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.

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.

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.

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.

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.