Abundance, Diversity, and Dynamics of Viruses on Microorganisms in Activated Sludge Processes

We examined the abundance of viruses on microorganisms in activated sludge and the dynamics of their community structure. Direct counting with epifluorescence microscopy and pulsed-field gel electrophoresis (PFGE) were applied to 20 samples from 14 full-scale wastewater treatment plants (wwtps) treating municipal, industrial, or animal wastewater. Furthermore, to observe the dynamics of viral community structure over time, a laboratory-scale sequencing batch reactor was operated for 58 days. The concentrations of virus particles in the wwtps, as quantified by epifluorescence microscopy, ranged from 4.2 × 10⁷ to 3.0 × 10⁹ mL⁻¹. PFGE, improved by the introduction of a higher concentration of Tris–EDTA buffer in the DNA extraction step, was successfully used to profile DNA viruses in the activated sludge. Most of the samples from different wwtps commonly had bands in the 40–70 kb range. In the monitoring of viral DNA size distribution in the laboratory-scale reactor, some bands were observed stably throughout the experimental period, some emerged during the operation, and others disappeared. Rapid emergence and disappearance of two intense bands within 6 days was observed. Our data suggest that viruses—especially those associated with microorganisms—are abundant and show dynamic behavior in activated sludge.     

The critical flux method for reduced filter membrane fouling when monitoring high‐solids digesters

Membrane fouling currently makes filtration of high‐solids anaerobic sludges difficult and this is discouraging online monitoring of volatile fatty acids and control of high‐solids digesters. The present study tests the critical flux approach to reduce membrane fouling. Filtration tests are performed on two sludges, filtered via a side‐stream off two full‐scale digesters. Sub‐critical flux operating conditions (for minimal cake layer formation) are identified for each of the sludges and the filtration units are operated at these conditions to assess longer term performance. Results for one of the sludges (co‐digested primary and secondary sludge) is found to be encouraging, showing that sufficient flux rates (up to 40 L m^?2 h^?1) can be readily sustained to allow longer term digester monitoring and control. Filtration performance for this sludge did not deteriorate significantly over the test period. Results for the other test sludge (digested thermally hydrolyzed waste activated sludge) were not as favorable and indicated that application may be limited for very high solids digesters (>5% total solids concentration). Differences in filtration behavior for the two test sludges were ascribed to the presence of complex soluble organics, the concentration of sludge solids and their particle size. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1059–1063, 2013     

Enhancement of the activated sludge process by activated carbon produced from surplus biological sludge

Surplus biological sludge from wastewater treatment operations was converted into activated carbon and then added to the aerated vessel of an activated sludge process treating phenol and glucose. The addition of activated carbon, either sludge-based or commercial, enhanced phenol removal from 58 to 98.7% and from 87 to 93% for COD with feed concentrations of 100 mg phenol l^–1 and 2500 mg COD l^–1. No differences were found between the activated sludge-activated carbon bench scale continuous reactors operating with either commercial or sludge-based activated carbon in spite of the higher adsorption capacity of the former.     

Efficiency evaluation of activated sludge treatments of wastewater from fiber board manufacturing

Summary Wastewater from fiber board manufacture consisting in a mixture of Pinus radiata, Eucaliptus globulus and Laureliopsis phillipiana (tepa) (3:1:1) has been studied in laboratory scale activated sludge reactors with organic load rate range of 50–1700 gCOD/m³.d. A stable operation at high organic load rate with hydraulic retention time of one day was achieved. Purification efficiencies up to 90 % of COD removal could be achieved in an activated sludge treatment of fiber board wastewater working with 1 day HRT for wood log cooking wastewater and with 4 days HRT when glueing wastewater is added to the cooking wastewater treatment. Suspended solids, color and phenol concentration were negligible in the efluent of the activated sludge system.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

Comparison of bacteriophage and enteric virus removal in pilot scale activated sludge plants

AIMS: The aim of this experimental study was to determine comparatively the removal of two types of bacteriophages, a somatic coliphage and an F-specific RNA phage and of three types of enteric viruses, hepatitis A virus (HAV), poliovirus and rotavirus during sewage treatment by activated sludge using laboratory pilot plants. METHODS AND RESULTS: The cultivable simian rotavirus SA11, the HAV HM 175/18f cytopathic strain and poliovirus were quantified by cell culture. The bacteriophages were quantified by plaque formation on the host bacterium in agar medium. In each experiment, two pilots simulating full-scale activated sludge plants were inoculated with viruses at known concentrations, and mixed liquor and effluent samples were analysed regularly. In the mixed liquor, liquid and solid fractions were analysed separately. The viral behaviour in both the liquid and solid phases was similar between pilots of each experiment. Viral concentrations decreased rapidly following viral injection in the pilots. Ten minutes after the injections, viral concentrations in the liquid phase had decreased from 1.0 +/- 0.4 log to 2.2 +/- 0.3 log. Poliovirus and HAV were predominantly adsorbed on the solid matters of the mixed liquor while rotavirus was not detectable in the solid phase. In our model, the estimated mean log viral reductions after 3-day experiment were 9.2 +/- 0.4 for rotavirus, 6.6 +/- 2.4 for poliovirus, 5.9 +/- 3.5 for HAV, 3.2 +/- 1.2 for MS2 and 2.3 +/- 0.5 for PhiX174. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that the pilots are useful models to assess the removal of infectious enteric viruses and bacteriophages by activated sludge treatment. Our results show the efficacy of the activated sludge treatment on the five viruses and suggest that coliphages could be an acceptable indicator of viral removal in this treatment system.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

Test of Porous Ceramic Material for the Immobilisation of Predominant Nitrifying Bacteria and for the Improvement of the AAO Process

The efficiency of nitrification in a fixed bed reactor was compared to that found in an activated sludge reactor to determine their sensitivity to changing loads and lower temperatures. Two structurally identical lab‐scale systems, using the anaerobic/anoxic/aerobic (AAO) process to remove nitrogen and phosphorus simultaneously, were operated in parallel with secondary clarifiers and sludge return. The first aerobic system was operated as an activated sludge reactor, the second system as a fixed bed reactor. The aerobic fixed bed reactor was filled with porous ceramic materials for the immobilisation of predominantly nitrifying bacteria. The removal efficiencies of 99 % NH₄⁺‐N, 90 % DOC, and 98 % PO₄^3–‐P for normal loads of 0.11 g/L d DOC, 0.06 g/L d NH₄⁺‐N, and 0.0054 g/L d PO₄^3–‐P were achieved for both systems. However, the system with an aerobic fixed bed reactor was characterised by the following advantages over the system with an activated sludge reactor: a shorter time to reach almost complete nitrification, a higher nitrification rate at higher loads of NH₄⁺‐N and a lower sensitivity of nitrification at lower temperatures down to 12 °C.     

Luminescent and hydrophilic LaF3–polymer nanocomposite for DNA detection

Luminescent LaF₃–Ce³⁺/Tb³⁺ nanocrystals have been successfully prepared via a simple wet chemical technique. For the next bioapplication, these nanoparticles dispersed in cyclohexane have also been functionalized with poly(St‐co‐MAA), based on a designed oil‐in‐water microemulsion system. These polymer‐coated nanospheres are water‐soluble and bioconjugable. Unlike semiconductor quantum dots, the as‐prepared lanthanum fluoride nanocrystals possess non‐size‐dependent emissions and completely stable photocycles. With functionalized LaF₃ nanospheres as fluorescence probes, a fluorescence method was developed for the rapid quantitative analysis of DNA, due to the quenching effect of fluorescence by the DNA. Under optimum conditions, the fluorescence intensity was proportional to the concentration of the introduced DNA over the range 2.5–35 µg/mL for calf thymus DNA (ctDNA) and 2.5–30 µg/mL for fish sperm DNA (fsDNA), respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

Rapid construction and characterization of synthetic antibody libraries without DNA amplification

We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V_H) and light (V_κ) libraries. Four high quality, chemically synthesized polynucleotides (90–140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10⁹ transformants could be synthesized within 1 day. Fusion to β‐lactamase and selection on ampicillin resulted in 3.7 × 10⁸ V_H and 6.9 × 10⁸ V_κ clones highly enriched for full‐length, in‐frame genes. High‐throughput 454 DNA sequencing of >250,000 V_H and V_κ genes from the pre‐ and post‐selection libraries revealed that, in addition to the expected reduction in reading‐frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V_H/V_κ‐β‐lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions. Biotechnol. Bioeng. 2010; 106: 347–357. © 2010 Wiley Periodicals, Inc.     

Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods

Aims: The study describes the development of simple and rapid DNA extraction method in combination with loop‐mediated isothermal amplification (LAMP) to detect enterotoxigenic Staphylococcus aureus in food samples. Methods and Results: In this study, isolation of genomic DNA of enterotoxigenic Staph. aureus from spiked milk, milk burfi, khoa, sugarcane juice and boiled rice was carried out by boiling the isolated sample pellets for 10 min with 1% Triton X‐100. The isolated DNA was evaluated by polymerase chain reaction (PCR) and LAMP method. The LAMP was found to be 100 times more sensitive than PCR. The LAMP assay was very specific for Staph. aureus, and the presence of other contaminating bacterial DNAs and food matrix did not interfere or inhibit the LAMP assay. Conclusions: The template DNA extraction method developed in this study for food samples is simple, rapid and cost‐effective. LAMP was found to be less sensitive to matrix effect of food, compared to PCR. Significance and Impact of the Study: The method is suitable for direct detection of Staph. aureus without any enrichment in contaminated food samples and hence finds its application in food safety analysis, in permutation with LAMP.     

Occurrence and reduction of human viruses,F‐specific RNA coliphage genogroups and microbial indicators at a full‐scale wastewater treatment plant in Japan

Aims

To evaluate and compare the reductions of human viruses and F‐specific coliphages in a full‐scale wastewater treatment plant based on the quantitative PCR (qPCR) and plate count assays.

Methods and Results

A total of 24 water samples were collected from four locations at the plant, and the relative abundance of human viruses and F‐RNA phage genogroups were determined by qPCR. Of the 10 types of viruses tested, enteric adenoviruses were the most prevalent in both influent and effluent wastewater samples. Of the different treatment steps, the activated sludge process was most effective in reducing the microbial loads. Viruses and F‐RNA phages showed variable reduction; among them, GI and GIII F‐RNA phages showed the lowest and the highest reduction, respectively.

Conclusions

Ten types of viruses were present in wastewater that is discharged into public water bodies after treatment. The variability in reduction for the different virus types demonstrates that selection of adequate viral indicators is important for evaluating the efficacy of wastewater treatment and ensuring the water safety.

Significance and Impact of the Study

Our comprehensive analyses of the occurrence and reduction of viruses and indicators can contribute to the future establishment of appropriate viral indicators to evaluate the efficacy of wastewater treatment.     

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

Aims: To develop a quick and accurate PCR‐based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. Methods and Results: The number of BbrY in faeces was detected by using strain‐specific quantitative real‐time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA‐intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10⁵–10⁹ cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (±1·5) log CFU g^?1 [mean (±SD)] of BbrY was detected in faeces by using strain‐specific transgalactosylated oligosaccharide–carbenicillin (T‐CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (±1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (±0·8) log cells per g. Conclusions: Strain‐specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Significance and Impact of the Study: Combination of strain‐specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.     

Estimating the Growth Rate of a Bacterial Species in a Complex Mixture by Hybridization of Genomic DNA

Abstract Advances in molecular techniques have enabled new approaches to identifying bacteria. However, once identified, there is no quantitative information on the in situ growth rate of the species, mainly because the technology has not been available. The quantitative incorporation of [methyl-³H]thymidine into dividing bacteria is coupled with a molecular (hybridization) method, to determine the growth rate of bacterial species in situ. The basis of this molecular method is a reverse gene probe—natural populations are labeled in situ with [methyl-³H]thymidine. The probe (³H-Tdr-DNA) is captured, using a hybridization procedure, on a positively charged nylon membrane on which is attached non-labeled target DNA. Two bacterial species, Bacillus cereus and Zoogloea ramigera, were used to demonstrate the principle in laboratory cultures and in a municipal activate sludge treatment process. The DNA of the dividing bacteria in activated sludge was radioactively labeled with [methyl-³H]thymidine, and the DNA of Z. ramigera was recovered using a DNA hybridization method. The recovered radioactively-labeled DNA was used to estimate the growth rate (0.03 × 10⁹ cells · ml⁻¹· h⁻¹) of Z. ramigera in situ. Simultaneously applying these two powerful molecular-based methods could potentially be used to study bacterial population dynamics in situ. Received: 10 September 1997; Accepted: 5 January 1998     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.     

A protocol for high-throughput extraction of DNA from rice leaves

Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less than 6 mo of storage at 4°C.     

Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants

To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full‐scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real‐time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year.     

The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2‐week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction.