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Wastewater Bioremediation From Filamentous Algae

Congress: 2015
Author(s): Michael Ross, Andrea J C Semiao, Michele Stanley, John Day
University of Edinburgh1, SAMS2

Keyword(s): Sub-theme 9: Water allocation among competing uses and users,
Abstract1. Introduction

Water is an invaluable asset for life on Earth, with "clean" freshwater being essential for maintaining human health and sanitation. Furthermore, it is a necessity for many industrial and agricultural processes. Only a small proportion of the water in Earth is available for human use and currently we are undergoing a water crisis, whereby 3 billion people do not have access to drinking water and lack adequate sanitation [1]. This problem is likely to be further exacerbated by a growing population, increased urbanization and industrialisation [2]. Clearly, we need to make the best use of the accessible water. An objective of the EU Horizon 2020 programme is that water, as a resource, should be recovered, re-used, and recycled [3]. Current wastewater treatment (WWT) practices are largely unsatisfactory. They tend to have either a high capital or operational cost, large area requirement, or an inefficient removal of nutrients, heavy metals, or other contaminants. This project aims to investigate the possibility of using the cosmopolitan, green, filamentous macro-alga Cladophora sp. as a tool for wastewater bioremediation, whilst concomitantly providing other services, such as bioenergy, heavy metal and water recovery [4]. Cladophora, being filamentous in nature would obviate the cost of harvesting, which is significant for micro-algal culture [5]. The feasibility of this bioremediation application was initially tested by trying to determine the upper and lower tolerance levels of Cladophora to varying abiotic conditions.

2. Materials and Methods

2.1 Macro-algal Species and Cultivation

There were two species of Cladophora used in this experiment (Figs. 1A&B); one marine in origin, C. parriaudii, and the other was a freshwater species isolated from the Black Forest in Germany. Fresh weight (FW) biomass was obtained via starter cultures of algae in an appropriate medium (F/2 Medium containing 33.5g/L Instant Ocean for the marine alga and Jaworski's Medium (JM) for freshwater species).

The experiments were performed in 6-well plates containing 5 mL of different media (see Table 1). The plates were placed in an incubated shaker at 60 µmol/m2/s-1 PAR with a 16h/8h Light/Dark cycle at 20°C throughout. An inoculum of 5±0.5mg of FW algae was added per well and incubated for 2-3 weeks.

Table 1. Experimental variables for determining the upper and lower tolerance levels of three species of Cladophora to three abiotic factors.

Once an appropriate biomass had been achieved, the Cladophora was harvested and blotted dry using filter paper.

2.2 Algal Growth Performance

The daily growth was measured by determining the % algal cover in each well. This was achieved by taking daily photographs and analysing the images using ImageJTM.
The water chemistry was analysed at the beginning and end of the experiment using ICP-MS, with the concentration of Nitrite/Nitrate, Ammonium, Phosphate, and Silicate measured in each aliquot.

3. Results and Discussion

The temporal growth of two different species of Cladophora was measured as it was exposed to the three variable abiotic factors tested. In general, Cladophora exhibited growth in almost all treatments, as indicated in Figure 2A-C. This set of experiments demonstrated that the Cladophora spp. used in this trial are very robust and capable of tolerating a wide range of variable environmental parameters.

The initial experiment (Fig 2A.) aimed to investigate osmotic tolerance of both Cladophora strains. C. parriaudii failed to grow in the freshwater treatment (0‰); however, for all of the other treatments the algal biomass doubled over the course of the experiment. Best growth was observed in the brackish and normal strength seawater (15 and 33.5‰). Conversely, the freshwater species of Cladophora failed to exhibit any real growth in the saline treatments and only the freshwater treatment resulted in any growth. The extreme changes in osmotic pressure may result in the algal cells becoming turgid or plasmolysed, respectively [6] This osmotic response, in conjunction with light exposure, is thought to damage the photosynthetic apparatus of Cladophora [7] and this will be explored in more detail in the future. Alternatively, the cells of the freshwater Cladophora would have likely plasmolysed which may have resulted in gross metabolic uncoupling, which hindered their cellular uptake of nitrates and phosphates.

The pH tolerance of Cladophora was tested in the second experiment as shown in Figure 2B. Both species showed similar growth patterns. An initial pH of 3 proved to be too extreme an environment for the macro-alga to grow, with a rapid biomass reduction over the first 2 days, with no recovery thereafter C. parriaudii grew best in slightly acidic to pH neutral conditions. The freshwater species of Cladophora exhibited better growth in the alkaline treatments. However, from day 8 onwards all treatments showed a reduction in % cover; this was possibly due to nutrient limitation.

The final experiment investigated the ability of Cladophora to grow in nutrient rich conditions and its potential for bioremediation (Figs 2C. and 3A-D.). The Cladophora exhibited greatest growth in the most nutrient rich treatments, with eight times the normal concentration of both nitrate and phosphate in relation to the normal F/2 and JM algal growth recipes. The marine Cladophora grown in the two lesser concentrated solutions may have become nutrient limited form day 9 onwards.

Water chemistry was performed for the final nutrient experiment for C. parriaudii only (Figs 3A-D.). In general, C. parriaudii was excellent at removing nutrients from its environment, especially phosphate. Despite this there was still a reasonable concentration of N remaining in the media (Figs. 3A&B.); this suggests that phosphate may have limited growth in this study.

Figure 2A-C. The growth of three species of Cladophora exposed to varying abiotic conditions; A -- salinity, B -- pH, and C -- nutrient concentration.

4. Conclusions

Cladophora is generally regarded as a robust, cosmopolitan genus, often associated with nutrient-rich or eutrophic waters. The findings from this study further substantiate this, demonstrating that the two representative species are capable of withstanding a wide range of salinities, nutrient concentrations, and pH levels. This study proposes that Cladophora could be a possible candidate for application as a new WWT tool. Its ability to reduce N and P from the culture media from greater-than algal bloom-forming concentrations, to almost negligible levels is promising. Further work from this study will focus uupon different types of waste media, specifically exploring the capacity of the alga to remove and recover heavy metals from water, and its potential as a feedstock for different types of bioenergy. 1. Water.org, 2014
2. AQUASTAT, 2006 & 2012
3. ec.europa.eu
4. Aitken, D. and Antizar-Ladislao, B. (2012) Achieving a Green Solution: Limitations and Focus Points for Sustainable Algal Fuels. Energies 5, 1613-1647.
5. Zulkifly, S.B., Graham, J.M., Young, E.B>, Mayer, R.J., Piotrowski, M.J., Smith, I., Graham, L.E., (2013) The genus Cladophora Kützing (Ulvophyceae) as a globally distributed ecological engineer. Journal of Phycology 49, 1-17.
6. Wiencke, C., Gorham, J., Tomos, D., Davenport, J. (1992) Incomplete Turgor Adjustment in Cladophora rupestris under Fluctuating Salinity Regimes. Estuarine, Coastal and Shelf Science 34, 413-427.
7. Wiencke, C. and Davenport, J. (1987) Respiration and phototsynthesis in the intertidal alga Cladophora rupestris (L.) Kütz. under fluctuating salinity regimes. Journal of Experimental Marine Biology and Ecology 114, 183-197.

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