Effect of arbuscular mycorrhizal fungi on plant biomass and the rhizosphere microbial community structure of mesquite grown in acidic lead/zinc mine tailings

Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse experiment was performed to determine the effects of three AMF inocula on biomass, shoot accumulation of heavy metals, and changes in the rhizosphere microbial community structure of the native plant Prosopis juliflora (mesquite). Plants were grown in an acidic lead/zinc mine tailings amended with 10% (w/w) compost amendment, which is slightly sub-optimal for plant growth in these tailings. After two months, AMF-inoculated plants showed increased dry biomass and root length (p < 0.05) and effective AMF colonization compared to controls grown in uninoculated compost-amended tailings. Mesquite shoot tissue lead and zinc concentrations did not exceed domestic animal toxicity limits regardless of whether AMF inoculation was used. The rhizosphere microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) profiles of the small subunit RNA gene for bacteria and fungi. Canonical correspondence analysis (CCA) of DGGE profiles showed that the rhizosphere fungal community structure at the end of the experiment was significantly different from the community structure in the tailings, compost, and AMF inocula prior to planting. Further, CCA showed that AMF inoculation significantly influenced the development of both the fungal and bacterial rhizosphere community structures after two months. The changes observed in the rhizosphere microbial community structure may be either a direct effect of the AMF inocula, caused by changes in plant physiology induced by AMF, or a combination of both mechanisms.     

Comparative effects of native filamentous and arbuscular mycorrhizal fungi in the establishment of an autochthonous, leguminous shrub growing in a metal-contaminated soil

The aim of this study was to assess the effectiveness of inoculation with a native arbuscular mycorrhizal (AM) fungus, Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, or a filamentous fungus, Penicillium aurantiogriseum Dierckx 1901, on the establishment of Coronilla juncea L. seedlings grown in a polluted, semiarid soil. For that, root and shoot biomass, nutrient uptake, mycorrhizal colonisation and nitrate reductase (NR) and phosphatase activities were analysed. Six months after planting, the shoot biomass of C. juncea was increased only by the inoculation with G. mosseae (by about 62% compared with non-mycorrhizal plants). The shoot NR and root acid phosphatase activities were increased more by inoculation with G. mosseae than with P. aurantiogriseum inoculation. The root NR activity and foliar nutrient contents were increased only by the inoculation with the AM fungus. The root Zn and Cu decreased with the AM fungus. In conclusion, the autochthonous AM fungus was an effective inoculant with regard to stimulating growth and alleviating heavy metal toxicity for plants growing on a soil contaminated by multiple heavy metals. Inoculation with an autochthonous, filamentous fungus does not seem to be a good strategy for phytoremediation of such problematic sites.     

Application of temporal temperature gradient gel electrophoresis for characterisation of fungal endophyte communities of Salix caprea L. in a heavy metal polluted soil

Fungal endophytes can affect the heavy metal uptake of their host plants and increase the tolerance of their host plants to heavy metal stress. Therefore, in the present study, a wide-range screening of the fungal endophyte communities was conducted to determine the fungal distribution and diversity on S. caprea roots on a metal polluted site. Fungal communities were screened using amplification with the 5.8S-ITS2-28S part of the rDNA operon, with the resulting amplicons analysed by temporal temperature gradient gel electrophoresis (TTGE) and sequencing. This technique is reproducible and shows good coverage of ascomycete and basidiomycete taxa, as 68% and 32% of all of the sequences, respectively. No clear shift in fungal ITS-TTGE profiles from S. caprea roots was seen along the secondary succession stages. Ascomycetes dominated the more polluted plots, while there was a greater diversity of basidiomycetes in the less polluted and control plots, suggesting greater tolerance of ascomycetes in comparison with basidiomycete fungi. The high diversity of DSEs was confirmed at the highly metal-enriched locations, with species belonging to the genera Phialophora, Phialocephala and Leptodontidium. Furthermore, the DSE colonisation of S. caprea roots and the frequency of the sequences showing affinity towards DSE genus Phialophora, showed good correspondence with soil Pb, Cd and plant-available P concentrations, possibly indicating that DSEs improve metal tolerance of willows to high heavy metal contamination.     

Enhanced removal of polychlorinated biphenyls from alfalfa rhizosphere soil in a field study: The impact of a rhizobial inoculum

Polychlorinated biphenyls (PCB) are persistent pollutants in soil environments where they continue to present considerable human health risks. Successful strategies to remediate contaminated soils are needed that are effective and of low cost. Bioremediation approaches that include the use of plants and microbial communities to promote degradation of PCB have significant potential but need further assessment under field conditions. The effects of growth of alfalfa (Medicago sativa L.) and inoculation with a symbiotic nitrogen fixing bacterium (Rhizobium meliloti) on the removal of polychlorinated biphenyls (PCB) from rhizosphere soil were evaluated in a field experiment. The initial PCB content of the soil ranged from 414 to 498 µg kg⁻¹. PCB removal for the rhizosphere soil was enhanced in the planted treatments, an average of 36% decrease in PCB levels compared to a 5.4% decrease in the unplanted soil, and further enhanced when plants were inoculated with the symbiotic Rhizobium (an average of 43% decrease) when evaluated at 90 days after planting. Plant biomass production was higher in the inoculated treatment. The total PCB content was increased from 3.30 µg kg⁻¹ to 26.72 µg kg⁻¹ in plant shoots, and from 115.07 µg kg⁻¹ to 142.23 µg kg⁻¹ in roots in the inoculated treatment compared to the planted treatment. Increased colony forming units (cfu) of total heterotrophic bacteria, biphenyl-degrading bacteria and fungi were observed in the rhizosphere of inoculated plants. PCB removal from the rhizosphere soil was not significantly correlated with the direct PCB uptake by the plants in any of the treatments but was significantly correlated with the stimulation of rhizosphere microflora. Changes in the soil microbial community structure in the planted and inoculated treatment were observed by profiling of bacterial ribosomal sequences. Some bacteria, such as Flavobacterium sp., may have contributed to the effective degradation of PCB and deserve further investigation.     

Bacterial community dynamics in horizontal flow constructed wetlands with different plants for high salinity industrial wastewater polishing

This study is focused on the diversity of bacterial communities from two series of horizontal subsurface flow constructed wetlands (CW) polishing high salinity tannery wastewater. Each series was planted with Arundo donax or Sarcocornia sp. in a substrate composed by expanded clay and sand. Chemical and biochemical oxygen demand removal efficiencies were similar in each series, varying between 58 and 67% (inlet COD 218 ± 28 mg L⁻¹) and 60 and 77% (inlet BOD₅ 37 ± 6 mg L⁻¹), respectively. High numbers of culturable bacteria were obtained from substrate and root samples - 5.75 × 10⁶-3.95 × 10⁸ CFU g⁻¹ recovered on marine agar and 1.72 × 10⁷-8.46 × 10⁸ CFU g⁻¹ on nutrient agar. Fifty bacterial isolates were retrieved from the CW, related phylogenetically to Firmicutes, Actinobacteria, Bacteroidetes, α-, β-, and γ-Proteobacteria. Changes in the bacterial communities, from roots and substrate of each series, related to the plant species, hydraulic loading rates and along CW operation were examined using denaturating gradient gel electrophoresis (DGGE). The clustering analysis suggested that a diverse and distinct bacterial community inhabits each series, which was related to the type of plant present in each CW.     

Phytoprotective influence of bacteria on growth and cadmium accumulation in the aquatic plant Lemna minor

Certain plants are known to accumulate heavy metals, and can be used in remediation of polluted soil or water. Plant-associated bacteria, especially those that are metal tolerant, may enhance the total amount of metal accumulated by the plant, but this process is still unclear. In this study, we investigated metal enhancement vs. exclusion by plants, and the phytoprotective role plant-associated bacteria might provide to plants exposed to heavy metal. We isolated cadmium-tolerant bacteria from the roots of the aquatic plant Lemna minor grown in heavy metal-polluted waters, and tested these isolates for tolerance to cadmium. The efficiency of plants to accumulate heavy metal from their surrounding environment was then tested by comparing L. minor plants grown with added metal tolerant bacteria to plants grown axenically to determine, whether bacteria associated with these plants increase metal accumulation in the plant.Unexpectedly, cadmium tolerance was not seen in all bacterial isolates that had been exposed to cadmium. Axenic plants accumulated slightly more cadmium than plants inoculated with bacterial isolates. Certain isolates promoted root growth, but overall, addition of bacterial strains did not enhance plant cadmium uptake, and in some cases, inhibited cadmium accumulation by plants. This suggests that bacteria serve a phytoprotective role in their relationship with Lemna minor, preventing toxic cadmium from entering plants.     

Antibiotic resistance in coagulase negative staphylococci isolated from wastewater and drinking water

This study reports the antibiotic resistance patterns of coagulase negative staphylococci (CNS) isolated from a drinking water treatment plant (WTP), a drinking water distribution network, responsible for supplying water to the consumers (WDN), and a wastewater treatment plant (WWTP), responsible for receiving and treating domestic residual effluents. Genotyping and the 16S rRNA gene sequence analysis demonstrated a higher diversity of species both in the WTP (6 species/19 isolates) and WWTP (12 species/47 isolates) than in the WDN (6 species/172 isolates). Staphylococcus pasteuri and Staphylococcus epidermidis prevailed in the WTP and WDN and Staphylococcus saprophyticus in the WWTP. Staphylococci with reduced susceptibility (resistance or intermediary phenotype) to beta-lactams, tetracycline, clindamycin and erythromycin were observed in all types of water and belonged to the three major species groups. The highest resistance rate was found against erythromycin, presumably due to the presence of the efflux pump encoded by the determinant msrA, detected in the majority of the resistant isolates. This study demonstrates that antibiotic resistant CNS may colonize different types of water, namely drinking water fulfilling all the quality standards.     

Improved tolerance of maize (Zea mays L.) to heavy metals by colonization of a dark septate endophyte (DSE) Exophiala pisciphila

Dark septate endophytes (DSE) are ubiquitous and abundant in stressful environments including heavy metal (HM) stress. However, our knowledge about the roles of DSE in improving HM tolerance of their host plants is poor. In this study, maize (Zea mays L.) was inoculated with a HM tolerant DSE strain Exophiala pisciphila H93 in lead (Pb), zinc (Zn), and cadmium (Cd) contaminated soils. E. pisciphila H93 successfully colonized and formed typical DSE structures in the inoculated maize roots. Colonization of E. pisciphila H93 alleviated the deleterious effects of excessive HM supplements and promoted the growth of maize (roots and shoots) under HM stress conditions, though it significantly decreased the biomass of inoculated maize under no HM stress. Further analysis showed that the colonization of E. pisciphila H93 improved the tolerance of maize to HM by restricting the translocation of HM ions from roots to shoots. This study demonstrated that under higher HM stress, such a mutual symbiosis between E. pisciphila and its host (maize) may be an efficient strategy to survive in the stressful environments.     

Impact of inocula and growth mode on the molecular microbial ecology of anaerobic ammonia oxidation (anammox) bioreactor communities

The composition of distinctly inoculated granular anammox and biofilm-based completely autotrophic nitrogen removal over nitrite (CANON) bioreactors was investigated from start-up through continuous long-term operation via denaturing gradient gel electrophoresis (DGGE) and sequencing. The granular anammox reactor was seeded with sludge from an operational anammox reactor in Strass, Austria. The CANON reactor was seeded with activated sludge from a local wastewater treatment plant in New York City. The principal anammox bacteria (AMX) shifted from members related to Kuenenia stuttgartiensis present in the initial inoculum to members related to Candidatus Brocadia fulgida during pre-enrichment (before this study) and to members related to Candidatus Brocadia sp. 40 (during this study) in the granular reactor. AMX related to C. Brocadia sp. 40 were also enriched from activated sludge in the CANON reactor. The estimated doubling times of AMX in the granular and CANON reactors were 5.3 and 8.9 days, respectively, which are lower than the value of 11 days, reported previously. Both the granular anammox and CANON reactors also fostered significant amounts of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). The fractions of AMX and two groups of NOB were generally similar in the granular anammox and CANON reactors. However, the diversity and fractions of AOB in the two reactors was markedly different. Therefore, it is suggested that the composition of the feed and extant substrate concentrations in the reactor likely select for the microbial community composition more than the inocula and reactor configuration. Further, such selection is not equivalent for all resident communities.     

Arbuscular mycorrhiza alters metal uptake and the physiological response of Coffea arabica seedlings to increasing Zn and Cu concentrations in soil

Studies on mycorrhizal symbiosis effects on metal accumulation and plant tolerance are not common in perennial crops under metal stress. The objective of this study was to evaluate the influence of mycorrhization on coffee seedlings under Cu and Zn stress. Copper (Cu) and zinc (Zn) uptake and some biochemical and physiological traits were studied in thirty-week old Coffea arabica seedlings, in response to the inoculation with arbuscular mycorrhizal fungi (AMF) and to increasing concentrations of Cu or Zn in soil. The experiments were conducted under greenhouse conditions in a 2 × 4 factorial design (inoculation or not with AMF and 0, 50, 150 and 450 mg kg^− 1 Cu or 0, 100, 300 and 900 mg kg^− 1 Zn). Non-mycorrhizal plants maintained a hampered and slow growth even in a soil with appropriate phosphorus (P) levels for this crop. As metal levels increased in soil, a greater proportion of the total absorbed metals were retained by roots. Foliar Cu concentrations increased only in non-mycorrhizal plants, reaching a maximum concentration of 30 mg kg^− 1 at the highest Cu in soil. Mycorrhization prevented the accumulation of Cu in leaves, and mycorrhizal plants showed higher Cu contents in stems, which indicated a differential Cu distribution in AMF-associated or non-associated plants. Zn distribution and concentrations in different plant organs followed a similar pattern independently of mycorrhization. In mycorrhizal plants, only the highest metal concentrations caused a reduction in biomass, leading to significant changes in some biochemical indicators, such as malondialdehyde, proline and amino acid contents in leaves and also in foliar free amino acid composition. Marked differences in these physiological traits were also found due to mycorrhization. In conclusion, AMF protected coffee seedlings against metal toxicity.     

Diversity and antibiotic resistance of Aeromonas spp. in drinking and waste water treatment plants

The taxonomic diversity and antibiotic resistance phenotypes of aeromonads were examined in samples from drinking and waste water treatment plants (surface, ground and disinfected water in a drinking water treatment plant, and raw and treated waste water) and tap water. Bacteria identification and intra-species variation were determined based on the analysis of the 16S rRNA, gyrB and cpn60 gene sequences. Resistance phenotypes were determined using the disc diffusion method.Aeromonas veronii prevailed in raw surface water, Aeromonas hydrophyla in ozonated water, and Aeromonas media and Aeromonas puntacta in waste water. No aeromonads were detected in ground water, after the chlorination tank or in tap water. Resistance to ceftazidime or meropenem was detected in isolates from the drinking water treatment plant and waste water isolates were intrinsically resistant to nalidixic acid. Most of the times, quinolone resistance was associated with the gyrA mutation in serine 83. The gene qnrS, but not the genes qnrA, B, C, D or qepA, was detected in both surface and waste water isolates. The gene aac(6’)-ib-cr was detected in different waste water strains isolated in the presence of ciprofloxacin. Both quinolone resistance genes were detected only in the species A. media. This is the first study tracking antimicrobial resistance in aeromonads in drinking, tap and waste water and the importance of these bacteria as vectors of resistance in aquatic environments is discussed.     

Addition of microbially-treated sugar beet residue and a native bacterium increases structural stability in heavy metal-contaminated Mediterranean soils

A mesocosm experiment was conducted to investigate the effect of the addition of Aspergillus niger-treated sugar beet waste, in the presence of rock phosphate, and inoculation with a native, metal-tolerant bacterium, Bacillus thuringiensis, on the stabilisation of soil aggregates of two mine tailings, with differing pH values, from a semiarid Mediterranean area and on the stimulation of growth of Piptatherum miliaceum. Bacterium combined with organic amendment enhanced structural stability (38% in acidic soil and 106% in neutral soil compared with their corresponding controls). Only the organic amendment increased pH, electrical conductivity, water-soluble C, water-soluble carbohydrates and plant growth, in both soils. While in neutral soil both organic amendment and bacterium increased dehydrogenase activity, only organic amendment had a significant effect in acidic soil. This study demonstrates that the use of P. miliaceum in combination with organic amendment and bacterium is a suitable tool for the stabilisation of the soil structure of degraded mine tailings, although its effectiveness is dependent on soil pH.     

Occurrence and diversity of Arcobacter spp. along the Llobregat River catchment, at sewage effluents and in a drinking water treatment plant

The presence of Arcobacter species in faecally contaminated environmental waters has previously been studied. However, the ability to eliminate Arcobacter during the water treatment processes that produce drinking water has been little studied. We have investigated the prevalence and diversity of Arcobacter spp. throughout the year at 12 sampling points in the Llobregat River catchment (Catalonia, Spain) including 3 sites at a drinking water treatment plant. Positive samples for Arcobacter spp., came predominantly from the most faecally polluted sites. Recovery rates from all sites were greater in the spring (91.7%) and summer (83.3%) than in autumn and winter (75.0% in both cases), but this trend was not statistically evaluated due to the limited number of samples. Among the 339 colonies analyzed, the most prevalent species by multiplex PCR and 16S rDNA restriction fragment length polymorphism were Arcobacter butzleri (80.2%), followed by Arcobacter cryaerophilus (19.4%) and Arcobacter skirrowii (0.3%). Isolates showed a high genotype diversity as determined by the enterobacterial repetitive intergenic consensus PCR. In fact, 91.2% (309/339) of the colonies had different genotypes, i.e. 248 of them among the 275 isolates of A. butzleri and 60 among the 63 isolates of A. cryaerophilus and 1 genotype of A. skirrowii. Arcobacter was never detected or isolated from finished drinking water, demonstrating that water treatment is effective in removing Arcobacter species.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

Large scale analysis of virulence genes in Escherichia coli strains isolated from Avalon Bay, CA

Contamination of recreational waters with Escherichia coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the β intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures.     

Improvement of growth of Eucalyptus globulus and soil biological parameters by amendment with sewage sludge and inoculation with arbuscular mycorrhizal and saprobe fungi

Sewage sludge is widely used as an organic soil amendment to improve soil fertility. We investigated the effects of sewage sludge (SS) application on certain biological parameters of Eucalyptus globulus Labill. The plant was either uninoculated or inoculated with saprobe fungi (Coriolopsis rigida and Trichoderma harzianum) or arbuscular mycorrhizal (AM) fungi (Glomus deserticola and Gigaspora rosea). Sewage sludge was applied to the surface of experimental plots at rates of 0, 2, 4, 6 and 8 g 100 g^− 1 of soil. Inoculation with both AM and saprobe fungi in the presence of SS was essential for the promotion of plant growth. The AM, saprobe fungi and SS significantly increased dry shoot weight. The AM fungi induced a significant increase in Fluorescein diacetate (FDA) activity but did not increase β-glucosidase activity. Addition of SS to AM-inoculated soil did not affect either FDA or α-glucosidase activities in plants from soil that was either uninoculated or inoculated with the saprobe fungi. SS increased β-glucosidase activity when it was applied at 4 g 100 g^− 1. SS negatively affected AM colonization as well as the mycelium SDH activity for both mycorrhizal fungi. SS increased Eucalyptus shoot biomass and enhanced its nutrient status. Inoculation of the soil with G. deserticola stimulated significant E. globulus growth and increases in shoot tissue content of N, P, K, Ca, Mg and Fe. Dual inoculation with G. deserticola and either of the saprobe fungi had positive effects on K, Ca, Mg and Fe contents. The application of 8 g 100 g^− 1 of SS had no positive effects on plant nutrition. The experimental setup provided a suitable tool for evaluating SS in combination with saprobe and AM fungi as a biological fertiliser for its beneficial effects on E. globulus plant growth.     

Multi-scale temporal and spatial variation in genotypic composition of Cladophora-borne Escherichia coli populations in Lake Michigan

High concentrations of Escherichia coli in mats of Cladophora in the Great Lakes have raised concern over the continued use of this bacterium as an indicator of microbial water quality. Determining the impacts of these environmentally abundant E. coli, however, necessitates a better understanding of their ecology. In this study, the population structure of 4285 Cladophora-borne E. coli isolates, obtained over multiple three day periods from Lake Michigan Cladophora mats in 2007-2009, was examined by using DNA fingerprint analyses. In contrast to previous studies that have been done using isolates from attached Cladophora obtained over large time scales and distances, the extensive sampling done here on free-floating mats over successive days at multiple sites provided a large dataset that allowed for a detailed examination of changes in population structure over a wide range of spatial and temporal scales. While Cladophora-borne E. coli populations were highly diverse and consisted of many unique isolates, multiple clonal groups were also present and accounted for approximately 33% of all isolates examined. Patterns in population structure were also evident. At the broadest scales, E. coli populations showed some temporal clustering when examined by year, but did not show good spatial distinction among sites. E. coli population structure also showed significant patterns at much finer temporal scales. Populations were distinct on an individual mat basis at a given site, and on individual days within a single mat. Results of these studies indicate that Cladophora-borne E. coli populations consist of a mixture of stable, and possibly naturalized, strains that persist during the life of the mat, and more unique, transient strains that can change over rapid time scales. It is clear that further study of microbial processes at fine spatial and temporal scales is needed, and that caution must be taken when interpolating short term microbial dynamics from results obtained from weekly or monthly samples.     

High diversity and differential persistence of fecal Bacteroidales population spiked into freshwater microcosm

Bacteroidales markers are promising indicators of fecal pollution and are now widely used in microbial source tracking (MST) studies. However, a thorough understanding of the persistence of Bacteroidales population after being released into environmental waters is lacking. We investigated the persistence of two host specific markers (HF183 and CF193) and temporal change of Bacteroidales population over 14 days in freshwater microcosms seeded with human or bovine feces. The concentrations of HF183/CF193 and Escherichia coli were determined using quantitative polymerase chain reaction (qPCR) and standard cultivation method, respectively. Shifts in the Bacteroidales population structure were fingerprinted using PCR-denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing analysis targeting both 16S rDNA and rRNA-transcribed cDNA. Both HF183 and CF193 decayed significantly faster than E. coli but the decay curves fit poorly with first-order model. High diversity of Bacteroidales population was observed for both microcosms, and persistence of different species in the population varied. Sequence analysis indicated that most of the bovine Bacteroidales populations in our study are unexplored. DGGE and decay curve indicated that RNA decayed faster than DNA, further supporting the use of rRNA as indicator of metabolically active Bacteroidales population. Evaluations with more realistic scenarios are warranted prior to extending the results of this study to real field settings.     

Production of Shiga-like toxins in viable but nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7, a causative agent of hemolytic uremic syndrome, can enter into a viable but nonculturable (VBNC) state when under stress. To date, it is unknown whether VBNC cells produce Shiga-like toxins (Stx). To address this question, we confirmed the expression of the stx1 and stx2 genes and the production of Stx in VBNC E. coli O157:H7 cells. To quantitatively assess the production of Stx in VBNC cells, we developed a Vero-cell microplate cytotoxicity assay based on the correspondence of the cytotoxicity of VBNC cells on Vero cells to the number of inoculated VBNC cells. Using this method, we found that all VBNC cells induced by river water, PBS buffer, deionized water, or chloraminated water retained the ability to produce Stx, and that they had differing levels of Stx. Both aged (19-month-old) VBNC cells induced by river water and fresh VBNC cells induced by chloraminated water showed very low half maximal inhibitory concentration (IC₅₀; 6.6 × 10⁴ and 7.1 × 10⁴ respectively), corresponding to higher levels of toxins produced than VBNC cells induced by deionized water and PBS buffer. VBNC cells originating from different isolates may vary in Stx production, and the VBNC cells from bovine isolates produced higher levels of Stx than those from clinical isolates. These results demonstrate a potential health risk of VBNC E. coli O157:H7 in environmental water and the importance of monitoring VBNC E. coli O157:H7.     

Halophyte filter beds for treatment of saline wastewater from aquaculture

The expansion of aquaculture and the recent development of more intensive land-based marine farms require efficient and cost-effective systems for treatment of highly nutrient-rich saline wastewater. Constructed wetlands with halophytic plants offer the potential for waste-stream treatment combined with production of valuable secondary plant crops. Pilot wetland filter beds, constructed in triplicate and planted with the saltmarsh plant Salicornia europaea, were evaluated over 88 days under commercial operating conditions on a marine fish and shrimp farm. Nitrogen waste was primarily in the form of dissolved inorganic nitrogen (TDIN) and was removed by 98.2 ± 2.2% under ambient loadings of 109-383 μmol l⁻¹. There was a linear relationship between TDIN uptake and loading over the range of inputs tested. At peak loadings of up to 8185 ± 590 μmol l⁻¹ (equivalent to 600 mmol N m⁻² d⁻¹), the filter beds removed between 30 and 58% (250 mmol N m⁻² d⁻¹) of influent TDIN. Influent dissolved inorganic phosphorus levels ranged from 34 to 90 μmol l⁻¹, with 36-89% reduction under routine operations. Dissolved organic nitrogen (DON) loadings were lower (11-144 μmol l⁻¹), and between 23 and 69% of influent DON was removed during routine operation, with no significant removal of DON under high TDIN loading. Over the 88-day study, cumulative nitrogen removal was 1.28 mol m⁻², of which 1.09 mol m⁻² was retained in plant tissue, with plant uptake ranging from 2.4 to 27.0 mmol N g⁻¹ dry weight d⁻¹. The results demonstrate the effectiveness of N and P removal from wastewater from land-based intensive marine aquaculture farms by constructed wetlands planted with S. europaea.