Toolbox for the sampling and monitoring of benthic cyanobacteria
Graphical abstract
Introduction
Planktonic cyanobacteria are associated with a range of water quality issues and are now recognised globally as a major risk factor for drinking water (Falconer and Humpage, 2005, Falconer and Humpage, 2006; Hudnell, 2008; Gaget et al., 2017b). Despite decades of research on growth, control and toxin production by pelagic cyanobacteria, there are relatively few studies evaluating the risk associated with benthic forms. Benthic cyanobacteria are organised in mats, representing an important part of the photosynthetic layer of epilithic biofilms (Quiblier et al., 2013). In these mats, thin filamentous microalgae and other microorganisms are arranged in a complex polysaccharidic matrix (Pierre et al., 2012). During high growth periods, photosynthetic activity increases and produces air bubbles, which become entrapped in the mat and cause some segments to detach and float. Floating mats can contribute to the ingestion of toxins by animals, which have occasionally proved lethal (Gugger et al., 2005; Mez et al., 1997). While this still represents an emerging issue, recent studies have demonstrated that cyanotoxins are widespread in benthic mats across Australia (Dasey et al., 2005; Seifert et al., 2007; Gaget et al., 2017c; Sendall and McGregor, 2018) and around the world (Cadel-Six et al., 2007; Gugger et al., 2005; Mez et al., 1997; Mohamed et al., 2006).
Benthic biofilms also produce a range of taste and odour compounds (hereafter referred to as T&O compounds) in a range of waterbodies (Izaguirre et al., 1995; Jüttner and Watson, 2007; Izaguirre and Taylor, 2007; Chen et al., 2010; Otten et al., 2016). Geosmin and 2-methylisoborneol (MIB) are the main taste and odour compounds of concern and cause water to smell and taste earthy/musty when present above 10 ng/L. These compounds can represent a considerable challenge to water treatment (Chen et al., 2010). Due to the very high sensitivity that humans have to these two compounds, the public may notice off-flavours before they are identified via laboratory analysis. Even if toxins are not present, the presence of T&O compounds can generate public doubt that the water is safe to drink because treatment does not appear to have been effective. Therefore, it is vitally important for the water industry to have early warning of the presence of cyanobacteria that have the potential to produce T&O compounds or toxins.
Despite the demonstration that benthic cyanobacteria can be a source of toxins and T&O in drinking water reservoirs (Gaget et al., 2017c), no recommended guideline for their survey exists, however, routine sampling of planktonic cyanobacteria is generally well established in the water industry (Chorus and Bartram, 1999; ADWG, 2011). In contrast, no routine techniques have been scientifically validated for the sampling of benthic forms. Conventional benthic sampling consists of the collection of sediment core samples (Savichtcheva et al., 2011; Karlson et al., 2012). While collecting sediment core samples gives a ‘close-to-reality’ picture of the biodiversity present, its practicality is limited in rocky areas, where benthic mats seem to thrive, and there is also a significant risk for cross-contamination between different layers of the core when extracting the sediment out of the sampler. This is particularly problematic when using analyses such as Next Generation Sequencing (NGS) (e.g. for community composition analysis or metagenomics). The ministry for the environment of New Zealand has published recommendations for the survey of benthic cyanobacteria, particularly in rivers (Wood et al., 2009). These guidelines give advice on areas where the sampling effort should be concentrated, (mainly in shallow shorelines or reaches) and on the use of underwater viewers to collect core samples. Wood et al. applied these recommendations by sampling 1 m2 quadrants inside a 10m × 10m grid in shallow areas (Wood et al., 2011). The technique was shown to be relevant only in very shallow water and required 10 sub-samples per collection site to be reliable, making it unsuitable for routine monitoring of reservoirs. The present study aimed to develop a reliable and easy-to-use sampling technique for the routine collection of benthic bacteria growing in epilithic biofilms. Cell counts under light microscopy, the traditional monitoring technique used for planktonic species, is not applicable to benthic mats due to the complexity of the matrix.
In this context, the present study aimed to 1) design and trial a novel and easy-to-use sampling method for benthic cyanobacteria, 2) test a range of existing molecular techniques on benthic mats to evaluate their validity as monitoring tools, 3) develop a qPCR assay for the specific detection of the MIB-synthase gene in cyanobacteria and 4) use the aforementioned tools to conduct a field survey in a South Australian reservoir. To address these aims, a sampler was designed and eight quantitative Polymerase Chain Reaction (qPCR) assays and NGS were developed or adapted to analyse benthic matrix samples. All of the techniques developed were validated during a year-long field survey and utilised to establish seasonal patterns of benthic growth and secondary metabolite production.
Section snippets
Design
The general sampling concept involved deploying artificial substrates in the reservoirs and allowing the development of microbial biofilms. To achieve proper colonisation, substrates need to be placed as close as possible to the surface of the sediment. Therefore, a supporting structure for the substrates that could be anchored to rest on the sediment to limit movement was designed and constructed. Rectangular frames (144 cm × 67 cm) were built using PVC pipes (DWV pipe, 100 mm × 6m, Vinidex,
Sampler deployment and incubation trial
The sampling frames were simple to deploy and sank appropriately. Pulling the central buoy allowed the sampling frame to be lifted easily and the collection of samples could be executed safely. At t0+2 weeks and t0+4 weeks, the biomass collected from the wooden substrates was somewhat comparable (Fig. 3). The biomass growing on the two other substrates (sandpaper and tile) increased progressively over time. At t0+8 weeks all substrates were colonised by the biofilm, and the wooden coupon
Discussion
The sampling frames were easy to use and allowed for safe recovery of benthic samples. This device has application to both research and routine monitoring by water utilities to sample benthic biofilms. The fact that the biomass collected from the wooden substrate after 2 and 4 weeks of incubation were somewhat comparable between each other demonstrates that the growth observed on this substrate until t0+4 weeks is not necessarily a reflection of its colonisation by the biofilm. This preliminary
Conclusion
The sampling device designed during this study worked effectively in the field and provides a new solution for routinely monitoring benthic biofilms. Substrates used to collect samples need to be incubated for at least 6–8 weeks prior to first sampling in order to allow for the biofilm to colonise the surface and a minimum of 3 replicate samples need to be collected to assess the microbial community adequately. Although the sampling system worked efficiently, it tended to underestimate the
Conflicts of interest
All authors listed for this publication hereby attest that none of the participants in this research had a conflict of interest in the research conducted and the herein published results.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We wish to thank the Australian Research Council (ARC), SA Water Corporation, Water Research Australia (WaterRA) and Sydney Catchment Authority for funding this work (ARC linkage project number: LP 120200587, Water RA project number #1059-11). We would also like to thank Dr Luis Arriola from the ACAD for his help in decomplexing the NGS data.
References (64)
- et al.
A multiplex PCR targeting hepato- and neurotoxigenic cyanobacteria of global significance
Harmful Algae
(2012) - et al.
First report of anatoxin-a producing cyanobacterium Aphanizomenon issatschenkoi in northeastern Germany
Toxicon
(2010) - et al.
Benthic cyanobacteria: a source of cylindrospermospin and microcystin in Australian drinking water reservoirs
Water Res.
(2017) - et al.
First report in a river in France of the benthic cyanobacterium Phormidium favosum producing anatoxin-a associated with dog neurotoxicosis
Toxicon
(2005) - et al.
Geosmin and 2-methylisoborneol production in a major aqueduct system
Water Sci. Technol.
(1995) - et al.
Benthic fauna affects recruitment from sediments of the harmful cyanobacterium Nodularia spumigena
Harmful Algae
(2012) - et al.
Development and application of a molecular assay to detect and monitor geosmin-producing cyanobacteria and actinomycetes in the Great Lakes
J. Great Lakes Res.
(2014) - et al.
Microcystin production in benthic mats of cyanobacteria in the Nile River and irrigation canals, Egypt
Toxicon
(2006) Isolation and purification of cyanobacteria
Methods Enzymol.
(1988)- et al.
First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium, Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck
Harmful Algae
(2007)
Cryptic diversity within the Scytonema complex: characterization of the paralytic shellfish toxin producer Heteroscytonema crispum, and the establishment of the family Heteroscytonemataceae (Cyanobacteria/Nostocales)
Harmful Algae
Development of solid phase adsorption toxin tracking (SPATT) for monitoring anatoxin-a and homoanatoxin-a in river water
Chemosphere
Seasonality and diversity patterns of microphytobenthos in a mesotrophic lake
Arch. Hydrobiol.
Australian Drinking Water Guidelines
Simple conditions for growth of unicellular blue-green algae on plates
J. Phycol.
Spatial and temporal variability in epilithic biofilm bacterial communities along an upland river gradient
FEMS Microbiol. Ecol.
Command-line Tools for Processing Biological Sequencing Data
High bacterial diversity in epilithic biofilms of oligotrophic mountain lakes
Microb. Ecol.
Different genotypes of anatoxin producing cyanobacteria coexist in the Tarn River, France
Appl. Environ. Microbiol.
QIIME allows analysis of high-throughput microbial community analysis sequencing data
PMC
Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms
ISME J.
An improved method for obtaining axenic clones of planktonic blue-green algae
J. Phycol.
In situ measurement of odor compound production by benthic cyanobacteria
J. Environ. Monit.
Toxic Cyanobacteria in Water. A Guide to Their Public Consequences, Monitoring and Management
Investigations into the taxonomy, toxicity and ecology of benthic cyanobacterial accumulations in Myall Lake, Australia
Mar. Freshw. Res.
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB
Appl. Environ. Microbiol.
Search and Clustering Orders of Magnitude Faster than BLAST
Health risk assessment of cyanobacterial (blue-green algal) toxins in drinking water
Int. J. Environ. Res. Public Health
Cyanobacterial (blue-green algal) toxins in water supplies: cylindrospermopsins
Environ. Toxicol.
From an environmental sample to a long lasting culture: the steps to better isolate and preserve cyanobacterial strains
J. Appl. Phycol.
DNA extraction from benthic cyanobacteria: comparative assessment and optimisation
J. Appl. Microbiol.
Isolation and characterization of the gene associated with geosmin production in cyanobacteria
Environ. Sci. Technol.
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