Second life of water and wastewater in the context of circular economy - Do the membrane bioreactor technology and storage reservoirs make the recycled water safe for further use?

In recent years water demand drastically increased which is particularly evident in tourism-burdened mountain regions. In these areas, climate neutral circular economy strategies to minimize human impact on the environment can be successfully applied. Among these strategies, treated wastewater reuse and retaining water in storage reservoirs deserve particular attention. This study aimed to determine if recycled water produced with two circular economy systems, namely membrane bioreactor treatment plant (MBR) with UV-light effluent disinfection and a storage reservoir, is safe enough for further use in green areas irrigation in summer and artificial snow production in winter. The assessment was based on the presence and concentration of antimicrobial agents, antibiotic resistant bacteria, antibiotic resistance genes, bacterial community composition and diversity. The treated water and wastewater was compared with natural water in their vicinity. Both systems fulfill the criteria set by the European Union in terms of reclaimed water suitable for reuse. Although the MBR/UV light wastewater treatment substantially reduced the numbers of E. coli and E. faecalis (from e.g. 32,000 CFU/100 ml to 20 CFU/100 ml and 15,000 CFU/100 ml to nearly 0 CFU/ml), bacteria resistant to ampicillin, aztreonam, cefepime, ceftazidime, ertapenem and tigecycline, as well as ESBL-positive and multidrug resistant E. coli were highly prevalent in MBR-treated wastewater (88.9 %, 55.6 %, 33.3 %, 22.2 % and 11.1 % and 44.4 and 55.6 %, respectively). Applying additional tertiary treatment technology is recommended. Retaining water in storage reservoirs nearly eliminated bacterial contaminants (e.g. E. coli dropped from 350 CFU/100 ml to 10 CFU/100 ml), antibiotic resistant bacteria, resistance genes (none detected in the storage reservoir) and antibiotics (only enrofloxacin detected once in the concentration of 3.20 ng/l). Findings of this study point to the limitations of solely culture-based assessment of reclaimed water and wastewater while they may prove useful in risk management and prevention in wastewater reuse.

Perhaps there were northern refugia in LGM? The phylogeographic structure of the thermophilic tree Carpinus betulus in the Carpathian region.

Carpinus betulus L., the hornbeam, is a component of lowland and highland forests in Europe. By examining the postglacial migratory history of thermophilic tree species, the study aimed to unravel their putative glacial microrefugia in the Carpathian region. The present study points to the two distinct genetic AFLP groups of C. betulus in the Carpathian region that represent different genetic lineages based on Bayesian analysis. They differed in Nei's gene diversity index h, and the analysis of molecular variance AMOVA showed a percentage variation of the populations between the groups of 13.74 %. Principal coordinate analysis (PCoA) of 368 AFLP tree samples confirmed the presence of two genetic groups. Ninety-five populations underwent principal component analysis (PCA) to show the main correlations between genetic diversity indices and bioclimatic/climate variables (WorldClim and Carpatclim). The generalized logistic model (GLM) showed the significance of Nei's genetic index h in delimiting genetic groups. The results of population-genetic and multivariate analyses determined that the two genetic groups nowadays are spatially diffused and do not show a clear geographic pattern, pointing to a genetic melting pot. We found ecological links between genetic diversity and bioclimatic characteristics, especially the precipitation in the coldest quarter - Bio19. The refugial Maxent model indicates a significant contribution of the Bio7 variable (both linked with a continental type of climate) to the occurrence of the species during the LGM in Europe. We suggest the relict character of hornbeam populations in a specific climatic-terrain niche in the northern part of the Carpathian Basin.

Climate, host and geography shape insect and fungal communities of trees.

Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries. Generalized dissimilarity models revealed similar relative importance of studied climatic, host-related and geographic factors on differences in tree-associated communities. Mean annual temperature, phylogenetic distance between hosts and geographic distance between locations were the major drivers of dissimilarities. The increasing importance of high temperatures on differences in studied communities indicate that climate change could affect tree-associated organisms directly and indirectly through host range shifts. Insect and fungal communities were more similar between closely related vs. distant hosts suggesting that host range shifts may facilitate the emergence of new pests. Moreover, dissimilarities among tree-associated communities increased with geographic distance indicating that human-mediated transport may serve as a pathway of the introductions of new pests. The results of this study highlight the need to limit the establishment of tree pests and increase the resilience of forest ecosystems to changes in climate.

Anthropogenic pollution gradient along a mountain river affects bacterial community composition and genera with potential pathogenic species.

Mountain regions in Poland are among the most frequently visited tourist destinations, causing a significant anthropogenic pressure put on the local rivers. In this study, based on numbers of 9 microorganisms, content of 17 antibiotics and 17 physicochemical parameters, we determined a pollution gradient in six sites along Bialka, a typical mountain river in southern Poland. The E.coli/Staphylococcus ratio varied evidently between polluted and non-polluted sites, indicating that the possible utility of this parameter in assessing the anthropogenic impact on river ecosystems is worth further investigation. Then, using next generation sequencing, we assessed the changes in bacterial community structure and diversity as a response to the pollution gradient. Proteobacteria and Bacteroidetes were the most abundant phyla in the majority of samples. Actinobacteria were the most abundant in the most pristine (groundwater) sample, while Firmicutes and Verrucomicrobia were more prevalent in polluted sites. Bacterial diversity at various levels increased with water pollution. Eleven bacterial genera potentially containing pathogenic species were detected in the examined samples, among which Acinetobacter, Rhodococcus, and Mycobacterium were the most frequent. At the species level, Acinetobacter johnsonii was most prevalent potential pathogen, detected in all surface water samples, including the pristine ones. Two bacterial taxa-genus Flectobacillus and order Clostridiales showed very distinct variation in the relative abundance between the polluted and non-polluted sites, indicating their possible potential as biomarkers of anthropogenic impact on mountain river waters.

Worldwide diversity of endophytic fungi and insects associated with dormant tree twigs.

International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees.

COVID-19 lockdown shows how much natural mountain regions are affected by heavy tourism.

Mountain areas in Poland are among the most frequented tourist destinations and such intensive tourism negatively affects the natural environment. The COVID-19 pandemic and the resulting lockdown restricted travel for a few months in 2020, providing a unique opportunity to observe the studied mountain environment without the impact of typical tourist traffic. This study is based on the determination of antibiotic content, hydrochemical parameters, enumeration of culturable bacterial water quality indicators, antimicrobial susceptibility tests together with extended spectrum beta-lactamase (ESBL) gene detection in waterborne E. coli and NGS-based bacterial community composition at six sites along the Bialka river valley (one of the most popular touristic regions in Poland) in three periods: in summer and winter tourist seasons and during the COVID-19 lockdown. The results of individual measurements showed decreased numbers of bacterial indicators of water contamination (e.g. numbers of E. coli dropped from 99 × 104 CFU/100 ml to 12 CFU/100 ml at the most contaminated site) and the share of antimicrobial resistant E. coli (total resistance dropped from 21% in summer to 9% during lockdown, share of multidrug resistant strains from 100 to 44%, and ESBL from 20% in summer to none during lockdown). Antibiotic concentrations were the highest during lockdown. The use of multivariate analysis (principal component analysis - PCA and heatmaps) revealed a clear pattern of tourism-related anthropogenic pressure on the water environment and positive impact of COVID-19 lockdown on water quality. PCA distinguished three major factors determining water quality: F1 shows strong effect of anthropogenic pressure; F2 describes the lockdown-related quality restoration processes; F3 is semi-natural and describes the differences between the most pristine and most anthropogenically-impacted waters.

Worldwide Genetic Structure Elucidates the Eurasian Origin and Invasion Pathways of Dothistroma septosporum, Causal Agent of Dothistroma Needle Blight.

Dothistroma septosporum, the primary causal agent of Dothistroma needle blight, is one of the most significant foliar pathogens of pine worldwide. Its wide host and environmental ranges have led to its global success as a pathogen and severe economic damage to pine forests in many regions. This comprehensive global population study elucidated the historical migration pathways of the pathogen to reveal the Eurasian origin of the fungus. When over 3800 isolates were examined, three major population clusters were revealed: North America, Western Europe, and Eastern Europe, with distinct subclusters in the highly diverse Eastern European cluster. Modeling of historical scenarios using approximate Bayesian computation revealed the North American cluster was derived from an ancestral population in Eurasia. The Northeastern European subcluster was shown to be ancestral to all other European clusters and subclusters. The Turkish subcluster diverged first, followed by the Central European subcluster, then the Western European cluster, which has subsequently spread to much of the Southern Hemisphere. All clusters and subclusters contained both mating-types of the fungus, indicating the potential for sexual reproduction, although asexual reproduction remained the primary mode of reproduction. The study strongly suggests the native range of D. septosporum to be in Eastern Europe (i.e., the Baltic and Western Russia) and Western Asia.

Heavy-metal pollution induces changes in the genetic composition and anatomical properties of photobionts in pioneer lichens colonising post-industrial habitats.

Certain lichens are effective colonisers of polluted sites. However, little is known about the tolerance of photobionts and the degree of mycobiont selectivity to photobionts relative to metal pollution. The present study recognises the genetic and anatomical diversity of Asterochloris photobionts in epigeic lichens, i.e. Cladonia cariosa, C. rei, and Diploschistes muscorum, in relation to a wide spectrum of soil pollution. In accordance with phylogenetic analysis, photobionts were clustered in 7 moderately- to well-supported clades, including 19 haplotypes. The mycobionts of all studied lichens demonstrated a low level of selectivity and were capable of associating with various Asterochloris lineages. This tendency was also expressed by the frequent (~25%) occurrence of multiple algal genotypes in a single thallus. This indicates that identified Asterochloris lineages are generally tolerant to heavy-metal pollution, and the low level of selectivity of mycobionts enables them to select the most suitable and/or available partner. The trend of increasing incidence of certain Asterochloris lineages and decreasing frequency of others along with increasing soil pollution was observed. This proves the superior adaptation of some photobionts to polluted sites. Such symbiotic plasticity constitute an adaptive feature necessary for the successful colonisation. High number of haplotypes at polluted sites could be the result of multiple introduction events from different areas during the initial stages of spontaneous succession. Regardless of the genetic pattern, Asterochloris cells were considerably smaller, and the density and compaction of cells in the algal layer were higher, in lichen specimens from polluted sites, indicating that photobiont characteristics may be closely dependent on heavy-metal pollution.

Lichen-forming fungi in postindustrial habitats involve alternative photobionts.

Mycobionts of many lichen genera appear to demonstrate strong selectivity in the choice of algal partner. The biological properties of a photobiont and its availability in an environment significantly determine the habitat requirements of lichens. Flexibility in photobiont choice extends the ecological amplitude of lichens; therefore, it may constitute an important adaptive strategy for colonization of extreme habitats. The photobiont inventory of the three epigeic lichens most resistant to soil pollution, i.e., Cladonia cariosa, C. rei, and the hyperaccumulator Diploschistes muscorum, was examined to verify whether and to what extent algal composition depends on the type of habitat and substrate enrichment with heavy metals. Photobionts Asterochloris and Trebouxia were identified in the studied lichen species; however, the presence of Trebouxia was directly related to anthropogenic sites with technogenic substrates, and the proportion of lichen specimens with these algae clearly depended on the level of heavy-metal soil pollution and the habitat type. The total number of algal haplotypes increased with increasing soil pollution, and the richness was associated more with soil pollution than with a given lichen species. Additionally, a large number of lichen individuals bearing multiple algal genotypes at polluted sites were recorded. Although Cladonia lichens were previously thought to be restricted to Asterochloris, they are able to start the relichenization process with Trebouxia under specific habitat conditions and to establish a stable association with these algae when colonization of disturbed sites takes place. Comparative analysis of the internal transcribed spacer (ITS) rDNA sequences revealed as many as 13 haplotypes of Trebouxia, and phylogenetic analysis grouped them into two different clades. Such a high level of genetic diversity indicates that Trebouxia is well adapted to metal pollution and could be an alternative photosynthetic partner for certain lichens, especially in polluted sites.

How much of antibiotics can enter surface water with treated wastewater and how it affects the resistance of waterborne bacteria: A case study of the Bialka river sewage treatment plant.

This study aimed to gain insight into the presence of antibiotics, occurrence of antimicrobial resistance and prevalence of extended-spectrum ß-lactamase (ESBL) genes in Escherichia coli in surface water, based on the example of the Bialka river, located in one of the most attractive tourist destinations in Poland. Water samples were collected in three sites: in the Tatra National Park (TNP), by the sewage discharge from the local treatment plant (STP) and c.a. 3 km downstream (DSTP). The analyses included determination of antibiotic content, enumeration of bacterial indicators of poor water quality, isolation and identification of Escherichia coli, which was subjected to antimicrobial susceptibility tests and assessment of ESBL-determining genes. Fourteen antimicrobials out of 24 tested were detected in river waters in varying concentrations. Trimethoprim and ofloxacin were most frequently detected. Most antibiotics were absent in the TNP, the highest numbers and the highest concentrations of antibiotics were observed by the STP discharge to decrease their content downstream. Culture-based tests of microbiological contamination showed similar results. Resistance to ampicillin was most frequent (64.5% strains), followed by cefazolin (50%). 20.6% of strains were ESBL-positive, while ESBL-determining gene, blaTEM was detected in 23.8% of E. coli strains. The largest percentage of antibiotic resistant and MDR E. coli strains was detected nearby the STP, indicating that malfunctioning STP may contribute largely to river water contamination downstream, also having significant environmental and economic impact.

The genetic relationships within Apiognomonia errabunda and related species.

Species of Apiognomonia are some of the most ubiquitous leaf-associated fungi of broad-leaved trees of the northern temperate zone. Especially widespread and diverse is Apiognomonia errabunda, found mostly on beech, oak, and linden. This species and its closest relatives are known for their complicated phylogenetic relationships that have caused considerable confusion in the past. In the present paper, we describe the results of a three-gene-based reconstruction of phylogenetic relationships between A. errabunda, A. veneta, and A. hystrix for 373 isolates. Using combined information from the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and intron regions of actin (ACT) and calmodulin (CAL) genes for 263 isolates, we confirmed the occurrence of host-associated lineages within A. errabunda. However, the clustering of isolates by host species was incomplete: some isolates occurred on the "wrong" hosts and a number of isolates carried mixed genetic profile, indicating substantial level of inter-host group recombination. In addition, a number of isolates were identified as putative, pending further verification, interspecies hybrids between the much more divergent A. errabunda and A. hystrix.

Modifications in the structure of the lichen Cladonia thallus in the aftermath of habitat contamination and implications for its heavy-metal accumulation capacity.

Phenotypic traits of lichens can be greatly modified by environmental factors. Granulose thalli on soil and podetia, densely covered with granules, referring to common and widespread lichen Cladonia cervicornis subsp. verticillata were found near zinc smelter. The granules are stratified, filled with fungal medulla and heavily encrusted with calcium oxalate weddellite crystals, not observed on regularly developed thalli of the species. Phylogenetic analysis revealed that deformed granulose forms belong to this taxon, showing that the phenotypic plasticity of the lichens of Cladonia can lead to the emergence of features that do not coincide with the taxonomic definition of the species. The heavy-metal accumulation capacity of both granulose and regular form of primary and secondary lichen thallus, in relation to the element content in corresponding substrate, was determined. Granulose-modified thalli accumulate greater amounts of heavy metals than regular ones, meaning that the bioaccumulation property of a given species may be greatly affected by morphological modifications. The granulose forms are also characterised by considerably higher ratios of Cd, Pb and As concentrations in lichen samples in relation to the corresponding substrates than regular ones. This means that collection of variously formed thalli should be avoided in biomonitoring sampling procedures. The results indicate that a substantial part of the element load, in particular zinc, in the examined lichen thalli collected near the smelter originates from atmospheric fallout.

Survival and Antibiotic Resistance of Bacteria in Artificial Snow Produced from Contaminated Water.

This study shows microbiological contamination of water in two main Podhale rivers, whose resources are used for the production of artificial snow, and the resulting snow contamination. Thirty-one E. coli strains were isolated from snow at two ski stations in the studied region, their antimicrobial resistance was determined, and the presence of extended spectrum ß-lactamase (ESBL) genes was searched for. The results indicate that the waters of both rivers are severely contaminated, resulting in the contamination of artificial snow with, among others, thermotolerant E. coli. E. coli isolated from snow were most frequently resistant to ampicillin (74.19%) and amoxicillin/clavulanate (51.61% isolates). Aminoglycosides and third generation cephalosporins were most efficient among the tested antimicrobials. Some bacterial strains were multidrug resistant and three strains exhibited the ESBL mechanism. Molecular analyses showed the presence of ESBL genes in the same three strains. Genetic variation among E. coli indicates that only some genotypes are able to survive the artificial snow production process.

The Yeast Fungus Trichosporon lactis Found as an Epizoic Colonizer of Dung Beetle Exoskeletons.

The study on the biology and biodiversity of coprophagous Scarabaeoidea carried out in the Polish Carpathians revealed the occurrence of unusual epizoic excrescences on various dung beetles species of the genus Onthophagus. The excrescences occur on the elytra, prothorax, and head of the studied beetles. Detailed research on this phenomenon determined that the fungus grew in the form of multicellular thalli. The ITS-based identification of fungal material collected from beetles' exoskeletons resulted in a 100 % match with Trichosporon lactis. Until now, only a yeast lifestyle/stage was known for this basidiomycete species. Therefore, in this paper, we describe a new substrate for growth of T. lactis and its unknown and intriguing relationship with dung beetles. The results obtained in this study open up numerous research possibilities on the new role of dung beetles in terrestrial ecosystems, as well as on using the physiological properties of T. lactis to restore soils.

The molecular marker-based comparison of Azotobacter spp. populations isolated from industrial soils of Cracow-Nowa Huta steelworks (southern Poland) and the adjacent agricultural soils.

The occurrence of Azotobacter spp., which has beneficial effects on plant development, is related to various soil properties, such as pH and fertility. This study evaluated the prevalence of Azotobacter spp. in industrial (H) and agricultural soils (P) in Nowa Huta, Cracow and determined the phenotypic and genetic diversity of these bacteria. The examined bacteria were present in 40% of H and in 50% of P soils. Taxonomic identification of the bacterial isolates indicated the presence of three species--A. salinestris, A. chroococcum and A. vinelandii. The genetic diversity, determined using two fingerprinting methods--Random Analysis of Polymorphic DNA (RAPD) and Rep-PCR (BOX) revealed high level of population diversity. In AMOVA analysis most of diversity was attributed to within-population variation (76-85%), and only 3.78-6.18% was associated with among-group H and P variation. Global test of differences revealed distinct population structure within bacterial strains isolated from H and P areas only for BOX markers (Fst = 0.05732, P = 0.00275). Phenetic analyses: UPGMA and DCA better discriminated H and P groups based on RAPD data. Both BOX and RAPD methods provided an insight into the genetic complexity of Azotobacter spp. variation in soils of different land-use types.