Phytoaccumulation of cadmium by Pelargonium × hortorum - tolerance and metal recovery.

The main aim of the present study was to assess the removal of cadmium (Cd) from contaminated soil by using Pelargonium × hortorum - an ornamental plant. Furthermore, the genotoxic impacts of Cd on plant was evaluated, and accumulated Cd in shoots were recovered as Cd-nanoparticles. For this purpose, a pot experiment was carried out with Cd (0-150 mg/kg) spiked soil. P. hortorum was grown for 24 weeks in a greenhouse. Subsequently, harvested root/shoot biomass and Cd concentration in root/shoot were determined. The micronucleus assay was performed to assess the genotoxicity of Cd within the selected plant. Accumulated Cd in shoots was recovered as Cd-nanoparticles and was characterized by SEM and XRD. Exposure to Cd exhibited a phytotoxic impact by reducing the plant biomass, but plant survived at higher Cd concentrations and the tolerance index was greater than 60% at a higher Cd level (150 mg/kg). Moreover, 257 mg/kg of Cd in aerial parts was observed, and maximum Cd uptake (120 mg plant-1) by P. hortorum was found at 150 mg/kg Cd. Plants exposed to Cd exhibited genotoxic impact by increasing the number of micronuclei by 59% at a higher Cd level (150 mg/kg) and the mitotic index was reduced by 20%. Furthermore, recovered nanoparticles were spherically shaped with an average size of 36.2-355 nm. The plant has potential for the removal of Cd and has exhibited good tolerance.

Graphene quantum dot and iron co-doped TiO2 photocatalysts: Synthesis, performance evaluation and phytotoxicity studies.

The present study reports the synthesis, photocatalytic decolorization of reactive black 5 dye and phytotoxicity of graphene quantum dots (GQDs) and iron co-doped TiO2 photocatalysts via modified sol gel method. GQDs were synthesized by direct pyrolysis of citric acid (CA). Scanning electron microscopy (SEM) and energy dispersion spectroscopy (EDS), Raman spectroscopy, atomic force microscopy (AFM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), diffuse reflectance spectroscopy (DRS), Brunauer-Emmett-Teller (BET) and photoluminescence spectroscopy (PL) were used to determine the physicochemical properties of the best performing photocatalysts. The results indicated improved physicochemical properties of GQD-0.1Fe-TiO2-300 with root mean square roughness (Rz) (33.82 nm), higher surface area (170.79 m2 g-1), pore volume (0.08 cm3 g-1), and bandgap (2.94 eV). Moreover, GQD-0.1Fe co-doping of TiO2 greatly improved the photocatalytic decolorization efficiency for RB5 dye. The photocatalytic reaction followed the pseudo first order reaction with gradual decrease in Kapp values for increment in RB5 concentration. The KC value was obtained as 2.45 mg L-1 min-1 while the KLH value was 0.45 L mg-1 indicating the heterogeneous reaction system followed the Langmuir-Hinshelwood isotherm and simultaneously occurring adsorption and photocatalytic processes. Photocatalytic reaction mechanism studies exhibited the holes and OH radicals as the main active species in the GQD-0.1Fe-TiO2-300 responsible for the decolorization of RB5. The proposed reaction pathway showed that both Fe-TiO2 and GQDs play important role in generation of electrons and holes. Additionally, GQD-0.1Fe-TiO2-300 were durable up to four cycles. Phytotoxicity assay displayed that treated water and best performing photocatalysts had no effect on Lycopersicon esculentum seed germination. Therefore, the proposed system can pave a viable solution for safe usage of dye loaded wastewater and effluent for irrigation after treatment.

Optimization of integrated phytoremediation system (IPS) for enhanced lead removal and restoration of soil microbial activities.

Improving phytoremediation efficiency in lead (Pb) contaminated soil through either bacterial or fungal inoculants have extensively been studied with different successes and limitations. In this study, co-application of bacteria and fungi have been investigated for development of an integrated phytoremediation system (IPS) for efficient Pb removal and restoration of soil microbial and enzymatic activities in degraded soil. For this purpose, Pb tolerant bacterial and fungal strains were firstly analyzed for antifungal and antibacterial activities through disc diffusion method. Afterwards, the co-inoculation studies were performed to investigate the effects on phytoavailability and uptake of Pb by Pelargonium hortorum through soil incubation and pot culture experiments, respectively. Results indicated significant (p < 0.05) antibacterial activity of Mucor spp. against bacterial species (Klebsiella variicola and K. quasipneumoniae). The highest significant increase in extractable Pb fraction (5.0-folds) was observed when soil was co-inoculated with Aspergillus flavus + Microbacterium paraoxydans compared to the control soil (un-inoculated soil) at 2000 mg Pb kg-1 concentration. Similarly, uptake results also indicated significantly higher Pb uptake in plants inoculated with A. flavus + M. paraoxydans. Soil microbial results indicated significant decrease in microbial health indicators and enzymatic activities with increasing Pb concentration and exposure time, as compared to control soil. A relatively severe decline was observed in soil respiration and dehydrogenase (DEH) activities by 2.8- and 2.5-folds, respectively at 2000 mg Pb kg-1 of soil. The optimized IPS was effective for restoring enzymatic activities in Pb contaminated soil and could be applied for sustainable restoration of Pb contaminated soil.

Enhanced phytoremediation of lead by soil applied organic and inorganic amendments: Pb phytoavailability, accumulation and metal recovery.

Chelation of lead (Pb) is an important factor in enhancing the Pb mobility thereby improving availability to promote phytoremediation of Pb from contaminated soil. The study was conducted to evaluate the effect of amendments in enhancing the phytoremediation of Pb in soil. For this purpose, soil was spiked to obtain desired Pb concentrations (0-1500 mg kg-1) and pots were filled. One month old and uniform seedlings of Pelargonium hortoum were transplanted into each pot. Five different amendments i.e. compost (0-10%), ammonium nitrate (0-10 mmol kg-1), TiO2NPs (0-100 mg kg-1), citric acid (0-10 mmol kg-1) and EDTA (0-5 mmol kg-1), were applied. Overall, ammonium nitrate, EDTA and citric acid application increased the Pb concentration, however, compost and TiO2NPs decreased the concentration in roots and shoots. At 1500 mg Pb kg-1, Pb concentration in shoots was increased by 0.9-, 0.6- & 0.8-folds and in roots by 1.8-, 1.3- & 1.7-folds upon EDTA, ammonium nitrate and citric acid application, respectively. TiO2NPs and compost application decreased Pb concentration by 29% & 35% in shoots and 25% & 51% in roots, respectively. At the highest level of Pb (1500 mg kg-1), plant biomass was increased by 26.6%, 19.5%, 17.9% and 18.4% upon application of compost, TiO2 NPs, ammonium nitrate and citric acid, respectively. However, EDTA reduced the plant dry biomass by 28.4%. The accumulated Pb content was recovered as Pb-nanoparticles, which were in anatase phase, size ranged between 98 and 276 nm. Among all the studied amendments, citric acid efficiently increased Pb phytoaccumulation without any toxicity.

Lead phytoextraction by Pelargonium hortorum: Comparative assessment of EDTA and DIPA for Pb mobility and toxicity.

Soil amendments like ethylene-diamine-tetraacetic-acid (EDTA) have extensively been used for enhancing lead (Pb) phytoextraction. But due to its toxic effects, environment friendly substitute is required. Therefore, the present study was conducted to investigate the effect of EDTA and Di-iso-propanol-amine (DIPA) to enhance Pb phytoavailability and uptake by Pelargonium hortorum along with comparative toxicities of both organic amendments. For this purpose, soil was spiked with Pb concentrations (0, 500, 750, 1000 and 1500 mg kg1) and amended with EDTA and DIPA at dosage levels (0, 1.5, 3, 5, 7.5, 10 mmol kg-1) for plantation of Pelargonium hortorum. Soil samples were extracted with MgCl2, plant samples were acid digested and analyzed for metal content. Biomass and root/shoot length of Pelargonium hortorum was decreased with increase in concentration of Pb and chelating agents. Phytoavailability of Pb at 1500 mg kg-1 with EDTA 10 mmol kg-1 was 0.3-folds in comparison to DIPA at the same dosage. Pelargonium hortorum plants amended with EDTA and DIPA at 10 mmol kg-1 with Pb 1000 mg kg-1 were found to uptake Pb 5.3-fold and 2.5-folds, respectively in comparison to Pb 1000 mg kg-1 alone. Pb uptake decreased at 1500 mg kg-1 with both chelating agents. The EDTA alone and in combination with 1500 mg Pb kg-1 showed maximum genotoxicity by reducing the mitotic index and increasing the micronuclei formation. EDTA+Pb showed maximum toxicity followed by Pb and DIPA. Overall, 10 mmol kg-1 of EDTA and DIPA performed better among all dosages in enhancing phytoavailability and uptake of Pb. DIPA showed less toxicity than that caused by EDTA, with comparable ability to promote Pb phytoextraction.

Lead availability and phytoextraction in the rhizosphere of Pelargonium species.

Availability of lead (Pb) in soil is a major factor controlling the phytoremediation efficiency of plants. This study was focused on investigating the plant-induced changes in rhizosphere and corresponding effect on bioavailable fraction of Pb and accumulation in different plant parts. For rhizosphere study, special cropping device was designed locally. Two Pb accumulator plants Stigmatocarpum criniflorum (L. f.) L. Bolus and Pelargonium × hortorum L.H. Bailey were grown in cropping device setup containing Pb spiked soil (500, 1000, 1500, and 2000 mg kg-1) for a period of 3 weeks. Further plants were also analyzed for Pb-induced oxidative stress. The results indicated higher ability of soil adjustment for Pb uptake by P. hortorum. The soil pH was (p < 0.05) decreased (ΔpH = - 0.22 pH), and dissolved organic carbon (DOC) content was significantly increased (by 1.7-fold) in rhizosphere of P. hortorum. The bioavailable fraction of Pb was twofold higher in rhizosphere of P. hortorum than S. criniflorum at the same soil Pb concentration (2000 mg kg-1). Maximum Pb concentration in root and shoot of S. criniflorum was 755 ± 99 and 207 ± 12 mg Pb/kg DW and for P. hortorum was 1281 ± 77 and 275 ± 7 mg Pb/kg DW. P. hortorum uptakes more Pb per plant by threefold compared with S. criniflorum. The oxidative stress results indicated higher Pb tolerance and suitability of P. hortorum for phytoextraction of Pb-contaminated soil.

Plant uptake and leaching potential upon application of amendments in soils spiked with heavy metals (Cd and Pb).

The aim of this study was to assess the effects of soil amendments (organic and inorganic) on phytoavailability and leaching of cadmium (Cd) and lead (Pb) during enhanced phytoextraction. The vertical column study was carried out to investigate the metal leaching meanwhile studying plant biomass and metals uptake for phytoextraction by Pelargonium hortroum. For this purpose, soil amendments at several concentration levels, such as ethylene diaminetetraacetic acid (EDTA 0, 4, 5 mmol kg-1), ammonium nitrate (0, 8, 10 mmol kg-1), citric acid (0, 8, 10 mmol kg-1), compost (0, 8%, 10%) and titanium dioxide nanoparticles (TNPs, 0, 80, 100 mg kg-1) were used. Results revealed that EDTA efficiently improved Cd and Pb accumulation (mg kg-1) in shoots and roots. Cd accumulation was significantly increased by 270%, 44%, 145%, and 6.4% in shoot and 94%, 19%, 48% and 14% in root upon EDTA, ammonium nitrate, citric acid and TNPs application, respectively compared to the control without amendment (WA). Similarly, Pb accumulation was significantly increased by 71%, 58% and 52% in shoot and 88.8%, 70.6% and 64.6% in root upon exposure to higher levels of EDTA, citric acid and ammonium nitrate, respectively when compared to the WA control. Application of TNPs reduced the Pb-accumulation in shoots and roots by 33% and 28%, respectively. Similarly, the Pb-accumulation in shoots and roots was decreased by 39% and 35%, respectively upon compost addition. Plant biomass was significantly increased upon application of soil amendments, with the exception of EDTA. The maximum Cd and Pb uptake was found in citric acid amended soil. Leachate analysis revealed that the concentrations of Cd and Pb were increased by 109% and 101% in leachates upon EDTA application as compared to other amendments. In comparison with other amendments, citric acid may be recommended as an environmentally friendly alternative for non-biodegradable EDTA for enhanced phytoextraction of Cd and Pb.

Fungi-assisted phytoextraction of lead: tolerance, plant growth-promoting activities and phytoavailability.

Lead (Pb) is known for its low mobility and persistence in soils. The main aim of the present study was to explore potential of different fungal strains to promote phytoextraction of Pb-contaminated soils. Five non-pathogenic fungal strains (Trichoderma harzianum, Penicillium simplicissimum, Aspergillus flavus, Aspergillus niger, and Mucor spp.) were tested for their ability to modify soil properties (pH and organic matter) and to increase Pb phytoavailability at varying concentrations. Lead tolerance of fungal strains followed the decreasing order as A. niger > T. harzianum > A. flavus > Mucor sp. > P. simplicissimum. Lead solubility induced by A. flavus and Mucor spp. was increased by 1.6- and 1.8-fold, respectively, as compared to the control soil (Pb added, without fungi). A. flavus and Mucor spp. lowered the soil pH by - 0.14 and - 0.13 units, in soils spiked with 2000 mg Pb kg-1. The maximum increase in the percentage of organic matter (OM) recorded was 1.7-fold for A. flavus at 500 mg Pb kg-1 soil. Plant growth-promoting assays confirmed the beneficial role of these fungal strains. Significantly high production of IAA (249 µg mL-1) and siderophores (61%) was observed with A. niger, and phosphate solubilization with P. simplicissimum (58 µg mL-1). Based on the results in Pb-contaminated soils, Pelargonium hortorum L. inoculated with Mucor spp. showed the potential to enhance phytoextraction of Pb by promoting Pb phytoavailability in soil and improving plant biomass production through plant growth-promoting activities.

Metal tolerant bacteria enhanced phytoextraction of lead by two accumulator ornamental species.

Microbially enhanced availability and phytoextraction is a promising technique for phytoremediation of lead (Pb). In this study, Pb resistant strains were isolated and investigated for potential effects on plant growth and Pb phytoextraction. Incubation experiments were carried for inoculated and un-inoculated soil containing different levels of Pb. Results revealed that 20% of the isolated bacteria could tolerate Pb up to 800 mg L-1. Five Pb resistant strains with plant growth promoting (PGP) abilities were evaluated for possible influence on water soluble Pb through soil incubation experiments and significant increase i.e. 1.85- and 1.49-folds in water soluble Pb was observed for NCCP-1848 and NCCP-1862 strains, respectively. Pot experiments indicated significantly higher uptake by Pelargonium hortorum than that by Mesembryanthemum criniflorrum at all levels of soil Pb concentrations with the highest increase (1.9-folds) in plants inoculated with NCCP-1848 followed by NCCP-1862 (1.8-folds increase) compared to the control (Pb and without bacterial strain). The strains NCCP-1848 and NCCP-1862 were identified by 16S rRNA gene sequencing as Microbacterium sp. and Klebsiella sp. The results of present study suggest that Pb resistant plant growth promoting bacteria can serve as an effective bio-inoculant through wide action spectrum for maximizing efficiency of phytoremediation system.

EDTA-assisted phytoextraction of lead and cadmium by Pelargonium cultivars grown on spiked soil.

The aim of this study was to assess EDTA-assisted Pb and Cd phytoextraction potential of locally grown Pelargonium hortorum and Pelargonium zonale. Plants were exposed to different levels of Pb (0-1500 mg kg-1) and Cd (0-150 mg kg-1) in the absence or presence of EDTA (0-5 mmol kg-1). P. hortorum and P. zonale accumulated 50.9% and 42.2% higher amount of Pb in shoots at 1500 mg kg-1 Pb upon addition of 5 mmol kg-1 EDTA. Plant dry biomass decreased 46.8% and 64.3% for P. hortorum and P. zonale, respectively at the combination of 1500 mg kg-1 Pb and 5 mmol kg-1 EDTA. In Cd and EDTA-treated groups, P. hortorum and P. zonale accumulated 2.7 and 1.6-folds more Cd in shoots at 4 and 2 mmol kg-1 EDTA, respectively, in 150 mg Cd kg-1 treatment. Plant dry biomass of P. hortorum and P. zonale was reduced by 46.3% and 71.3%, respectively, in soil having 150 mg Cd kg-1 combined with 5 mmol kg-1 EDTA. Translocation factor and enrichment factor of both plant cultivars at all treatment levels were >1. Overall, the performance of P. hortorum was better than that of P. zonale for EDTA-assisted phytoextraction of Pb and Cd.

Data on rhizosphere pH, phosphorus uptake and wheat growth responses upon TiO2 nanoparticles application.

In this study, the data sets and analyses provided the information on the characterization of titanium dioxide nanoparticles (TiO2 NPs), and their impacts on rhizosphere pH, and soil-bound phosphorus (P) availability to plants together with relevant parameters. For this purpose, wheat (Triticum aestivum L.) was cultivated in the TiO2 NPs amended soil over a period of 60 days. After harvesting, the soil and plants were analyzed to examine the rhizosphere pH, P availability in rhizosphere soil, uptake in roots and shoots, biomass produced, chlorophyll content and translocation to different plant parts monitored by SEM and EDX techniques in response to different dosages of TiO2 NPs. The strong relationship can be found among TiO2 NPs application, P availability, and plant growth.

Bioturbation effects on bioaccumulation of cadmium in the wetland plant Typha latifolia: A nature-based experiment.

The development of efficient bioremediation techniques to reduce aquatic pollutant load in natural sediment is one of the current challenges in ecological engineering. A nature-based solution for metal bioremediation is proposed through a combination of bioturbation and phytoremediation processes in experimental indoor microcosms. The invertebrates Tubifex tubifex (Oligochaeta Tubificidae) was used as an active ecological engineer for bioturbation enhancement. The riparian plant species Typha latifolia was selected for its efficiency in phyto-accumulating pollutants from sediment. Phytoremediation efficiency was estimated by using cadmium as a conservative pollutant known to bio-accumulate in plants, and initially introduced in the overlying water (20µg Cd/L of cadmium nitrate - Cd(NO3)2·4H2O). Biological sediment reworking by invertebrates' activity was quantified using luminophores (inert particulates). Our results showed that bioturbation caused by tubificid worms' activity followed the bio-conveying transport model with a downward vertical velocity (V) of luminophores ranging from 16.7±4.5 to 18.5±3.9cm·year-1. The biotransport changed the granulometric properties of the surface sediments, and this natural process was still efficient under cadmium contamination. The highest value of Cd enrichment coefficient for plant roots was observed in subsurface sediment layer (below 1cm to 5cm depth) with tubificids addition. We demonstrated that biotransport changed the distribution of cadmium across the sediment column as well as it enhanced the pumping of this metal from the surface to the anoxic sediment layers, thereby increasing the bioaccumulation of cadmium in the root system of Typha latifolia. This therefore highlights the potential of bioturbation as a tool to be considered in future as integrated bioremediation strategies of metallic polluted sediment in aquatic ecosystems.

Toxicity effects of an environmental realistic herbicide mixture on the seagrass Zostera noltei.

Worldwide seagrass declines have been observed due to multiple stressors. One of them is the mixture of pesticides used in intensive agriculture and boat antifouling paints in coastal areas. Effects of mixture toxicity are complex and poorly understood. However, consideration of mixture toxicity is more realistic and ecologically relevant for environmental risk assessment (ERA). The first aim of this study was to determine short-term effects of realistic herbicide mixture exposure on physiological endpoints of Zostera noltei. The second aim was to assess the environmental risks of this mixture, by comparing the results to previously published data. Z. noltei was exposed to a mixture of four herbicides: atrazine, diuron, irgarol and S-metolachlor, simulating the composition of typical cocktail of contaminants in the Arcachon bay (Atlantic coast, France). Three stress biomarkers were measured: enzymatic activity of glutathione reductase, effective quantum yield (EQY) and photosynthetic pigment composition after 6, 24 and 96 h. Short term exposure to realistic herbicide mixtures affected EQY, with almost 100% inhibition for the two highest concentrations, and photosynthetic pigments. Effect on pigment composition was detected after 6 h with a no observed effect concentration (NOEC) of 1 µg/L total mixture concentration. The lowest EQY effect concentration at 10% (EC10) (2 µg/L) and pigment composition NOEC with an assessment factor of 10 were above the maximal field concentrations along the French Atlantic coast, suggesting that there are no potential short term adverse effects of this particular mixture on Z. noltei. However, chronic effects on photosynthesis may lead to reduced energy reserves, which could thus lead to effects at whole plant and population level. Understanding the consequences of chemical mixtures could help to improve ERA and enhance management strategies to prevent further declines of seagrass meadows worldwide.

Antisense expression of a NBS-LRR sequence in sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.): evidence for a dual role in plant development and fungal resistance.

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.

Local infiltration of high- and low-molecular-weight RNA from silenced sunflower (Helianthus annuus L.) plants triggers post-transcriptional gene silencing in non-silenced plants.

Using grafting procedures, we have characterized post-transcriptional gene silencing (PTGS) in transgenic sunflower expressing beta-glucuronidase (GUS) activity. Silencing was observed as early as 2 weeks after grafting of non-silenced scions on to silenced rootstock. Transmission of the systemic signal occurs solely from stock to scion, is independent of the physiological age of the rootstock and is not heritable. Furthermore, we report, for the first time in plants, an easy and low-cost method of activating RNA silencing by infiltration of purified RNA from silenced plants. Local application of total RNA derived from silenced sunflower plants to leaves of non-silenced plants induces PTGS in newly developed leaves above the point of infiltration, as shown by reduced GUS activity and mRNA levels. Silenced plants contain 21-23-nucleotide RNAs hybridizing to transgene target sequences, in contrast with leaves of non-silenced plants. However, de novo production of GUS-specific short RNA in non-silenced plants can be activated by leaf infiltration of low-molecular-weight RNAs isolated from leaves of silenced plants. Significant levels were detected as early as 2 weeks after infiltration, peaked at 3 weeks and declined 5 weeks after infiltration. Our results provide evidence that RNA infiltration in sunflower induces transient silencing and is not transmitted to offspring. This approach could be of major use in dissecting the mechanisms involved in PTGS.

Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia.

The mechanism of nitrate reductase (NR) regulation under long-term anoxia in roots of whole plants and the putative role of nitrate in anoxia tolerance have been addressed. NR activity in tomato roots increased significantly after 24 h of anaerobiosis and increased further by 48 h, with a concomitant release of nitrite into the culture medium. Anoxia promoted NR activation through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation. After 24 h of anoxia, the total amount of NR increased slightly up to 48 h. However, NR-mRNA levels remained constant between 0 h and 24 h of root anoxia and decreased after 48 h. This is probably due to the inhibition of NR degradation and the accumulation of its native form. NR was slightly dephosphorylated in the absence of oxygen and nitrate. Under anoxia, NR dephosphorylation was modulated by nitrate-controlled NR activity. In addition, the presence of nitrate prevents anoxic symptoms on leaves and delays wilting by 48 h during root anoxia. In the absence of nitrate, plants withered within 24 h, as they did with tungstate treatment, an inhibitor of NR activity. Thus, anoxia tolerance of tomato roots could be enhanced by nitrate reduction.