Francis L. de los Reyes

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently-Aerated Reactors

ABSTRACT The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of the reactors performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.

Role of filamentous microorganisms in activated sludge foaming: relationship of mycolata levels to foaming initiation and stability.

The relationship between the levels of mycolic acid-containing actinomycetes (mycolata), Gordonia spp. and Gordonia amarae, and foam initiation and stability was characterized using: (1) batch tests involving addition of G. amarace cells to activated sludge, (2) analysis of a full-scale activated sludge plant that experienced seasonal foaming, and (3) a study of lab-scale activated sludge reactors augmented with G. amarae. Using batch tests, threshold Gordonia levels for foam formation and foam stability were determined to be approximately 2 x 10(8) microm ml(-1) and 1 x 10(9) microm ml(-1), respectively. In the full-scale plant, the levels of Gordonia spp. and G. amarae increased during the course of foaming, and the foam formation threshold of 2 x 10 microm ml(-1) corresponded to the onset of foaming. This value was also verified in lab-scale reactor washout experiments, where decreasing mycolata levels were observed during the course of foam dissipation. The foam stability threshold of 1 x 10(9) micorm ml(-1) was verified in lab-scale reactor studies. The increase in the levels of Gordonia spp. and G. amarae in the full-scale plant corresponded to an increase in temperature, suggesting that the growth of Gordonia spp. was favored during warmer periods.

Evidence for Fat, Oil, and Grease (FOG) Deposit Formation Mechanisms in Sewer Lines

The presence of hardened and insoluble fats, oil, and grease (FOG) deposits in sewer lines is a major cause of line blockages leading to sanitary sewer overflows (SSOs). Despite the central role that FOG deposits play in SSOs, little is known about the mechanisms of FOG deposit formation in sanitary sewers. In this study, FOG deposits were formed under laboratory conditions from the reaction between free fatty acids and calcium chloride. The calcium and fatty acid profile analysis showed that the laboratory-produced FOG deposit displayed similar characteristics to FOG deposits collected from sanitary sewer lines. Results of FTIR analysis showed that the FOG deposits are metallic salts of fatty acid as revealed by comparisons with FOG deposits collected from sewer lines and pure calcium soaps. Based on the data, we propose that the formation of FOG deposits occurs from the aggregation of excess calcium compressing the double layer of free fatty acid micelles and a saponification reaction between aggregated calcium and free fatty acids.

Effect of spatial differences in microbial activity, pH, and substrate levels on methanogenesis initiation in refuse.

ABSTRACT The initiation of methanogenesis in refuse occurs under high volatile fatty acid (VFA) concentration and low pH (5.5 to 6.25), which generally are reported to inhibit methanogenic Archaea. One hypothesized mechanism for the initiation of methanogenesis in refuse decomposition is the presence of pH-neutral niches within the refuse that act as methanogenesis initiation centers. To provide experimental support for this mechanism, laboratory-scale landfill reactors were operated and destructively sampled when methanogenesis initiation was observed. The active bacterial and archaeal populations were evaluated using RNA clone libraries, RNA terminal restriction fragment length polymorphism (T-RFLP), and reverse transcription-quantitative PCR (RT-qPCR). Measurements from 81 core samples from vertical and horizontal sections of each reactor showed large spatial differences in refuse pH, moisture content, and VFA concentrations. No pH-neutral niches were observed prior to methanogenesis. RNA clone library results showed that active bacterial populations belonged mostly to Clostridiales, and that methanogenic Archaea activity at low pH was attributable to Methanosarcina barkeri. After methanogenesis began, pH-neutral conditions developed in high-moisture-content areas containing substantial populations of M. barkeri. These areas expanded with increasing methane production, forming a reaction front that advanced to low-pH areas. Despite low-pH conditions in >50% of the samples within the reactors, the leachate pH was neutral, indicating that it is not an accurate indicator of landfill microbial conditions. In the absence of pH-neutral niches, this study suggests that methanogens tolerant to low pH, such as M. barkeri, are required to overcome the low-pH, high-VFA conditions present during the anaerobic acid phase of refuse decomposition.

Determining the limits of anaerobic co-digestion of thickened waste activated sludge with grease interceptor waste.

Anaerobic co-digestion of thickened waste activated sludge (TWAS) with grease interceptor waste (GIW) from a food service establishment was conducted in lab scale semi-continuous digesters. GIW included the entire contents of the grease interceptor (GI) including fat, oil, and grease (FOG), food residuals, and associated wastewater. GIW was added in step increases to identify the maximum methane production and the corresponding threshold input of GIW that led to inhibition of methanogenesis. The experiment was performed at mesophilic conditions (37 °C) with a solids retention time (SRT) of 20 days. The highest GIW addition rate achieved without digester failure was 20% (v/v), or 65.5% (w/w) of volatile solids (VS) added, enhancing the methane yield from 0.180 to 0.752 m3(CH4)/kg(VS added), biogas production from 2.2 × 10(-3) to 1.4 × 10(-2) m(3)/d, and methane content from 60.2% to 70.1%. The methane yield of 0.752 m3(CH4)/kg(VS added) is the highest value reported to date for co-digestion of GIW. Stepwise increases in co-substrate addition led to better microbial acclimation and reduced the GIW inhibitory effect. The limit for GIW addition leading to an inhibited digestion process was identified to be between 20 and 40% (v/v) or 65.5 and 83.5% (w/w) of VS added. The results show the significant benefits of anaerobic co-digestion of GIW and the positive impacts of gradual addition of GIW.

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

ABSTRACT Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates B. atrophaeus and S. marcescens in building debris and leachate.

Mechanisms of Fat, Oil and Grease (FOG) deposit formation in sewer lines

FOG deposits in sewer systems have recently been shown to be metallic salts of fatty acids. However, the fate and transport of FOG deposit reactant constituents and the complex interactions during the FOG deposit formation process are still largely unknown. In this study, batch tests were performed to elucidate the mechanisms of FOG deposit formation that lead to sanitary sewer overflows (SSOs). We report the first formation of FOG deposits on a concrete surface under laboratory conditions that mimic the formation of deposits in sewer systems. Results showed that calcium, the dominant metal in FOG deposits, can be released from concrete surfaces under low pH conditions and contribute to the formation process. Small amounts of additional oil to grease interceptor effluent substantially facilitated the air/water or pipe surface/water interfacial reaction between free fatty acids and calcium to produce surface FOG deposits. Tests of different fatty acids revealed that more viscous FOG deposit solids were formed on concrete surfaces, and concrete corrosion was accelerated, in the presence of unsaturated FFAs versus saturated FFAs. Based on all the data, a comprehensive model was proposed for the mechanisms of FOG deposit formation in sewer systems.

Relationship of Species-Specific Filament Levels to Filamentous Bulking in Activated Sludge

ABSTRACT To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 × 108 μm ml−1 resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the “substrate diffusion limitation” hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.

Characterization of filamentous foaming in activated sludge systems using oligonucleotide hybridization probes and antibody probes

A quantitative method was developed for estimating Gordona mass in activated sludge foam and mixed liquor samples. The technique involves in situ hybridization with a genus-specific fluorescently labeled oligonucleotide probe calibrated on pure cultures of Gordona . The immunofluorescent technique of Hernandez et al. was modified to allow staining with fluorescently labeled antibody and hybridization probes. The results of this technique were compared to those from membrane hybridization studies using radioactively-labeled oligonucleotide probes. Quantitative membrane hybridizations, in situ hybridizations, and antibody staining resulted in significantly different levels of Gordona in activated sludge foam, activated sludge mixed liquor, return activated sludge, and anaerobic digester sludge. Simultaneous staining with labeled antibodies and oligonucleotide probes provide a definitive identification for Gordona , and represents a new approach for in situ studies of this organism9s role in foaming.

Comparison of Bacteria and Archaea communities in municipal solid waste, individual refuse components, and leachate

Refuse decomposition in landfills is a microbially mediated process that occurs primarily under anaerobic conditions. Because of limited moisture conditions, hydraulic transport as a means of cellular translocation within the landfill appears limited, especially during the initial stages of decomposition. Thus, microbial communities within the incoming refuse serve as a primary source of facultative and obligate anaerobic microorganisms that initiate refuse decomposition. Fresh residential refuse was collected five times over 26 months, and microbial communities in these samples were compared with those in individual refuse components and decomposed refuse. Bacterial and archaeal community structures were determined using T-RFLP. The Bacterial microbial community richness was correlated (r(2) = 0.91) with seasonal differences in ambient air temperature. Analysis of the results shows that fresh refuse is most likely not the source of methanogens in landfills. Microbial communities in the solid and leachate phases were different, indicating that both matrices must be considered when characterizing microbial diversity within a landfill.