Kevin G. Robinson

Quantification of Nitrosomonas oligotropha-Like Ammonia- Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

ABSTRACT Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus Nitrospira. The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ± 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% ± 0.28% of the biosludge population in the municipal WWTP and 0.37% ± 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.

Micellar solubilization of polynuclear aromatic hydrocarbons in coal tar-contaminated soils

Solubilization of PAHs from a coal tar-contaminated soil obtained from a manufactured gas plant (MGP) site was evaluated using nonionic polyoxyethylene surfactants at dosages greater than cmc. Up to 25% of Soxhlet-extractable PAHs could be solubilized at surfactant loadings of 0.3 g/g of oil in 16 days in completely stirred batch reactors. Longer periods were required to reach equilibrium at higher surfactant dosages. Raoult`s law satisfactorily described the partitioning of constituent PAHs between the weathered coal tar and the micellar solution. An equilibrium model was developed to predict the solubilization of PAHs from coal tar-contaminated soils for given properties of the soil, surfactant, and component PAHs. The model predicted solubilization of constituent PAHs reasonably well at low surfactant dosages. At extremely high surfactant dosages, the model failed to reliably predict solubilization. Presumably, mass transfer mass transfer limitations prevented the attainment of equilibrium during the duration (380h) of solubilization experiments. 25 refs., 6 figs., 4 tabs.

Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

ABSTRACT The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

Emergence of Competitive Dominant Ammonia-Oxidizing Bacterial Populations in a Full-Scale Industrial Wastewater Treatment Plant

ABSTRACT Ammonia-oxidizing bacterial populations in an industrial wastewater treatment plant were investigated with amoA and 16S rRNA gene real-time PCR assays. Nitrosomonas nitrosa initially dominated, but over time RI-27-type ammonia oxidizers, also within the Nitrosomonas communis lineage, increased from below detection to codominance. This shift occurred even though nitrification remained constant.

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.

Limited impact of free ammonia on Nitrobacter spp. inhibition assessed by chemical and molecular techniques.

Free ammonia has long been identified as a nitrite oxidation inhibitor. However, past attempts to use this compound to eliminate nitrite oxidation and thereby promote more efficient nitrogen removal strategies during biological wastewater treatment have often failed. Additionally, contradictory results exist in the literature where direct measurements of free ammonia inhibition of nitrite oxidation have been reported. In this study, suspended biomass samples (nitrifier enriched activated sludge) were collected from a bench scale nitrification reactor with Nitrobacter spp. as the dominant nitrite oxidizer and subjected to batch respirometric experiments designed to quantify free ammonia inhibition of nitrite oxidization. A variety of data including ammonia, nitrite, and nitrate conversion rates, oxygen consumption rates, and Nitrobacter ribosomal RNA transcript abundance, a molecular indicator of growth activity, were used to assess nitrite oxidation and growth activity. Both the traditional and molecular activity assessments indicated that free ammonia had a limited inhibitory effect on Nitrobacter spp. In fact, the pH changes necessary to induce high free ammonia concentrations (>10mg-N/L) had a demonstrably more important inhibiting effect on nitrite oxidation than free ammonia. In contrast, during high ammonia oxidizing activity (5.3mg-N/L/h), low nitrite oxidation rates (0.2mg-N/L/h) and severely impaired Nitrobacter spp. growth activity, indicated by a low abundance of the Nitrobacter spp. ribosomal gene transcript relative to the ribosomal gene (0.08), were measured. The findings suggest that pH changes and ammonia oxidizing bacteria activity are more important factors limiting Nitrobacter spp. mediated nitrite oxidation, rather than the free ammonia concentration.

Public attitudes and risk perception toward land application of biosolids within the south-eastern United States

A descriptive-correlational study of biosolids recycling was conducted in the south-eastern United States to assess current knowledge, attitudes and risk perceptions of participants in two communities that land apply biosolids as part of their waste management programs. One community, Amelia County VA, has been outspoken against biosolids recycling in the past, whereas the second community, Knoxville, TN region, has voiced few concerns about biosolids recycling. Additionally, gender differences within the entire study population were assessed. A 45-question telephone survey, utilizing a 4-point Likert scale, was developed and administered to 311 randomly selected adults in the two regions. Commonalities identified during the study revealed key risk perceptions by the public regarding biosolids regulations, treatment, and application. Given current perceptions and knowledge, respondents felt that the benefits derived from biosolids recycling do not offset the perceived health and safety risks. However, as distance between application and personal property increased, a decrease in opposition of biosolids reuse became evident for all respondents. Survey participants were dissatisfied with the level of stakeholder involvement in research and decision-making processes concerning biosolids. The outspoken Amelia County residents perceived greater health risks due to inadequate treatment of biosolids and odorous emissions during the application process than the less engaged Knox Metro respondents. Significant gender differences were observed with sampled females perceiving greater risks to health and safety from biosolids recycling than males. There was also indication that decisions and risks were not sufficiently communicated to the public, leading to respondents being inadequately informed about biosolids land application in both communities. Community-specific outreach programs must address these public risk perceptions and the differences in perception caused by gender and issue awareness to assist solid waste managers in developing and implementing successful biosolids land application systems that are acceptable to the public.

Fate of 2,4,6-trichloro-(14C)-phenol bound to dissolved humic acid

Abstract Batch microcosms were utilized to evaluate binding of 2,4,6-trichlorophenol (TCP) to dissolved humic acid (HA). Biodegradation of 2,4,6-TCP was then evaluated after addition of a bacterial culture previously acclimated to the substrate. Gel chromatography (size exclusion) was used to separate HA-bound 2,4,6-TCP from free (unbound) 2,4,6-TCP by molecular size before quantitative analysis. Dissolved HA can interact with free 2,4,6-TCP, forming an HA/TCP association in solution. Equilibrium isotherms of 2,4,6-TCP for dissolved HA are linear for 2,4,6-TCP concentrations up to 1000 μg/l and HA concentrations up to 100 mg/l. Distribution coefficients of 2,4,6-TCP for HA vary with dissolved humic concentration. Coefficients decreased with increasing HA concentration indicating a change in HA sorption characteristics with concentration. Acclimated bacteria, Pseudomonas aeruginosa , can mineralize up to 60% of the total 2,4,6-TCP in solution, however, total mineralization decreased with increasing HA concentration. Overall mineralization in HA-containing solutions was 5–15% less than in HA-free solutions. Biodegradation of free 2,4,6-TCP was very rapid while biodegradation of HA-bound 2,4,6-TCP was much reduced and appeared to be desorption controlled.

Transcription levels (amoA mRNA-based) and population dominance (amoA gene-based) of ammonia-oxidizing bacteria

A population shift of ammonia-oxidizing bacteria (AOB) was described within a bench-scale activated sludge process treating an industrial wastewater in a previous report (Kuo et al. in Environ Eng Sci 23:507–520, 2006). In this investigation, transcriptional levels (amoA mRNA-based) of the three AOB groups (i.e., RI-27, B2-3, and Nitrosomonas nitrosa) identified in the treatment process were determined by quantitative real-time reverse transcription (RT-PCR) assays to circuitously evaluate AOB ammonia-oxidizing activity and to assess the presumed correlation between cellular activity and the dominant (greatest number) AOB population. Results demonstrated that the AOB group with higher amoA mRNA levels dominated the overall AOB population in the wastewater treatment process. Although AOB population dominance did not correlate well with transcripts at a normalized cellular level (amoA mRNA/DNA ratio), overall amoA mRNA levels did reflect the activity of distinct AOB groups under different N-loading conditions. Thus, an additional molecular parameter (amoA mRNA) was successfully utilized to assess timely shifts in AOB population structure that may impact nitrification treatment performance.

Molecular indicators of Nitrobacter spp. population and growth activity during an induced inhibition event in a bench scale nitrification reactor

The Nitrobacter spp. ribosomal RNA gene (rDNA) and transcript (rRNAt) abundance were quantified in a bench scale nitrification reactor during baseline periods of high nitrification efficiency and an intervening staged inhibition event. The transcript to gene ratio (rRNAt/rDNA) was highly sensitive to changes in the reactor nitrite oxidation rate. During high nitrification efficiency, the rRNAt/rDNA metric displayed a range from 0.68 to 2.01 with one-sided (α=0.10) lower and upper prediction intervals of 0.70 and 1.78, respectively. When nitrification was inhibited by disabling the reactor pH control system, this activity metric declined an order of magnitude to ≈ 0.05, well below the lower prediction interval reflecting high nitrification efficiency. The decline was rapid (2h) and preceded a significant drop in reactor nitrification performance, which occurred as ammonia accumulated. The rRNAt/rDNA ratio remained low (≈ 0.05) for several days after the pH control system was re-enabled at a setpoint of 8.0, which otherwise induced rapid oxidation of accumulated ammonia and produced high free ammonia concentrations. The timing of a subsequent increase in the rRNAt/rDNA ratio, which transiently exceeded the upper prediction interval established during the baseline period of high nitrification efficiency, was not coincidental with resumption of pH control at 7.2 that lowered free ammonia concentrations to non-inhibitory levels. Rather, nitrite oxidation resumed and the rRNAt/rDNA ratio increased only after oxidation of accumulated ammonia was complete, which was coincidental with reduced reactor oxygen demand. In summary, the Nitrobacter rRNAt/rDNA activity metric reflected timely and easily recognizable changes in nitrite oxidation activity, illustrating that molecular data can be used to diagnose poor biological wastewater treatment performance.