Ecological indicators and biological resources for hydrocarbon rhizoremediation in a protected area.

Spillage from oil refineries, pipelines, and service stations consistently leads to soil, food and groundwater contamination. Bacterial-assisted phytoremediation is a non-invasive and sustainable solution to eliminate or decrease the concentration of xenobiotic contaminants in the environment. In the present study, a protected area interested by a fuel discharge was considered to assess a bioremediation intervention. From the spill point, a plume of contamination flowed South-West into the aquifer, eventually reaching a wetland area. Soils, groundwaters and plants belonging to the species Scirpus sylvaticus (L.) were sampled. In the majority of the soil samples, concentrations of total petroleum hydrocarbons, both C ≤ 12 and C > 12, exceeded legal limits set forth in Directive 2000/60/EC. The analysis of diatom populations, used as ecological indicators, evidenced morphology alterations and the presence of Ulnaria ulna and Ulnaria biceps species, previously detected in hydrocarbon-polluted waters. Tests for phytotoxicity and phytodegradation, carried out in soil mesocosms, planted with Zea mays and Helianthus annuus, demonstrated that both species significantly contributed to the removal of total petroleum hydrocarbons. Removal of C ≤ 12 and C > 12 petroleum hydrocarbons was in the range of 80%-82% for Z. mays and 71%-72% for H. annuus. Microbial communities inhabiting high organic carbon and vegetated soils were more active in hydrocarbon degradation than those inhabiting subsoils, as evidenced by soil slurry experiments. The abundance of functional genes encoding toluene-benzene monooxygenase (tbmD) and alkane hydroxylase (alkB), quantified in environmental samples, confirmed that the plant rhizosphere recruited a microbial community with higher biodegradation capacity. Bacterial strains isolated from the sampling site were able to grow on model hydrocarbons (hexane, hexadecane and o-, m-, p-xylene) as sole carbon and energy sources, indicating that a natural bio-attenuation process was on-going at the site. The bacterial strains isolated from rhizosphere soil, rhizoplane and endosphere showed plant growth promoting traits according to in vitro and in vivo tests on Z. mays and Oryza sativa, allowing to forecast a possible application of bacterial assisted rhizoremediation to recover the protected area.

Microbial communities in paddy soils: differences in abundance and functionality between rhizosphere and pore water, the influence of different soil organic carbon, sulfate fertilization and cultivation time, and contribution to arsenic mobility and speciation.

Abiotic factors and rhizosphere microbial populations influence arsenic accumulation in rice grains. Although mineral and organic surfaces are keystones in element cycling, localization of specific microbial reactions in the root/soil/pore water system is still unclear. Here, we tested if original unplanted soil, rhizosphere soil and pore water represented distinct ecological microniches for arsenic-, sulfur- and iron-cycling microorganisms and compared the influence of relevant factors such as soil type, sulfate fertilization and cultivation time. In rice open-air-mesocosms with two paddy soils (2.0% and 4.7% organic carbon), Illumina 16S rRNA gene sequencing demonstrated minor effects of cultivation time and sulfate fertilization that decreased Archaea-driven microbial networks and incremented sulfate-reducing and sulfur-oxidizing bacteria. Different compartments, characterized by different bacterial and archaeal compositions, had the strongest effect, with higher microbial abundances, bacterial biodiversity and interconnections in the rhizosphere vs pore water. Within each compartment, a significant soil type effect was observed. Higher percentage contributions of rhizosphere dissimilatory arsenate- and iron-reducing, arsenite-oxidizing, and, surprisingly, dissimilatory sulfate-reducing bacteria, as well as pore water iron-oxidizing bacteria in the lower organic carbon soil, supported previous chemistry-based interpretations of a more active S-cycling, a higher percentage of thioarsenates and lower arsenic mobility by sorption to mixed Fe(II)Fe(III)-minerals in this soil.

Pollution and edaphic factors shape bacterial community structure and functionality in historically contaminated soils.

Studies about biodegradation potential in soils often refer to artificially contaminated and simplified systems, overlooking the complexity associated with contaminated sites in a real context. This work aims to provide a holistic view on microbiome assembly and functional diversity in the model site SIN Brescia-Caffaro (Italy), characterized by historical and uneven contamination by organic and inorganic compounds. Here, physical and chemical analyses and microbiota characterization were applied on one-hundred-twenty-seven soil samples to unravel the environmental factors driving bacterial community assembly and biodegradation potential in three former agricultural fields. Chemical analyses showed a patchy distribution of metals, metalloids and polychlorinated biphenyls (PCB) and allowed soil categorization according to depth and area of collections. Likewise, the bacterial community structure, described by molecular fingerprinting and 16S rRNA gene analyses, was significantly different according to collection site and depth. Pollutant concentrations (i.e., hexachloro-biphenyls, arsenic and mercury), nitrogen content and parameters related to soil texture were identified as main drivers of microbiota assembly, being significantly correlated to bacterial community composition. Moreover, bacteria putatively involved in the aerobic degradation of PCBs were enriched over the total bacterial community in topsoils, where the highest activity was recorded using fluorescein hydrolysis as proxy. Metataxonomic analyses revealed the presence of bacteria having metabolic pathways related to PCB degradation and tolerance to heavy metals and metalloids in the topsoil samples collected in all areas. Overall, the provided dissection of soil microbiota structure and its degradation potential in the SIN Brescia-Caffaro can contribute to target specific areas for rhizoremediation implementation. Metagenomics studies could be implemented in the future to understand if specific degradative pathways are present in historically polluted sites characterized by the co-occurrence of multiple classes of contaminants.

Adaptation of Microbial Communities to Environmental Arsenic and Selection of Arsenite-Oxidizing Bacteria From Contaminated Groundwaters.

Arsenic mobilization in groundwater systems is driven by a variety of functionally diverse microorganisms and complex interconnections between different physicochemical factors. In order to unravel this great ecosystem complexity, groundwaters with varying background concentrations and speciation of arsenic were considered in the Po Plain (Northern Italy), one of the most populated areas in Europe affected by metalloid contamination. High-throughput Illumina 16S rRNA gene sequencing, CARD-FISH and enrichment of arsenic-transforming consortia showed that among the analyzed groundwaters, diverse microbial communities were present, both in terms of diversity and functionality. Oxidized inorganic arsenic [arsenite, As(III)] was the main driver that shaped each community. Several uncharacterized members of the genus Pseudomonas, putatively involved in metalloid transformation, were revealed in situ in the most contaminated samples. With a cultivation approach, arsenic metabolisms potentially active at the site were evidenced. In chemolithoautotrophic conditions, As(III) oxidation rate linearly correlated to As(III) concentration measured at the parental sites, suggesting that local As(III) concentration was a relevant factor that selected for As(III)-oxidizing bacterial populations. In view of the exploitation of these As(III)-oxidizing consortia in biotechnology-based arsenic bioremediation actions, these results suggest that contaminated aquifers in Northern Italy host unexplored microbial populations that provide essential ecosystem services.

Exposure to different arsenic species drives the establishment of iron- and sulfur-oxidizing bacteria on rice root iron plaques.

Iron- and sulfur-oxidizing bacteria inhabiting rice rhizoplane play a significant role on arsenic biogeochemistry in flooded rice paddies, influencing arsenic translocation to rice grains. In the present study, the selective pressure of arsenic species on these microbial populations was evaluated. Rice roots from continuously flooded plants were incubated in iron sulfide (FeS) gradient tubes and exposed to either arsenate or arsenite. The biomass developed in the visible iron-oxidation band of the enrichments was analyzed by Scanning Electron Microscopy and Energy-Dispersive Spectroscopy (SEM-EDS) and the bacterial communities were characterized by 16S rRNA gene sequencing. Different Proteobacteria communities were selected depending on exposure to arsenate and arsenite. Arsenate addition favored the versatile iron-oxidizers Dechloromonas and Azospira, associated to putative iron (hydr)oxide crystals. Arsenite exposure decreased the diversity in the enrichments, with the development of the sulfur-oxidizer Thiobacillus thioparus, likely growing on sulfide released by FeS. Whereas sulfur-oxidizers were observed in all treatments, iron-oxidizers disappeared when exposed to arsenite. These results reveal a strong impact of different inorganic arsenics on rhizospheric iron-oxidizers as well as a crucial role of sulfur-oxidizing bacteria in establishing rice rhizosphere communities under arsenic pressure.

Exploring Biodiversity and Arsenic Metabolism of Microbiota Inhabiting Arsenic-Rich Groundwaters in Northern Italy.

Arsenic contamination of groundwater aquifers is an issue of global concern. Among the affected sites, in several Italian groundwater aquifers arsenic levels above the WHO limits for drinking water are present, with consequent issues of public concern. In this study, for the first time, the role of microbial communities in metalloid cycling in groundwater samples from Northern Italy lying on Pleistocene sediments deriving from Alps mountains has been investigated combining environmental genomics and cultivation approaches. 16S rRNA gene libraries revealed a high number of yet uncultured species, which in some of the study sites accounted for more of the 50% of the total community. Sequences related to arsenic-resistant bacteria (arsenate-reducing and arsenite-oxidizing) were abundant in most of the sites, while arsenate-respiring bacteria were negligible. In some of the sites, sulfur-oxidizing bacteria of the genus Sulfuricurvum accounted for more than 50% of the microbial community, whereas iron-cycling bacteria were less represented. In some aquifers, arsenotrophy, growth coupled to autotrophic arsenite oxidation, was suggested by detection of arsenite monooxygenase (aioA) and 1,5-ribulose bisphosphate carboxylase (RuBisCO) cbbL genes of microorganisms belonging to Rhizobiales and Burkholderiales. Enrichment cultures established from sampled groundwaters in laboratory conditions with 1.5 mmol L-1 of arsenite as sole electron donor were able to oxidize up to 100% of arsenite, suggesting that this metabolism is active in groundwaters. The presence of heterotrophic arsenic resistant bacteria was confirmed by enrichment cultures in most of the sites. The overall results provided a first overview of the microorganisms inhabiting arsenic-contaminated aquifers in Northern Italy and suggested the importance of sulfur-cycling bacteria in the biogeochemistry of arsenic in these ecosystems. The presence of active arsenite-oxidizing bacteria indicates that biological oxidation of arsenite, in combination with arsenate-adsorbing materials, could be employed for metalloid removal.

Impacts of dietary silver nanoparticles and probiotic administration on the microbiota of an in-vitro gut model.

Ingestion of silver nanoparticles (AgNPs) is inevitable linked to their widespread use in food, medicines and other consumer products. However, their effects on human microbiota at non-lethal concentrations remain poorly understood. In this study, the interactions among 1 µg mL-1 AgNPs, the intestinal microbiota, and the probiotic Bacillus subtilis (BS) were tested using in-vitro batch fermentation models inoculated with human fecal matter. Results from metagenomic investigations revealed that the core bacterial community was not affected by the exposure of AgNPs and BS at the later stage of fermentation, while the proportions of rare species changed drastically with the treatments. Furthermore, shifts in the Firmicutes/Bacteriodetes (F/B) ratios were observed after 24 h with an increase in the relative abundance of Firmicutes species and a decrease in Bacteroidetes in all fermentation cultures. The co-exposure to AgNPs and BS led to the lowest F/B ratio. Fluorescent in-situ hybridization analyses indicated that non-lethal concentration of AgNPs negatively affected the relative percentage of Faecalibacterium prausnitzii and Clostridium coccoides/Eubacterium rectales taxa in the fermentation cultures after 24 h. However, exposure to single and combined treatments of AgNPs and BS did not change the overall diversity of the fecal microflora. Functional differences in cell motility, translation, transport, and xenobiotics degradation occurred in AgNPs-treated fermentation cultures but not in AgNPs+BS-treated samples. Compared to the control samples, treated fecal cultures showed no significant statistical differences in terms of short-chain fatty acids profiles, cytotoxic and genotoxic effects on Caco-2 cell monolayers. Overall, AgNPs did not affect the composition and diversity of the core fecal microflora and its metabolic and toxic profiles. This work indicated a chemopreventive role of probiotic on fecal microflora against AgNPs, which were shown by the decrease of F/B ratio and the unaltered state of some key metabolic pathways.

Bioelectrochemical Nitrogen fixation (e-BNF): Electro-stimulation of enriched biofilm communities drives autotrophic nitrogen and carbon fixation.

A new approach to microbial electrosynthesis is proposed, aimed at producing whole biomass from N2 and inorganic carbon, by electrostimulation of complex microbial communities. On a carbon-based conductor under constant polarization (-0.7 V vs SHE), an electroactive biofilm was enriched with autotrophic nitrogen fixing microorganims and led to biomass synthesis at higher amounts (up to 18 fold), as compared to controls kept at open circuit (OC). After 110 days, the electron transfer had increased by 30-fold, as compared to abiotic conditions. Metagenomics evidenced Nif genes associated with autotrophs (both Archaea and Bacteria) only in polarized biofilms, but not in OC. With this first proof of concept experiment, we propose to call this promising field 'bioelectrochemical nitrogen fixation' (e-BNF): a possible way to 'power' biological nitrogen fixation, organic carbon storage and soil fertility against desertification, and possibly a new tool to study the development of early prokaryotic life in extreme environments.

Rice Paddy Nitrospirae Carry and Express Genes Related to Sulfate Respiration: Proposal of the New Genus "Candidatus Sulfobium".

Nitrospirae spp. distantly related to thermophilic, sulfate-reducing Thermodesulfovibrio species are regularly observed in environmental surveys of anoxic marine and freshwater habitats. Here we present a metaproteogenomic analysis of Nitrospirae bacterium Nbg-4 as a representative of this clade. Its genome was assembled from replicated metagenomes of rice paddy soil that was used to grow rice in the presence and absence of gypsum (CaSO4·2H2O). Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression of this pathway in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia (DNRA), with expression of its first step being detected in bulk soil without gypsum amendment. The relative abundances of Nbg-4 were similar under both treatments, indicating that Nbg-4 maintained stable populations while shifting its energy metabolism. Whether Nbg-4 is a strict sulfate reducer or can couple sulfur oxidation to DNRA by operating the pathway of dissimilatory sulfate reduction in reverse could not be resolved. Further genome reconstruction revealed the potential to utilize butyrate, formate, H2, or acetate as an electron donor; the Wood-Ljungdahl pathway was expressed under both treatments. Comparison to publicly available Nitrospirae genome bins revealed the pathway for dissimilatory sulfate reduction also in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we propose for Nbg-4 the name "Candidatus Sulfobium mesophilum," gen. nov., sp. nov.IMPORTANCE Rice paddies are indispensable for the food supply but are a major source of the greenhouse gas methane. If it were not counterbalanced by cryptic sulfur cycling, methane emission from rice paddy fields would be even higher. However, the microorganisms involved in this sulfur cycling are little understood. By using an environmental systems biology approach with Italian rice paddy soil, we could retrieve the population genome of a novel member of the phylum Nitrospirae This microorganism encoded the full pathway of dissimilatory sulfate reduction and expressed it in anoxic paddy soil under sulfate-enriched conditions. Phylogenomics and comparison to the results of environmental surveys showed that such microorganisms are actually widespread in freshwater and marine environments. At the same time, they represent an undiscovered genus within the little-explored phylum Nitrospirae Our results will be important for the design of enrichment strategies and postgenomic studies to further understanding of the contribution of these novel Nitrospirae spp. to the global sulfur cycle.

A study of microbial communities on terracotta separator and on biocathode of air breathing microbial fuel cells.

Recently, terracotta has attracted interest as low-cost and biocompatible material to build separators in microbial fuel cells (MFCs). However, the influence of a non-conductive material like terracotta on electroactive microbiological communities remains substantially unexplored. This study aims at describing the microbial pools developed from two different seed inocula (bovine and swine sewage) in terracotta-based air-breathing MFC. A statistical approach on microbiological data confirmed different community enrichment in the MFCs, depending mainly on the inoculum. Terracotta separators impeded the growth of electroactive communities in contact with cathodes (biocathodes), while a thick biofilm was observed on the surface (anolyte-side) of the terracotta separator. Terracotta-free MFCs, set as control experiments, showed a well-developed biocathode, Biocathode-MFCs resulted in 4 to 6-fold higher power densities. All biofilms were analyzed by high-throughput Illumina sequencing applied to 16S rRNA gene. The results showed more abundant (3- to 5-fold) electroactive genera (mainly Geobacter, Pseudomonas, Desulfuromonas and Clostridia MBA03) in terracotta-free biocathodes. Nevertheless, terracotta separators induced only slight changes in anodic microbial communities.

Influence of water management on the active root-associated microbiota involved in arsenic, iron, and sulfur cycles in rice paddies.

In recent years, the role of microorganisms inhabiting rice rhizosphere in promoting arsenic contamination has emerged. However, little is known concerning the species and metabolic properties involved in this phenomenon. In this study, the influence of water management on the rhizosphere microbiota in relation to arsenic dissolution in soil solution was tested. Rice plants were cultivated in macrocosms under different water regimes: continuous flooding, continuous flooding with a 2-week period drainage before flowering, and dry soil watered every 10 days. The active bacterial communities in rhizosphere soil and in rhizoplane were characterized by 16S rRNA pyrosequencing. An in-depth analysis of microbial taxa with direct or indirect effects on arsenic speciation was performed and related contribution was evaluated. Continuous flooding promoted high diversity in the rhizosphere, with the plant strongly determining species richness and evenness. On the contrary, under watering the communities were uniform, with little differences between rhizosphere soil and rhizoplane. Arsenic-releasing and arsenite-methylating bacteria were selected by continuous flooding, where they represented 8% of the total. On the contrary, bacteria decreasing arsenic solubility were more abundant under watering, with relative abundance of 10%. These values reflected arsenic concentrations in soil solution: 135 µg L-1 and negligible in continuous flooding and under watering, respectively. When short-term drainage was applied before flowering, intermediate conditions were achieved. This evidence strongly indicates an active role of the rhizosphere microbiota in driving arsenic biogeochemistry in rice paddies, influenced by water management, explaining amounts and speciation of arsenic often found in rice grains.

Influences of dissolved oxygen concentration on biocathodic microbial communities in microbial fuel cells.

Dissolved oxygen (DO) at cathodic interface is a critical factor influencing microbial fuel cells (MFC) performance. In this work, three MFCs were operated with cathode under different DO conditions: i) air-breathing (A-MFC); ii) water-submerged (W-MFC) and iii) assisted by photosynthetic microorganisms (P-MFC). A plateau of maximum current was reached at 1.06±0.03mA, 1.48±0.06mA and 1.66±0.04mA, increasing respectively for W-MFC, P-MFC and A-MFC. Electrochemical and microbiological tools (Illumina sequencing, confocal microscopy and biofilm cryosectioning) were used to explore anodic and cathodic biofilm in each MFC type. In all cases, biocathodes improved oxygen reduction reaction (ORR) as compared to abiotic condition and A-MFC was the best performing system. Photosynthetic cultures in the cathodic chamber supplied high DO level, up to 16mgO2L-1, which sustained aerobic microbial community in P-MFC biocathode. Halomonas, Pseudomonas and other microaerophilic genera reached >50% of the total OTUs. The presence of sulfur reducing bacteria (Desulfuromonas) and purple non-sulfur bacteria in A-MFC biocathode suggested that the recirculation of sulfur compounds could shuttle electrons to sustain the reduction of oxygen as final electron acceptor. The low DO concentration limited the cathode in W-MFC. A model of two different possible microbial mechanisms is proposed which can drive predominantly cathodic ORR.

Phylogenetic Structure and Metabolic Properties of Microbial Communities in Arsenic-Rich Waters of Geothermal Origin.

Arsenic (As) is a toxic element released in aquatic environments by geogenic processes or anthropic activities. To counteract its toxicity, several microorganisms have developed mechanisms to tolerate and utilize it for respiratory metabolism. However, still little is known about identity and physiological properties of microorganisms exposed to natural high levels of As and the role they play in As transformation and mobilization processes. This work aims to explore the phylogenetic composition and functional properties of aquatic microbial communities in As-rich freshwater environments of geothermal origin and to elucidate the key microbial functional groups that directly or indirectly may influence As-transformations across a natural range of geogenic arsenic contamination. Distinct bacterial communities in terms of composition and metabolisms were found. Members of Proteobacteria, affiliated to Alpha- and Betaproteobacteria were mainly retrieved in groundwaters and surface waters, whereas Gammaproteobacteria were the main component in thermal waters. Most of the OTUs from thermal waters were only distantly related to 16S rRNA gene sequences of known taxa, indicating the occurrence of bacterial biodiversity so far unexplored. Nitrate and sulfate reduction and heterotrophic As(III)-oxidization were found as main metabolic traits of the microbial cultivable fraction in such environments. No growth of autotrophic As(III)-oxidizers, autotrophic and heterotrophic As(V)-reducers, Fe-reducers and oxidizers, Mn-reducers and sulfide oxidizers was observed. The ars genes, involved in As(V) detoxifying reduction, were found in all samples whereas aioA [As(III) oxidase] and arrA genes [As(V) respiratory reductase] were not found. Overall, we found that As detoxification processes prevailed over As metabolic processes, concomitantly with the intriguing occurrence of novel thermophiles able to tolerate high levels of As.

Gypsum amendment to rice paddy soil stimulated bacteria involved in sulfur cycling but largely preserved the phylogenetic composition of the total bacterial community.

Rice paddies are indispensable for human food supply but emit large amounts of the greenhouse gas methane. Sulfur cycling occurs at high rates in these water-submerged soils and controls methane production, an effect that is increased by sulfate-containing fertilizers or soil amendments. We grew rice plants until their late vegetative phase with and without gypsum (CaSO4 ·2H2 O) amendment and identified responsive bacteria by 16S rRNA gene amplicon sequencing. Gypsum amendment decreased methane emissions by up to 99% but had no major impact on the general phylogenetic composition of the bacterial community. It rather selectively stimulated or repressed a small number of 129 and 27 species-level operational taxonomic units (OTUs) (out of 1883-2287 observed) in the rhizosphere and bulk soil, respectively. Gypsum-stimulated OTUs were affiliated with several potential sulfate-reducing (Syntrophobacter, Desulfovibrio, unclassified Desulfobulbaceae, unclassified Desulfobacteraceae) and sulfur-oxidizing taxa (Thiobacillus, unclassified Rhodocyclaceae), while gypsum-repressed OTUs were dominated by aerobic methanotrophs (Methylococcaceae). Abundance correlation networks suggested that two abundant (>1%) OTUs (Desulfobulbaceae, Rhodocyclaceae) were central to the reductive and oxidative parts of the sulfur cycle.