Bioaugmentation of Thauera mechernichensis TL1 for enhanced polyhydroxyalkanoate production in mixed microbial consortia for wastewater treatment.

Polyhydroxyalkanoate (PHA) is a fully biodegradable bioplastic. To foster a circular economy, the integration of PHA production into wastewater treatment facilities can be accomplished using mixed microbial consortia. The effectiveness of this approach relies greatly on the enrichment of PHA-accumulating microorganisms. Hence, our study focused on bioaugmenting Thauera mechernichensis TL1 into mixed microbial consortia with the aim of enriching PHA-accumulating microorganisms and enhancing PHA production. Three sequencing batch reactors-SBRctrl, SBR2.5%, and SBR25%-were operated under feast/famine conditions. SBR2.5% and SBR25% were bioaugmented with T. mechernichensis TL1 at 2.5%w/w of mixed liquor volatile suspended solids (MLVSS) and 25%w/w MLVSS, respectively, while SBRctrl was not bioaugmented. SBR2.5% and SBR25% achieved maximum PHA accumulation capacities of 56.3 %gPHA/g mixed liquor suspended solids (MLSS) and 50.2 %gPHA/gMLSS, respectively, which were higher than the 25.4 %gPHA/gMLSS achieved by SBRctrl. The results of quantitative polymerase chain reaction targeting the 16S rRNA gene specific to T. mechernichensis showed higher abundances of T. mechernichensis in SBR2.5% and SBR25% compared with SBRctrl in the 3rd, 17th, and 31st cycles. Fluorescence in situ hybridization, together with fluorescent staining of PHA with Nile blue A, confirmed PHA accumulation in Thauera spp. The study demonstrated that bioaugmentation of T. mechernichensis TL1 at 2.5%w/w MLVSS is an effective strategy to enhance PHA accumulation and facilitate the enrichment of PHA-accumulating microorganisms in mixed microbial consortia. The findings could contribute to the advancement of PHA production from wastewater, enabling the transformation of wastewater treatment plants into water and resource recovery facilities.

Dietborne Toxicity of Tebuconazole to a Benthic Crustacean, Heterocypris incongruens and Its Relative Contribution to the Overall Effects under Food-Water Equilibrium Partitioning.

Tebuconazole (TEB), a widely used and persistent pesticide, has garnered attention due to its frequent detection in sediments worldwide. This widespread occurrence has raised concerns about potential dietborne toxicity to benthic crustaceans, as they may ingest contaminated particles in their habitat. While bioaccumulation studies indicate the importance of TEB ingestion for benthic crustaceans, limited data exist on direct dietborne toxicity testing. This study investigated the diet-related toxicity of TEB by subjecting a benthic ostracod, Heterocypris incongruens, to a 6 day toxicity test under dietary and combined exposures. Subsequently, the importance of dietary exposure for TEB toxicity was uncovered, followed by quantification of relative dietborne toxicity contributions using a modified concentration-additive model. Results revealed that the dietary route was more toxicologically significant than the aqueous route in equilibrium. The dietborne lethal concentration (LC50) for TEB on H. incongruens was 200 (170-250) mg/kg, with an 80% relative dietborne toxicity contribution. To gain comprehensive insights into dietborne significance, toxicity data were collected from previous studies involving different pollutants to calculate relative contributions. Finally, the correlation between dietborne toxicity and the partitioning coefficient was analyzed to understand the pollutant behavior and its toxic impact when ingested through the diet.

Selection of formulated sediment and feeding condition for 10-day spiked-sediment toxicity test with estuarine amphipod Grandidierella japonica.

This study was performed to select formulated sediment with appropriate feeding conditions for the evaluation of estuarine sediment toxicity. Grandidierella japonica, which inhabits seawater worldwide, was examined for 10-day survival, and the repeatability was evaluated under nine test conditions. The formulated sediment feeding condition combined with the highest survival rate was applied to evaluate fluoranthene toxicity in the sediment. The KB-T10 feeding condition showed the highest survival rate of 93.5%, with sufficient repeatability (4.6%). In the fluoranthene spiked-sediment toxicity test, the estimated LC50 (95% significance level) was successfully achieved under the KB-T10 condition but not under the OE-T10 condition. Therefore, we propose the use of TetraMin® (1 mg/org./day) and Kemble sediment in 10-day static spiked-sediment toxicity tests with G. japonica. Further improvements are needed for OECD sediment in seawater to increase the survival rate and prevent chemical loss due to overlying water renewal.

Effect of prolonged sludge retention times on the performance of membrane bioreactor and microbial community for leachate treatment under restricted aeration.

Leachate treatment is challenging owing to the complex composition of pollutants. This study investigated the treatment performance of a membrane bioreactor (MBR) and the microbial community structure corresponding to the effect of prolonged sludge retention times (SRTs) under restricted aeration. In the present study, a pilot-scale MBR was designed to treat leachate after being pretreated with an anaerobic filter for continuous operation for 240 days. The experimental results showed that removal performance of over 90% was achieved for biochemical oxygen demand, total Kjeldahl nitrogen, ammonia-nitrogen, and suspended solids when the MBR was operated at SRTs of 150-300 days. The results on microbial communities revealed that Proteobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Chloroflexi, and Actinobacteria were the major phyla. Furthermore, ammonia-oxidizing bacteria belonging to Nitrosomonadaceae were considered to play a vital role in the ammonia-nitrogen removal. A high abundance of Rhizobiales was detected on the biofilm of the membrane, which could be the key driver of bio-fouling. The dynamic changes in the microbial community indicate steady performance of MBR and can act as an indicator of membrane bio-fouling. The results of our study highlight that MBR can be viably operated in long SRTs under restricted aeration for leachate treatment with technical, economic, and environmental feasibility for resource recovery.

Determining the Relative Importance of Dietborne and Waterborne Toxicity of 4-tert-Butylphenol and 4-tert-Octylphenol to the Benthic Crustacean, Heterocypris incongruens.

Organic pollutants with high solid-water equilibrium partition coefficients are adsorbed into solid particles and are easily ingested by benthic organisms, potentially causing dietborne toxicity. Whether dietborne toxicity is more important than waterborne toxicity for such chemicals remains to be determined. In this study, we identify the most relevant uptake route for the toxicity of two alkylphenols, 4-tert-butylphenol (4tBP), and 4-tert-octylphenol (4tOP). To achieve this, 6-day toxicity tests under two exposure conditions, namely dietary exposure (clean water + contaminated food) and combined exposure (contaminated water + contaminated food) were conducted on a benthic ostracod, Heterocypris incongruens. The toxicologically important exposure routes were confirmed by the consistency of dietary and aqueous dose-response relationships under different exposure conditions. During the test, frequent renewal of water and food was performed to reduce variability in the exposure conditions. The results showed that, under the equilibrium condition, the dietary exposure route was toxicologically more important than the aqueous route for 4tBP, whereas the waterborne exposure route was more important than the dietary exposure route for 4tOP. This study provides a novel approach to identify the most relevant uptake pathways for chemical toxicity, which better explains the importance of exposure routes in toxicity effects.

Development and application of a metabolomic tool to assess exposure of an estuarine amphipod to pollutants in the environment.

Identifying major adverse effects on aquatic organisms in environmental samples is still challenging, and metabolomic approaches have been utilized as non-target screening techniques in the context of ecotoxicology. While existing methods have focused on statistical tests or univariate analysis, there is the need to further explore a multivariate analytical method that captures synergetic effects and associations among metabolites and toxicants. Here we show a new tool for screening sediment toxicity in the environment. First, we constructed predictive models using the metabolomic profiles and the result of exposure tests, to discriminate the toxic effects of target substances. The developed models were then applied to sediment samples collected from an actual urban area that contain chromium, nickel, copper, zinc, cadmium, fluoranthene, nicotine, and osmotic stress, incorporated with exposure tests of the benthic amphipod Grandidierella japonica. As a result, the fitted models showed high predictive power (Q2 > 0.71) and could detect toxicants from mixed chemical samples across a wide range of concentrations in test datasets. The application of the constructed models to river sediment and road dust samples indicated that almost all target substances were less toxic compared with the effects at LC50 levels. Only zinc showed slight increasing trends among samples, suggesting that the proposed method can be used for prioritization of toxicants. The present work made a direct connection between chemical exposures and metabolomic responses, and draws attention to the need for further studies on interactive mechanisms of metabolites in toxicological assessments.

Exploring the operating factors controlling Kouleothrix (type 1851), the dominant filamentous bacterial population, in a full-scale A2O plant.

This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.

Effect of calcite saturation state on the growth and mortality of Heterocypris incongruens and a proposal for an reference artificial sediment in the sediment toxicity test ISO14371.

Since the establishment of the whole-contact sediment toxicity test using Heterocypris incongruens in 2012, this user-friendly method has been widely used for the assessment of many kinds of solid samples, such as sediment, soil, compost, and sewage sludge. There are two endpoints for this method: mortality and growth inhibition. Currently, there is no standard toxicity threshold established for the endpoint of growth inhibition. However, there is evidence showing that the calcite saturation state of the overlying water, which might be different among various samples, influences the growth and survival of H. incongruens. Thus, it is necessary to characterize the influence of the calcite saturation state to ensure that the test results are reliable and comparable among different samples. In the present study, we created artificial sediments comprised of quartz sand and 0-20% calcite particulates to manipulate the calcite saturation state in the overlying water and performed the test using H. incongruens for 6 d. The results show that a low calcite saturation state inhibited the growth of H. incongruens but had no effect on survival rate. The growth of ostracods was linearly related to the calcite saturation index of the overlying water. Additionally, a formula for artificial sediment consisting of 10% calcite and 90% quartz sand for the H. incongruens toxicity test was proposed. This study helps to distinguish H. incongruens growth inhibition caused by calcite saturation from that caused by toxicity.

Candidates of quorum sensing bacteria in activated sludge associated with N-acyl homoserine lactones.

This study aimed to explore candidates of microbial groups which is associated with quorum sensing in activated sludge. Activated sludge samples were collected from three wastewater treatment plants (WWTP) to analyze N-acyl homoserine lactone (AHL) by Fourier-transform mass spectrometry (FTMS) and 16S rRNA-based microbial community. Among activated sludge samples taken at 3 WWTPs in different seasons, 2 AHL species of N-3-hydroxyoctanoyl-l-homoserine lactone and N-3-hydroxydecanoyl-l-homoserine lactone were detected in the range of ranged of 0.1 ng/L to 1.6 ng/L. The detected AHL species were not dependent on treatment systems nor seasons. From microbial community analysis, population abundance of one strain in Verrucomicrobia and two strains in Holophagaceae had high correlation with AHL concentration in activated sludge. Comamonadaceae had also moderately correlated population with AHL concentrations among quorum sensing bacteria reported previously.

Whole transcriptome analysis of an estuarine amphipod exposed to highway road dust.

Urban road dust can potentially have adverse effects on aquatic and benthic ecosystems if discharged into receiving waters; however, little is known about the mode of action of road dust toxicity within aquatic organisms. With an aim to reveal the biological effects of road dust on benthic crustacean species, we performed a de novo transcriptome analysis of the estuarine amphipod Grandidierella japonica exposed to road dust collected from highways around Tokyo. A transcriptome analysis by Illumina HiSeq 2500 identified differentially expressed genes related to the gamma-aminobutyric acid (GABA) signaling pathway, oxidative damage, and cuticle metabolism. Among these, a GABAB receptor subunit showed down-regulation in the road dust treatment, but a constant expression in the treatment of road dust with a carbonaceous resin XAD-4, which can reduce the acute toxicity of road dust to G. japonica. These results and the time course expressions of the related genes were partially confirmed by quantitative PCR (qPCR) experiments. Although the linkage between acute lethal toxicity and the molecular initiating events induced by road dust was still unclear, our findings provide lines of evidence to identify the causative toxicants in urban road dust.

A Microcosm Study of Surface Sediment Environmental DNA: Decay Observation, Abundance Estimation, and Fragment Length Comparison.

Interpretation of environmental DNA (eDNA) is a major problem hindering the application of this emerging technology for environmental monitoring. The decay characteristics and bioabundance estimation of different DNA fragment lengths are largely unknown, especially for eDNA captured from surface sediments. An estuarine amphipod, Grandidierella japonica, was used as the target species in this study. We conducted a lab-scale experiment using DNA extraction to clarify the effect of bacteria on eDNA decay. We also conducted a microcosm experiment using amphipods to clarify interpretations of information regarding eDNA decay and bioabundance estimation by using two fragments with different lengths (126 and 358 bp). It was found that the bacteria concentration accelerated eDNA decay, and long fragments were more susceptible to bacteria, thus decaying faster, than short fragments in the exponential decay period. The fresh eDNA (collected within 24 h of removing the amphipods) was more indicative of bioabundance than old eDNA (collected 240-480 h after removing amphipods), and short fragments better reflected bioabundance than long fragments. Finally, we compared the half-life of eDNA in surface sediment with that in a water sample and found that the temporal scales of surface sediment and water are similar (days to weeks). Our results suggest that surface sediment also has the potential to monitor the environment at a temporal scale similar to water.

Duration of life-cycle toxicity tests with the ostracod Heterocypris incongruens.

An acute sediment toxicity test using the ostracod Heterocypris incongruens is user friendly and has high sensitivity; however, a life-cycle test using this species has not been developed. The most challenging problem when developing a life-cycle test is that the egg development time varies greatly and is sometimes too long (> 150 d) to monitor. It is desirable to shorten the duration of life-cycle toxicity tests including the observation period of egg development while preserving the ecological relevance of the net reproductive rate (R0 ), an endpoint in the life-cycle test. Therefore, in the present study, we suggest a practical test duration for R0 using population growth rate (λ) as a measure of ecological relevance. We collected a range of life history characteristics of the ostracod by combining data from the literature and experimental results, constructed population matrix models, and calculated λ for 20 life history patterns. The results showed that a longer test period (> 150 d) did not increase the correlation coefficients between R0 and λ. Rather, a shorter test duration resulted in R0 being highly correlated with λ. Our results suggest that a life-cycle toxicity test using the ostracod can provide an ecologically relevant toxicity endpoint, even if the test is abandoned after approximately 50 d and unhatched eggs remain. Environ Toxicol Chem 2017;36:3443-3449. © 2017 SETAC.

Development of a chronic sediment toxicity test using the benthic ostracod Heterocypris incongruens and their application to toxicity assessments of urban road dust.

This study reports on the development and application of a chronic sediment toxicity test using the benthic ostracod Heterocypris incongruens. H. incongruens reproduction parameters such as egg production, first day of brooding, egg-laying ratio, and hatching ratio were examined in this study. The test was first validated by determining the repeatability of the test method under seven control performances. The results showed good test repeatability of most endpoints, with coefficient of variation (CV) results below 15%. However, lifetime egg production, hatching ratio, and the reproductive rate were highly variable, with CVs ranging from 29.5% to 51.9%. Next, an application example of the proposed chronic method was performed using a series of urban road dust (URD) samples diluted with a reference sediment and compared to a 6 d H. incongruens toxicity test. The results of the proposed chronic test showed a statistically significant difference in the first day of brooding at 6.25% URD which did not exhibit significant mortality and growth inhibition in 6 d toxicity test.

Application of cDNA-AFLP to biomarker exploration in a non-model species Grandidierella japonica.

Biomarkers of exposure can be used to identify specific contaminants that are adversely affecting aquatic organisms. However, it remains prohibitively costly to investigate multiple novel biomarkers of exposure in a non-model species, despite the development of next-generation sequencing technology. In this study, we focused on the use of cDNA-amplified fragment length polymorphism (AFLP) as a cost-effective biomarker discovery tool to test whether it could identify biomarkers of exposure in the non-model amphipod species Grandidierella japonica. Loci were identified that were differentially expressed in amphipods exposed to reference chemicals (Cu, Zn, and nicotine) and to an environmental sample (road dust) at sublethal concentrations. Eight loci were shown to respond consistently to nicotine at different concentrations, but not to Cu or Zn. Some of the loci also responded to an environmental road dust sample containing nicotine. These findings suggest that loci identified using cDNA-AFLP could be used as biomarkers of nicotine exposure in environmental samples with complex matrices. Further studies with other organisms and toxicants are needed, but we have demonstrated that the use of cDNA-AFLP to identify biomarkers for ecotoxicological studies of non-model species is at least feasible.

Causes of highway road dust toxicity to an estuarine amphipod: Evaluating the effects of nicotine.

Urban road dust can potentially have adverse effects on ecosystems if it is discharged into receiving waters. This study investigated the causes of highway road dust toxicity by performing sediment toxicity identification evaluation (TIE) tests with an estuarine amphipod, Grandidierella japonica. In addition to metals and polycyclic aromatic hydrocarbons, which are traditionally considered to be the major toxicants in road runoff, we focused on dissolved nicotine as a causative toxicant. The sediment TIE results suggested that organic contaminants contributed to the majority of toxicity, and that the contribution of unionized nicotine to the toxicity was the highest among the chemicals considered. However, additional mortality tests with 48-h pulsed nicotine exposure demonstrated that exposure to nicotine at the same concentration as the baseline level in TIE tests did not cause significant 10-day amphipod mortality. Thus, the road dust toxicity could not be explained only by unionized nicotine, thereby suggesting contributions from joint effects of the measured toxicants and the presence of other unmeasured factors.

Alleviation of membrane fouling in a submerged membrane bioreactor with electrochemical oxidation mediated by in-situ free chlorine generation.

The control of membrane fouling is still the biggest challenge that membrane bioreactor (MBR) for wastewater treatment faces with. In this report, we evince that an in-situ electrochemical free chlorine generation is effective for membrane fouling mitigation. An electrochemical oxidation (EO) apparatus with perforated Ti/IrO2 anodes and Ti/Pt cathodes was integrated into a conventional MBR with microfiltration module (EO-MBR). The membrane fouling characteristics of EO-MBR fed with synthetic wastewater were monitored for about 2 months in comparison to control MBRs. In the EO-MBR at a direct current density of 0.4 mA/cm(2), the frequency of membrane fouling when the trans-membrane pressure (TMP) reached 30 kPa was effectively reduced by 40% under a physical membrane cleaning regime. The evolution patterns of TMP together with hydraulic resistance analysis based on resistance-in-series model indicated that the electrochemically generated active chlorine alleviated the physically irremovable membrane fouling. Further analysis on extracellular polymeric substances (EPS) of sludge cake layer (SCL) revealed significant reductions of protein contents in soluble EPS and fluorescence emission intensities from humic acids and other fluorophores in bound EPS, which in-turn would decrease the hydrophobic accumulation of organic foulants on membrane pores. The chlorine dosage from the EO apparatus was estimated to be 4.7 mg Cl2/g MLVSS/day and the overall physicochemical properties (bio-solids concentration, floc diameter, zeta-potential) as well as the microbial activity in terms of specific oxygen utilization rate and removal efficiency of dissolved organic carbon (>97%) were not affected significantly. A T-RFLP (terminal restriction fragment length polymorphism) analysis suggested noticeable shifts in microbial community both in mixed liquor and sludge cake layer. Consequently, our electrochemical chlorination would be an efficient fouling control strategy in membrane-based water treatment processes where additional electricity consumption and cathodic scale deposition are not of serious concerns.

Seawater-driven forward osmosis for enriching nitrogen and phosphorous in treated municipal wastewater: effect of membrane properties and feed solution chemistry.

Seawater-driven forward osmosis (FO) is considered to be a novel strategy to concentrate nutrients in treated municipal wastewater for further recovery as well as simultaneous discharge of highly purified wastewater into the sea with low cost. As a preliminary test, the performance of FO membranes in concentrating nutrients was investigated by both batch experiments and model simulation approaches. With synthetic seawater as the draw solution, the dissolved organic carbon, phosphate, and ammonia in the effluent from a membrane bioreactor (MBR) treating municipal wastewater were 2.3-fold, 2.3-fold, and 2.1-fold, respectively, concentrated by the FO process with approximately 57% of water reduction. Most of the dissolved components, including trace metals in the MBR effluent, were highly retained (>80%) in the feed side, indicating high water quality of permeate to be discharged. The effect of membrane properties on the nutrient enrichment performance was investigated by comparing three types of FO membranes. Interestingly, a polyamide membrane possessing a high negative charge demonstrated a poor capability of retaining ammonia, which was hypothesized because of an ion exchange-like mechanism across the membrane prompted by the high ionic concentration of the draw solution. A feed solution pH of 7 was demonstrated to be an optimum condition for improving the overall retention of nutrients, especially for ammonia because of the pH-dependent speciation of ammonia/ammonium forms. The modeling results showed that higher than 10-fold concentrations of ammonia and phosphate are achievable by seawater-driven FO with a draw solution to feed solution volume ratio of 2:1. The enriched municipal wastewater contains nitrogen and phosphorous concentrations comparable with typical animal wastewater and anaerobic digestion effluent, which are used for direct nutrient recovery.

A study of the relationship among sludge retention time, bacterial communities, and hydrolytic enzyme activities in inclined plate membrane bioreactors for the treatment of municipal wastewater.

Inclined plate membrane bioreactors (ip-MBRs) have been proposed as a highly effective method in wastewater treatment. With the help of settling enhancer inclined plates, dense excess sludge can be kept in the mainstream of the process, and consequently, suitable sludge mass can be maintained in the membrane tank. In this study, the relationship among sludge retention time (SRT), bacterial communities, and hydrolytic enzyme activities was investigated. Two identical bench-scale ip-MBRs were operated 1 year in real municipal wastewater treatment. Multidimensional scaling (MDS) plots of terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed similar changes in the bacterial communities in terms of bacterial members and abundance over time in both the reactors, which was primarily caused by the changes of wastewater composition. However, the impact of SRT revealed significant differences in the dominant bacterial communities when both the reactors were operated with a largely different SRT (infinite SRT and SRT of 20 days). The sequences of bacterial 16S rRNA gene were classified into six libraries of A-F. The largest group of sequences belonged to the phylum Proteobacteria. The phylum Bacteroidetes was dominant in the seed sludge retrieved from the conventional activated sludge (CAS) as Flavobacterium-like bacterium was dominantly observed. Under the MBR operation (libraries B-F), bacterial communities belonging to the phyla Proteobacteria and Chloroflexi were dominant. Most of them may be responsible for protein degradation because aminopeptidase activity increased in proportion with the abundance of these bacteria.

Sensitive and substrate-specific detection of metabolically active microorganisms in natural microbial consortia using community isotope arrays.

The goal of this study was to develop and validate a novel fosmid-clone-based metagenome isotope array approach - termed the community isotope array (CIArray) - for sensitive detection and identification of microorganisms assimilating a radiolabeled substrate within complex microbial communities. More specifically, a sample-specific CIArray was used to identify anoxic phenol-degrading microorganisms in activated sludge treating synthetic coke-oven wastewater in a single-sludge predenitrification-nitrification process. Hybridization of the CIArray with DNA from the (14) C-phenol-amended sample indicated that bacteria assimilating (14) C-atoms, presumably directly from phenol, under nitrate-reducing conditions were abundant in the reactor, and taxonomic assignment of the fosmid clone end sequences suggested that they belonged to the Gammaproteobacteria. The specificity of the CIArray was validated by quantification of fosmid-clone-specific DNA in density-resolved DNA fractions from samples incubated with (13) C-phenol, which verified that all CIArray-positive probes stemmed from microorganisms that assimilated isotopically labeled carbon. This also demonstrated that the CIArray was more sensitive than DNA-SIP, as the former enabled positive detection at a phenol concentration that failed to yield a 'heavy' DNA fraction. Finally, two operational taxonomic units distantly related to marine Gammaproteobacteria were identified to account for more than half of 16S rRNA gene clones in the 'heavy' DNA library, corroborating the CIArray-based identification.

Specificity of randomly generated genomic DNA fragment probes on a DNA array.

The use of randomly generated DNA fragment sequences as probes on DNA arrays offers a unique potential for exploring unsequenced microorganisms. In this study, the detection specificity was evaluated with respect to probe-target sequence similarity using genomic DNAs of four Pseudomonas strains. Genome fragments averaging 2000 bp were found to be specific enough to discriminate 85-90% similarity under highly stringent hybridization conditions. Such stringent conditions compromised signal intensities; however, specific signals remained detectable at the highest stringency (at 75 °C hybridization) with negligible false negatives. These results suggest that, without any probe design or selection, genomic fragments can provide a reasonable specificity for microbial diagnostics or species delineation by DNA-DNA similarities.