Respirometric evaluation of side-stream treatment of reject water as a source of nitrifying bacteria for main-stream activated sludge bioreactors.

A laboratory-scale bioreactor study was conducted to characterize differences in nitrification function in main-stream reactors due to bioaugmentation from side-stream reactors treating reject water. The objective was to evaluate how configuration of a suspended growth side-stream bioreactor impacts nitrification function in the main-stream bioreactor. A bioaugmentation effect was not observed in main-stream reactors operated at warm temperatures. Complete oxidation of ammonia to nitrate was observed in the bioaugmented and control main-stream reactors although nitrite accumulation was observed in each case. Furthermore, respirometry did not reveal superior kinetics in bioaugmented reactors operated at warm temperatures. At cold temperatures bioaugmentation may have stabilized ammonia oxidation in main-stream reactor B2 bioaugmented from a PFR side-stream. Complete ammonia oxidation was observed for most of cold period of operation in the main-stream bioreactor B2. Furthermore, respirometry revealed a higher rate of ammonia oxidation and more stable nitrite oxidation compared with the control bioreactor.

Ecological engineering of bioaugmentation from side-stream nitrification.

Wastewater treatment relies on careful integration of environmental engineering with microbial ecology. This would seem to be particularly the case when attempting to enhance survivability of organisms introduced from outside the main-stream reactor, i.e. bioaugmentation. Molecular biology tools were utilised in this study to assist in understanding the mechanisms of successful bioaugmentation. Molecular fingerprinting showed that side-stream reactor configuration strongly influenced ammonia-oxidising bacteria (AOB) community structure. In both lab-scale and full-scale systems, AOB communities in the side-stream and main-stream were very similar. The experimental systems revealed that a PFR side-stream produced greater diversity of AOB than a CSTR side-stream in a PFR main-stream system, whereas the full-scale side-stream resulted in essentially an AOB monoculture. Phylogenetic analysis revealed less diversity than molecular fingerprinting perhaps due to biases in the cloning/transformation procedure.

Modelling the competition of planktonic and sessile aerobic heterotrophs for complementary nutrients in biofilm reactor.

A comprehensive, simplified microbial biofilm model was developed to evaluate the impact of bioreactor operating parameters on changes in microbial population abundance. Biofilm simulations were conducted using three special cases: fully penetrated, internal mass transfer resistance and external mass transfer resistance. The results of model simulations showed that for certain operating conditions, competition for growth limiting nutrients generated oscillations in the abundance of planktonic and sessile microbial populations. These oscillations resulted in the violation of the competitive exclusion principle where the number of microbial populations was greater than the number of growth limiting nutrients. However, the operating conditions which impacted microbial community diversity were different for the three special cases. Comparing the results of model simulations for dispersed-growth, biofilms and bioflocs showed that oscillations and microbial community diversity were a function of competition as well as other key features of the ecosystem. The significance of the current study is that it is the first to examine competition as a mechanism for controlling microbial community diversity in biofilm reactors.

An ecology-based analysis of irreversible biofouling in membrane bioreactors.

To provide the first step towards a microbial ecology-based understanding of irreversible membrane biofouling, four laboratory-scale membrane bioreactors (MBRs) were operated to investigate the identity of bacterial populations highly correlated with irreversible membrane biofouling. The conventional MBR was divided into two separate experimental units. Unit one consisted of four suspended-growth, activated sludge, sequencing batch bioreactors treating a synthetic paper mill wastewater. Unit two consisted of a microfiltration membrane cell. Amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA) was used to compare the predominant bacterial populations in samples of mixed liquor and irreversibly bound to the membrane surface. The results of ARDRA showed a significant difference between the planktonic and sessile bacterial communities suggesting that irreversible biofouling of microfiltration membranes may be more highly correlated to specific bacterial populations rather than the total, bulk concentration of biomass. A custom-built mini-flow cell and light microscopy were used to visualise the early formation of biofilms by two pure cultures (Escherichia coli and Acinetobacter calcoaceticus) on membrane surfaces. The results confirmed that A. calcoaceticus was able to enhance the initiation of biofilm formation on microfiltration membranes.

Using microbial genomics to evaluate the effectiveness of silver to prevent biofilm formation.

Increasingly, monovalent silver cations are being considered as an alternative biocide in water distribution systems. The objective of this study was to understand how bacteria respond when challenged with silver. The approach used included cultivation of Escherichia coli in planktonic and sessile phases, exposing biomass to a solution of silver nitrate and evaluating genetic responses using commercial Affymetrix microarrays. Experimental results showed that lower pH enhanced silver toxicity in a dose-dependent manner. Sessile biomass demonstrated resistance to silver, and the microarray results indicated that the genetic mechanism for silver resistance was similar to the mechanism for copper resistance including upregulation of efflux pumps as well as upregulation of metal oxidoreductases. The gene, copA, a P-type ATPase efflux flux, was upregulated in response to silver exposure, and the gene of CusCFBA, a Cu(I) efflux pump, was also upregulated. The gene of CueO, a robust cuprous oxidase, was also upregulated and may have reduced silver toxicity through oxidation of silver ions. This study is significant because it provides baseline data to understand the genetic response of bacteria to silver biocides.

Microbial community development in a laboratory-scale nitrifying activated sludge system with input from a side-stream bioreactor treating digester supernatant.

Three laboratory-scale activated sludge treatment trains were operated to investigate the effect on biodiversity in plug flow (PFR) main-stream sewage treatment from input of biomass from side-stream reactors treating anaerobic digester supernatant. One train had a completely mixed (CSTR) side-stream reactor, one a PFR side-stream reactor, and the third train was a control that did not receive input from a side-stream reactor. Restriction endonucleases were used to digest polymerase chain reaction-amplified ammonia monooxygenase subunit A (amoA) genes in monthly samples from each reactor. Restriction fragment banding patterns from polyacrylimide gel electrophoresis indicated that the structure of the ammonia oxidizing bacteria (AOB) populations in all five reactors stabilized by the fourth month of operation and then did not vary subsequently. Furthermore, a dendrogram generated using the Jaccard distance showed that the AOB in each side-stream reactor was most similar to the main-stream reactor in the same train indicating that the AOB population in the side-stream reactor exerts a strong influence on the population in the main-stream reactor. Sequencing results indicated that Nitrosomonas europea, an r-strategist, was the dominant AOB in the PFR side-stream reactor, while Nitrosomonas europea and Nitrosomonas marina, a marine bacterium, were strongly represented in the CSTR side-stream reactor.

Elevated precursor 16S rRNA levels suggest the presence of growth inhibitors in wastewater.

Conventional activated sludge systems require bacteria to grow to avoid washout through decay and routine solids wasting. Recently we developed a procedure targeting precursor 16S ribosomal RNA to measure the in situ growth activity of phylogenetically defined microbial populations, and this procedure was used to study the growth of bacteria in activated sludge systems. The current study significantly expands this previous work by quantifying levels of precursor 16S ribosomal RNA within individual cells of pure cultures of bacteria exposed to various culture conditions. Initially, three ranges (Type I, Type II, and Type III) of precursor 16S ribosomal RNA levels were defined by whole cell fluorescence in situ hybridization of a pure culture of Acinetobacter calcoaceticusT prepared in three culture conditions. Low levels of precursor 16S ribosomal RNA (Type I) corresponded to a stationary phase culture prepared overnight in Luria-Bertani medium. Intermediate levels of precursor 16S ribosomal RNA (Type II) corresponded to a culture transferred into fresh Luria-Bertani medium, and high levels of precursor 16S ribosomal RNA (Type III) corresponded to a culture treated with the growth inhibiting antibiotic chloramphenicol. Subsequently, the abundance of individual cells of each Type were measured in four different pure cultures after exposure to 0.45-microm filtered primary effluent collected from four different conventional activated sludge treatment plants in Cincinnati, OH, USA. Individual cells of each Type were observed in the culture of A. calcoaceticusT exposed to each of the four primary effluents. Only Type I cells were observed in cultures of A. johnsoniiT, A. johnsonii strain 210a, and Escherichia coliT exposed to each of the four primary effluents. These results suggest that the growth of A. calcoaceticusT was inhibited by an unidentified component of filtered primary effluent present in each of the four wastewaters; whereas the growth of A. johnsoniiT, A. johnsonii strain 210a, and E. coliT were not inhibited. These results have significance for understanding the growth of phylogenetically defined microbial populations within activated sludge treatment systems. If the pattern of elevated p16S rRNA levels observed in A. calcoaceticusT is prevalent in many microbial populations in activated sludge systems, this may have implications for preventing washout of critical microbial populations that may be experiencing growth inhibition.

Who eats what? Classifying microbial populations based on diurnal profiles of rRNA levels.

Identifying the relationships between various bacterial populations and the substrates they consume is central to the understanding of population dynamics and to the development of process control in activated sludge. However, linking a heterotrophic population to its activity in situ is difficult because ribosomal RNA (rRNA) techniques, while allowing the rapid identification of populations, provide little information about their heterotrophic activity. Activated sludge models describe biodegradation kinetics by classifying substrates into two types: readily and slowly degradable substrates. Assuming that bacterial populations specialize in degrading one type of substrate, their growth rate should be affected differently if the COD loading rate varies diurnally as for a municipal activated sludge system. Modeling results suggested that the growth rates of populations consuming readily degradable substrates vary according to variations in COD loading rate. On the other hand, the growth rates of populations consuming slowly degradable substrates do not change despite the variation in COD loading rate. Since the cellular rRNA level is positively correlated with the growth rate, we hypothesized that the rRNA levels of some populations in municipal activated sludge should increase throughout the day, while they should stay constant for other populations. This hypothesis was verified by monitoring the rRNA level of Acinetobacter (a model population consuming readily degradable substrates) and Gordonia (a model population consuming slowly degradable substrates) in the mixed liquor of a full-scale municipal activated sludge reactor for three weeks.

Quantifying the impact of wastewater micronutrient composition on in situ growth activity of Acinetobacter spp.

Batch growth studies with pure cultures of Acinetobacter johnsoniiT and Acinetobacter johnsonii strain 210 on various media formulations were used to examine the effects of micronutrient composition on the growth rate of microbial populations in wastewater treatment systems. On nutrient rich Luria-Bertani medium, both strains of A. johnsonii grew with a doubling time of approximately 30 min. On a defined, minimal medium with acetate as the sole carbon source, the doubling time of A. johnsoniiT was 9.62 h and the doubling time of strain 210a was 2.6 h. Using a synthetic wastewater as a growth medium, the type strain had a doubling time of 56 h and strain 210a had a doubling time of 9.62 h. The concentration and redox state of iron appeared to be the principle growth limiting factors with higher doubling times occurring in media containing ferric iron as compared to ferrous iron. Additionally, grab samples from batch growth experiments were analyzed with oligonucleotide hybridization probes targeting the mature 16S ribosomal RNA (rRNA) and precursor 16S rRNA of Acinetobacter spp. Results showed that the precursor 16S rRNA levels responded more rapidly and to a greater extent than total 16S rRNA levels to changes in the micronutrient composition of the growth media. Precursor 16S rRNA levels increased in both strains when overnight cultures were diluted with fresh media and when micronutrient supplements were added to growing cultures. Our results show that the micronutrient composition of the influent wastewater can have a significant impact on the microbial community structure in wastewater treatment systems.

Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems.

Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457-4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3' region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems.