Site Specific Relationships between COVID-19 Cases and SARS-CoV-2 Viral Load in Wastewater Treatment Plant Influent.

Wastewater based epidemiology (WBE) has become an important tool during the COVID-19 pandemic, however the relationship between SARS-CoV-2 RNA in wastewater treatment plant influent (WWTP) and cases in the community is not well-defined. We report here the development of a national WBE program across 28 WWTPs serving 50% of the population of Scotland, including large conurbations, as well as low-density rural and remote island communities. For each WWTP catchment area, we quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. Daily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation (ρ > 0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from 25 cases for larger plants to a single case in smaller plants. SARS-CoV-2 viral RNA load can be used to predict the number of cases detected in the WWTP catchment area, with a clear statistically significant relationship observed above site-specific case thresholds.

Seagrasses in tropical Australia, productive and abundant for decades decimated overnight.

Seagrass ecosystems provide unique coastal habitats critical to the life cycle of many species. Seagrasses are a major store of organic carbon. While seagrasses are globally threatened and in decline, in Cairns Harbour, Queensland, on the tropical east coast of Australia, they have flourished. We assessed seagrass distribution in Cairns Harbour between 1953 and 2012 from historical aerial photographs, Google map satellite images, existing reports and our own surveys of their distribution. Seasonal seagrass physiology was assessed through gross primary production, respiration and photosynthetic characteristics of three seagrass species, Cymodocea serrulata, Thalassia hemprichii and Zostera muelleri. At the higher water temperatures of summer, respiration rates increased in all three species, as did their maximum rates of photosynthesis. All three seagrasses achieved maximum rates of photosynthesis at low tide and when they were exposed. For nearly six decades there was little change in seagrass distribution in Cairns Harbour. This was most likely because the seagrasses were able to achieve sufficient light for growth during intertidal and low tide periods. With historical data of seagrass distribution and measures of species production and respiration, could seagrass survival in a changing climate be predicted? Based on physiology, our results predicted the continued maintenance of the Cairns Harbour seagrasses, although one species was more susceptible to thermal disturbance. However, in 2011 an unforeseen episodic disturbance - Tropical Cyclone Yasi - and associated floods lead to the complete and catastrophic loss of all the seagrasses in Cairns Harbour.

Enumerating viruses by using fluorescence and the nature of the nonviral background fraction.

Bulk fluorescence measurements could be a faster and cheaper way of enumerating viruses than epifluorescence microscopy, flow cytometry, or transmission electron microscopy (TEM). However, since viruses are not imaged, the background fluorescence compromises the signal, and we know little about its nature. In this paper the size ranges of nucleotides that fluoresce in the presence of SYBR gold were determined for wastewater and a range of freshwater samples using a differential filtration method. Fluorescence excitation-emission matrices (FEEMs) showed that >70% of the SYBR fluorescence was in the <10-nm size fraction (background) and was not associated with intact viruses. This was confirmed using TEM. The use of FEEMs to develop a fluorescence-based method for counting viruses is an approach that is fundamentally different from the epifluorescence microscopy technique used for enumerating viruses. This high fluorescence background is currently overlooked, yet it has had a most pervasive influence on the development of a simple fluorescence-based method for quantifying viral abundance in water.

Fluorescence instrument for in situ monitoring of viral abundance in water, wastewater and recycled water.

In a world of advanced molecular methods quantifying viruses in water remains one of the most inefficient and costly. Using a general molecular DNA/RNA probe - SYBR Gold combined with differential filtration a fast, cost effective and sensitive method is presented to determine the concentration of viruses in water in situ or on-line. The approach differentiates the nucleotide size fractions that are stained with SYBR Gold to show only those associated with Viral DNA and RNA. There was a linear relationship between the fluorescence maxima for SYBR Gold added to wastewater and viral numbers determined with direct counting using epifluorescent microscopy (r(2)=0.97) and for a range of diverse natural water samples (r(2)=0.86). The method was applied to water from the tropics and Antarctica, marine and freshwater environments where natural viral abundances ranged from 10(6) to 10(8) virusesmL(-1). The method takes into account the background fluorescence that represented 70% of total fluorescence and any auto-fluorescence due to other dissolved organic carbon. While DNAse II lowered the background fluorescence associated with free DNA and RNA it could not be eliminated. The technique presented is suitable for monitoring in situ viral numbers in natural water bodies and engineered water treatment processes. This on-line viral monitoring design has the potential to replace human viral pathogen indicators.

Environmental influences on virus-host interactions in an Australian subtropical reservoir.

Viral and prokaryotic interactions in freshwaters have been investigated worldwide but there are few temporal studies in the tropics and none in the sub-tropics. In this 10-month study, we examined temporal changes in virus-host interactions and viral life cycles (lytic versus lysogenic) in relation to the prevailing environmental conditions in a subtropical water reservoir (Wivenhoe) in southeast Queensland, Australia. Heterotrophic prokaryotes and picocyanobacteria were positively correlated with concentrations of viruses throughout the study, indicating the presence of both bacteriophages and cyanophages in the reservoir. The percentage of heterotrophic prokaryotes and picocyanobacteria containing intracellular viruses (FVIC) ranged between 0.2% and 2.4% and did not vary significantly over the 10-month study, whereas lysogenic heterotrophic prokaryotes were only detected in the drier months of June and July. Spearman rank correlation analysis showed that the oxidative-reduction potential (ORP) of the water reservoir influenced the concentrations of viruses, heterotrophic prokaryotes and picocyanobacteria significantly, with low ORP offering a favourable environment for these components. There was a negative relationship between FVIC and rainfall suggesting the associated run-off altered virus-host interactions. Overall, our study provides novel information and inferences on how virus-host interactions in subtropical freshwaters might respond to changes in precipitation predicted to occur with global climate change.

Bacterial activity in plant (Schoenoplectus validus) biofilms of constructed wetlands.

Biofilm-bacterial communities have been exploited in the treatment of wastewater in 'fixed-film' processes. Our understanding of biofilm dynamics requires a quantitative knowledge of bacterial growth-kinetics in these microenvironments. The aim of this paper was to apply the thymidine assay to quantify bacterial growth without disturbing the biofilm on the surfaces of emergent macrophytes (Schoenoplectus validus) of a constructed wetland. The isotope was rapidly and efficiently taken-up and incorporated into dividing biofilm-bacteria. Isotope diffusion into the biofilm did not limit the growth rate measurement. Isotope dilution was inhibited at >12 µM thymidine. Biofilm-bacterial biomass and growth rates were not correlated to the plant surface area (r(2) < 0.02). The measurements of in situ biofilm-bacterial growth rates both displayed, and accommodated, the inherent heterogeneity of the complex wetland ecosystem. Biofilm-bacterial respiratory activities, measured using the redox dye CTC, and growth rates were measured simultaneously. The dye did not interfere with bacterial growth. Biofilm-bacterial specific growth rates ranged from 1.4 ± 0.6 d(-1) to 3.3 ± 1.3 d(-1). In the constructed wetlands of this study biofilm-bacterial specific growth rates, compared to those of natural ecosystems, could be markedly improved through changes in wetland design that increased bacterial respiration while minimising biofilm growth.

Sensitive and meaningful measures of bacterial metabolic activity using NADH fluorescence.

Successful biological wastewater treatment depends on bacterial metabolic activity. Commercial fluorimeters are designed to monitor this activity using the native fluorescence of Nicotinamide Adenine Dinucleotide [NADH]. However, fluorescence measurements in wastewater treatment plants remain scarce due to difficulties with interpreting fluorescence data. This paper shows that fluorescence probe measurements taken from wastewater do not represent bacterial cell metabolic activity because intracellular NADH is likely swamped by the stable extracellular NADH fraction. Thus, a simple filtration/extraction/centrifugation method was developed to collect the bacterial cells, extract the intracellular NADH using heat treatment in Tris buffer and collect the purified intracellular NADH fraction. NADH standards were used to quantify NADH from the unknown wastewater samples where limits of detection were between 1 nmol mL(-1) and 0.35 micromol mL(-1). Fluorescence of [NADH] greater than 0.35 micromol mL(-1) was self-quenched. At high pH's NADH was stable outside the cell. NADH was stable at neutral and basic pH ranges of pH 7 to 11, but declined proportionally below a pH of 7. Since commercially available fluorescence probes used for measuring NADH are more likely detecting extracellular NADH, separating bacterial cells from water samples followed by NADH extraction was essential to distinguish intracellular and extracellular [NADH]. Here we have proposed three simple steps to meaningful measures of bacterial metabolic activity based on the autofluorescence of NADH. The three simple steps to getting it right are Future development of an on-line monitoring system based on these three steps is achievable with a little ingenuity.

A quantitative measure of nitrifying bacterial growth.

Nitrifying bacteria convert ammonia (NH3) to nitrate (NO3-) in a nitrification reaction. Methods to quantitatively separate the growth rate of these important bacterial populations from that of the dominant heterotrophic bacteria are important to our understanding of the nitrification process. The changing concentration of ammonia is often used as an indirect measure of nitrification but ammonification processes generate ammonia and confound this approach while heterotrophs remove nitrate via denitrification. Molecular probe methods can tell us what proportion of the microbial community is nitrifying bacteria but not their growth rate. The technique proposed here was able to quantify the growth rate of the nitrifying bacterial populations amidst complex ecological processes. The method incubates [methyl-3H] thymidine with water samples in the presence and absence of an inhibitor of nitrification-thiourea. The radioactively labeled DNA in the growing bacteria was extracted. The rate of incorporation of the label into the dividing bacterial DNA was used to determine bacterial growth rate. Total bacterial community growth rates in full-scale and pilot-scale fixed-film nitrifying reactors and an activated sludge reactor were 2.1 x 10(8), 4.1 x 10(8) and 0.4 x 10(8)cell ml(-1)d(-1), respectively; the growth rate of autotrophic-nitrifying bacteria was 0.7 x 10(8), 2.6 x 10(8) and 0.01 x 10(8)cell ml(-1)d(-1), respectively. Autotrophic-nitrifying bacteria contributed 30% and 60% of the total bacterial community growth rate in the nitrifying reactors whereas only 2% was observed in the activated sludge reactor that was not designed to nitrify. The rates of ammonia loss from the nitrifying reactors corresponded to the rate of growth of the nitrifying bacteria. This method has the potential to more often identify factors that enhance or limit nitrifying processes in both engineered and natural aquatic environments.

A rapid, direct measurement of bacterial growth rate in anaerobic wastewater treatment.

Reliable design and operation of biological wastewater treatment systems demand robust models of biological degradation processes. However, methods to directly measure key bacterial growth kinetics have not been readily available. Those methods that are available rely on the classic measurement of aerobic respiration using oxygen uptake take rates. This paper shows how the thymidine assay can be used as a rapid and direct measurement of bacterial specific growth rates (mu) in situ for an anaerobic treatment process, independent of aerobic respiration. A filtration-based assay is applied and evaluated a dispersed-phase high-rate anaerobic treatment process, with results obtained in less than an hour. The chemical oxygen demand (COD) biomass in the reactor was 0.52 kg COD m(-3) and the specific growth rate of these anaerobic bacteria was 0.8 +/- 0.2 d(-1). It took the bacterial populations 21.6 hours to double. This is an important advancement from existing methods that use aerobic respiration as a pseudo measurement of bacterial specific growth rates. The method allows rapid and direct measures of microbial growth rates for anaerobic treatment processes.

Identification of cyanophage Ma-LBP and infection of the cyanobacterium Microcystis aeruginosa from an Australian subtropical lake by the virus.

Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium Microcystis aeruginosa (host). M. aeruginosa was isolated, cultured, and characterized from a subtropical monomictic lake-Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the M. aeruginosa host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h(-1); generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of M. aeruginosa cell lysis (r(2) = 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the Podoviridiae group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 x 10(4) cyanophage x ml(-1), representing 0.23% of the natural viral population of 2.46 x 10(7) x ml(-1). Our results showed that this cyanophage could be a major natural control mechanism of M. aeruginosa abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production.