Biostimulation of sulfate reduction for in-situ metal(loid) precipitation at an industrial site in Flanders, Belgium.

A 30-month pilot study was conducted to evaluate the potential of in-situ metal(loid) removal through biostimulation of sulfate-reducing processes. The study took place at an industrial site in Flanders, Belgium, known for metal(loid) contamination in soil and groundwater. Biostimulation involved two incorporations of an organic substrate (emulsified vegetable oil) as electron donor and potassium bicarbonate to raise the pH of the groundwater by 1-1.5 units. The study focused on the most impacted permeable fine sand aquifer (8-9 m below groundwater level) confined by layers of non-permeable clay. The fine sands exhibited initially oxic conditions (50-200 mV), an acidic pH of 4.5 and sulfate concentrations ranging from 600 to 800 mg/L. At the central monitoring well, anoxic conditions (-200 to -400 mV) and a pH of 5.9 established shortly after the second substrate and reagent injection. Over the course of 12 months, there was a significant decrease in the concentration of arsenic (from 2500 to 12 µg/L), nickel (from 360 to <2 µg/L), zinc (from 78,000 to <2 µg/L), and sulfate (from 930 to 450 mg/L). Low levels of metal(loid)s were still present after 34 months (end of study). Mineralogical analysis indicated that the precipitates formed were amorphous in nature. Evidence for biologically driven metal(loid) precipitation was provided by compound specific stable isotope analysis of sulfate. In addition, changes in microbial populations were assessed using next-generation sequencing, revealing stimulation of native sulfate-reducing bacteria. These results highlight the potential of biostimulation for long-term in situ metal(loid) plume treatment/containment.

Phytostabilization of metal(loid)s by ten emergent macrophytes following a 90-day exposure to industrially contaminated groundwater.

Better understanding of macrophyte tolerance under long exposure times in real environmental matrices is crucial for phytoremediation and phytoattenuation strategies for aquatic systems. The metal(loid) attenuation ability of 10 emergent macrophyte species (Carex riparia, Cyperus longus, Cyperus rotundus, Iris pseudacorus, Juncus effusus, Lythrum salicaria, Menta aquatica, Phragmites australis, Scirpus holoschoenus, and Typha angustifolia) was investigated using real groundwater from an industrial site, over a 90-day exposure period. A "phytobial" treatment was included, with 3 plant growth-promoting rhizobacterial strains. Plants exposed to the polluted water generally showed similar or reduced aerial biomass compared to the controls, except for C. riparia. This species, along with M. aquatica, exhibited improved biomass after bioaugmentation. Phytoremediation mechanisms accounted for more than 60% of As, Cd, Cu, Ni, and Pb removal, whilst abiotic mechanisms contributed to ∼80% removal of Fe and Zn. Concentrations of metal(loid)s in the roots were generally between 10-100 times higher than in the aerial parts. The macrophytes in this work can be considered "underground attenuators", more appropriate for rhizostabilization strategies, especially L. salicaria, M. aquatica, S. holoschoenus, and T. angustifolia. For I. pseudacorus, C. longus, and C. riparia; harvesting the aerial parts could be a complementary phytoextraction approach to further remove Pb and Zn. Of all the plants, S. holoschoenus showed the best balance between biomass production and uptake of multiple metal(loid)s. Results also suggest that multiple phytostrategies may be possible for the same plant depending on the final remedial aim. Phytobial approaches need to be further assessed for each macrophyte species.

Physiological and transcriptome profiling of Chlorella sorokiniana: A study on azo dye wastewater decolorization.

Over decades, synthetic dyes have become increasingly dominated by azo dyes posing a significant environmental risk due to their toxicity. Microalgae-based systems may offer an alternative for treatment of azo dye effluents to conventional physical-chemical methods. Here, microalgae were tested to decolorize industrial azo dye wastewater (ADW). Chlorella sorokiniana showed the highest decolorization efficiency in a preliminary screening test. Subsequently, the optimization of the experimental design resulted in 70% decolorization in a photobioreactor. Tolerance of this strain was evidenced using multiple approaches (growth and chlorophyll content assays, scanning electron microscopy (SEM), and antioxidant level measurements). Raman microspectroscopy was employed for the quantification of ADW-specific compounds accumulated by the microalgal biomass. Finally, RNA-seq revealed the transcriptome profile of C. sorokiniana exposed to ADW for 72 h. Activated DNA repair and primary metabolism provided sufficient energy for microalgal growth to overcome the adverse toxic conditions. Furthermore, several transporter genes, oxidoreductases-, and glycosyltransferases-encoding genes were upregulated to effectively sequestrate and detoxify the ADW. This work demonstrates the potential utilization of C. sorokiniana as a tolerant strain for industrial wastewater treatment, emphasizing the regulation of its molecular mechanisms to cope with unfavorable growth conditions.

Mass Transfer Limitation during Slow Anaerobic Biodegradation of 2-Methylnaphthalene.

While they are theoretically conceptualized to restrict biodegradation of organic contaminants, bioavailability limitations are challenging to observe directly. Here we explore the onset of mass transfer limitations during slow biodegradation of the polycyclic aromatic hydrocarbon 2-methylnaphthalene (2-MN) by the anaerobic, sulfate-reducing strain NaphS2. Carbon and hydrogen compound specific isotope fractionation was pronounced at high aqueous 2-MN concentrations (60 µM) (εcarbon = -2.1 ± 0.1‰/εhydrogen = -40 ± 7‰) in the absence of an oil phase but became significantly smaller (εcarbon = -0.9 ± 0.3‰/εhydrogen = -6 ± 3‰) or nondetectable when low aqueous concentrations (4 µM versus 0.5 µM) were in equilibrium with 80 or 10 mM 2-MN in hexadecane, respectively. This masking of isotope fractionation directly evidenced mass transfer limitations at (sub)micromolar substrate concentrations. Remarkably, oil-water mass transfer coefficients were 60-90 times greater in biotic experiments than in the absence of bacteria (korg-aq2-MN = 0.01 ± 0.003 cm h-1). The ability of isotope fractionation to identify mass transfer limitations may help study how microorganisms adapt and navigate at the brink of bioavailability at low concentrations. For field surveys our results imply that, at trace concentrations, the absence of isotope fractionation does not necessarily indicate the absence of biodegradation.

Mechanistic Dichotomy in Bacterial Trichloroethene Dechlorination Revealed by Carbon and Chlorine Isotope Effects.

Tetrachloroethene (PCE) and trichloroethene (TCE) are significant groundwater contaminants. Microbial reductive dehalogenation at contaminated sites can produce nontoxic ethene but often stops at toxic cis-1,2-dichloroethene ( cis-DCE) or vinyl chloride (VC). The magnitude of carbon relative to chlorine isotope effects (as expressed by ΛC/Cl, the slope of δ13C versus δ37Cl regressions) was recently recognized to reveal different reduction mechanisms with vitamin B12 as a model reactant for reductive dehalogenase activity. Large ΛC/Cl values for cis-DCE reflected cob(I)alamin addition followed by protonation, whereas smaller ΛC/Cl values for PCE evidenced cob(I)alamin addition followed by Cl- elimination. This study addressed dehalogenation in actual microorganisms and observed identical large ΛC/Cl values for cis-DCE (ΛC/Cl = 10.0 to 17.8) that contrasted with identical smaller ΛC/Cl for TCE and PCE (ΛC/Cl = 2.3 to 3.8). For TCE, the trend of small ΛC/Cl could even be reversed when mixed cultures were precultivated on VC or DCEs and subsequently confronted with TCE (ΛC/Cl = 9.0 to 18.2). This observation provides explicit evidence that substrate adaptation must have selected for reductive dehalogenases with different mechanistic motifs. The patterns of ΛC/Cl are consistent with practically all studies published to date, while the difference in reaction mechanisms offers a potential answer to the long-standing question of why bioremediation frequently stalls at cis-DCE.

Chlorinated Electron Acceptor Abundance Drives Selection of Dehalococcoides mccartyi (D. mccartyi) Strains in Dechlorinating Enrichment Cultures and Groundwater Environments.

Dehalococcoides mccartyi (D. mccartyi) strains differ primarily from one another by the number and identity of the reductive dehalogenase homologous catalytic subunit A (rdhA) genes within their respective genomes. While multiple rdhA genes have been sequenced, the activity of the corresponding proteins has been identified in only a few cases. Examples include the enzymes whose substrates are groundwater contaminants such as trichloroethene (TCE), cis-dichloroethene (cDCE) and vinyl chloride (VC). The associated rdhA genes, namely tceA, bvcA, and vcrA, along with the D. mccartyi 16S rRNA gene are often used as biomarkers of growth in field samples. In this study, we monitored an additional 12 uncharacterized rdhA sequences identified in the metagenome in the mixed D. mccartyi-containing culture KB-1 to monitor population shifts in more detail. Quantitative PCR (qPCR) assays were developed for 15 D. mccartyi rdhA genes and used to measure population diversity in 11 different sub-cultures of KB-1, each enriched on different chlorinated ethenes and ethanes. The proportion of rdhA gene copies relative to D. mccartyi 16S rRNA gene copies revealed the presence of multiple distinct D. mccartyi strains in each culture, many more than the two strains inferred from 16S rRNA analysis. The specific electron acceptor amended to each culture had a major influence on the distribution of D. mccartyi strains and their associated rdhA genes. We also surveyed the abundance of rdhA genes in samples from two bioaugmented field sites (Canada and United Kingdom). Growth of the dominant D. mccartyi strain in KB-1 was detected at the United Kingdom site. At both field sites, the measurement of relative rdhA abundances revealed D. mccartyi population shifts over time as dechlorination progressed from TCE through cDCE to VC and ethene. These shifts indicate a selective pressure of the most abundant chlorinated electron acceptor, as was also observed in lab cultures. These results also suggest that reductive dechlorination at contaminated sites is brought about by multiple strains of D. mccartyi whether or not the site is bioaugmented. Understanding the driving forces behind D. mccartyi population selection and activity is improving predictability of remediation performance at chlorinated solvent contaminated sites.

Bioremediation of chlorinated ethenes in fractured bedrock and associated changes in dechlorinating and nondechlorinating microbial populations.

The use of enhanced in situ anaerobic bioremediation (EISB) and bioaugmentation in fractured bedrock is limited compared to its use in granular media. We evaluated EISB for the treatment of trichloroethene (TCE)-impacted groundwater in fractured carbonate rock at a site in Southern Ontario, Canada, with cool average groundwater temperature (∼ 13 °C). Borehole-connectivity, contaminant concentrations, and groundwater properties were investigated. Changes in dechlorinating and nondechlorinating populations (fermenters, acetogens, methanogens, and sulfate reducers) were assessed via quantitative PCR (qPCR). During biostimulation with ethanol, concentrations of TCE daughter products cis-dichloroethene (cDCE) and vinyl chloride (VC) decreased in association with an enrichment of vcrA (VC reductive dehalogenase)-carrying Dehalococcoides, whereas ethene production was only moderate. Following bioaugmentation with the mixed dechlorinating culture KB-1, greater concentrations of chloride-a product of dechlorination-was observed in most wells; in addition, ethene production increased significantly in monitoring well locations that had strong hydraulic connectivity to the groundwater recirculation system, while Dehalococcoides and vcrA concentrations did not appreciably vary. Interestingly, increases of 3-4 orders of magnitude of an ethanol-fermenting Bacteroidetes population also present in KB-1 were correlated to improved conversion to ethene, an observation which suggests there could be a causal relationship-for example, better syntrophy and/or synergy among bacterial populations.

Activity of native hydrolytic enzymes and their association with the cell wall of three ectomycorrhizal fungi.

The ecological and biogeochemical relevance of hydrolytic enzymes associated with the fungal cell wall has been poorly studied in ectomycorrhizal (ECM) fungi. We used a modified sequential extraction procedure to investigate the activity of various hydrolytic enzymes (ß-glucosidase, acid-phosphatase, leucine-aminopeptidase, chitinase, xylanase and glucuronidase) and their association with the cell wall of three ECM fungi (Rhizopogon roseolus, Paxillus involutus and Piloderma croceum). Fungi were grown on C-rich solid medium under three different P concentrations (3.7, 0.37 and 0.037 mM). The sequential extraction procedure classifies enzymes as: (a) cytosolic, (b) loosely bound, (c) hydrophobically bound, (d) ionically bound and (e) covalently bound. Results showed that for the same fungus absolute enzymatic activity was affected by P concentration, whilst enzymatic compartmentalization among the cytosol and the cell wall fractions was not. The association of enzymes with the cell wall was fungus- and enzyme-specific. Our data indicate also that enzymes best known for being either extracellular or cytosolic or both, do act in muro as well. The ecological implications of cell wall-bound enzymes and the potential applications and limitations of sequential extractions are further discussed.

Monitoring biodegradation of ethene and bioremediation of chlorinated ethenes at a contaminated site using compound-specific isotope analysis (CSIA).

Chlorinated ethenes are commonly found in contaminated groundwater. Remediation strategies focus on transformation processes that will ultimately lead to nontoxic products. A major concern with these strategies is the possibility of incomplete dechlorination and accumulation of toxic daughter products (cis-1,2-dichloroethene (cDCE), vinyl chloride (VC)). Ethene mass balance can be used as a direct indicator to assess the effectiveness of dechlorination. However, the microbial processes that affect ethene are not well characterized and poor mass balance may reflect biotransformation of ethene rather than incomplete dechlorination. Microbial degradation of ethene is commonly observed in aerobic systems but fewer cases have been reported in anaerobic systems. Limited information is available on the isotope enrichment factors associated with these processes. Using compound-specific isotope analysis (CSIA) we determined the enrichment factors associated with microbial degradation of ethene in anaerobic microcosms (ε = -6.7‰ ± 0.4‰, and -4.0‰ ± 0.8‰) from cultures collected from the Twin Lakes wetland area at the Savannah River site in Georgia (United States), and in aerobic microcosms (ε = -3.0‰ ± 0.3‰) from Mycobacterium sp. strain JS60. Under anaerobic and aerobic conditions, CSIA can be used to determine whether biotransformation of ethene is occurring in addition to biodegradation of the chlorinated ethenes. Using δ(13)C values determined for ethene and for chlorinated ethenes at a contaminated field site undergoing bioremediation, this study demonstrates how CSIA of ethene can be used to reduce uncertainty and risk at a site by distinguishing between actual mass balance deficits during reductive dechlorination and apparent lack of mass balance that is related to biotransformation of ethene.

Phytostabilization of semiarid soils residually contaminated with trace elements using by-products: sustainability and risks.

We investigated the efficiency of various by-products (sugarbeet lime, biosolid compost and leonardite), based on single or repeated applications to field plots, on the establishment of a vegetation cover compatible with a stabilization strategy on a multi-element (As, Cd, Cu, Pb and Zn) contaminated soil 4-6 years after initial amendment applications. Results indicate that the need for re-treatment is amendment- and element-dependent; in some cases, a single application may reduce trace element concentrations in above-ground biomass and enhance the establishment of a healthy vegetation cover. Amendment performance as evaluated by % cover, biomass and number of colonizing taxa differs; however, changes in plant community composition are not necessarily amendment-specific. Although the translocation of trace elements to the plant biotic compartment is greater in re-vegetated areas, overall loss of trace elements due to soil erosion and plant uptake is usually smaller compared to that in bare soil.

Abundance and diversity of n-alkane-degrading bacteria in a forest soil co-contaminated with hydrocarbons and metals: a molecular study on alkB homologous genes.

Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C(5) and C(16) at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of hydrocarbon-degrading bacteria to the contamination at the molecular level.

A comparative study of most probable number (MPN)-PCR vs. real-time-PCR for the measurement of abundance and assessment of diversity of alkB homologous genes in soil.

The utilization of quantitative PCR (qPCR) approaches such as MPN-qPCR and real-time-qPCR for in situ assessment of functional genes yields substantial quantitative and qualitative differences. We show this by targeting the alkB gene related to biodegradation of aliphatic alkanes in a set of environmental samples with differing hydrocarbon content.

MPN- and real-time-based PCR methods for the quantification of alkane monooxygenase homologous genes (alkB) in environmental samples.

Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

Long-term impact of acid resin waste deposits on soil quality of forest areas II. Biological indicators.

In this study, we evaluated the effects of two acid resin deposits on the soil microbiota of forest areas by means of biomass, microbial activity-related estimations and simple biological ratios. The determinations carried out included: total DNA yield, basal respiration, intracellular enzyme activities (dehydrogenase and catalase) and extracellular enzyme activities involved in the cycles of C (beta-glucosidase and chitinase), N (protease) and P (acid-phosphatase). The calculated ratios were: total DNA/total N; basal respiration/total DNA; dehydrogenase/total DNA and catalase/total DNA. Total DNA yield was used to estimate soil microbial biomass. Results showed that microbial biomass and activity were severely inhibited in the deposits, whilst resin effects on contaminated zones were variable and site-dependant. Correlation analysis showed no clear effect of contaminants on biomass and activities outside the deposits, but a strong interdependence with natural organic matter related parameters such as total N. In contrast, by using simple ratios we could detect more stressful conditions in terms of organic matter turnover and basal metabolism in contaminated areas compared to their uncontaminated counterparts. These results stress that developed ecosystems such as forests can buffer the effects of pollutants and preserve high functionality via natural attenuation mechanisms, but also that acid resins can be toxic to biological targets negatively affecting soil dynamics. Acid resin deposits can therefore act as contaminant sources adversely altering soil processes and reducing the environmental quality of affected areas despite the solid nature of these wastes.

Long-term impact of acid resin waste deposits on soil quality of forest areas I. Contaminants and abiotic properties.

Acid resins are residues characterised by elevated concentrations of hydrocarbons and trace elements, which were produced by mineral oil industries in Central Europe during the first half of the last century. Due to the lack of environmental legislation at that time, these wastes were dumped into excavated ponds in public areas without further protection. In this work, the long-term effects of such resin deposits on soil quality of two forest areas (Bayern, Germany) were assessed. We evaluated the distribution and accumulation of contaminants in the surroundings of the deposits, where the waste was disposed of about 60 years ago. General soil chemical properties such as pH, C, N and P content were also investigated. Chemical analysis of resin waste from the deposits revealed large amounts of potential contaminants such as hydrocarbons (93 g kg(-1)), As (63 mg kg(-1)), Cd (24 mg kg(-1)), Cu (1835 mg kg(-1)), Pb (8100 mg kg(-1)) and Zn (873 mg kg(-1)). Due to the location of the deposits on a hillside and the lack of adequate isolation, contaminants have been released downhill despite the solid nature of the waste. Five zones were investigated in each site: the deposit, three affected zones along the plume of contamination and a control zone. In affected zones, contaminants were 2 to 350 times higher than background levels depending on the site. In many cases, contaminants exceeded the German environmental guidelines for the soil-groundwater path and action levels based on extractable concentrations. Resin contamination yielded larger total C/total N ratios in affected zones, but no clear effect was observed on absolute C, N and P concentrations. In general, no major acidification effect was reported in affected zones.

Trace element availability and plant growth in a mine-spill contaminated soil under assisted natural remediation I. Soils.

We evaluated the effects of different amendments and/or a plant cover on reclamation of a trace element contaminated soil. Seven treatments were established: four organic (leonardite (LEO), litter (LIT), municipal waste compost (MWC), biosolid compost (BC)), one inorganic (sugar beet lime (SL)) and two controls (control without amendment but with Agrostis (CTRP) and control without amendment and without Agrostis (CTR)). Results showed that total organic C was significantly higher in organic treatments in all samplings. Water-soluble C was lower in CTR compared to other treatments, but no significant differences were observed between organic treatments and SL and CTR. SL, BC and MWC treatments increased soil pH and reduced 0.01 M CaCl2-extractable Cd, Cu and Zn concentrations more efficiently, especially in the first 2 years. At the end of the experiment 0.01 M CaCl2-extractable trace element concentrations were similar in all treatments. 0.01 M CaCl2-extractable As and Pb were below the detection limit. Addition of amendments showed no clear reduction in 0.05 M EDTA-extractable trace element concentrations and some amendments even increased 0.05 M EDTA-extractable As and Cu with time. Pseudo-total trace element concentrations were higher for As in controls. On the other hand, mean values of Cu and Zn were higher in MWC treatment. BC and SL treatments also showed higher Zn mean concentration than controls. No amendment effect was observed for Cd and Pb.

Trace element availability and plant growth in a mine-spill-contaminated soil under assisted natural remediation II. Plants.

In this second part, we evaluated the effects of different amendments on plant growth (Agrostis stolonifera L.), and trace element accumulation and removal by plants in a trace element (As, Cd, Cu, Pb and Zn) contaminated soil. Description of the various treatments is given in Part I of this work. The plants were grown for consecutive periods (2002, 2003, 2004), 5 months each and harvested twice in each period. Results showed that plant growth was enhanced and trace element concentrations in plant were reduced in SL, MWC, BC and LEO treatments in the first period. No significant differences were observed in subsequent periods. This seemed to be related with changes in soil pH. Removal of trace elements was higher in SL, MWC, BC and LEO treatments due to higher biomass production in the first period. In following years no significant differences between treatments were found. Data from Part I of this study were also used to compare trace element bioavailable concentrations extracted with 0.01 M CaCl2 and 0.05 M EDTA with trace elements in plant. We observed that 0.01 M CaCl2 was more suitable for determination of bioavailable concentrations and that extraction with EDTA overestimated biovailability of trace elements in amended treatments, especially in those where composts were added.