Do chromium-resistant bacterial symbionts of hyperaccumulator Callitriche cophocarpa support their host in phytobial remediation of water?

Callitriche cophocarpa Sendtn. is a macrophyte widely distributed in aquatic systems of the temperate climate zone and a known hyperaccumulator of chromium. Ten pure symbiotic bacterial isolates of C. cophocarpa were obtained and identified. Three of the isolates showed the highest resistance to Cr(VI): Microbacterium sp. (Ct1), Aeromonas sp. (Ct3) and Acinetobacter sp. (Ct6). Acinetobacter sp. (Ct6) was able to survive up to a concentration of 104 mg/L (2 mM). The isolates were also able to effectively detoxify Cr(VI) by reducing it to Cr(III). We tested whether inoculation of plants with a consortium consisting of Ct1, Ct3 and Ct6 affects: (1) the phytoextraction of chromium from leachates, (2) the physiological state of plants after Cr(VI) treatment. The solutions were landfill leachates and contained 10.7 mg/L of Cr(VI) - an amount 530 times exceeding the legal limits. We influenced the plants with Cr in two steps, each lasting for 10 days, first using mature shoots and then apical ones. The highest Cr content concomitant with the highest bioconcentration factor (BCF) were found in the inoculated plants: 1274 and 119 mg/kg dry mass (d.m.), respectively. The physiological status of the plants was assessed by biometric tests and advanced chlorophyll fluorescence analyses. The photosynthetic activity of mature shoots was influenced by Cr(VI) more negatively than that of young apical shoots. The inoculation with the bacterial consortium significantly reduced the negative effect of Cr(VI) on mature organs. In some cases the inoculated mature plants exhibited photosynthetic activity that was even higher than in the control plants. The results unequivocally show a beneficial effect of C. cophocarpa inoculation with the tested isolates resulting in a significant improvement of the phytoremediation properties of this aquatic chromium hyperaccumulator.

Algae from Cr-containing infiltrate bioremediation for valorised chemical production - Seasonal availability, composition, and screening studies on hydrothermal conversion.

Integrating bioremediation of toxic wastewater with value-added production is increasing interest, but - due to some essential problems - it is hardly applied in industrial practice. The aim of the study was an annual observation of the taxonomic and biochemical composition of various Cr-resistant algal communities grown in the existing Cr-containing infiltrate treatment system, selection of the most suitable algal biomass for infiltrates bioremediation and chromium-loaded algae conversion under mild subcritical conditions. Considering continuous availability and relatively constant chemical composition, Cladophora sp. was selected for utilisation in the chromium bioremediation system, simultaneously as a waste biomass source suitable for hydrothermal conversion. Screening studies conducted in a continuous pilot plant confirmed the possibility of selective extraction of saccharides and their separation from the metals remaining in the solid residual. The negligible concentration of metals in the obtained sugar-rich aqueous phase is essential for its further use in biotechnological processes.

Exploring a unique water ecosystem under long-term exposure to hexavalent chromium - An in situ study of natural diatom (Bacillariophyceae) communities.

This paper reports the third in a series of three studies of a unique aquatic phytobial consortium that developed in a polluted ditch situated near an old chromium waste landfill. The ditch is a specific ecological niche having increased loads of several chemical compounds, including extreme hexavalent chromium concentrations up to two thousand times the allowed limit (0.02 mg dm-3 in Poland) in the more polluted section B; the moderate concentrations in section A are twice the limit. We focus on the microscopic algae group of diatoms, one of the most important components of the phytobial consortium, and continue our novel attempt to analyze the bioremediation potential of the entire consortium under those environmental conditions. We used numerical methods to analyze differences in diatom biodiversity between sections A and B, and assessed the relations between diatoms and selected water chemistry properties, including hexavalent chromium, chlorides and sulphates, the latter two known to positively influence the resistance of algae to the impact of hexavalent chromium. We noted 37 diatom taxa in section A of the ditch and 30 in section B. The most frequently observed diatoms were cosmopolitan taxa and/or characteristic taxa of saline waters. Sulphates were the most important factor influencing the diatom composition in the ditch, explaining 52% of the total variability, followed by chlorides (30%) and hexavalent chromium (9%). Gomphonema acuminatum, Melosira varians and Nitzschia frustulum var. frustulum were found to be most resistant to hexavalent chromium and were selected for further experimental studies on their biotechnological usefulness.

VCF2CAPS-A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset.

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS-a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters-selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software-VCF2CAPS-fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Natural community of macroalgae from chromium-contaminated site for effective remediation of Cr(VI)-containing leachates.

The natural macroalgal community, which developed in the unique and extremely Cr(VI)-polluted aquatic reservoir situated near a historical chromium-waste landfill, was studied in order to recognize the main mechanisms of Cr(VI) detoxification by the algal species. The conducted taxonomic analysis revealed mixed composition of the filamentous forms of algae and showed that three species of Tribonema, namely T. vulgare, T. microchloron and T. viride, which have not been studied before with regard to the mechanisms of Cr(VI) removal, are likely responsible for the effective bioremediation of this highly Cr(VI)-polluted habitat. The studied algal community, with the ability to grow in extremely high concentrations of Cr(VI), i.e. up to ca. 6150 times the upper limit for surface water, exhibited hyperaccumulative properties for chromium (max 16230 mg/kg dry weight) under the given environmental conditions. We found that the main mechanism of Cr(VI) detoxification was reduction followed by Cr(III) biosorption - feasibly by ion exchange and complexation mechanisms - and that the excellent efficiency of chromium reduction under the given, unfavorable weakly alkaline conditions indicates the biological origin of this process. It was concluded that the examined reservoir inhabited by the algal community can be used, after some modifications, as a simple cost-effective "bioreactor" allowing the reduction of chromium concentration to the desired level. Moreover, the conducted studies are also essential to obtain in-depth knowledge and should also be helpful in the relevance of the community for its further application as a potential biosorbent of Cr(VI) on a global scale.

Unique biocenosis as a foundation to develop a phytobial consortium for effective bioremediation of Cr(VI)-polluted waters and sediments.

This paper analyzes a unique, aquatic phytobial biocenosis that has been forming naturally for over 20 years and operating as a filter for Cr(VI)-polluted groundwater. Our study presents a thorough taxonomic analysis of the biocenosis, including filamentous algae, vascular plants, and microbiome, together with the analysis of Cr accumulation levels, bioconcentration factors and other environmentally-significant parameters: siderophore production by bacteria, biomass growth of the plants or winter hardiness. Among 67 species identified in the investigated reservoir, 13 species were indicated as particularly useful in the bioremediation of Cr(VI)-polluted water and sediment. Moreover, three species of filamentous algae, Tribonema sp., and three easily culturable bacterial species were for the first time shown as resistant to Cr concentration up to 123 mg/dm3, i.e. 6150 times over the permissible level. The work presents a modern holistic phytobial consortium indispensable for the remediation of Cr(VI)-contaminated aquatic environment in temperate zones worldwide.

Sourcing and Propagation of Pontechium maculatum for Horticulture and Species Restoration.

Pontechium maculatum, a species of ornamental, apicultural, health and medicinal value, is threatened in some Central European countries including Poland. Its propagation using seeds or in vitro techniques is needed for multiple applications including conservation. Generative propagation efficacy of P. maculatum plants representing different genetic resources (received from botanical gardens in Germany and in Poland) propagated from seeds or in tissue culture was assessed. Moreover, an efficient technique of propagation of P. maculatum using in vitro shoot culture from seedlings was elaborated for the first time. The highest propagation efficacy was noted for German plants of seed origin. The ability of seeds to germinate was similar for all plants; however, seeds were in a state of dormancy, which was broken by GA3. After two years of storage, the seeds still retained the ability to germinate though seeds from propagated in vitro plants germinated more poorly than those from seed-originated plants. The ploidy assessment showed that some in vitro-origin plants had altered DNA content. The results indicate that efficacy of generative propagation of P. maculatum is resource dependent. Furthermore, results suggest that cultivation in vitro influenced some generative features of examined species, which makes this way of P. maculatum propagation a valuable source of genetic variation and a potential breeding tool.

The use of Callitriche cophocarpa Sendtn. for the reclamation of Cr-contaminated freshwater habitat: benefits and limitations.

This work is the first attempt to evaluate suitability of Callitriche cophocarpa Sendtn. (water-starwort) to remove Cr under real-world conditions. Our earlier laboratory-scale studies demonstrated outstanding hyperaccumulation properties of this aquatic higher plant (macrophyte) toward chromium in solution. We introduced C. cophocarpa plants into the watershed with sediments heavily polluted (on average 1400 mg/kg d.w. of Cr) by a tannery. The plants grew vigorously and exhibited no physiological or anatomical disorders. Based on chemical fractionations of bottom sediments, we found low Cr bioavailability. The element was strongly associated with the sediments and could be classified into the following fractions (%): oxidizable III (68.2) > residual IV (28.8) > reducible II (1.6) > exchangeable I (1.4). Despite this, Cr content in plant organs at the contaminated sites was 33 up to 83 times greater than in the control leaf/stem and roots, respectively. Altering redox potential during, i.e., sediment deposition on land may change chemical forms of bound metals in a solid phase, and thus further increase Cr phytoextraction by plants. With this in mind, we concluded that the species, being an outstanding Cr accumulator under laboratory conditions, can be useful in the reclamation of Cr-polluted sediments under controlled, oxidizing conditions.

Callitriche cophocarpa biomass as a potential low-cost biosorbent for trivalent chromium.

The present study focused on the use of the dry mass of the macrophyte Callitriche cophocarpa as an effective biosorbent for chromium removal from concentrated solutions, typical for industrial effluents. In order to evaluate the usability of C. cophocarpa as the Cr(III) sorbent, its detailed physicochemical characterization has been performed as well as the preliminary adsorption studies. The biosorbent was characterized by specific surface area (SSA), porosity, total organic carbon (TOC), inorganic content as well as the cation exchange capacity (CEC), dominant exchangeable cations and anion exchange capacity (AEC), point of zero charge (pHpzc) and buffering capacity. The effect of the initial chromium concentration, solution pH and co-existing anions on the sorption effectiveness have been investigated. Based on theoretical isotherm models, the maximum adsorption capacity of the dry C. cophocarpa has been determined as 77.1 mg Cr(III)/g. Finally, the strength of Cr-binding onto the plant biomass has been evaluated using the BCR extraction method, stating that chromium was strongly and - under environmental conditions - irreversibly bound to the plant biomass.

Callitriche cophocarpa (water starwort) proteome under chromate stress: evidence for induction of a quinone reductase.

Chromate-induced physiological stress in a water-submerged macrophyte Callitriche cophocarpa Sendtn. (water starwort) was tested at the proteomic level. The oxidative stress status of the plant treated with 1 mM Cr(VI) for 3 days revealed stimulation of peroxidases whereas catalase and superoxide dismutase activities were similar to the control levels. Employing two-dimensional electrophoresis, comparative proteomics enabled to detect five differentiating proteins subjected to identification with mass spectrometry followed by an NCBI database search. Cr(VI) incubation led to induction of light harvesting chlorophyll a/b binding protein with a concomitant decrease of accumulation of ribulose bisphosphate carboxylase (RuBisCO). The main finding was, however, the identification of an NAD(P)H-dependent dehydrogenase FQR1, detectable only in Cr(VI)-treated plants. The FQR1 flavoenzyme is known to be responsive to oxidative stress and to act as a detoxification protein by protecting the cells against oxidative damage. It exhibits the in vitro quinone reductase activity and is capable of catalyzing two-electron transfer from NAD(P)H to several substrates, presumably including Cr(VI). The enhanced accumulation of FQR1 was chromate-specific since other stressful conditions, such as salt, temperature, and oxidative stresses, all failed to induce the protein. Zymographic analysis of chromate-treated Callitriche shoots showed a novel enzymatic protein band whose activity was attributed to the newly identified enzyme. We suggest that Cr(VI) phytoremediation with C. cophocarpa can be promoted by chromate reductase activity produced by the induced quinone oxidoreductase which might take part in Cr(VI) â†’ Cr(III) bioreduction process and thus enable the plant to cope with the chromate-generated oxidative stress.

From laboratory to field studies - The assessment of Biscutella laevigata suitability to biological reclamation of areas contaminated with lead and cadmium.

The aim of the work was to evaluate the usefulness of the in vitro multiplication of Biscutella laevigata calamine ecotype for in situ reclamation of post-flotation wastes polluted with Pb and Cd. The experiment was conducted on three steps: (i) plant shoots' production under in vitro condition, (ii) establishment of the material in greenhouse experiment, and finally (iii) field cultivation directly on the mining-waste heap of Olkusz Ore-Bearing Region, Poland. This region is known to be one of the most chemically-degraded area in central Europe. The laboratory-set in vitro analysis enabled to obtain the high-quality plant shoots, which multiply the most effectively (with growth tolerance index 130-150%) on medium containing 5.0µM CdCl2 and 0.5mM Pb(NO3)2. These plants were used for the next two ex vitro experiments. Several biometric and physiological analysis (i.e. of photosystem II activity Fv/Fm and PI, photosynthetic pigment contents) were done to indicate plant physiological status during these experiments. The main novelty of the work was to prove that in vitro-multiplied shoots of B. laevigata - the representative of native flora from Olkusz Ore-Bearing Region - can be successfully implemented in situ for the restoration of these degraded area. Moreover, the addition of sewage sludge as a source of organic compounds significantly improved plants' growth and development what is especially important due to the lack of other legal solutions for the management of the sewage sludge in some countries.

Accumulation patterns of Cr in Callitriche organs--qualitative and quantitative analysis.

The aims of this study were both the qualitative and quantitative analysis of chromium accumulation in the shoots of Callitriche cophocarpa. This globally distributed, submersed macrophyte exhibits outstanding Cr phytoremediation capacity in an aquatic environment. Cr was applied separately for 7 days at two stable forms as Cr(VI) and Cr(III), known from their diverse physicochemical properties and toxicities. The maps of Cr depositions in young leaves, mature leaves, and stems were obtained by micro X-ray fluorescence spectroscopy (µXRF). The detailed analysis of XRF maps was done based on Image-Pro PLUS (Media Cybernetics) software. Cr was accumulated either in trichomes or vascular bundles in respect to the element speciation and the plant organ. The concentration of Cr significantly increased in the following order: Cr(VI) mature leaves < Cr(VI) young leaves = Cr(VI) stems < Cr(III) young leaves ≤ Cr(III) mature leaves ≤ Cr(III) stems. The observed differences in distribution and accumulation of Cr were correlated with the different reduction potential of Cr(VI) by particular plant organs. The reduction of Cr(VI) is considered the main detoxification mechanism of the highly toxic Cr(VI) form. The unique L-band electron resonance spectrometer (L-band EPR) was applied to follow the reduction of Cr(VI) to Cr(III) in the studied material.

Potential for chromium (VI) bioremediation by the aquatic carnivorous plant Utricularia gibba L. (Lentibulariaceae).

The aquatic carnivorous plant Utricularia gibba has one of the smallest known genomes among flowering plants, and therefore, it is an excellent model organism for physiological and developmental studies. The main aim of our work was to check whether the ubiquitous U. gibba might be useful for the phytoremediation of the highly toxic and mobile hexavalent chromium in waters. Plants were incubated for 1 week in a 50 µM (2.6 mg dm(-3)) Cr(VI) solution in laboratory conditions. Our results revealed that the plant exhibits a very high accumulation capacity for Cr. The accumulation level was higher than 780 mg kg(-1) and a bioconcentration factor >300. On the other hand, the plants showed a low tolerance to the elevated Cr concentration, which was expressed in a significant decrease of the photosystem II activity. However, the most pronounced negative influence of chromate was found on the morphology and activity of the traps. Due to its high accumulation capacity, we suggest that U. gibba may be efficient in the removal of chromate over a short time scale. It can also provide a new molecular resource for studying the mechanisms of Cr(VI) detoxification.

Chromium distribution in shoots of macrophyte Callitriche cophocarpa Sendtn.

The aim of the study was the analysis of Cr distribution in shoots of the macrophyte Callitriche cophocarpa by means of two X-ray-based techniques: micro X-ray fluorescence (µXRF) and electron probe X-ray microanalysis (EPXMA). Plants were treated with 100 µM (5.2 mg l(-1)) chromium solutions for 7 days. Cr was introduced independently at two speciations as Cr(III) and Cr(VI), known for their diverse physicochemical properties and different influence on living organisms. A comparative analysis of Cr(III)-treated plants by EPXMA and µXRF demonstrated high deposition of Cr in epidermal glands/hairs localized on leaves and stems of the plant shoots. Cr in Cr(III)-treated plants was recorded solely in glands/hairs, and the element was not present in any other structures. On the other hand, Cr in Cr(VI)-treated group of plants was rather found in vascular bundles. Moreover, the concentration of Cr in Cr(VI)-treated plants was significantly lower than in plants incubated in Cr(III) solution. The results obtained in this work suggest differences in chromium uptake, transport and accumulation dependent on the oxidative state of the element.

Phytoremediation of water polluted by thallium, cadmium, zinc, and lead with the use of macrophyte Callitriche cophocarpa.

The objective of the present work was to study the phytoremediation capacity of Callitriche cophocarpa concerning water contaminated with thallium (Tl), cadmium (Cd), zinc (Zn), and lead (Pb) derived from the natural environment. We found that after a 10-day incubation period, shoots of C. cophocarpa effectively biofiltrated the water so that it met (for Cd, Zn, and Pb) appropriate quality standards. The order of accumulation of the investigated elements by shoots (mg kg(-1) dry weight) were as follows: Zn (1120) < Tl (251) < Cd (71) < Pb (35). The order of bioconcentration factors were as follows: Cd (1177) < Tl (1043) < Zn (718) < Pb (597). According to Microtox bioassay, C. cophocarpa significantly eradicated polluted water toxicity. During the experiment, the physiological status of plants was monitored by taking measurements of photosystem II activity (maximum efficiency of PSII, photochemical fluorescence quenching, nonphotochemical fluorescence quenching, and quantum efficiency of PSII), photosynthetic pigment contents, and shoot morphology. Plants exposed to metallic pollution did not exhibit significant changes in their physiological status compared with the control. This work is potentially applicable to the future use of C. cophocarpa in the phytoremediation of polluted, natural watercourses.

Study on chromium-binding capacity of Callitriche cophocarpa in an aquatic environment.

The aim of the present study was to investigate the binding strength of chromium (Cr) ions to aquatic macrophyte Callitriche cophocarpa. Shoots of the plants were incubated in a natural water solution containing Cr(III) or Cr(VI) at a concentration ranging from 0.5 to 4 mM under laboratory conditions. We found that C. cophocarpa has an extremely high capacity to bind Cr. The average level of accumulation reached 28,385 or 7,315 mg kg(-1) dry weight for plants incubated with Cr(III) or Cr(VI), respectively. Shoots incubated in a 0.5 mM concentration of Cr(III) for 5 days removed almost 100 % of the metal from solution. The major pool of the bound Cr(III) ions follows the strongest mechanism of metal-binding to an organic matter. In contrast, we found that only 25 % of Cr(VI) ions are bound into the metallo-organic compounds and 57 % of Cr(VI) exists in an easily remobilizable form. Activity of a photosynthetic electron transport (as F V/F M) was evaluated with respect to the Cr-binding mechanism. Our results contribute to the development of knowledge on processes controlling bioremediation of heavy-metallic compounds in aquatic systems.

Chromium(VI) bioremediation by aquatic macrophyte Callitriche cophocarpa Sendtn.

Callitriche cophocarpa (water-starwort)--aquatic widespread macrophyte--was found to be an excellent chromium accumulator. The plants were exposed to various chromium(VI) concentration ranging from 50 to 700 microM in a hydroponic culture up to ca. 3 weeks. Physiological conditions of shoots were monitored via measuring potential photosynthesis quantum efficiency (F(v)/F(m)) and photosynthetic pigment contents. Additionally, the structure of leaves was analyzed using optical and atomic force microscopy (AFM). It has been shown that plants grown in 50 microM Cr(VI) solution exhibited photosynthetic activity and shoot and leaf morphology similar to control plants. Moreover, at the same time the average Cr concentration in their shoots reached about 470 mg kg(-1)d.w. after 10d and up to 1000 mg kg(-1)d.w. after 3 weeks of culture while in control plants did not exceed a few mgkg(-1)d.w. Our results point to Callitriche cophocarpa as a very promising species to be used in the investigation of chromium(VI) phytoremediation mechanisms as well as a good candidate for wastewaters remediation purpose.

Acquisition of plastid movement responsiveness to light during mesophyll cell differentiation.

A culture of Nicotiana tabacum leaf protoplasts, regenerating in vitro, was used to study light-induced plastid translocations. Experiments were carried out for 5 months starting with protoplasts, through single cells, microcolonies and callus to the differentiated mesophyll of regenerated plants. Although the actin cytoskeleton was fully developed at every stage of culture, blue light-mediated directional movements of chloroplasts were observed only after the full differentiation of callus into leaf tissues. These chloroplast rearrangements were similar to those observed in control plants grown from seeds. Under strong blue light, chloroplasts gathered at the cell walls parallel to the light direction (profile position); under weak blue light, they gathered at the walls perpendicular to the light direction (face position). No light-dependent plastid arrangements were found in undifferentiated cell cultures even after cell wall recovery. A characteristic pattern of plastids in the dividing cells was independent of light signals. Only trace chloroplast responses to strong blue light were detected in the first leaves regenerating from callus. We hypothesize that factors which control the developmental status of a cell, i.e. division and differentiation, take over the control of plastid redistribution from light signals.