaminated soil. C. odorata with M. luteus showed the highest Pb accumulation (513.7 mg kg(-1)) and uptake (7.7 mg plant(-1)), and the highest reduction percentage of TPHs (52.2%). The higher TPH degradation in vegetated soils indicated the interaction between the rhizosphere microorganisms and plants. The results suggested that C. odorata together with M. luteus and other rhizosphere microorganisms is a promising candidate for the removal of Pb and TPHs in co-contaminated soils.

Phytoremediation potential of charophytes: bioaccumulation and toxicity studies of cadmium, lead and zinc.

The ability for usage of common freshwater charophytes, Chara aculeolata and Nitella opaca in removal of cadmium (Cd), lead (Pb) and zinc (Zn) from wastewater was examined. C. aculeolata and N. opaca were exposed to various concentrations of Cd (0.25 and 0.5 mg/L), Pb (5 and 10 mg/L) and Zn (5 and 10 mg/L) solutions under hydroponic conditions for 6 days. C. aculeolata was more tolerant of Cd and Pb than N. opaca. The relative growth rate of N. opaca was drastically reduced at high concentrations of Cd and Pb although both were tolerant of Zn. Both macroalgae showed a reduction in chloroplast, chlorophyll and carotenoid content after Cd and Pb exposure, while Zn exposure had little effects. The bioaccumulation of both Cd and Pb was higher in N. opaca (1544.3 microg/g at 0.5 mg/L Cd, 21657.0 microg/g at 10 mg/L Pb) whereas higher Zn accumulation was observed in C. aculeolata (6703.5 microg/g at 10 mg/L Zn). In addition, high bioconcentration factor values (> 1000) for Cd and Pb were observed in both species. C. aculeolata showed higher percentage of Cd and Pb removal (> 95%) than N. opaca and seemed to be a better choice for Cd and Pb removal from wastewater due to its tolerance to these metals.

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 by hybridization probe based real-time PCR.

Quantitative detection of the oil-degrading bacterium Acinetobacter sp. strain MUB1 was performed using the SoilMaster() DNA Extraction Kit (Epicentre, Madison, Wisconsin) and hybridization probe based real-time PCR. The detection target was the alkane hydroxylase gene (alkM). Standard curve construction showed a linear relation between log values of cell concentrations and real-time PCR threshold cycles over five orders of magnitude between 5.4+/-3.0x10(6) and 5.4+/-3.0x10(2)CFUml(-1) cell suspension. The detection limit was about 540CFUml(-1), which was ten times more sensitive than conventional PCR. The quantification of Acinetobacter sp. strain MUB1 cells in soil samples resulted in 46.67%, 82.41%, and 87.59% DNA recovery with a detection limit of 5.4+/-3.0x10(4)CFUg(-1) dry soil. In this study, a method was developed for the specific, sensitive, and rapid quantification of the Acinetobacter sp. strain MUB1 in soil samples.

Batch and continuous packed column studies of cadmium biosorption by Hydrilla verticillata biomass.

The removal of heavy metal ions by the nonliving biomass of aquatic macrophytes was studied. We investigated Cd biosorption by dry Hydrilla verticillata biomass. Data obtained in batch experiments indicate that H. verticillata is an excellent biosorbent for Cd. Cd was rapidly adsorbed and such adsorption reached equilibrium within 20 min. The initial pH of the solution affected Cd sorption efficiency. Results obtained from the other batch experiments conformed well to those obtained using the Langmuir model. The maximum adsorption capacity q(max) for H. verticillata was 15.0 mg/g for Cd. The breakthrough curve from the continuous flow studies shows that H. verticillata in the fixed-bed column is capable of decreasing Cd concentration from 10 to a value below the detection limit of 0.02 mg/l. The presence of Zn ions affected Cd biosorption. It can be concluded that H. verticillata is a good biosorbent for treating wastewater with a low concentration of Cd contaminants.

Alterations of organ histopathology and metallothionein mRNA expression in silver barb, Puntius gonionotus during subchronic cadmium exposure.

Common silver barb, Puntius gonionotus, exposed to the nominal concentration of 0.06 mg/L Cd for 60 d, were assessed for histopathological alterations (gills, liver and kidney), metal accumulation, and metallothionein (MT) mRNA expression. Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae, vacuolization in hepatocytes, and prominent tubular and glomerular damage in the kidney. In addition, kidney accumulated the highest content of cadmium, more than gills and liver. Expression of MT mRNA was increased in both liver and kidney of treated fish. Hepatic MT levels remained high after fish were removed to Cd-free water. In contrast, MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water. The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.

Toxicity and bioaccumulation of cadmium and lead in Salvinia cucullata.

The toxicity and accumulation of heavy metals, cadmium (Cd) and lead (Pb) in aquatic fern, Salvinia cucullata were studied. Plants were cultured in Hoagland's medium which was supplemented with 0.5,1,2, and 4 mg/l of Cd and 5, 10, and 40 mg/l of Pb and were separately harvested after 2,4,6, and 8 days. The toxicity symptoms of Cd and Pb to S. cucullata showed chlorosis on leaves. There were significant derceases in the relative growth, biomass productivity and total chlorophyll content when the exposure time and concentration were increased. The accumulation study showed the significant increases of both metals when the exposure time and concentration were increased. The roots of S. cucullata had higher Cd and Pb contents than leaves suggesting that the metals were bound to the root cells and were partially transported to the leaves.

Ultrastructural alterations in the liver and kidney of white sea bass, Lates calcarifer, in acute and subchronic cadmium exposure.

Ultrastructural alterations in the liver and kidney of 3-month-old white sea bass, Latescalcarifer, after cadmium exposure were studied by transmission electron microscopy (TEM). One group of fish was exposed to a cadmium concentration of 10 mg/L (acute) for 96 h in a static system, and another group was exposed to cadmium concentrations of 0.8 and 3 mg/L cadmium (subchronic) for 3 months in a recirculation closed system. Ultrastructural alterations observed in the hepatocytes included mitochondrial condensation, swelling, and lysis. The rough endoplasmic reticulum (RER) showed dilation, fragmentation, and vesiculation. After subchronic exposure there were numerous large lipid droplets and abundant stored glycogen. Ultrastructural alterations observed in the proximal tubules of the kidney included nuclear degeneration, condensation, and massive swelling of the mitochondria; RER fragmentation and vesiculation. Disorganized brush borders and increased numbers of large hydropic vacuoles and lysosomes were also observed.

Biosorption of cadmium and chromium in duckweed Wolffia globosa.

The biosorption of cadmium (Cd) and chromium (Cr) by using dried Wolffia globosa biomass were investigated using batch technique. The effects of concentration and pH solution on the adsorption isotherm were measured by determining the adsorption isotherm at initial metal concentrations from 10 to 400 mg/L and pH 4 to 7 for Cd, and pH 1.5 to 6 for Cr. The adsorption equilibria were found to follow Langmuir models. The maximum adsorption capacity (Xm) at pH 7 in W. globosa-Cd system was estimated to be 80.7 mg/g, while the maximum removal achieved at pH 4, pH 5, and pH 6 were 35.1, 48.8, and 65.4 mg/g, respectively. The Xm at pH 1.5 in W. globosa--Cr system was estimated to be 73.5 mg/g, while the maximum removal achieved at pH 3, pH 5, and pH 6 were 47.4, 33.1, and 12.9 mg/g, respectively. The effects of contact times on Cd and Cr sorption indicated that they were absorbed rapidly and more efficiently at lower concentrations.

Phytoaccumulation and phytotoxicity of cadmium and chromium in duckweed Wolffia globosa.

The phytoaccumulation and phytotoxicity of heavy metals, cadmium (Cd), and chromium (Cr) on a common duckweed, Wolffia globosa, were studied. W. globosa were cultured in 3% Hoagland's nutrient medium, which was supplemented with 1, 2, 4, and 8 mg/L of Cd and Cr and were separately harvested after 3, 6, 9, and 12 days. The accumulation of Cd and Cr in W. globosa showed significant increases when the exposure time and metal concentration were increased. The effects of Cd and Cr on the biomass productivity and total chlorophyll content in W. globosa indicated that there were significant decreases in the biomass productivity and total chlorophyll content when the exposure time and metal concentration were increased.