Measuring photosynthesis of both oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) fluorometry in wastewater ponds

Authors

P. Chandaravithoon, R. J. Ritchie & J. W. Runcie

Oxygenic photosynthesis can be measured easily using O₂ or CO₂ gas exchange, oxygen electrodes, Winkler titration, ¹⁴CO₂-fixation and by PAM (pulse amplitude modulation) fluorometry. PAM estimates the photosynthetic electron transport rate (ETR) by measuring the variable fluorescence of chlorophyll (Chl) a (> 695 nm) induced by absorption of blue or red light. Anoxygenic photosynthetic bacteria (APB) do not use water as an electron source and are typically photoheterotrophic rather than photoautotrophic and so ¹⁴CO₂ fixation is a misleading estimate of photosynthetic electron transport in APB photosynthesis. In vivo bacteriochlorophyll a (BChl a) absorbs blue light similar to Chl a but its characteristic longer-wavelength absorption is in the infrared and fluorescence is at > 800 nm. Blue light-induced PAM fluorescence can be used to measure the ETR in purple non-sulphur anoxygenic photobacteria and purple sulphur photobacteria because their RC-2 type BChl a complexes fluoresce similarly to PSII but at longer wavelengths than Chl a. Conventional PAM fluorometers using blue light cannot readily distinguish between oxygenic and RC-2 type anoxygenic photosynthesis because they use a simple > 700 nm highpass filter in front of the detector diode. We modified one fluorometer to use a 695–750-nm bandpass filter to measure Chl a fluorescence from PS-II, representing oxygenic photosynthesis. Similarly, we modified another fluorometer to use a highpass filter (> 830 nm) to measure BChl a fluorescence, representing anoxygenic photosynthesis. However, the fluorescence bands of Chl a and BChl a were found to be too wide to unambiguously distinguish between oxygenic and anoxygenic photosynthesis purely by fluorometry. Treatment with the specific PS-II inhibitor DCMU (Diuron) did enable discrimination of the two types of photosynthesis in a mixture of oxygenic and anoxygenic organisms. Ecological niches made up of both oxygenic and anoxygenic organisms such as microbial mats and hypereutrophic environments such as sewage ponds, wastewater ponds and prawn farm ponds are much more common than often realized. Anoxygenic photosynthesis in such systems is significant yet largely unquantified.

 

Internal loading in stormwater ponds as a phosphorus source to downstream waters

Authors

Vinicius J. Taguchi Tyler A. Olsen Poornima Natarajan Ben D. Janke John S. Gulliver Jacques C. Finlay Heinz G. Stefan

We assessed the prevalence and causes of sediment phosphorus (P) release within urban stormwater ponds, a process that may reduce P removal by sedimentation. Data collected from surface water of 98 urban stormwater ponds in Minnesota showed that nearly 40% had median summer total P concentrations in excess of average stormwater runoff (0.38 mg L⁻¹), implying effects of internal loading. We sampled seven ponds more intensively and found four were strongly stratified with persistent hypolimnetic anoxia, despite mean depths <2 m. Sediment core incubations revealed that, unlike in most lakes, both labile organic P (NaOH minus persulfate extractions) and redox‐sensitive P (NH₄Cl and Na₂S₂O₄ extractions) contribute to P release. Together, these analyses suggest P accumulated in stormwater ponds is highly susceptible to internal release and potentially contributes to downstream eutrophication. Understanding how frequently these conditions occur and how they affect different P forms is vital to improving pond design and management.

Flow cytometry for rapid characterisation of microbial community dynamics in waste stabilisation ponds

Authors

Liah X. Coggins, Irma Larma, Amy Hinchliffe, Ruben Props, Anas Ghadouani

Algal and bacterial communities play a major role in the treatment performance and efficiency of waste stabilisation ponds (WSPs); however, the study of these WSP microbial communities has been challenging. Flow cytometry (FCM) has been used widely as a rapid, culture-independent method of characterising algae and/or bacteria in a range of freshwater and marine environments, and in conventional wastewater treatment processes, but its application to WSP wastewater has been underexplored. In this study, a method for the characterisation of both algal and bacterial microbial populations in WSP wastewater is presented and standardised, using cultures and field samples. We show that SYTO 16 dye is more effective than SYBR Green I for the concurrent detection of both algae and bacteria in samples. Through gating and phenotypic diversity analysis, the FCM results show both spatial and temporal shifts in pond microbial communities. The ability to rapidly determine the spatiotemporal shifts in pond populations is not only important for the improvement of pond operation and monitoring strategies, but also for the planning and management. Flow cytometry has the potential to become a diagnostic tool for ponds to assess treatment performance and determine the most optimal operating conditions.

Cyanobacteria in small water bodies: The effect of habitat and catchment area conditions

Authors

Anna Kozak, Sofia Celewicz-Gołdyn, Natalia Kuczyńska-Kippen

Cyanobacteria are an important component of microalgae communities in aquatic ecosystems, however, their response to environmental factors in different habitats and catchment areas of small water bodies is still not well recognised. We examined ponds from two types of surroundings (field vs. forest) in order to find the best triggers for the distribution of cyanobacteria species, and analysed different habitats (open water and macrophyte-dominated zones) to find the habitat preferences of cyanobacteria species.

Our results underline the important role of habitats in the determination of the abundance of cyanobacteria species in small water bodies. Cyanobacteria as a group preferred macrophyte-dominated sites with stable water column conditions, which to lesser extent were inhabited by representatives of other systematic groups of algae. The co-occurrence of many cyanobacteria species and zooplankton in the studied ponds may have indirectly resulted from biotic interactions in the food web. In the open water a positive relationship between zooplankton and cyanobacteria suggests stimulation of their development through the elimination of smaller edible taxa or by nutrient resupply through zooplankton excretion.

The type of catchment area also impacted the cyanobacterial community. Field ponds with significantly higher values of pH and NO₃ were characterised by a higher abundance of cyanobacteria compared with ponds within the forest catchment. A positive relationship between pH and cyanobacteria indicates that they raise pH during photosynthesis. However, some species were negatively associated with water temperature and occurred exclusively only in forest ponds.

The study revealed that cyanobacteria in small water bodies can be a valuable indicator of important ecosystem conditions. Despite the fact that their prevalence in agricultural ponds may confirm their potential as an indicator of pollution, their high diversity associated with macrophytes contributes to an increase of overall landscape biodiversity.

Prospects and challenges of environmental DNA (eDNA) monitoring in freshwater ponds

Authors

Lynsey R. Harper, Andrew S. Buxton, Helen C. Rees, Kat Bruce, Rein Brys, David Halfmaerten, Daniel S. Read, Hayley V. Watson, Carl D. Sayer, Eleanor P. Jones, Victoria Priestley, Elvira Mächler, Cesc Múrria,Sandra Garcés-Pastor,Cecilia Medupin, Katherine Burgess, Gillian Benson, Neil Boonham, Richard A. Griffiths, Lori Lawson Handley, Bernd Hänfling

Environmental DNA (eDNA) analysis is a rapid, non-invasive, cost-efficient biodiversity monitoring tool with enormous potential to inform aquatic conservation and management. Development is ongoing, with strong commercial interest, and new uses are continually being discovered. General applications of eDNA and guidelines for best practice in freshwater systems have been established, but habitat-specific assessments are lacking. Ponds are highly diverse, yet understudied systems that could benefit from eDNA monitoring. However, eDNA applications in ponds and methodological constraints specific to these environments remain unaddressed. Following a stakeholder workshop in 2017, researchers combined knowledge and expertise to review these applications and challenges that must be addressed for the future and consistency of eDNA monitoring in ponds. The greatest challenges for pond eDNA surveys are representative sampling, eDNA capture, and potential PCR inhibition. We provide recommendations for sampling, eDNA capture, inhibition testing, and laboratory practice, which should aid new and ongoing eDNA projects in ponds. If implemented, these recommendations will contribute towards an eventual broad standardisation of eDNA research and practice, with room to tailor workflows for optimal analysis and different applications. Such standardisation will provide more robust, comparable, and ecologically meaningful data to enable effective conservation and management of pond biodiversity.

Diversity of potential antibiotic-resistant bacterial pathogens and the effect of suspended particles on the spread of antibiotic resistance in urban recreational water

Authors

Tingting Fang, Hui Wang, Qijia Cui, Matt Rogers, Peiyan Dong

Evidence of the increasing incidence of antibiotic resistance in watersheds has attracted worldwide attention. Limited in formation is available on the occurrences of health-related antibiotic-resistant bacterial pathogens (ARBPs) in recreational waters. The effects of certain environmental factors (e.g., suspended particles) on the spread of resistance also has not been characterized to date. In this study, a combination of culture and molecular methods was employed to comprehensively investigate the patterns of microbial resistance to representative antibiotics in samples from three recreational lakes in Beijing. The antibiotic resistance index (ARI) based on the gradient concentration assay revealed that samples showed high resistance to penicillin-G, moderate resistance to ampicillin, vancomycin and erythromycin and low resistance to ceftriaxone, gentamycin, tetracycline and chloramphenicol. Antibiotic-resistant bacteria (ARB) were cultured and collected, and the diversity of potential ARBP species was further explored using next-generation sequencing (NGS). The results showed that most of the identified ARBPs were environmental opportunistic pathogens with emerging clinical concerns, e.g., the multidrug-resistant Acinetobacter junii. Furthermore, particle-attached (PA) fractions presented higher ARI values than free-floating (FL) fractions did, indicating that the PA fractions were more resistant to selected antibiotics. And the NGS results revealed that the PA fractions showed higher similarity in the screened ARB community compositions in comparison with the FL fractions, primarily due to a protective effect provided by the particles. Accordingly, ARBPs could persist for a longer time in protective particle matrices. However, quantification of antibiotic-resistant genes (ARGs) by qPCR showed no significant abundance differences between the two fractions. Overall, these findings suggest a potential health risk from the prevalence of ARBPs in recreational waters and provides a better understanding of the contribution of particles in the spread of antibiotic resistance in aquatic systems, with implications for the control of excessive suspended particles by water management.

High methylmercury formation in ponds fueled by fresh humic and algal derived organic matter

Authors

Sonia Herrero, Ortega Núria Catalán, Erik Björn, Hannes Gröntoft, Torfi Geir Hilmarsson, Stefan Bertilsson, Pianpian Wu, Kevin Bishop, Oded Levanoni, Andrea G. Bravo

Neurotoxic methylmercury causes adverse effects to ecosystem viability and human health. Previous studies have revealed that ponding alters natural organic matter (NOM) composition and increase methylmercury concentrations in rivers, especially in the first years after flooding. Here, we investigate the influence of NOM composition (i.e., sources and degradation status) on mercury methylation rate constants in nine boreal beaver ponds of different ages across Sweden. We show that increased methylmercury concentrations in surface waters is a consequence of enhanced mercury methylation in the pond sediments. Moreover, our results reveal that during the first years after the initial flooding, mercury methylation rates are fueled by the amount of fresh humic substances released from the flooded soils and by an increased production of algal‐derived NOM triggered by enhanced nutrient availability. Our findings indicate that impoundment‐induced changes in NOM composition control mercury methylation processes, causing the raise in MeHg levels in ponds.

Hydrogen peroxide treatment promotes chlorophytes over toxic cyanobacteria in a hyper-eutrophic aquaculture pond

Authors

Zhen Yang, Riley P. Buley, Edna G. Fernandez-Figueroa, Mario U. G. Barros, Soorya Rajendran, Alan E. Wilson

Controlling blooms of toxigenic phytoplankton, including cyanobacteria, is a high priority for managers of aquatic systems that are used for drinking water, recreation, and aquaculture production. Although a variety of treatment approaches exist, hydrogen peroxide (H2O2) has the potential to be an effective and ecofriendly algaecide given that this compound may select against cyanobacteria while not producing harmful residues. To broadly evaluate the effectiveness of H2O2 on toxigenic phytoplankton, we tested multiple concentrations of H2O2 on (1) four cyanobacterial cultures, including filamentous Anabaena, Cylindrospermopsis, and Planktothrix, and unicellular Microcystis, in a 5-day laboratory experiment and (2) a dense cyanobacterial bloom in a 7-day field experiment conducted in a nutrient-rich aquaculture pond. In the laboratory experiment, half-maximal effective concentrations (EC50) were similar for Anabaena, Cylindrospermopsis, and Planktothrix (average EC50 = 0.41 mg L−1) but were ∼10x lower than observed for Microcystis (EC50 = 5.06 mg L−1). Results from a field experiment in an aquaculture pond showed that ≥1.3 and ≥ 6.7 mg L−1 of H2O2 effectively eliminated Planktothrix and Microcystis, respectively. Moreover, 6.7 mg L−1 of H2O2 reduced microcystin and enhanced phytoplankton diversity, while causing relatively small negative effects on zooplankton abundance. In contrast, 20 mg L−1 of H2O2 showed the greatest negative effect on zooplankton. Our results demonstrate that H2O2 can be an effective, rapid algaecide for controlling toxigenic cyanobacteria when properly dosed.

Agricultural Freshwater Pond Supports Diverse and Dynamic Bacterial and Viral Populations

Authors

Jessica Chopyk, Sarah Allard, Daniel J. Nasko, Anthony Bui, Emmanuel F. Mongodin and Amy R. Sapkota

Agricultural ponds have a great potential as a means of capture and storage of water for irrigation. However, pond topography (small size, shallow depth) leaves them susceptible to environmental, agricultural, and anthropogenic exposures that may influence microbial dynamics. Therefore, the aim of this project was to characterize the bacterial and viral communities of pond water in the Mid-Atlantic United States with a focus on the late season (October–December), where decreasing temperature and nutrient levels can affect the composition of microbial communities. Ten liters of freshwater from an agricultural pond were sampled monthly, and filtered sequentially through 1 and 0.2 μm filter membranes. Total DNA was then extracted from each filter, and the bacterial communities were characterized using 16S rRNA gene sequencing. The remaining filtrate was chemically concentrated for viruses, DNA-extracted, and shotgun sequenced. Bacterial community profiling showed significant fluctuations over the sampling period, corresponding to changes in the condition of the pond freshwater (e.g., pH, nutrient load). In addition, there were significant differences in the alpha-diversity and core bacterial operational taxonomic units (OTUs) between water fractions filtered through different pore sizes. The viral fraction was dominated by tailed bacteriophage of the order Caudovirales, largely those of the Siphoviridaefamily. Moreover, while present, genes involved in virulence/antimicrobial resistance were not enriched within the viral fraction during the study period. Instead, the viral functional profile was dominated by phage associated proteins, as well as those related to nucleotide production. Overall, these data suggest that agricultural pond water harbors a diverse core of bacterial and bacteriophage species whose abundance and composition are influenced by environmental variables characteristic of pond topology and the late season.

An environmentally friendly approach for mitigating cyanobacterial bloom and their toxins in hypereutrophic ponds: Potentiality of a newly developed granular hydrogen peroxide-based compound

Authors

Amit Kumar Sinha, Michael A. Eggleton, Rebecca T. Lochmann

Cyanobacterial blooms and their associated toxins are growing issues for many aquatic resources, and pose a major threat to human health and ecological welfare. To control cyanobacterial blooms and their toxins, the efficacy of a newly developed granular compound (sodium carbonate peroxyhydrate ‘SCP’, trade name ‘PAK® 27’ algaecide) containing hydrogen peroxide (H2O2) as the active ingredient was investigated. First, the dose efficacy of the SCP that corresponded to 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0 and 8.0 mg/L H2O2 was tested for 10 days in small-scale tanks installed in 0.1-acre experimental hypereutrophic ponds dominated by blooms of the toxic cyanobacterium Planktothrix sp. SCP ranging from 2.5–4.0 mg/L H2O2 selectively killed Planktothrix sp. without major impacts on either eukaryotic phytoplankton (e.g., diatom Synedra sp., green algae Spirogyra sp. and Cladophora sp.) or zooplankton (e.g., rotifers Brachionus sp. and cladocerans Daphnia sp.). Based on these results, SCP at 2.5 mg/L and 4.0 mg/L H2O2 were homogeneously introduced into entire water volume of the experimental ponds in parallel with untreated control ponds. The dynamics of cyanobacterium Planktothrix sp., microcystins (commonly occurring cyanotoxins), eukaryotic phytoplankton, zooplankton, and water quality parameters were measured daily for 10 days and followed by a weekly sampling for 6 weeks. Temporal analysis indicated that Planktothrix sp. blooms collapsed remarkably in both 2.5 mg/L and 4.0 mg/L H2O2 treatments. Both treatments also were accompanied by an overall reduction in the total microcystin concentration. At 2.5 mg/L H2O2, the growth of eukaryotic phytoplankton (Synedra and Cladophora sp.) increased, but these populations along with zooplankton (Brachionus and Daphnia sp.) were suppressed at 4.0 mg/L H2O2. The longevity of 2.5 and 4.0 mg/L H2O2 treatment effects were up to 5 weeks. In addition, the added granular algaecide degraded within a few days, thereby leaving no long-term traces of H2O2 in the environment.