James E. Alleman

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater

ABSTRACT The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domainBacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the β subdivision of the divisionProteobacteria (β-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive andSynergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, includingCytophaga-Flavobacterium-Bacteroides,Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, andProteobacteria (α-proteobacteria, β-proteobacteria, γ-proteobacteria and δ-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity—a function of both the total number of species present (richness) and their relative distribution (evenness).

Stability of the bacterial communities supported by a seven-stage biological process treating pharmaceutical wastewater as revealed by PCR-DGGE

The stabilities of the bacterial community structures supported by seven full-scale biological reactors treating pharmaceutical wastewater were investigated by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA gene fragments. Effluent quality from this treatment process was consistently high with respect to BOD5 (<30 mgl(-1)), soluble COD (<500 mgl(-1)), and total ammonia (< 5 mgl(-1) as N) concentrations. Long-term community structure stability was studied by comparing the similarity of PCR-DGGE fingerprints from samples collected 87 days apart between which the influent wastewater characteristics were relatively stable. The Dice index (Cs) of similarity was moderately high for the first four reactors (Cs = 0.61-0.77) and very high for the last three reactors (Cs = 0.89-0.91). Short-term community structure stability was studied by comparing PCR-DGGE fingerprints from samples collected 15 days apart between which the influent wastewater characteristics changed significantly, while the effluent quality remained consistently high. The bacterial community composition of each of the seven bioreactors showed a moderate community shift (Cs = 0.70-0.76). Short-term variability in influent wastewater composition, therefore, affected a greater community shift than did long-term operation treating a wastewater of relatively consistent composition. These results indicate that functionally stable wastewater treatment bioreactors have stable microbial community structures under normal operating conditions but are able to adapt in response to perturbations to sustain high effluent quality.

Aerobic Biological Treatment of a Pharmaceutical Wastewater:: Effect of Temperature on COD Removal and Bacterial Community Development

The effect of temperature was studied on the efficiency of soluble COD removal and bacterial community development during the aerobic biological treatment of a pharmaceutical wastewater. Using wastewater and bacterial inoculum obtained from the full-scale facility treating this wastewater, batch laboratory cultures were operated at 5 degrees C intervals from 30 degrees C to 70 C. Following four culture transfers to allow for bacterial acclimation, residual soluble COD levels were measured and bacterial community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments. Soluble COD removal efficiency declined as temperature increased from 30 degrees C (62%) to 60 degrees C (38%). Biological treatment of this wastewater failed to occur at temperatures higher than 60 C. Gradual shifts in bacterial community structure were detected as temperature increased, including a concomitant reduction in the number of different bacterial populations. The impact of temperature on a two-stage biological treatment process was also compared. Better soluble COD removal was achieved when both reactors were operated at 30 degrees C compared to a system where the two stages were consecutively operated at 55 degrees C and 30 degrees C. These results indicate that operation of aerobic biological wastewater treatment reactors at elevated temperatures can have adverse effects on process performance.

Storage-induced denitrification using sequencing batch reactor operation

Abstract As a biochemical reduction of oxidized-nitrogen species, the denitrification reaction requires the availability of an electron-donor substrate. This requirement is typically satisfied by the provision of an organic carbon supplement or, possibly, a reactor design which utilizes raw-waste organic carbon components. Bacterial storage can also provide the necessary electron source. However, in the instance of conventional wastewater treatment facilities, the limited availability of such cellular reserve material results in a markedly reduced denitrification capacity. Bench-scale studies of storage induced denitrification were conducted using a sequencing batch reactor. By operating the reactor in a mode conducive to the development of cellular storage, the system maintained a consistent 92+% reduction in total nitrogen without a carbon supplement. Depletion of the cellular glycogen reserve was observed during the denitrification reaction.

Effects of wastewater disinfection on waterborne bacteria and viruses.

Wastewater disinfection is practiced with the goal of reducing risks of human exposure to pathogenic microorganisms. In most circumstances, the efficacy of a wastewater disinfection process is regulated and monitored based on measurements of the responses of indicator bacteria. However, inactivation of indicator bacteria does not guarantee an acceptable degree of inactivation among other waterborne microorganisms (e.g., microbial pathogens). Undisinfected effluent samples from several municipal wastewater treatment facilities were collected for analysis. Facilities were selected to provide a broad spectrum of effluent quality, particularly as related to nitrogenous compounds. Samples were subjected to bench-scale chlorination and dechlorination and UV irradiation under conditions that allowed compliance with relevant discharge regulations and such that disinfectant exposures could be accurately quantified. Disinfected samples were subjected to a battery of assays to assess the immediate and long-term effects of wastewater disinfection on waterborne bacteria and viruses. In general, (viable) bacterial populations showed an immediate decline as a result of disinfectant exposure; however, incubation of disinfected samples under conditions that were designed to mimic the conditions in a receiving stream resulted in substantial recovery of the total bacterial community. The bacterial groups that are commonly used as indicators do not provide an accurate representation of the response of the bacterial community to disinfectant exposure and subsequent recovery in the environment. UV irradiation and chlorination/dechlorination both accomplished measurable inactivation of indigenous phage; however, the extent of inactivation was fairly modest under the conditions of disinfection used in this study. UV irradiation was consistently more effective as a virucide than chlorination/dechlorination under the conditions of application, based on measurements of virus (phage) diversity and concentration. Taken together, and when considered in conjunction with previously published research, the results of these experiments illustrate several important limitations of common disinfection processes as applied in the treatment of municipal wastewaters. In general, it is not clear that conventional disinfection processes, as commonly implemented, are effective for control of the risks of disease transmission, particularly those associated with viral pathogens. Microbial quality in receiving streams may not be substantially improved by the application of these disinfection processes; under some circumstances, an argument can be made that disinfection may actually yield a decrease in effluent and receiving water quality. Decisions regarding the need for effluent disinfection must account for site-specific characteristics, but it is not clear that disinfection of municipal wastewater effluents is necessary or beneficial for all facilities. When direct human contact or ingestion of municipal wastewater effluents is likely, disinfection may be necessary. Under these circumstances, UV irradiation appears to be superior to chlorination in terms of microbial quality and chemistry and toxicology. This advantage is particularly evident in effluents that contain appreciable quantities of ammonia-nitrogen or organic nitrogen.

Thermophilic aerobic treatment of a synthetic wastewater in a membrane-coupled bioreactor

Synthetic wastewater containing -lactose and gelatin was treated in a thermophilic membrane-coupled bioreactor (MBR). Thermophilic (>45°C) treatment represents a potentially advantageous process for high-temperature as well as high-strength industrial wastewaters susceptible to reactor autoheating. Thermophilic systems, however, generally support a nonflocculating biomass that resists conventional methods of cell separation from the treated wastewater. MBRs were applied to thermophilic treatment systems because bacterial cells can be retained regardless of cell aggregation. Thermophilic aerobic MBRs were successfully operated at high levels of biocatalyst and produced a better effluent quality than analogous thermophilic bioreactors without cell recycle. At a hydraulic residence time (HRT) of 13.1 h, the chemical oxygen demand (COD) of the membrane eluate improved from 760 mg l−1 (without cell recycle) to 160 mg l−1 (with cell recycle). Bacterial community shifts were detected by denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) -amplified 16S rRNA gene fragments — 6 of 13 bands disappeared within 2 days of MBR operation. A concomitant 40–50% reduction in physiological indicators of cell reactivity (RNA:protein; ATP:protein) was also observed. The specific activity of β-galactosidase and aminopeptidase, however, increased by 10–25%, indicating that there is a definite advantage to MBR operation at the highest biomass level possible. Nucleotide sequence analysis of 16S rDNA clones identified phylotypes from the low-G+C Gram-positive division and the β- and γ-subdivisions of Proteobacteria. Journal of Industrial Microbiology & Biotechnology (2001) 26, 203–209.

Effects of elevated temperature on bacterial community structure and function in bioreactors treating a synthetic wastewater

The impact of elevated temperature on bacterial community structure and function during aerobic biological wastewater treatment was investigated. Continuous cultures, fed a complex growth medium containing gelatin and α-lactose as the principal carbon and energy sources, supported mixed bacterial consortia at temperatures ranging from 25–65°C. These temperature- and substrate-acclimated organisms were then used as inocula for batch growth experiments in which the kinetics of microbial growth and substrate utilization, efficiency of substrate removal, and mechanism of substrate removal were compared as functions of temperature. Bacterial community analysis by denaturing gradient gel electrophoresis (DGGE) revealed that distinct bacterial consortia were supported at each temperature. The efficiency of substrate removal declined at elevated temperatures. Maximum specific growth rates and the growth yield increased with temperature from 25–45°C, but then decreased with further elevations in temperature. Thus, maximum specific substrate utilization rates did not vary significantly over the 40°C temperature range (0.64 ± 0.04 mg COD mg−1 dry cell mass h−1). A comparison of the degradation of the protein and carbohydrate portions of the feed medium revealed a lag in α-lactose uptake at 55°C, whereas both components were utilized simultaneously at 25°C. Journal of Industrial Microbiology & Biotechnology (2000) 24, 140–145.

Scanning electron microscope evaluation of rotating biological contactor biofilm

Abstract The use of rotating biological contactor (RBC) systems for wastewater treatment has recently drawn considerable interest. This attraction likely systems from the purported energy efficiency offered by these fixed-film biological reactors. A number of existing RBC applications have, however, encountered significant filamentous surface growth, particularly in cases involving high system loadings and/or reduced oxygen tension. In this study, scanning electron microscopy (SEM) was used to evaluate the composition of such an RBC film. The observed biofilm community was believed to comprise a stratified layering of Beggiatoa and Desulfovibrio microorganisms, both of which are involved in the metabolism of sulfur species.

Effects of disinfectants on wastewater effluent toxicity

Wastewater disinfection is most commonly achieved by halogenation, ozonation or ultraviolet (UV) irradiation. All of these processes are known to be capable of inducing chemical changes among non-microbial aqueous constituents, which can lead to changes in effluent toxicity behavior. Undiluted effluent samples from seven wastewater treatment facilities were exposed to commonly-applied disinfectants (chlorination/dechlorination, ozonation and UV irradiation) and assayed for their toxicity behavior; Ceriodaphnia dubia survival and reproduction were used as measures of chronic toxicity. The results of these assays confirmed the ability of all the disinfectants to alter toxicity response behavior. The toxicity responses demonstrated site-specificity and strong temporal variations. In general, when undisinfected effluent did not display toxicity, no toxicity was observed following any of the disinfection procedures; when toxicity was displayed in the undisinfected effluent, all disinfectants demonstrated the ability to alter toxicity, in most cases resulting in an increase. The changes in toxicological response attributable to disinfectant exposure followed the trend: chlorination/dechlorination > ozonation > UV irradiation, but substantial variations from this trend were evident in individual samples.

Light induced Nitrosomonas inhibition

The purpose of this study was to evaluate and verify the impact of light exposure on an enriched Nitrosomonas culture. Resting cells maintained under aerobic conditions without an exogenous ammonium-nitrogen source were fully inhibited within a 10 min period of ambient light contact (i.e. fluorescent and indirect natural room light). These cells were, however, protected against this inhibitive phenomenon during active respiration periods and during anoxic exposure conditions. Recovery, albiet partial, from such light induced inhibition was initiated after a “dark” contact period of approx. 2.5–3 h without exogenous ammonium presence. Research involving enriched or pure Nitrosomonas cultures (e.g. batch bioassay testing) should consequently be completed under environmental conditions designed to obviate this problem.