Combined bioaugmentation and biostimulation techniques in bioremediation of pentachlorophenol contaminated forest soil.

Pentachlorophenol (PCP) is quite persistent in the environment and severely affects different ecosystems including forest soil. The main objective of this work was to study different bioremediation processes of artificially PCP (100 mg kg-1) contaminated forest soil (Sc). In fact, we used bioaugmentation by adding two different bacterial consortia B1 and B2, biostimulation procedures by amendments based on forest compost (FC), municipal solid waste compost (MC), sewage sludge (SS), and phosphate, and their combined treatments. Soil physical and chemical properties, residual PCP, soil microbial biomass carbon, soil respiration and some enzymatic activities at zero time and after 30 d of incubation, were evaluated. A net reduction of PCP, 71% of the initial concentration, after 30 d-incubation occurred in the sample Sc+B1+FC, as the best performance among all treatments, due to natural attenuation, immobilization of PCP molecules in the forest soil through organic amendments, and the action of the exogenous microbial consortium B1. The single application of FC or B1 led to a depletion of PCP concentration of 52% and 41%, respectively. Soil microbial biomass carbon decreased in PCP contaminated soil but it increased when organic amendment also in combination with microbial consortia was carried out as bioremediation action. Soil respiration underwent no changes in contaminated soil and increased under FC based bioremediation treatment. These results demonstrate that the combined treatments of biostimulation and bioaugmentation might be a promising process for remediation of PCP contaminated soil.

Removal of pentachlorophenol from contaminated wastewater using phytoremediation and bioaugmentation processes.

The phytoremediation procedure was conducted by Lemna gibba (L) and Typha angustifolia (T) and the bioaugmentation procedure used P. putida HM627618. The ability of the selected P. putida HM627618 to tolerate and remove PCP (200 mg L-1) was measured by high performance liquid chromatography analysis and optical density at 600 nm. Five different experiments were conducted in secondary treated wastewater for PCP testing removal (100 mg L-1) including two phytoremediation assays (T + PCP; L + PCP), three bioaugmentation-phytoremediation assays (T + B + PCP; L + B + PCP; L + T + B + PCP) and a negative control assay with PCP. Various analytical parameters were determined in this study such as bacterial count, chlorophylls a and b, COD, pH and PCP content. The main results showed that the average PCP removal by P. putida HM627618 was around 87.5% after 7 days of incubation, and 88% of PCP removal was achieved by treatment (T + B) after 9 days. During these experiments, pH, COD and chloride content showed a net increase in all treatments. The chlorophylls a and b in case of (T) and (L) Chlorophylls a and b for T and L phytoremediation showed a decrease with a value less than 10 µg/mg of fresh material after 20 days of cultivation.

Surfactant efficiency on pentachlorophenol-contaminated wastewater enhanced by Pseudomonas putida AJ 785569.

This study aims to evaluate the effect of three surfactants on the removal of PCP (800 mg L-1) from Secondary Treated Wastewater (STWW) by Pseudomonas putida AJ 785569. The effect of surfactants [sodium lauryl sulfate (SDS) as anionic, Tween 80 (TW80) as non-anionic and cetyltrimethylammonium bromide (CTAB) as cationic] is tested about the three following aspects: (1) bacterial growth, (2) bacterial biofilm formation or development and (3) PCP rate removal. The results showed that strain P. putida AJ 785569 could adsorb around 30 mg L-1 and remove 600 mg L-1 of PCP within 168 h of incubation. The SDS developed the growth of bacteria and the removal of PCP. This PCP removal in mineral salt medium (MSM) is around 760 mg L-1 (95% degradation) higher than the ones registered with CTAB and TW80 with a value 506.75 (63% degradation) and 364.1 mg L-1 (45% degradation), respectively. The obtained results of chloride concentration showed an important relation with PCP removal during incubation with an important value. Monitoring the development of bacterial biofilm, in MSM medium added with PCP (100 mg L-1) by strain P. putida AJ 785569, showed a significant increase in the optical density value from 0.9 to 4 at λ = 595 nm, a modification of strain P. putida AJ 785569's morphotype, density and color colonies.

Community-level genetic profiles of actinomycetales in long-term biowaste-amended soils.

Actinomycetales is an order of actinobacteria that have an important role in the decomposition of organic matter. Their abundance and distribution can reflect a good level of soil fertility as well as biological activity. In this research study, actinomycetal diversity in soil was investigated under various field treatments with biowastes. Initially, unvegetated agricultural soil plots of 4 m2 had been annually amended with increasing rates of municipal solid waste compost (MSWC at 40, 80 and 120 t ha-1 year-1) and farmyard manure (FM at 40 and 120 t ha-1 year-1) for eight consecutive years. Control consisted of unamended soil and all treatments were distributed in four randomized complete blocks. At the end of the experimental period, total DNA was extracted from fresh topsoil samples (0-20 cm) then nested PCR-DGGE sequencing method was applied to assess the long-term effect of treatments on the diversity of actinomycetes. Analytical outcomes revealed the presence of ten actinomycetal families with Streptomycetaceae, Pseudonocardiaceae and Nocardioidaceae being the most dominant regardless to changes in experimental conditions. Besides, the long-term accumulation of both biowastes in soil affected the diversity of actinomycetal communities in different ways including contribution, stimulation or inhibition. Interestingly, soil treated with MSWC at an equivalent rate of 40 t ha-1 year-1 was likely to provide optimal growth conditions for major identified genera because it showed the highest actinomycetal diversity as compared to the rest of the treatments.

Phosphorus and ammonium removal characteristics from aqueous solutions by a newly isolated plant growth-promoting bacterium.

An indigenous plant growth-promoting bacterium isolated from Peganum Harmala rhizosphere in the arid ecosystem was found to solubilize and accumulate phosphates. This isolate was identified as Pseudomonas sp. (PHR6) by partial 16S rRNA gene sequence analysis. Controlled batch experiments on nutrients removal by this isolate in mineral medium showed relatively high efficiencies after 24 h of aerobic incubation with average values of 117.59 and 335.38 mg gVSS-1 for phosphorus (P-PO4) and nitrogen (N-NH4), respectively. Furthermore, the strain performed heterotrophic nitrification ranging from 48.81% to 84.24% of the total removed nitrogen. On the other hand, the experimental results showed that a short idle period (24 h) significantly enhanced P accumulation (up to 95%) and N assimilation (up to 50%) of the total removed amounts. However, long idle period (20 days) revealed firstly aerobic phosphorous release phase succeeded by another removal one within 24 h of incubation. Overall, the idle treatment enhances P removal efficiency from the mineral liquid medium without significant effects on N-NH4 removal performance. The isolated strain showed also significant nutrient removal ability from synthetic wastewater providing an accumulated fraction of 98% from the total removed phosphorus amount. This study highlights the potential contribution of the selected rhizobacterium PHR6 to both environmental nutrient recycling and pollution control especially regarding phosphorus.

Characterization of Pseudomonas aeruginosa isolated from various environmental niches: New STs and occurrence of antibiotic susceptible "high-risk clones".

The aim of this study was to investigate the antimicrobial phenotypes, major virulence factors, and the molecular typing of 66 P. aeruginosa isolates collected from various sources: human patients and hospital environment, raw milk, poultry meat, chicken/sheep fecal samples, wastewater, thermal water, and seawater. All isolates, except one, were susceptible to all tested antibiotics. exoA, lasB, rhlR, and lasR genes were harbored by 60 isolates. Forty-six, 18, and 2 isolates amplified exoS, exoU, and exoS+exoU genes, respectively. Twenty-one isolates showed high elastase and pigment production. The PFGE typing identified 26 pulsotypes. Some pulsotypes included isolates from different environmental niches and areas. Twelve selected isolates were typed by MLST and eight different STs were found, three of them were new. Our results highlighted the dissemination of some clones amongst different settings and the occurrence of antibiotic susceptible 'high-risk clones' that might be very harmful when acquiring genes encoding antibiotic resistance.

UPLC-MS/MS analysis of antibiotics in pharmaceutical effluent in Tunisia: ecotoxicological impact and multi-resistant bacteria dissemination.

The UPLC MS/MS analysis showed the presence of the two antibiotics in the pharmaceutical industry discharges during 3 months; norfloxacin and spiramycin which were quantified with the mean concentrations of 226.7 and 84.2 ng mL-1, respectively. Sixteen resistant isolates were obtained from the pharmaceutical effluent and identified by sequencing. These isolates belong to different genera, namely Citrobacter, Acinetobacter, Pseudomonas, Delftia, Shewanella, and Rheinheimera. The antibiotic resistance phenotypes of these isolates were determined (27 tested antibiotics-discs). All the studied isolates were found resistant to amoxicillin and gentamicin, and 83.33% of isolates were resistant to ciprofloxacin. Multiple antibiotic resistances were revealed against ß-lactams, quinolones, and aminoglycosides families. Our overall results suggest that the obtained bacterial isolates may constitute potential candidates for bioremediation and can be useful for biotechnological applications. Genotoxic effects were assessed by a battery of biotests; the pharmaceutical wastewater was genotoxic according to the bacterial Vitotox test and micronuclei test. Genotoxicity was also evaluated by the comet test; the tail DNA damages reached 38 and 22% for concentrated sample (10×) and non-concentrated sample (1×), respectively. However, the histological sections of kidney and liver's mice treated by pharmaceutical effluent showed normal histology and no visible structural effects or alterations as cytolysis, edema, or ulcerative necrosis were observed. Residual antibiotics can reach water environment through wastewater and provoke dissemination of the antibiotics resistance and induce genotoxic effects.

Detection of Aichi virus genotype B in two lines of wastewater treatment processes.

Enteric viruses are released in important quantities into the environment where they can persist for a very long time. At very low doses, they can cause human gastroenteritis, and are responsible for a substantial number of waterborne diseases. The aims of this study were multiple: firstly, to study the circulation of Aichi viruses (AiV) in wastewater sampled at the scale of a pilot wastewater treatment plant; secondly, to evaluate the performance of two wastewater treatment procedures, as natural oxidizing lagoons and rotating Biodisks, concerning the AiV removal; and finally, to determine the different type of AiV genotype found during this study. Hence, the pilot wastewater treatment plant is principally irrigated by the wastewater of three neighbouring clinics. Wastewater samples were collected during 2011 from the two lines of biological treatment procedures. AiV detection in wastewater were achieved using the Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, and the identification of AiV genotype was realized by the direct sequencing of PCR products. The result revealed that AiV strains were identified in 50% (n = 51) of the wastewater samples. A significant increase of the AiV detection frequency was registered from upstream to downstream of the five ponds constituting the natural oxidizing lagoon process, and at the exit of the rotating Biodisks procedure. All detected AiV strains showed the highest nucleotide sequence identity to genotype B that has been recently observed in patients in Asia. This finding represented the first Tunisian survey that revealed and mentioned the first detection of AiV genotype B in sewage and by the same argued for a noticeable resistance or survival of this type of virus in the two lines of treatment considered.

Designation of pathogenic resistant bacteria in the Sparusaurata sea collected in Tunisia coastlines: Correlation with high performance liquid chromatography-tandem mass spectrometry analysis of antibiotics.

Vibrio is characterized by a large number of species and some of them are human pathogens causing gastro intestinal and wound infections through the ingestion or manipulation of contaminated fishes including Vibrio parahaemolyticus and Vibrio alginolyticus. In this study, we reported the phenotypic and molecular characterization of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from wild and farm sea bream (Sparus aurata L.) along the Tunisian coast from December 2015 to April 2016. Therefore, the antibiograms indicate a difference between farmed and wild fish. Resistance against amoxicillin antibiotic appears for the bacteria isolated from wild fish, while those from aquaculture farming presented sensitivity to amoxicillin and resistance to antibiotics colistin and fusidic acid. The chloramphenicol antibiotic exhibited a high sensitivity in all isolated bacteria. In fact, traces of amoxicillin in the organs of the fish from Hergla farm were detected by UPLC-MS/MS analysis during December 2016 to April 2016. In addition, antibiotics were detected in January 2014 with high concentration of norfloxacin 2262 ng/g in fish from Hergla coast. The results obtained in this work indicated that the use and presence of antibiotics in water impacts on the occurrence of resistant bacteria and the detection of antibiotic in fish.

Nitrifying-denitrifying filters and UV-C disinfection reactor: a combined system for wastewater treatment.

Biological treatment systems use the natural processes of ubiquitous organisms to remove pollutants and improve the water quality before discharge to the environment. In this paper, the nitrification/denitrification reactor allowed a reduction in organic load, but offered a weak efficiency in nitrate reduction. However, the additions of the activated sludge in the reactor improve this efficiency. A decrease of [Formula: see text] values from 13.3 to 8 mg/l was noted. Nevertheless, sludge inoculation led to a net increase of the number of pathogenic bacteria. For this reason, a UV-C pilot reactor was installed at the exit of the biological nitrification-denitrification device. Thus, a fluence of 50 mJ.cm-2 was sufficient to achieve values of 20 MPN/100 ml for fecal coliform and 6 MPN/100 ml for fecal streptococci, conforms to Tunisian Standards of Rejection. On the other hand, the DGGE approach has allowed a direct assessment of the bacterial community changes upon the treated wastewater.

Biofilms in bioremediation and wastewater treatment: characterization of bacterial community structure and diversity during seasons in municipal wastewater treatment process.

The bacterial community structure and diversity were assessed at the scale of rotating biodisk procedure (RB) in a semi-industrial pilot plant. As well, the Salmonella community was particularly monitored, and the effects of ultraviolet (UV-C254) on the bacterial community were studied. The identification of dominant bacteria revealed the presence of beneficial and useful species that could play an important role in the process of wastewater purification. Several species as Enterobacter agglomerans, Cronobacter sakazakii, and Pantoea agglomerans known for their bioremediation activities were revealed in the majority of biofilm samples. Common detection of Salmonella community provides evidence that the RB system did not seriously affect Salmonella. Furthermore, the investigation on the (UV)-C254 inactivation of the whole bacterial community, in secondary treated wastewater, showed variable UV resistance results. No Salmonella detection was registered at a dose of around 1440 mW s cm-2 since a total disappearance of Salmonella was recorded.

Isolation and characterization of antibiotic-resistant bacteria from pharmaceutical industrial wastewaters.

Contamination of surface waters in underdeveloped countries is a great concern. Treated and untreated wastewaters have been discharged into rivers and streams, leading to possible waterborne infection outbreaks which may represent a significant dissemination mechanism of antibiotic resistance genes among pathogenic bacterial populations. The present study aims to determine the multi-drug resistance patterns among isolated and identified bacterial strains in a pharmaceutical wastewater effluent in north Tunisia. Fourteen isolates were obtained and seven of them were identified. These isolates belong to different genera namely, Pseudomonas, Acinetobacter, Exiguobacterium, Delftia and Morganella. Susceptibility patterns of these isolates were studied toward commonly used antibiotics in Tunisia. All the identified isolates were found to have 100% susceptibility against colistin sulfate and 100% resistance against amoxicillin. Among the 11 antibiotics tested, six patterns of multi-drug resistance were obtained. The potential of the examined wastewater effluent in spreading multi-drug resistance and the associated public health implications are discussed.

Comparison of five molecular subtyping methods for differentiation of Salmonella Kentucky isolates in Tunisia.

Salmonellosis is one of the most common causes of food-borne infection worldwide. In the last decade, Salmonella enterica serovar Kentucky has shown an increase in different parts of the world with the concurrent emergence of multidrug-resistant isolates. These drug-resistant types spread from Africa and the Middle East to Europe and Asia. Although S. Kentucky serovar is of potential human relevance, there is currently no standardized fingerprinting method for it, in Tunisia. In the present study, a collection of 57 Salmonella Kentucky isolates were analyzed using plasmid profiling, pulsed-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC) fingerprinting, and Random Amplification of Polymorphic DNA. Plasmid profiling showed a discriminatory index (D) of 0.290, and only 9 out of 57 (16%) isolates carried plasmids, which represents a limitation of this technique. Fingerprinting of genomic DNA by PFGE and ribotyping produced 4 and 5 patterns, respectively. Distinct PFGE patterns (SX1, SX2, SX3, and SX4) were generated for only 28 strains out of 57 (49.1%) with a D value of 0.647. RAPD fingerprinting with primers RAPD1 and RAPD2 produced 4 and 20 patterns, respectively. ERIC fingerprinting revealed 14 different patterns with a high discriminatory index (D) of 0.903. When the methods were combined, the best combination of two methods was ERIC-2 with RAPD2. These results indicates that a single method cannot be relied upon for discriminating between S. Kentucky strains, and a combination of typing methods such ERIC2 and RAPD2 allows further discrimination.

Genetic and functional heterogeneities among fluorescent Pseudomonas isolated from environmental samples.

Fluorescent Pseudomonas from diverse environmental samples including wastes were identified and screened for the solubilization of tricalcium phosphate, indole-3-acetic acid (IAA), production and inhibition of extracellular N-acylhomoserine lactone (AHLs) and characterized for their siderophores. Genotypic analysis by amplified rDNA restriction analysis (ARDRA) and BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) typing resulted respectively in 14 ARDRA types and 24 different BOX-types with diverse incidence among the analyzed strains. Based on 16S rRNA sequence analysis the isolates were assigned to P. aeruginosa, P. otitidis, P. plecoglossicida, P. mosselii, P. monteilii, P. koreensis, P. taiwanenesis, P. frederiksbergensis and P. graminis. Of the 66 isolates, 56 (84.85%) isolates solubilized tri-calcium phosphate (TCP), 53 (80.30%) isolates produced plant growth hormone IAA, 62 (94%) produced bacteriocin and 34 (52%) isolates produced extracellular N-acylhomoserine lactone while 30 (45%) isolates were able to interfere with N-acylhomoserine lactone. Isolates were clustered into 17 siderotypes and (59)Fe cross-incorporation experiments permitted assignment of all siderotypes but two into well-defined siderovars.