Low-dissolved-oxygen nitrification in tropical sewage: an investigation on potential, performance and functional microbial community.

Intensive aeration for nitrification is a major energy consumer in sewage treatment plants (STPs). Low-dissolved-oxygen (low-DO) nitrification has the potential to lower the aeration demand. However, the applicability of low-DO nitrification in the tropical climate is not well-understood. In this study, the potential of low-DO nitrification in tropical setting was first examined using batch kinetic experiments. Subsequently, the performance of low-DO nitrification was investigated in a laboratory-scale sequential batch reactor (SBR) for 42 days using real tropical sewage. The batch kinetic experiments showed that the seed sludge has a relatively high oxygen affinity. Thus, the rate of nitrification was not significantly reduced at low DO concentrations (0.5 mg/L). During the operation of the low-DO nitrification SBR, 90% of NH4-N was removed. The active low-DO nitrification was mainly attributed to the limited biodegradable organics in the sewage. Fluorescence in-situ hybridisation and 16S rRNA amplicon sequencing revealed the nitrifiers were related to Nitrospira genus and Nitrosomonadaceae family. Phylogenetic analysis suggests 47% of the operational taxonomic units in Nitrospira genus are closely related to a comammox bacteria. This study has demonstrated active low-DO nitrification in tropical setting, which is a more sustainable process that could significantly reduce the energy footprint of STPs.

Agreement between amoA gene-specific quantitative PCR and fluorescence in situ hybridization in the measurement of ammonia-oxidizing bacteria in activated sludge.

Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.

Nitrification in hybrid bioreactors treating simulated domestic wastewater.

AIM: To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). METHODS AND RESULTS: Three bench-scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5-day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2.5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia-oxidizing bacteria, AOB; nitrite-oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB-NOB activity were less apparent, resulting in incomplete nitrification. CONCLUSIONS: Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.

Production of hydrogen from domestic wastewater in a pilot-scale microbial electrolysis cell.

Addressing the need to recover energy from the treatment of domestic wastewater, a 120-L microbial electrolysis cell was operated on site in Northern England, using raw domestic wastewater to produce virtually pure hydrogen gas (100 ± 6.4 %) for a period of over 3 months. The volumetric loading rate was 0.14 kg of chemical oxygen demand (COD) per cubic metre per day, just below the typical loading rates for activated sludge of 0.2-2 kg COD m(-3) day(-1), at an energetic cost of 2.3 kJ/g COD, which is below the values for activated sludge 2.5-7.2 kJ/g COD. The reactor produced an equivalent of 0.015 LH(2)L(-1) day(-1), and recovered around 70 % of the electrical energy input with a coulombic efficiency of 55 %. Although the reactor did not reach the breakeven point of 100 % electrical energy recovery and COD removal was limited, improved hydrogen capture and reactor design could increase the performance levels substantially. Importantly, for the first time, a 'proof of concept' has been made, showing that this technology is capable of energy capture as hydrogen gas from low strength domestic wastewaters at ambient temperatures.

Determination of the internal chemical energy of wastewater.

The wastewater industry is facing a paradigm shift, learning to view domestic wastewater not as a waste stream which needs to be disposed of but as a resource from which to generate energy. The extent of that resource is a strategically important question. The only previous published measurement of the internal chemical energy of wastewater measured 6.3 kJ/L. It has long been assumed that the energy content in wastewater relates directly to chemical oxygen demand (COD). However there is no standard relationship between COD and energy content. In this study a new methodology of preparing samples for measuring the internal chemical energy in wastewater is developed, and an analysis is made between this and the COD measurements taken. The mixed wastewater examined, using freeze-drying of samples to minimize loss of volatiles, had 16.8 kJ/L, while the domestic wastewater tested had 7.6 kJ/L nearly 20% higher than previously estimated. The size of the resource that wastewater presents is clearly both complex and variable but is likely to be significantly greater than previously thought. A systematic evaluation of the energy contained in wastewaters is warranted.

Nitrification-denitrification in waste stabilisation ponds: a mechanism for permanent nitrogen removal in maturation ponds.

A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.

Enhanced estrogen removal in activated sludge processes through the optimization of the hydraulic flow pattern.

The removal of ß-estradiol (E2) and α-ethinylestradiol (EE2) in biological wastewater treatment plants (WWTP) would need to be improved in order to comply with prospective Environmental Quality Standards (EQS) of 0.4 and 0.035 ng.L-1 respectively. The effluent concentration of a micropollutant in an activated sludge process is a function of the removal rate, the hydraulic retention time (HRT) and the flow pattern, which is usually overlooked. In order to better understand this aspect, we carried out tracer studies in eight WWTPs in the UK and found that relatively modest changes in aeration tanks would translate into tangible improvements in their flow pattern. We further evaluated the degradation rates for E1 (estrone), E2, E3 (estriol) and EE2 in each WWTP and we estimated that the modification of the flow pattern would be sufficient to place effluent concentrations of E2 (23.2 L∙gVSS-1∙d-1

Coupled virus - bacteria interactions and ecosystem function in an engineered microbial system.

Viruses are thought to control bacterial abundance, affect community composition and influence ecosystem function in natural environments. Yet their dynamics have seldom been studied in engineered systems, or indeed in any system, for long periods of time. We measured virus abundance in a full-scale activated sludge plant every week for two years. Total bacteria and ammonia oxidising bacteria (AOB) abundances, bacterial community profiles, and a suite of environmental and operational parameters were also monitored. Mixed liquor virus abundance fluctuated over an order of magnitude (3.18 × 108-3.41 × 109 virus's mL-1) and that variation was statistically significantly associated with total bacterial and AOB abundance, community composition, and effluent concentrations of COD and NH4+- N and thus system function. This suggests viruses play a far more important role in the dynamics of activated sludge systems than previously realised and could be one of the key factors controlling bacterial abundance, community structure and functional stability and may cause reactors to fail. These findings are based on statistical associations, not mechanistic models. Nevertheless, viral associations with abiotic factors, such as pH, make physical sense, giving credence to these findings and highlighting the role that physical factors play in virus ecology. Further work is needed to identify and quantify specific bacteriophage and their hosts to enable us to develop mechanistic models of the ecology of viruses in wastewater treatment systems. However, since we have shown that viruses can be related to effluent quality and virus quantification is simple and cheap, practitioners would probably benefit from quantifying viruses now.

Presence and activity of ammonia-oxidising bacteria detected amongst the overall bacterial diversity along a physico-chemical gradient of a nitrifying wastewater treatment plant.

We wished to discover if we could gain greater insights into how biological treatment plants function by contrasting the presence and activity of the most abundant Bacteria in plug flow and completely mixed activated sludge plants. Presence was assessed by amplifying 16S rRNA gene fragments (using PCR) and activity by amplifying native 16S rRNA, using reverse-transcriptase PCR (RT-PCR), using Bacteria-specific primers. The amplified sequences were compared using denaturing gradient gel electrophoresis (DGGE). The plug flow plant exhibited a strong physico-chemical gradient with an initial anoxic zone, whilst the two completely mixed reactors did not. Similarities were observed between the profile of the banding pattern for presence and activity. However, in the plug flow reactor one prominent band was detected in the active population (16S rRNA) but was absent from the corresponding profile of the 16S rRNA gene. Sequencing of this band revealed its identity as a Nitrosomonas-like sequence. The intensity of the 16S rRNA sequenced varied along the physico-chemical gradient of the plug-flow reactor in a manner that coincided with the growth of ammonia-oxidising bacteria (AOB) and the loss of ammonia. This band was also absent from the completely mixed reactors, although significant numbers of AOB were detected in all systems ( approximately 10(6)-10(8)cells ml(-1)) by fluorescence in situ hybridisation (FISH). An abundant and highly active AOB population was present in the anoxic zone of the plug-flow reactor where up to 60% of the total ammonia was removed. An examination of nitrogen removal/production rates, together with the above data, reveal that complex nitrogen removal processes occur in this system. These data also enabled the calculation of a specific in situ growth rate for the AOB as 0.12h(-1).

A universal threshold concept for hydrophobic mycolata in activated sludge foaming.

Recent studies using quantitative fluorescence in situ hybridization (FISH) have supported the principle that there are mycolata concentration thresholds, above which foaming is likely to occur. In this study, we surveyed 14 wastewater treatment plants (WWTPs) in the UK, using quantitative FISH, to establish that the principle of a mycolata threshold (2 x 10(6) mycolata cells ml(-1) mixed liquor suspended solids) is an empirical though widely held value. In addition, we designed, optimized and applied probes for members of the less hydrophobic mycolata genera Corynebacterium and Dietzia, to show that these organisms dominated the mycolata populations in two non-foaming WWTPs where the mycolata concentrations were above the threshold value. We propose that the mycolata threshold value is only applicable to hydrophobic members of the mycolata.

Temperature, inocula and substrate: Contrasting electroactive consortia, diversity and performance in microbial fuel cells.

The factors that affect microbial community assembly and its effects on the performance of bioelectrochemical systems are poorly understood. Sixteen microbial fuel cell (MFC) reactors were set up to test the importance of inoculum, temperature and substrate: Arctic soil versus wastewater as inoculum; warm (26.5°C) versus cold (7.5°C) temperature; and acetate versus wastewater as substrate. Substrate was the dominant factor in determining performance and diversity: unexpectedly the simple electrogenic substrate delivered a higher diversity than a complex wastewater. Furthermore, in acetate fed reactors, diversity did not correlate with performance, yet in wastewater fed ones it did, with greater diversity sustaining higher power densities and coulombic efficiencies. Temperature had only a minor effect on power density, (Q10: 2 and 1.2 for acetate and wastewater respectively): this is surprising given the well-known temperature sensitivity of anaerobic bioreactors. Reactors were able to operate at low temperature with real wastewater without the need for specialised inocula; it is speculated that MFC biofilms may have a self-heating effect. Importantly, the warm acetate fed reactors in this study did not act as direct model for cold wastewater fed systems. Application of this technology will encompass use of real wastewater at ambient temperatures.

Towards the design of diversity: stochastic models for community assembly in wastewater treatment plants.

We believe that the engineering of wastewater treatment systems would be improved if we could predict and manipulate the composition, that is, the diversity of such systems. This ability would complement our established ability to predict the size of treatment communities. The theoretically based design of diversity in wastewater treatment systems is held by some to be unnecessary and by others to be impossible. In this paper we point to important phenomena in wastewater treatment, such as acclimation, adaptation and functional redundancy that would benefit from the ability to confidently design diversity. We set out a rationale and a mathematical framework for a stochastic approach to the design of diversity and show that, in principle, this approach works with two important functional groups in wastewater treatment. The implications for design and theory are briefly discussed.

Flow cytometric quantification of viruses in activated sludge.

Viruses may play a critical role in the microbial dynamics of activated sludge systems; however the difficulty of their quantification makes long term and large scale studies costly, timely and challenging. Thus a flow cytometric protocol was optimised and employed to determine virus abundance in activated sludge samples. The best flow cytometry signature and highest virus count was obtained by separating the indigenous floc-associated viruses using Tween 80 and sodium pyrophosphate, diluting the sample with Tris-EDTA and staining with SYBR Green II. Using the optimised protocol viral concentrations from 25 activated sludge plants were determined, with average concentrations of 2.35 × 109 mL⁻¹ observed. Direct counts by transmission electron microscopy were highly correlated with flow cytometric counts (p = <0.05 and r² = 0.77), with concentrations from both quantification methods comparable at the order of magnitude level. The high counting efficiency, ease of preparation and rapidity and reproducibility of analysis makes flow cytometric quantification of viruses in activated sludge ideal for routine investigation and thus invaluable in unravelling the complexity of phage host interactions in such systems.

Fate of Cryptosporidium oocysts in an immobilised titanium dioxide reactor with electric field enhancement.

We have undertaken simple proof of principle experiments to find out if electric field enhanced photo-oxidation using immobilised titanium dioxide will damage Cryptosporidium oocysts. Using a simple Petri dish reactor and two forms of immobilised titanium catalyst (sol-gel and thermal-film) we have tested the ability of this technology to affect Cryptosporidium oocysts permeability assessed by propidium iodide exclusion. Test and control reactor runs were significantly different (P = 0.007). The thermal-film reactor had the greatest effect (approximately 27% of the seed) and was statistically distinguishable from the sol-gel reactor and the controls. The sol-gel reactor showed an increase in oocyst permeability, but was not statistically distinguishable from one of the controls. The enhanced performance of the thermal film reactor is attributed to the superior conversion of photochemical holes to hydroxyl radicals at the surface of this catalyst.

Are filamentous mycolata important in foaming?

In a previous study, we showed that there was a significant increase in mycolata numbers associated with foaming events at a wastewater treatment site possessing three activated sludge plants. In this paper, we demonstrate that branched filamentous mycolata were a minor proportion of the mycolata morphotypes present in those activated sludge plants, accounting for less than 21% of the mycolata population in the mixed liquor and foam samples examined. In most samples examined, the number of filamentous mycolata was negligible compared to the number of other mycolata morphotypes present. Furthermore, filamentous mycolata did not contribute to any of the significant differences in mycolata concentration observed between foaming and non-foaming situations (P<0.01). These findings suggest that conventional microscopic examination for monitoring mycolata populations in foaming plants may be misleading and should be used with caution.

Variable photosynthetic characteristics in waste stabilisation ponds.

Algae play several key roles in waste stabilisation ponds. A model has been developed to predict algal concentration in waste stabilisation ponds, in which the relationship between photosynthesis and underwater light availability is central. One equation was selected from several alternative expressions that describe this relationship. The selected equation consisted of four photosynthetic parameters. A field sampling programme was designed to investigate the relationships between the photosynthetic parameters and the pond environment. Although initial regression analyses were unsuccessful, distinct diurnal variations were revealed in two key photosynthetic parameters, related to an inverse variation in chlorophyll a concentration. This led to the derivation of a dynamic feedback hypothesis which challenges the classic assumption in algal modelling of constant photosynthetic parameters.

The effect of C/N ratio on ammonia oxidising bacteria community structure in a laboratory nitrification-denitrification reactor.

A laboratory scale reactor operated as a single sludge, denitrification-nitrification bioreactor (DNB), was fed a synthetic wastewater. The effect of the C/N ratio of the influent on the structure of beta-proteobacterial autotrophic ammonia-oxidizing bacterial (AOB) communities was determined by DGGE analysis of 16S rRNA gene fragments amplified using a range of AOB-selective primers. Fluorescence in situ hybridisation (FISH) was used to determine quantitative changes in the AOB communities. When operated at a C/N ratio of 2 the DNB was effective in nitrogen removal and nitrification was measured at approximately 1.0 mg NH4+-N/g dry wt/h. Altering the C/N ratio to 5 resulted in a 50% reduction in nitrification rates. Nitrification was restored to its original level when the C/N ratio was returned to 2. AOB were detected by DGGE analysis of samples from the DNB under all operating conditions but the changes in C/N ratio and nitrification rates were accompanied by changes in the community structure of the AOB. However, quantitative FISH analysis indicated that beta-proteobacterial AOB were only present in high numbers (ca. 10(8) cells/ml) under the original operating conditions with a C/N ratio of 2. Beta-proteobacterial AOB could not be detected by FISH when the C/N ratio was 5. When nitrification activity was restored by returning the C/N ratio to 2, beta-proteobacterial AOB were still not detected and it is likely that either beta-proteobacterial AOB were not responsible for ammonia oxidation or that beta-proteobacterial AOB that did not contain the target sites for the range of 4 AOB selective probes used, were present in the reactor.

A comparitive study of ammonia-oxidizing bacteria in lab-scale industrial wastewater treatment reactors.

The diversity and community structure of the beta-proteobacterial ammonia oxidising bacteria (AOB) in a range of different lab-scale industrial wastewater treatment reactors were compared. Three of the reactors treat waste from mixed domestic and industrial sources whereas the other reactor treats waste solely of industrial origin. PCR with AOB selective primers was combined with denaturing gradient ge electrophoresis to allow comparative analysis of the dominant AOB populations and the phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing or direct sequencing of bands excised from DGGE gels. Different AOB were found within and between different reactors. All AOB sequences identified were grouped within the genus Nitrosomonas. Within the lab-scale reactors there appeared to be selection for a low diversity of AOB and predominance of a single AOB population. Furthermore, the industrial input in both effluents apparently selected for salt tolerant AOB, most closely related to Nitrosococcus mobilis and Nitrosomonas halophila.

A comparison of autotrophic ammonia-oxidizing bacteria in full- and laboratory-scale wastewater treatment reactors.

Lab-scale reactors are commonly used to simulate full-scale plants as they permit the effects of defined experimental perturbations to be evaluated. Ideally, lab- and full-scale reactors should possess similar microbial populations. To determine this we compared the diversity of the beta-proteobacterial autotrophic ammonia-oxidising bacteria (AOB) in a full-scale and lab-scale biological aerated filter (BAF) using PCR with AOB selective primers combined with denaturing gradient gel electrophoresis (DGGE). PCR amplified 16S rRNA gene fragments from the nitrification unit of the lab-and full-scale BAF were subjected to cloning and sequencing to determine the phylogenetic affiliation of the AOB. A high degree of comparability between the lab-and full-scale BAF was observed with respect to AOB populations. However minor differences were apparent. The importance of these minor constituents in the overall performance of the reactor is unknown. Nonetheless the lab-scale reactor in this study did appear to reflect the dominant AOB community within the full-scale equivalent.

Factors affecting current production in microbial fuel cells using different industrial wastewaters.

This study evaluated how different types of industrial wastewaters (bakery, brewery, paper and dairy) affect the performance of identical microbial fuel cells (MFCs); and the microbial composition and electrochemistry of MFC anodes. MFCs fed with paper wastewater produced the highest current density (125 ± 2 mA/m(2)) at least five times higher than dairy (25 ± 1 mA/m(2)), brewery and bakery wastewaters (10 ± 1 mA/m(2)). Such high current production was independent of substrate degradability. A comprehensive study was conducted to determine the factor driving current production when using the paper effluent. The microbial composition of anodic biofilms differed according to the type of wastewater used, and only MFC anodes fed with paper wastewater showed redox activity at -134 ± 5 mV vs NHE. Electrochemical analysis of this redox activity indicated that anodic bacteria produced a putative electron shuttling compound that increased the electron transfer rate through diffusion, and as a result the overall MFC performance.