Polyhydroxyalkanoate as a slow-release carbon source for in situ bioremediation of contaminated aquifers: from laboratory investigation to pilot-scale testing in the field

A pilot-scale study aiming to evaluate the potential use of poly-3-hydroxy-butyrate (PHB) as an electron donor source for in situ bioremediation of chlorinated hydrocarbons in groundwater was conducted. Compared with commercially available electron donors, PHB offers a restricted fermentation pathway (i.e., through acetic acid and molecular hydrogen) by avoiding the formation of any residual carbon that could potentially spoil groundwater quality. The pilot study was carried out at an industrial site in Italy, heavily contaminated by different chlorinated aliphatic hydrocarbons (CAHs). Prior to field testing, PHB was experimentally verified as a suitable electron donor for biological reductive dechlorination processes at the investigated site by microcosm studies carried out on site aquifer material and measuring the quantitative transformation of detected CAHs to ethene. Owing to the complex geological characteristics of the aquifer, the use of a groundwater circulation well (GCW) was identified as a potential strategy to enable effective delivery and distribution of electron donors in less permeable layers and to mobilise contaminants. A 3-screened, 30-m-deep GCW coupled with an external treatment unit was installed at the site. The effect of PHB fermentation products on the in situ reductive dechlorination processes were evaluated by quantitative real-time polymerase chain reaction (qPCR). The results from the first 4 months of operation clearly demonstrated that the PHB fermentation products were effectively delivered to the aquifer and positively influenced the biological dechlorination activity. Indeed, an increased abundance of Dehalococcoides mccartyi (up to 6.6 fold) and reduced CAH concentrations at the installed monitoring wells were observed

Effects of the feeding solution composition on a reductive/oxidative sequential bioelectrochemical process for perchloroethylene removal

Chlorinated aliphatic hydrocarbons (CAHs) are common groundwater contaminants due to their improper use in several industrial activities. Specialized microorganisms are able to perform the reductive dechlorination (RD) of high-chlorinated CAHs such as perchloroethylene (PCE), while the low-chlorinated ethenes such as vinyl chloride (VC) are more susceptible to oxidative mechanisms performed by aerobic dechlorinating microorganisms. Bioelectrochemical systems can be used as an effective strategy for the stimulation of both anaerobic and aerobic microbial dechlorination, i.e., a biocathode can be used as an electron donor to perform the RD, while a bioanode can provide the oxygen necessary for the aerobic dechlorination reaction. In this study, a sequential bioelectrochemical process constituted by two membrane-less microbial electrolysis cells connected in series has been, for the first time, operated with synthetic groundwater, also containing sulphate and nitrate, to simulate more realistic process conditions due to the possible establishment of competitive processes for the reducing power, with respect to previous research made with a PCE-contaminated mineral medium (with neither sulphate nor nitrate). The shift from mineral medium to synthetic groundwater showed the establishment of sulphate and nitrate reduction and caused the temporary decrease of the PCE removal efficiency from 100% to 85%. The analysis of the RD biomarkers (i.e., Dehalococcoides mccartyi 16S rRNA and tceA, bvcA, vcrA genes) confirmed the decrement of reductive dechlorination performances after the introduction of the synthetic groundwater, also characterized by a lower ionic strength and nutrients content. On the other hand, the system self-adapted the flowing current to the increased demand for the sulphate and nitrate reduction, so that reducing power was not in defect for the RD, although RD coulombic efficiency was less

Assessment of long-term fermentability of pha-based materials from pure and mixed microbial cultures for potential environmental applications

The use of polyhydroxyalkanoates (PHA) as slow-release electron donors for environmental remediation represents a novel and appealing application that is attracting considerable attention in the scientific community. In this context, here, the fermentation pattern of different types of PHA-based materials has been investigated in batch and continuous-flow experiments. Along with commercially available materials, produced from axenic microbial cultures, PHA produced at pilot scale by mixed microbial cultures (MMC) using waste feedstock have been also tested. As a main finding, a rapid onset of volatile fatty acids (VFA) production was observed with a low-purity MMC-deriving material, consisting of microbial cells containing 56% (on weight basis) of intracellular PHA. Indeed, with this material a sustained, long-term production of organic acids (i.e., acetic, propionic, and butyric acids) was observed. In addition, the obtained yield of conversion into acids (up to 70% gVFA/gPHA) was higher than that obtained with the other tested materials, made of extracted and purified PHA. These results clearly suggest the possibility to directly use the PHA-rich cells deriving from the MMC production process, with no need of extraction and purification procedures, as a sustainable and effective carbon source bringing remarkable advantages from an economic and environmental point of view

Biomonitoring of chlorinated solvents-degrading bacteria in contaminated soil and groundwater

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Assessment of Long-Term Fermentability of PHA-Based Materials from Pure and Mixed Microbial Cultures for Potential Environmental Applications

The use of polyhydroxyalkanoates (PHA) as slow-release electron donors for environmental remediation represents a novel and appealing application that is attracting considerable attention in the scientific community. In this context, here, the fermentation pattern of different types of PHA-based materials has been investigated in batch and continuous-flow experiments. Along with commercially available materials, produced from axenic microbial cultures, PHA produced at pilot scale by mixed microbial cultures (MMC) using waste feedstock have been also tested. As a main finding, a rapid onset of volatile fatty acids (VFA) production was observed with a low-purity MMC-deriving material, consisting of microbial cells containing 56% (on weight basis) of intracellular PHA. Indeed, with this material a sustained, long-term production of organic acids (i.e., acetic, propionic, and butyric acids) was observed. In addition, the obtained yield of conversion into acids (up to 70% gVFA/gPHA) was higher than that obtained with the other tested materials, made of extracted and purified PHA. These results clearly suggest the possibility to directly use the PHA-rich cells deriving from the MMC production process, with no need of extraction and purification procedures, as a sustainable and effective carbon source bringing remarkable advantages from an economic and environmental point of view

Metagenomic Analysis Reveals Microbial Interactions at the Biocathode of a Bioelectrochemical System Capable of Simultaneous Trichloroethylene and Cr(VI) Reduction

Bioelectrochemical systems (BES) are attractive and versatile options for the bioremediation of organic or inorganic pollutants, including trichloroethylene (TCE) and Cr(VI), often found as co-contaminants in the environment. The elucidation of the microbial players' role in the bioelectroremediation processes for treating multicontaminated groundwater is still a research need that attracts scientific interest. In this study, 16S rRNA gene amplicon sequencing and whole shotgun metagenomics revealed the leading microbial players and the primary metabolic interactions occurring in the biofilm growing at the biocathode where TCE reductive dechlorination (RD), hydrogenotrophic methanogenesis, and Cr(VI) reduction occurred. The presence of Cr(VI) did not negatively affect the TCE degradation, as evidenced by the RD rates estimated during the reactor operation with TCE (111±2 μeq/Ld) and TCE/Cr(VI) (146±2 μeq/Ld). Accordingly, Dehalococcoides mccartyi, the primary biomarker of the RD process, was found on the biocathode treating both TCE (7.82E+04±2.9E+04 16S rRNA gene copies g−1 graphite) and TCE/Cr(VI) (3.2E+07±2.37E+0716S rRNA gene copies g−1 graphite) contamination. The metagenomic analysis revealed a selected microbial consortium on the TCE/Cr(VI) biocathode. D. mccartyi was the sole dechlorinating microbe with H2 uptake as the only electron supply mechanism, suggesting that electroactivity is not a property of this microorganism. Methanobrevibacter arboriphilus and Methanobacterium formicicum also colonized the biocathode as H2 consumers for the CH4 production and cofactor suppliers for D. mccartyi cobalamin biosynthesis. Interestingly, M. formicicum also harbors gene complexes involved in the Cr(VI) reduction through extracellular and intracellular mechanisms

Microbial Community Changes in a Chlorinated Solvents Polluted Aquifer Over the Field Scale Treatment With Poly-3-Hydroxybutyrate as Amendment

This study investigated the organohalide-respiring bacteria (OHRB) and the supporting microbial populations operating in a pilot scale plant employing poly-3-hydroxybutyrate (PHB), a biodegradable polymer produced by bacteria from waste streams, for the in situ bioremediation of groundwater contaminated by chlorinated solvents. The bioremediation was performed in ground treatment units, including PHB reactors as slow release source of electron donors, where groundwater extracted from the wells flows through before the re-infiltration to the low permeability zones of the aquifer. The coupling of the biological treatment with groundwater recirculation allowed to drastically reducing the contamination level and the remediation time by efficiently stimulating the growth of autochthonous OHRB and enhancing the mobilization of the pollutants. Quantitative PCR performed along the external treatment unit showed that the PHB reactor may efficiently act as an external incubator to growing Dehalococcoides mccartyi, known to be capable of fully converting chlorinated ethenes to innocuous end-products. The slow release source of electron donors for the bioremediation process allowed the establishment of a stable population of D. mccartyi, mainly carrying bvcA and vcrA genes which are implicated in the metabolic conversion of vinyl chloride to harmless ethene. Next generation sequencing was performed to analyze the phylogenetic diversity of the groundwater microbiome before and after the bioremediation treatment and allowed the identification of the microorganisms working closely with organohalide-respiring bacteria

Freshwater plastisphere: a review on biodiversity, risks, and biodegradation potential with implications for the aquatic ecosystem health

The plastisphere, a unique microbial biofilm community colonizing plastic debris and microplastics (MPs) in aquatic environments, has attracted increasing attention owing to its ecological and public health implications. This review consolidates current state of knowledge on freshwater plastisphere, focussing on its biodiversity, community assembly, and interactions with environmental factors. Current biomolecular approaches revealed a variety of prokaryotic and eukaryotic taxa associated with plastic surfaces. Despite their ecological importance, the presence of potentially pathogenic bacteria and mobile genetic elements (i.e., antibiotic resistance genes) raises concerns for ecosystem and human health. However, the extent of these risks and their implications remain unclear. Advanced sequencing technologies are promising for elucidating the functions of plastisphere, particularly in plastic biodegradation processes. Overall, this review emphasizes the need for comprehensive studies to understand plastisphere dynamics in freshwater and to support effective management strategies to mitigate the impact of plastic pollution on freshwater resources

Enhancing the anaerobic biodegradation of petroleum hydrocarbons in soils with electrically conductive materials

: Anaerobic bioremediation is a relevant process in the management of sites contaminated by petroleum hydrocarbons. Recently, interspecies electron transfer processes mediated by conductive minerals or particles have been proposed as mechanisms through which microbial species within a community share reducing equivalents to drive the syntrophic degradation of organic substrates, including hydrocarbons. Here, a microcosm study was set up to investigate the effect of different electrically conductive materials (ECMs) in enhancing the anaerobic biodegradation of hydrocarbons in historically contaminated soil. The results of a comprehensive suite of chemical and microbiological analyses evidenced that supplementing the soil with (5% w/w) magnetite nanoparticles or biochar particles is an effective strategy to accelerate the removal of selected hydrocarbons. In particular, in microcosms supplemented with ECMs, the removal of total petroleum hydrocarbons was enhanced by up to 50% relative to unamended controls. However, chemical analyses suggested that only a partial bioconversion of contaminants occurred and that longer treatment times would have probably been required to drive the biodegradation process to completion. On the other hand, biomolecular analyses confirmed the presence of several microorganisms and functional genes likely involved in hydrocarbon degradation. Furthermore, the selective enrichment of known electroactive bacteria (i.e., Geobacter and Geothrix) in microcosms amended with ECMs, clearly pointed to a possible role of DIET (Diet Interspecies Electron Transfer) processes in the observed removal of contaminants

Biorisanamento in situ di sorgenti storiche da DNAPL. Pozzi a ricircolazione per la ottimale distribuzione di donatori di elettroni e mobilizzazione di contaminanti da zone a bassa permeabilità

In this paper, an example, on a full scale and on the Italian territory, of the design and management of an in situ bioremediation intervention and the mobilization of chlorinated solvents, in a historically contaminated industrial site characterized by a complex hydrogeological conformation is reported. In particular, the presence of historical accumulations of chlorinated substances in layers with very limited permeability, with consequent slow back diffusion phenomena, has been addressed with the use of groundwater circulation wells, IEG-GCW®, which allow the mobilization and external treatment of contaminants retained in portions of the aquifer that cannot be attacked with traditional pumping systems. Furthermore, the occurrence of reductive dechlorination phenomena limited by the electron donor deficiency was addressed by using recirculation as a system for their continuous distribution, generated by the fermentation of a biodegradable polymer (PHA). The aim of the work is also to underline the relevance of a 3D hydrogeochemical model for the representation and understanding of the contamination dynamics and decontamination mechanisms in a highly contaminated industrial site. A multi-phase approach was followed allowing the management and release of data during the various phases of remediation, from the characterization of the site, through the conduct of a pilot test, up to full-scale remediation, thus allowing to monitor, analyze and manipulate the information in 4D space-time. Multi-source and multi-temporal scenarios reveal the impact of current hydraulic dynamics and describe the decontamination mechanisms in relation to the interventions implemented over time, quantifying the overall performance of the strategies adopted in terms of reducing the concentrations of contaminants present in groundwater. The results illustrate the effectiveness of the recirculation system in the progressive reduction of the mass of contaminants in the secondary sources identified, both by mobilization of contaminants and by enhancement of the in situ biological reductive dechlorination processes. It also confirms the usefulness in the management of interventions and in the representation of the results of the integrated georeferenced model