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Subarctic ecosystems provide key services to local communities, yet they are threatened by pollution caused by spills and disposal of petroleum waste. Finding solutions for the remediation and restoration of subarctic soils is valuable for reasons related to human and ecosystem health, as well as environmental justice.

Background Although fertilization and crop rotation practices are commonly used worldwide in agriculture to maximize crop yields, their long-term effect on the structures of soil microorganisms is still poorly understood. This study investigated the long-term impact of fertilization and crop rotation on soil microbial diversity and the microbial community structure in four different locations with three soil types. Since 1996, manure (MF; 330 kg N/ha), sewage sludge (SF; 330 and SF3x; 990 kg N/ha), and NPK (NPK; 330 kg N/ha) fertilizers were periodically applied to the soils classified as chernozem, luvisol and cambisol, which are among the most abundant or fertile soils used for agricultural purposes in the world. In these soils, potato (Solanum tuberosum L.), winter wheat (Triticum aestivum L.), and spring barley (Hordeum vulgare L.) were rotated every three years. Results Soil chemistry, which was significantly associated with location, fertilization, crop rotation, and the interaction of fertilization and location, was the dominant driver of soil microbial communities, both prokaryotic and fungal. A direct effect of long-term crop rotation and fertilization on the structure of their communities was confirmed, although there was no evidence of their influence on microbial diversity. Fungal and bacterial communities responded differently to fertilization treatments; prokaryotic communities were only significantly different from the control soil (CF) in soils treated with MF and SF3x, while fungal communities differed across all treatments. Indicator genera were identified for different treatments. These taxa were either specific for their decomposition activities or fungal plant pathogens. Sequential rotation of the three crops restricted the growth of several of the indicator plant pathogens. Conclusions Long-term fertilization and crop rotation significantly altered microbial community structure in the soil. While fertilization affected soil microorganisms mainly through changes in nutrient profile, crop rotations lead to the attraction and repulsion of specific plant pathogens. Such changes in soil microbial communities need to be considered when planning soil management.

Background The extreme conditions of thermal springs constitute a unique aquatic habitat characterized by low nutrient contents and the absence of human impacts on the microbial community composition. Thus, these springs may host phylogenetically novel microorganisms with potential use in biotechnology. With this hypothesis in mind, we examined the microbial composition of four thermal springs of the world-renowned spa town of Karlovy Vary (Carlsbad), Czechia, which differ in their temperature and chemical composition. Results Microbial profiling using 16S rRNA gene sequencing revealed the presence of phylogenetically novel taxa at various taxonomic levels, spanning from genera to phyla. Many sequences belonged to novel classes within the phyla Hydrothermae, Altiarchaeota, Verrucomicrobia, and TA06. Cultivation-based methods employing oligotrophic media resulted in the isolation of 44 unique bacterial isolates. These include strains that withstand concentrations of up to 12% NaClw/v in cultivation media or survive a temperature of 100 °C, as well as hitherto uncultured bacterial species belonging to the genera Thermomonas, Paenibacillus, and Cellulomonas. These isolates harbored stress response genes that allow them to thrive in the extreme environment of thermal springs. Conclusions Our study is the first to analyze the overall microbial community composition of the renowned Karlovy Vary thermal springs. We provide insight into yet another level of uniqueness of these springs. In addition to their unique health benefits and cultural significance, we demonstrate that these springs harbor phylogenetically distinct microorganisms with unusual life strategies. Our findings open up avenues for future research with the promise of a deeper understanding of the metabolic potential of these microorganisms.

In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.

The creamy white to beige, aerobic, non-motile, ovoid to rod-shaped, Gram-stain-negative strain, Cd-10(T), was isolated from heavy-metal-contaminated sludge from a decantation basin of a heavy metal processing factory based on its ability to tolerate CdCl2 in the cultivation medium. In the reconstruction of its phylogeny based on 16S rRNA gene sequences, strain Cd-10(T) clustered with species of the genera Gemmobacter, Xinfangfangia, Tabrizicola and Rhodobacter within the family Rhodobacteraceae. Its 16S rRNA gene sequence exhibited 96.32% pairwise similarity to the type strain of Xinfangfangia soli, 95.3 % to that of Gemmobacter intermedius, followed by Tabrizicola fusiformis (95.10 %), Rhodobacter sediminis (94.88 %), Gemmobacter nectariphilus and Rhodobacter capsulatus (both 94.81%). The major respiratory quinone was Q-10 accompanied by Q-9, the fatty acid profile consisted predominantly of C-18:1 omega 7C, C-18:0, C-16:0 and C-16:1 omega 7c, the major polar lipids were phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine and diphosphatidylglycerol. An analysis of the percentage of conserved proteins deduced from draft or complete genomic sequences of strain Cd-10(T) and representatives of its closest relatives suggested that strain Cd-10(T) is a member of a novel genus within the Rhodobacteraceae family for which we propose the name Pseudogemmobacter. Strain Cd-10(T) (=DSM 103618(T)=NCCB 100645(T)) is the type strain of Pseudogemmobacter bohemicus gen. nov., sp. nov., the type species of the genus Pseudogemmobacter gen. nov.

The involvement of bacterial aromatic ring-hydroxylating dioxygenases (ARHDs) in the degradation of aromatic pollutants, such as polychlorinated biphenyls (PCBs), has been well studied. However, there is considerable speculation as to the origin of this ability. One hypothesis is centered on a connection between the ability to degrade aromatic pollutants and the necessity of soil bacteria to cope with and/or utilize secondary plant metabolites (SPMs). To investigate this connection, we researched the involvement of biphenyl 2,3-dioxygenase (BPDO), an ARHD essential for the degradation of PCBs, in the metabolism of SPMs in the soil bacterium Pseudomonas alcaliphila JAB1, a versatile degrader of PCBs. We demonstrated the ability of the strain JAB1 to transform a variety of SPMs, namely the flavonoids apigenin, flavone, flavanone, naringenin, fisetin, quercetin, morin, and catechin, caffeic acid, trans-cinnamic acid, and the monoterpenes (S)-limonene and (R)-carvone. Of those, the transformation of flavone, flavanone, and (S)-limonene was conditioned by the activity of JAB1-borne BPDO and thus was researched in more detail, and we found evidence for the limonene monooxygenase activity of the BPDO. Furthermore, the bphA gene in the strain JAB1 was demonstrated to be induced by a wide range of SPMs, with monoterpenes being the strongest inducers of the SPMs tested. Thus, our findings contribute to the growing body of evidence that ARHDs not only play a role in the catabolism of aromatic pollutants, but also of natural plant-derived aromatics, and this study supports the hypothesis that ARHDs participate in ecological processes mediated by SPMs.

Certain industrial chemicals accumulate in the environment due to their recalcitrant properties. Bioremediation uses the capability of some environmental bacteria to break down these chemicals and attenuate the pollution. One such bacterial strain, designated Pvy, was isolated from sediment samples from a lagoon in Romania located near an oil refinery due to its capacity to degrade dibenzofuran (DF). The genome sequence of the Pvy strain was obtained using an Oxford Nanopore MiniION platform. According to the consensus 16S rRNA gene sequence that was compiled from six 16S rRNA gene copies contained in the genome and orthologous average nucleotide identity (OrthoANI) calculation, the Pvy strain was identified as Pseudomonas veronii, which confirmed the identification obtained with the aid of MALDI-TOF mass spectrometry and MALDI BioTyper. The genome was analyzed with respect to enzymes responsible for the overall biodegradative versatility of the strain. The Pvy strain was able to derive carbon from naphthalene (NP) and several aromatic compounds of natural origin, including salicylic, protocatechuic, p-hydroxybenzoic, trans-cinnamic, vanillic, and indoleacetic acids or vanillin, and was shown to degrade but not utilize DF. In total seven loci were found in the Pvy genome, which enables the strain to participate in the degradation of these aromatic compounds. Our experimental data also indicate that the transcription of the NP-dioxygenase α-subunit gene (ndoB), carried by the plasmid of the Pvy strain, is inducible by DF. These features make the Pvy strain a potential candidate for various bioremediation applications.

Endophytic bacteria often promote plant growth and protect their host plant against pathogens, herbivores, and abiotic stresses including drought, increased salinity or pollution. Current agricultural practices are being challenged in terms of climate change and the ever-increasing demand for food. Therefore, the rational exploitation of bacterial endophytes to increase the productivity and resistance of crops appears to be very promising. However, the efficient and larger-scale use of bacterial endophytes for more effective and sustainable agriculture is hindered by very little knowledge on molecular aspects of plant-endophyte interactions and mechanisms driving bacterial communities in planta. In addition, since most of the information on bacterial endophytes has been obtained through culture-dependent techniques, endophytic bacterial diversity and its full biotechnological potential still remain highly unexplored. In this study, we discuss the diversity and role of endophytic populations as well as complex interactions that the endophytes have with the plant and vice versa, including the interactions leading to plant colonization. A description of biotic and abiotic factors influencing endophytic bacterial communities is provided, along with a summary of different methodologies suitable for determining the diversity of bacterial endophytes, mechanisms governing the assembly and structure of bacterial communities in the endosphere, and potential biotechnological applications of endophytes in the future.

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-to-genome blast distance phylogeny (GBDP) algorithm and digital DNA–DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds.

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-togenome blast distance phylogeny (GBDP) algorithm and digital DNA–DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds, This research was funded by GREENER-H2020 (EU), grant number 826312, and MICINN/FEDER EU, grant number RTI2018-0933991- B-I00. D.G.-S. was supported by a MECD FPU fellowship program, grant number FPU14/03965. P.S.-L. was supported by the MICINN FPU fellowship program, grant number FPU18/02169. E.B.-R. was supported by the MECD FPU fellowship program, grant number FPU16/05513. J.S., T.C. and O.U. acknowledge Czech Science Foundation grant number 17–00227S, which enabled PCB co-metabolism experiments to be undertaken

Soil microbial communities are shaped by many different biotic and abiotic factors, whose effects, however, are not fully understood. In this study, our objective was to analyze microbial communities in the root zone of Tamarix parviflora grown in pristine, nutrient-rich or legacy contaminated, nutrient-poor soil and how these communities change in response to increased soil salinity (a possible stress factor) and soil inoculation with allochthonous bacteria. Our results reveal significant differences in microbial community structure between horticultural and contaminated soils. When controlling for the effect of soil, microbial community structure was significantly affected by salinization, however inoculation with allochthonous bacteria did not significantly change the total community. The genera Arthrobacter, Bacillus, Burkholderia, Dyella and Ktedonobacter, among others, were significantly enriched in the contaminated root zone, whereas populations in the root zone of horticultural soil were found to be more diverse. In this type of soil, representatives of genera such as Flavobacterium, Ignavibacterium, Sediminibacterium, Terrimonas, Chitinophaga and Hydrotalea were significantly enriched. Regardless of soil type, members of genera Bradyrhizobium, Parachlamydia, Dongia, Vampirovibrio, Flavisolibacter and Rhodomicrobium were enriched in non-salinized soil, while the genus Acidothermus was enriched in salinized soil. Phospholipid-derived fatty acid (PLFA) analysis showed higher bacterial and fungal biomass in horticultural soil as compared to contaminated soil, demonstrating that soil nutrition is one of the most important factors with respect to microbial richness.

Petroleum hydrocarbons (PHCs) continue to be among the most common pollutants in soil worldwide. Phytoremediation has become a sustainable way of dealing with PHC contamination. We conducted the off-site phytoremediation of PHC-polluted soil from an oil tanker truck accident, where poplars were used for the phytoremediation of the oil-polluted soil in a boreal climate during a seven-year treatment. The succession of bacterial communities over the entire phytoremediation process was monitored using microbial ecological tools relying on high-throughput 16S rRNA gene sequencing. Upon the successful depletion of PHCs from soils, endophytic communities were analyzed in order to assess the complete plant-associated microbiome after the ecological recovery. The rhizosphere-associated soil exhibited different bacterial dynamics than unplanted soil, but both soils had a bacterial community succession through the years, with diversity being negatively correlated with PHC concentration. In the relatively short growing season in North Europe, seasonal variations in environmental conditions were identified that contributed to the dynamics of bacterial communities. Overall, our study proved that phytoremediation using poplar trees can be used to assist in the removal of PHCs from soils in boreal climate conditions and provides new insight into the succession patterns of bacterial communities associated with these plants.

In this work, the effect of In-Situ Chemical Oxidation (ISCO) using peroxydisulfate (PDS) on chloroethenes-degrading microbial consortium in the presence of perchloroethene (PCE; tetrachloroethene) was investigated. Degradation of PCE was examined using PDS without an activation, activated with iron Fe(II) chelated by citric acid (CA), and microbial consortium derived from chloroethenes-contaminated site in liquid and sand microcosms. Two different molar ratios of PCE/PDS/(Fe(II)+CA) (1/8/1.6 and 1/16/3.2) were tested. The PCE removal efficiency was the highest in the bacteria-free microcosms. An expected increase in the PCE removal efficiency by coupling PDS and microbial consortium was not confirmed. Surprisingly, the reduced capacity of PDS to remove PCE in the systems containing both PDS and microbial consortium was observed indicating that indigenous microbes may reduce the efficiency of PDS during a remediation. High-throughput 16S rRNA gene sequencing analysis revealed negative effect of PDS on organohalide-respiring bacteria (OHRB), which were not detected after 19 days of the experiment, unlike in biotic control. On the other hand, amplicon sequence variants (ASVs) affiliated with genera Brevundimonas and Pseudomonas that have been described for their capability of aerobic cometabolic/metabolic degradation of chloroethenes (CEs) were among the most frequently detected ASVs after the PDS treatment. Results further showed that the sole Fe(II)-CA affected the diversity of the microbial consortium. Overall, results of this study provide new insight into the coupling ISCO using PDS with in situ bioremediation of CEs.

Fertilization is a worldwide agricultural practice used in agronomy to increase crop yields. Fertilizer application influences overall soil characteristics, including soil microbial community composition and metabolic processes mediated by microbial enzymatic activity. Changes in the structure of microbial communities and their metabolic activity after long-term fertilization were studied in this research. We hypothesized that the different types of fertilization regimes affect nutrient levels in the soil which subsequently influence the metabolic processes and microbial diversity and community structure. Manure (MF, 330 kg N/ha), sewage sludge at two application doses (SF, 330 kg N/ha and SF3x, 990 kg N/ha) and chemical (NPK, N-P-K nutrients in concentrations of 330-90-300 kg/ha) fertilizers have been applied regularly to an experimental field since 1996. The microbial diversity increased in all soils amended with both organic (MF, SF, SF3x) and chemical (NPK) fertilizers. The shifts in microbial communities were observed, which were mainly caused by less abundant genera that were mostly associated with one or more fertilization treatment(s). Fertilization also influenced soil chemistry and the activity of &beta, xylosidase, &beta, N-acetylglucosaminidase (NAG), acid phosphatase and FDA-hydrolases. Specifically, all fertilization treatments were associated with a higher activity of &beta, xylosidase and lower NAG activity. Only the NPK treatment was associated with a higher activity of acid phosphatase.

An understanding of how fertilization influences endophytes is crucial for sustainable agriculture, since the manipulation of the plant microbiome could affect plant fitness and productivity. This study was focused on the response of microbial communities in the soil and tubers to the regular application of manure (MF, 330 kg N/ha), sewage sludge (SF, 330 and SF3x, 990 kg N/ha), and chemical fertilizer (NPK, 330-90-300 kg N-P-K/ha). Unfertilized soil was used as a control (CF), and the experiment was set up at two distinct sites. All fertilization treatments significantly altered the prokaryotic and fungal communities in soil, whereas the influence of fertilization on the community of endophytes differed for each site. At the site with cambisol, prokaryotic and fungal endophytes were significantly shifted by MF and SF3 treatments. At the site with chernozem, neither the prokaryotic nor fungal endophytic communities were significantly associated with fertilization treatments. Fertilization significantly increased the relative abundance of the plant-beneficial bacteria Stenotrophomonas, Sphingomonas and the arbuscular mycorrhizal fungi. In tubers, the relative abundance of Fusarium was lower in MF-treated soil compared to CF. Although fertilization treatments clearly influenced the soil and endophytic community structure, we did not find any indication of human pathogens being transmitted into tubers via organic fertilizers.

Pollution by heavy metals (HM) represents a serious threat for both the environment and human health. Due to their elemental character, HM cannot be chemically degraded, and their detoxification in the environment mostly resides either in stabilization in situ or in their removal from the matrix, e.g., soil. For this purpose, phytoremediation, i.e., the application of plants for the restoration of a polluted environment, has been proposed as a promising green alternative to traditional physical and chemical methods. Among the phytoremediation techniques, phytoextraction refers to the removal of HM from the matrix through their uptake by a plant. It possesses considerable advantages over traditional techniques, especially due to its cost effectiveness, potential treatment of multiple HM simultaneously, no need for the excavation of contaminated soil, good acceptance by the public, the possibility of follow-up processing of the biomass produced, etc. In this review, we focused on three basic HM phytoextraction strategies that differ in the type of plant species being employed: natural hyperaccumulators, fast-growing plant species with high-biomass production and, potentially, plants genetically engineered toward a phenotype that favors efficient HM uptake and boosted HM tolerance. Considerable knowledge on the applicability of plants for HM phytoextraction has been gathered to date from both lab-scale studies performed under controlled model conditions and field trials using real environmental conditions. Based on this knowledge, many specific applications of plants for the remediation of HM-polluted soils have been proposed. Such studies often also include suggestions for the further processing of HM-contaminated biomass, therefore providing an added economical value. Based on the examples presented here, we recommend that intensive research be performed on the selection of appropriate plant taxa for various sets of conditions, environmental risk assessment, the fate of HM-enriched biomass, economical aspects of the process, etc.

Chlorophenols (CPs), including pentachlorophenol (PCP), are chemicals of concern due to their toxicity and persistence. Here we describe a successful reactor-based remediation of CP-contaminated soil and assess changes in the toxicity patterns and bacterial communities during the remediation. The remediation consisted of separating half of the contaminated soil to be ground (samples M) in order to test whether the grinding expedited the remediation, the other half was left unground (samples P). Both soils were mixed with wastewater treatment sludge to increase their bacterial diversity and facilitate the degradation of CPs, and the resultant mixtures were placed in 2 bioreactors, M and P, operated for 16 months under anaerobic conditions to favor dehalogenation and for an additional 16 months under aerobic conditions to achieve complete mineralization. Samples were taken every 4 months for toxicity and microbial analyses. The results showed a 64% removal of total CPs (ΣCPs) in reactor P after just 18 months of remediation, whereas similar depletion in reactor M occurred after ∼25 months, indicating that the grinding decelerated the remediation. By the end of the experiment, both reactors achieved 93.5–95% removal. The toxicity tests showed a decrease in toxicity as the remediation progressed. The succession of bacterial communities over time was significantly associated with pH, anaerobic/aerobic phase and the concentration of the majority of CP congeners. Our data indicate that the supplementation of contaminated soil with sludge and further incubation in pilot-scale bioreactors under consecutive anaerobic-aerobic conditions proved to be effective at the remediation of CP-contaminated soil.

Many ecological experiments are based on the extraction and downstream analyses of microorganisms from different environmental samples. Due to its high throughput, cost-effectiveness and rapid performance, Matrix Assisted Laser Desorption/Ionization Mass Spectrometry with Time-of-Flight detector (MALDI-TOF MS), which has been proposed as a promising tool for bacterial identification and classification, could be advantageously used for dereplication of recurrent bacterial isolates. In this study, we compared whole-cell MALDI-TOF MS-based analyses of 49 bacterial cultures to two well established bacterial identification and classification methods based on complete 16S rRNA gene sequence analyses: a phylotype-based approach, grounded on closest type strain assignment, and a sequence similarity-based approach involving a 98.65% sequence similarity threshold, which has been found to best delineate bacterial species. Culture classification using reference-based MALDI-TOF MS was comparable to that yielded by phylotype assignment up to the genus level. At the species level, agreement between 16S rRNA gene analysis and MALDI-TOF MS was found to be limited, potentially indicating that spectral reference databases need to be improved. We also evaluated the mass spectral similarity technique for species-level delineation which can be used independently of reference databases. We established optimal mass spectral similarity thresholds which group MALDI-TOF mass spectra of common environmental isolates analogically to phylotype- and sequence similarity-based approaches. When using a mass spectrum similarity approach, we recommend a mass range of 4 to 10 kDa for analysis, which is populated with stable mass signals and contains the majority of phylotype-determining peaks. We show that a cosine similarity (CS) threshold of 0.79 differentiate mass spectra analogously to 98.65% species-level delineation sequence similarity threshold, with corresponding precision and recall values of 0.70 and 0.73, respectively. When matched to species-level phylotype assignment, an optimal CS threshold of 0.92 was calculated, with associated precision and recall values of 0.83 and 0.64, respectively. Overall, our research indicates that a similarity-based MALDI-TOF MS approach can be routinely used for efficient dereplication of isolates for downstream analyses, with minimal loss of unique organisms. In addition, MALDI-TOF MS analysis has further improvement potential unlike 16S rRNA gene analysis, whose methodological limits have reached a plateau.

Despite wastewater treatment, sewage sludge is often contaminated with multiple pollutants. Their impact on the phylogenetic composition and diversity of prokaryotic communities in sludge samples remains largely unknown. In this study, we analyzed the phylogenetic structure of bacterial communities and diversity in sludge from six waste water treatment plants (WWTPs) and linked this information with the pollutants identified in these samples: eight potentially toxic metals (PTMs) and four groups of organic pollutants [polychlorinated biphenyls (PCBs), polyromantic hydrocarbons (PAHs), brominated flame retardants (BFRs) and organochlorine pesticides (OCPs)]. Alpha diversity measures and the distribution of dominant phyla varied among the samples, with the community from the thermophilic anaerobic digestion (TAD)-stabilized sample from Prague being the least rich and the least diverse and containing on average 36% of 16S rRNA gene sequence reads of the thermotolerant genus Coprothermobacter of the class Clostridia (phylum Firmicutes). Using weighted UniFrac distance-based redundancy analysis (dbRDA), we found that a collection of 5 PTMs: Cr, Cu, Ni, Pb, Zn, and a pair of BFRs: hexabromocyclododecane (HBCD) and tribromodiphenyl ethers (triBDEs) were significantly associated with the bacterial community structure in mesophilic anaerobic digestion (MAD)-stabilized samples, whereas PCBs were observed to be marginally significant. Altogether, 85% of the variance in bacterial community structure could be ascribed to these pollutants. The data presented here contribute to a greater understanding of the ecological effects of combined pollution on the composition and diversity of bacterial communities, hence have the potential to aid in predicting ecosystem functions and/or disruptions associated with pollution.

Pesticides are a class of xenobiotics intentionally released into the environment. Hexachlorobenzene (HCB) was used as a fungicide from 1945, leaving behind many contaminated sites. Very few studies have examined the biodegradation of HCB or the fate of HCB-derived carbon. Here we report that certain bacterial populations are capable of deriving carbon from HCB in contaminated soil under aerobic conditions. These populations are primarily Proteobacteria, including Methylobacterium and Pseudomonas , which predominated as detected by stable isotope probing (SIP) and 16S rRNA gene amplicon pyrosequencing. Due to the nature of SIP, which can be used as a functional method solely for assimilatory processes, it is not possible to elucidate whether these populations metabolized directly HCB or intermediates of its metabolism produced by different populations. The possibility exists that HCB is degraded via the formation of pentachlorophenol (PCP), which is further mineralized. With this in mind, we designed primers to amplify PCP 4-monooxygenase-coding sequences based on the available pcpB gene sequence from Methylobacterium radiotolerans JCM 2831. Based on 16S rRNA gene analysis, organisms closely related to this strain were detected in 13 C-labeled DNA. Using the designed primers, we were able to amplify pcpB genes in both total community DNA and 13 C-DNA. This indicates that HCB might be transformed into PCP before it gets assimilated. In summary, this study is the first report on which bacterial populations benefit from carbon originating in the pesticide HCB in a contaminated soil.

Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provides researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation.

Despite decades of research there is limited understanding of how vegetation impacts the ability of microbial communities to process organic contaminants in soil. Using a combination of traditional and molecular assays, we examined how phytoremediation with willow and/or fertilization affected the microbial community present and active in the transformation of diesel contaminants. In a pot study, willow had a significant role in structuring the total bacterial community and resulted in significant decreases in diesel range organics (DRO). However, stable isotope probing (SIP) indicated that fertilizer drove the differences seen in community structure and function. Finally, analysis of the total variance in both pot and SIP experiments indicated an interactive effect between willow and fertilizer on the bacterial communities. This study clearly demonstrates that a willow native to Alaska accelerates DRO degradation, and together with fertilizer, increases aromatic degradation by shifting microbial community structure and the identity of active naphthalene degraders.

Plant-microbe interactions are of particular importance in polluted soils. This study sought to determine how selected plants (horseradish, black nightshade and tobacco) and NPK mineral fertilization shape the structure of soil microbial communities in legacy contaminated soil and the resultant impact of treatment on the soil microbial community functional potential. To explore these objectives, we combined shotgun metagenomics and 16S rRNA gene amplicon high throughput sequencing with data analysis approaches developed for RNA-seq. We observed that the presence of any of the selected plants rather than fertilization shaped the microbial community structure, and the microbial populations of the root zone of each plant significantly differed from one another and/or from the bulk soil, whereas the effect of the fertilizer proved to be insignificant. When we compared microbial diversity in root zones versus bulk soil, we observed an increase in the relative abundance of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria or Bacteroidetes, taxa which are commonly considered copiotrophic. Our results thus align with the theory that fast-growing, copiotrophic, microorganisms which are adapted to ephemeral carbon inputs are enriched in the vegetated soil. Microbial functional potential indicated that some genetic determinants associated with signal transduction mechanisms, defense mechanisms or amino acid transport and metabolism differed significantly among treatments. Genetic determinants of these categories tend to be overrepresented in copiotrophic organisms. The results of our study further elucidate plant-microbe relationships in a contaminated environment with possible implications for the phyto/rhizoremediation of contaminated areas.

Aerobic mineralization of PCBs, which are toxic and persistent organic pollutants, involves the upper (biphenyl, BP) and lower (benzoate, BZ) degradation pathways. The activity of different members of the soil microbial community in performing one or both pathways and their synergistic interactions during PCB biodegradation, are not well understood. This study investigates BP and BZ biodegradation and subsequent carbon flow through the microbial community in PCB-contaminated soil. DNA stable isotope probing (SIP) was used to identify the bacterial guilds involved in utilizing 13C-biphenyl (unchlorinated analogue of PCBs) and/or 13C-benzoate (product/intermediate of BP degradation and analogue of chlorobenzoates). By performing SIP with two substrates in parallel, we reveal microbes performing the upper (BP) and/or lower (BZ) degradation pathways and heterotrophic bacteria involved indirectly in processing carbon derived from these substrates (i.e. through crossfeeding). Substrate mineralization rates and shifts in relative abundance of labeled taxa suggest that BP and BZ biotransformations were performed by microorganisms with different growth strategies: BZ-associated bacteria were fast growing, potentially copiotrophic organisms, while microbes that transform BP were oligotrophic, slower growing, organisms. Our findings provide novel insight into the functional interactions of soil bacteria active in processing biphenyl and related aromatic compounds in soil, revealing how carbon flows through a bacterial community.

The aim of the study was to investigate how selected natural compounds (naringin, caffeic acid, and limonene) induce shifts in both bacterial community structure and degradative activity in long-term polychlorinated biphenyl (PCB)-contaminated soil and how these changes correlate with changes in chlorobiphenyl degradation capacity. In order to address this issue, we have integrated analytical methods of determining PCB degradation with pyrosequencing of 16S rRNA gene tag-encoded amplicons and DNA-stable isotope probing (SIP). Our model system was set in laboratory microcosms with PCB-contaminated soil, which was enriched for 8 weeks with the suspensions of flavonoid naringin, terpene limonene, and phenolic caffeic acid. Our results show that application of selected plant secondary metabolites resulted in bacterial community structure far different from the control one (no natural compound amendment). The community in soil treated with caffeic acid is almost solely represented by Proteobacteria, Acidobacteria, and Verrucomicrobia (together over 99 %). Treatment with naringin resulted in an enrichment of Firmicutes to the exclusion of Acidobacteria and Verrucomicrobia. SIP was applied in order to identify populations actively participating in 4-chlorobiphenyl catabolism. We observed that naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. and Pseudoxanthomonas spp. None of these populations were detected among 4-chlorobiphenyl utilizers in non-amended soil. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was lowest after treating the soil with naringin. Addition of caffeic acid resulted in comparable decrease of total PCBs with non-amended soil; however, higher substituted congeners were more degraded after caffeic acid treatment compared to all other treatments. Finally, it appears that plant secondary metabolites have a strong effect on the bacterial community structure, activity, and associated degradative ability.

During the second half of the last century a large amount of substances toxic for higher organisms was released to the environment. Physicochemical methods of pollutant removal are difficult and prohibitively expensive. Using biological systems such as microorganisms, plants, or consortia microorganisms–plants is easier, cheaper, and more environmentally friendly. The aim of this study was to isolate, characterize and identify microorganisms from contaminated soil and to find out the effect of plants on microbial diversity in the environment. Microorganisms were isolated by two approaches with the aim to find all cultivable species and those able to utilise biphenyl as a sole source of carbon and energy. The first approach was direct extraction and the second was isolation of bacteria after enrichment cultivation with biphenyl. Isolates were biochemically characterized by NEFERMtest 24 and then the composition of ribosomal proteins in bacterial cells was determined by MALDI-TOF mass spectrometry. Ribosomal proteins can be used as phylogenetic markers and thus MALDI-TOF MS can be exploited also for taxonomic identification because the constitution of ribosomal proteins in bacterial cells is specific for each bacterial species. Identification of microorganisms using this method is performed with the help of database Bruker Daltonics MALDI BioTyper. Isolated bacteria were analyzed from the point of the bphA gene presence. Bacteria with detected bphA gene were then taxonomically identified by 16S rRNA sequence. The ability of two different plant species, tobacco (Nicotiana tabacum) and nightshade (Solanum nigrum), to accumulate PCBs was studied as well. It was determined that various plant species differ in the PCBs accumulation from the contaminated soil. Also the content of PCBs in various plant tissues was compared. PCBs were detected in roots and aboveground biomass including leaves and berries.

Given that the degradation of aromatic pollutants in anaerobic environments such as sediment is generally very slow, aeration could be an efficient bioremediation option. Using stable isotope probing (SIP) coupled with pyrosequencing analysis of 16S rRNA genes, we identified naphthalene-utilizing populations in aerated polyaromatic hydrocarbon (PAH)-polluted sediment. The results showed that naphthalene was metabolized at both 10 and 20°C following oxygen delivery, with increased degradation at 20°C as compared to 10°C-a temperature more similar to that found in situ. Naphthalene-derived (13)C was primarily assimilated by pseudomonads. Additionally, Stenotrophomonas, Acidovorax, Comamonas, and other minor taxa were determined to incorporate (13)C throughout the measured time course. The majority of SIP-detected bacteria were also isolated in pure cultures, which facilitated more reliable identification of naphthalene-utilizing populations as well as proper differentiation between primary consumers and cross-feeders. The pseudomonads acquiring the majority of carbon were identified as Pseudomonas veronii and Pseudomonas gessardii. Stenotrophomonads and Acidovorax defluvii, however, were identified as cross-feeders unable to directly utilize naphthalene as a growth substrate. PAH degradation assays with the isolated bacteria revealed that all pseudomonads as well as Comamonas testosteroni degraded acenaphthene, fluorene, and phenanthrene in addition to naphthalene. Furthermore, P. veronii and C. testosteroni were capable of transforming anthracene, fluoranthene, and pyrene. Screening of isolates for naphthalene dioxygenase genes using a set of in-house designed primers for Gram-negative bacteria revealed the presence of such genes in pseudomonads and C. testosteroni. Overall, our results indicated an apparent dominance of pseudomonads in the sequestration of carbon from naphthalene and potential degradation of other PAHs upon aeration of the sediment at both 20 and 10°C.

Bacteria and bacterial communities in sites contaminated with polychlorinated biphenyls have been extensively studied in the past decades. However, there are still major gaps in the knowledge of environmental processes, especially in the behavior of previously described bacteria in vitro, their real degradation abilities and the enzymes that are involved in the degradation processes. In this work we analyzed actively degrading bacterial populations by stable isotope probing with (13)C biphenyl and (13)C-4-chlorobiphenyl as labeled substrates in the environment of sediment contaminated with polychlorinated biphenyls. We performed analysis of populations which degrade biphenyl and 4-chlorobiphenyl at concentrations similar to those of the original site. Several bacterial genera were revealed to actively participate in biphenyl and 4-chlorobiphenyl removal, some of which had not previously been described to take part in this process. We also found there are few differences in the communities metabolizing biphenyl and 4-chlorobiphenyl. Analysis of the genes responsible for substrate removal proved most of the genes to be closely related to Pseudomonas pseudoalcaligenes KF707 genes giving bacteria the ability of transforming di-para-chlorinated biphenyls.