J Appl Phycol https://doi.org/10.1007/s10811-017-1314-0
Assessment of selected macroalgae for use in a biological hybrid system for treating sulphur in acid mine drainage (AMD) Paul J. Oberholster 1,2,3 & Po-Hsun Cheng 1 & Anna-Maria Botha 4 Liesl Hill 5
&
Philip Hobbs 5 &
Received: 21 May 2017 / Revised and accepted: 15 October 2017 # Springer Science+Business Media B.V. 2017
Abstract Filamentous algae biomass is very limited in aerobic constructed wetlands and totally absent in anaerobic constructed wetlands. Its contribution to the removal of sulphur from acid mine drainage will therefore rarely reach a significant level if not used in association with secondary algae treatment ponds as part of a biological hybrid treatment system. Although a high sulphur concentration has a lower environmental impact than dissolved metals and acidity, it does have an adverse impact on water quality. The current study employed selected macroalgal species under laboratory conditions to determine the bioaccumulation of sulphur (S) and other important algal growth elements such as calcium (Ca), magnesium (Mg) and phosphorus (P) from acid mine drainage (AMD) water and treated constructed wetland water at different pH values. Following exposure of the macroalgae to AMD and treated constructed wetland water for 192 h, Microspora tumidula showed the highest bioaccumulation of S and P which occurred at a pH of 5. Oedogonium crassum showed the highest bioaccumulation of Ca and Mg at a pH of 7. The results also showed that the accumulation efficiency of Mg by all three macroalgal species increased
* Anna-Maria Botha
[email protected]
1
CSIR Natural Resources and the Environment, PO Box 320, Stellenbosch 7599, South Africa
2
Department of Earth Sciences, University of the Western Cape, Private Bag X17, Bellville 7535, South Africa
3
Department of Botany and Zoology, University of Stellenbosch, Private Bag X1, Matieland, Stellenbosch 7601, South Africa
4
Department of Genetics, University of Stellenbosch, Private Bag X1, Matieland, Stellenbosch 7601, South Africa
5
CSIR Natural Resources and the Environment, PO Box 395, Pretoria 0001, South Africa
significantly as the pH increased. The filamentous species Oedogonium crassum and Klebsormidium klebsii showed very little increase in chl-a (mg g−1 fw) and ash free dry weight (mg g−1 AFDW) after exposure to the mine and treated constructed wetland water after 192 h at all four pH ranges in comparison to the species M. tumidula. However, the species M. tumidula show significant increases in both chl-a (mg g−1 fw) and mg g−1 AFDW at pH values 5, 6.14 and 7. The study established that the species M. tumidula is a good candidate for use in a biological hybrid system for treating sulphur-rich acid mine drainage. Keywords Acid mine drainage . Wetland treatment . Bioaccumulation . Filamentous macroalgae . Nonmetal elements
Introduction South Africa holds approximately 11% of the global recoverable hard coal reserves (Prevost 1997), and is the world’s sixth largest coal producer with 220 million tonnes per year (DME 2004). The Permian coal deposits of the Witbank, Highveld and Ermelo coal fields in the northern (and shallowest) portion of the main Karoo Basin form the largest conterminous area of active coal mining in South Africa. Coal has been mined in these fields for over a century, and has made a significant contribution to the South African economy through international export and, internally, in support of power generation. On the other hand, the impacts of coal mining activities on ecosystem services have been well documented and comprehensively reviewed elsewhere (de Klerk et al. 2016). The impact differs depending on the material extracted, the mining method used and differences in topography, geology, soil and climate. Mine dewatering activities in the Witbank coal field alone result in the discharge of approximately 50 million litres
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per day of mine water into the upper Olifants River catchment (Maree et al. 2004). Coal mining has significant impacts on the environment, even if it is assumed that post-mining reclamation is performed according to current regulations. The problem of abandoned mines is emphasised in the findings of the AGSA (2009) report which determined that there are approximately 6000 listed abandoned mines in South Africa. The cost to rehabilitate the impacts of acid mine drainage (AMD) due to these abandoned mines is estimated at ZAR30 billion (Oberholster et al. 2013). It is known that AMD originating from mining activities contains high concentrations of dissolved metals and sulphur with low pH values (Oberholster et al. 2013). Although chemical treatment has shown successful results in the treatment of AMD, the main disadvantages include high capital and maintenance and operational costs, membrane fouling and the large volumes of sludge generated. This has led to a growing interest in an alternative process such as passive treatment. The use of constructed wetlands for treatment of AMD has developed rapidly in recent years, and provides a low operational cost approach to the long-term treatment of AMD (Sheoran and Sheoran 2006). However, the drawbacks of constructed wetlands include the need for large surface areas to accommodate high flows, and concerns for the long-term stability of precipitated metals. Furthermore, the metal sequestration capability of macrophytes in surface and subsurface constructed wetlands for AMD treatment can be lost in temperate regions during winter months when macrophyte and microbial metabolic processes are reduced due to lower daily temperatures (Sheoran 2004). Kadlec and Reddy (2001) showed that the response of microbial processes was much greater to changes at the lower end of the temperature scale (< 15 °C) than in the optimal range 20– 35 °C. Furthermore, Rees and Bowell (1999) showed in a decade-long study using sulphur isotope analysis of the pore water of a constructed wetland that sulphur reduction was occurring in the wetland beds, but also that subsequent metal sulphide oxidation offset the treatment with the net effect that in the long-term sulphur attenuation would not occur. Alternatively, macroalgae have been reported to be quite efficient in the removal of heavy metals from AMD in constructed wetlands. According to Kleinmann and Hedin (1993), the accumulation by algae of iron (Fe) and manganese (Mn), calculated on a dry weight basis, is quite impressive. Kepler (1986) reported concentrations as high as 56,000 mg Mn kg−1 dry weight of samples of the filamentous cyanobacterium Oscillatoria. However, algal biomass in aerobic-constructed wetlands is very limited, and totally absent in anaerobic-constructed wetlands (Kleinmann and Hedin 1993). Its contribution to metal removal will therefore rarely reach a significant level if not used in association with secondary algae treatment ponds that form part of a biological hybrid system as suggested by Oberholster et al. (2014). However, it is also known that low temperatures during winter months limit metabolic and photosynthetic rates in algae
as some of the enzymes involved in these processes are temperature dependent (Davison 1991). Nevertheless, according to Lawton et al. (2014), most macroalgae are capable of acclimating to variable temperatures; however, this ability is expected to be higher in algae native to habitats with large annual variations. Lawton et al. (2014) demonstrated that some isolates of the genus Oedogonium are better able to tolerate lower temperatures than others. Schabhüttl et al. (2013) examines the temperaturedependent differences in growth rate related to three taxonomic groups. The authors reported that green algae and diatoms showed a trend to perform better at lower temperature, while cyanobacteria showed stronger responses with increase in temperature. A recent study by Oberholster et al. (2017) reported high (98 and 92 mg m−2 chl-a) benthic filamentous algae biomass during mid-winter conditions which correlated positively with a low pH values of < 3.5. The latter benthic algae biomass was dominated by the species Ulothrix punctata and Klebsormidium acidophilum. Although Oberholster et al. (2014) showed in a laboratory study mimicking winter conditions that certain selected macroalgae species can possibly be used in a biological hybrid system to reduce metal concentrations, very little is known about their potential to reduce sulphur concentrations in AMD effluent, as well as their association with P, Mn and Ca as growth elements. Although a lower environmental impact is attributed to sulphur compared to dissolved metals and acidity, lowering sulphate concentrations has generally not been considered a priority. Sulphate can cause various kinds of problems which include the following; altered taste of water, digestion troubles in animals and humans, soil acidification and corrosion of metals (Silva et al. 2010). Nevertheless, increasingly stringent regulations such as the European Water Framework Directive and the South African Department of Water and Sanitation guidelines require that sulphate concentrations do not exceed an upper limit to allow discharge of mine water (Johnson 2014; DWA 1996). In order to determine the bioaccumulation in macroalgae of sulphur and other important algal growth elements (Ca, Mg and P) from mine water, the species Microspora tumidula, Oedogonium crassum and Klebsormidium klebsii were exposed to acid mine drainage for 192 h at pH levels of 3, 5, 6.14 and 7 under laboratory conditions. The pH levels 3, 5 and 7 were selected on the basis of possible variation in the treatment potential of the constructed wetland affected by seasonal changes, e.g. a drop in temperature causing the die-off of upper parts of macrophytes and the reduction in microbial activity.
Materials and methods Study area and sampling methodology Duplicate water column samples were collected of the effluent from a decanting coal mine and discharge of the constructed
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surface flow wetland in the vicinity of the Boesman Spruit stream near the town of Carolina in the Ermelo coal field, South Africa (Fig. 1). Boshoff et al. (1991) report a sulphur content in the range 0.4 to 1.6% S (mean of 0.9%) in this coal field. The pH and electrical conductivity (EC) of the decanting stream and discharge of the constructed wetland were measured in situ using a Hach sension 156 portable multiparameter probe (USA) during the winter season. Surface decanting mine and discharge constructed wetland water samples were collected in two 5-L acid clean polyethylene containers for chemical analyses and macroalgae exposure. Each bottle was filled completely and carefully capped so as to minimise the presence of air and any consequent oxidation of the samples. All samples were kept on ice and in the dark during transportation to the laboratory. The Standard Methods for the Examination of Water and Wastewater (APHA 1992) were used to determine S, Mg, Ca and P concentrations. Exposure of axenic macroalgae to AMD The axenic macroalgae cultures Microspora tumidula, Oedogonium crassum and Klebsormidium klebsii previously established by Oberholster et al. (2014) were filtered, and their wet and ash-free dry biomass determined prior to inoculation of the algae culture broth (Sigma-Aldrich Chemie GmbH, Switzerland). The algae broth had the following chemical
composition, per litre: ammonium chloride 0.05 g; calcium chloride 0.058 g; dipotassium phosphate 0.25 g; ferric chloride 0.003 g; magnesium sulphate 0.513 g. Different stock axenic macroalgae cultures (100 mg) were poured into 900 mL of the sterilised algae culture broth. The selected macroalgal growth cultures were prepared in triplicate Erlenmeyer flasks. The flasks were shaken at 100 rpm under eight tubular cool white fluorescent lamps providing ∼ 120 μmol photons m−2 s−1 illumination. Light was set to 12:12-h light–dark cycles. The growth rate of algal biomass was expressed relative to total chlorophyll and ashfree dry mass, over a 192-h incubation period following the standard method of Porra et al. (1989) for chlorophyll and Steinman and Lamberti (1996) for ash-free dry biomass analyses. Exposures of the macroalgal cultures were conducted in triplicate, with stock macroalgae samples of M. tumidula, O. crassum and K. klebsii exposed to acid mine water with an average pH of 3.4 collected from the Carolina site (Table 1), as well as to AMD from this site treated with NaOH to a pH of 5 and 7. As a control, the macroalgae were exposed to treated water collected in mid-winter from the constructed wetland with a pH value of 6.14. Samples were collected and analysed at the start and end of the 192-h experiment. ICP-MS analysis For inductively coupled plasma mass spectrometry (ICPMS) analysis, both control and exposed macroalgae were
Fig. 1 Map of study area showing Carolina study site and regional drainage pattern
J Appl Phycol Table 1 Physico-chemical characteristics of mine water and treated water from the constructed wetland near the Carolina site. Mine water was sampled twice (n = 2) and constructed wetland water once in the winter (n = 1)
Characteristics: Concentration (mg L−1)
Mine water decant
Constructed wetland discharge
Calcium
(Ca)
265
200
Magnesium Sulphur
(Mg) (S)
208 1940
45 862
Phosphorus
(P)
Aluminium
(Al)
14
Iron Manganese
(Fe) (Mn)
pH
(−log10αH+)
38 104 3.4
centrifuged at 13,000×g for 2 min at 4 °C. The macroalgae pellets of each exposure were collected and washed with sterile Milli-Q water and centrifuged at 13,000×g for 1 min at 4 °C. The washing step was repeated three times, while excess supernatant was removed using a sterile pipette. The macroalgae samples were weighed before adding 2 mL concentrated 69% (v/v) HNO3 to each vial and left for cold digestion for 24 h. After 24 h, 1 mL of concentrated 37% (v/v) HCl acid was added. The vials were placed in a water bath at 60 °C until all visible particles dissolved. Samples were allowed to cool to room temperature (25 °C). The digested macroalgae were decanted into an ICP tube and made up to a final volume of 10 mL with Milli-Q water. The vials were cleaned, dried and weighed. Macroalgae biomass was calculated by subtracting the mass of the empty vial from the mass of the vial containing the macroalgae. The digested samples of macroalgae were filtered through a 0.45-μm syringe filter into an ICP-MS sample cup. The sample was analysed for S, P, Ca and Mg using an Agilent 7500cx quadrupole inductively coupled mass spectrometer equipped with Mass hunter software version G7200. The mass and volume of the macroalgae were used to convert the results from μg L−1 to μg kg−1.
Statistical analysis of cultured macroalgal experiments All variables, except pH, were log-transformed to normalise distributions. Two-way ANOVA was used to determine physicochemical and biological differences (i) among algae and (ii) different pH values having different temporal variations. Homogeneity of variances and normality of data were checked prior to data analysis. If significant differences were observed (p < 0.05), the ANOVA analysis was followed by a Tukey b test. All these statistical analyses were done with Xlstat software version 18.07, Addinosoft 2016. Principal component analyses (PCA) were performed on the macroalgae differences based on S, Ca, Mg and P accumulation and the corresponding time measured.
0.05
0.05 11 22 80.7 6.14
Results Water chemistry The water chemistry of the decanting mine and wetland treated water is presented in Table 1. High concentrations of sulphur which were much higher than the South African Water Quality Guideline value of 400 mg L−1 for domestic water supplies (DWAF 1996a) was observed in the decanting mine and in the wetland treated water samples. The average pH of the collected water from the decanting coal mine was 3.4 and 6.14 for the discharge treated wetland water. Both the decanting mine and discharge constructed wetland water samples were associated with high concentrations of Al and Mn. The Al and Mn concentrations exceeded the South African Water Quality Guideline values of 0.005 and 180 μg L−1 respectively, for aquatic ecosystems (DWAF 1996b). Levels of S, Mg, Ca and P accumulation by various algae under laboratory conditions The accumulation of S in macroalgae after 192 h of exposure to the mine and constructed wetland discharge water showed that the greatest accumulation occurred at a pH of 5 by M. tumidula (Fig. 2d). This species was also the best accumulator of P at a pH value of 5 (Fig. 2c). Oedogonium crassum showed the greatest accumulation of Mg and Ca at a pH of 7 (Figs. 2a, 2b). The relative bioaccumulation of S by the macroalgae at all four pH levels was M. tumidula > O. crassum > K. klebsii (Fig. 2a–d), whereas calcium accumulated in O. crassum at the pH levels 5, 6.14 and 7, and in M. tumidula at a pH of 7. No bioaccumulation of Ca was observed in K. klebsii. The accumulation of Mg in all three macroalgal species decreased significantly with a decrease in pH (Fig. 2b). Chlorophyll-a concentration (mg g−1 fw) and ash-free dry weight (mg g−1 AFDW) in relationship with different pH values The chlorophyll-a (mg g−1 fw) and ash-free dry weight (mg g−1 AFDW) data are summarised in Fig. 3a, b. From
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Fig. 2 Accumulation of S, Mg, Ca and P by K. klebsii, O. crassum and M. tumidula at pH levels of 3, 5, 6.14 and 7
the data, it was evident that the algae M. tumidula shows the highest chl-a (mg g−1 fw) and mg g−1 AFDW concentrations after 192 h of exposure at a pH value of 7, while the lowest concentrations where measured for the species M. tumidula
and K. klebsii at a pH value of 3. Chlorophyll-a and AFDW were generally much lower at a pH of 3 in comparison to the pH ranges 5, 6.14 and 7 in all three algae exposed.
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Fig. 3 Chlorophyll-a concentration and ash-free dry weight of M. tumidala, O. crassum and K. klebsii at pH levels of 3, 5, 6.14 and 7
PCA of different algae at different pH conditions Principal component analysis shows a distinct clustering of the macroalgae in association with S, P, Ca and Mg as well as the three pH levels (Fig. 4). The ordination plot describes 76.97% of the variation in the data, with 47.31% described on the first axis and 29.66% on the second axis. Microspora tumidula shows a close association with chl-a, S, P and the pH ranges of 5, 6.14 and 7 while O. crassum is associated with Ca and Mg at pH ranges of 6.14 and 7.
Discussion The control of sulphate in mine waters primarily employs one of two methodologies (Bowell 2004), namely (a) removal through membrane separation, or (b) removal by precipitation through ion exchange, permeable reactive barriers, formation of insoluble mineral precipitate or biological reduction. Although microbially driven sulphate and iron reduction is a biological process that occurs naturally in constructed wetland sediments through increasing the pH, this process loses efficacy at lower temperatures such as characterise winter seasons in temperate and cold climates.
Studies by Bender and Phillips (1993, 2004) and Wildeman et al. (1994) showed that microbial mats mainly comprising the cyanobacteria Oscillatoria sp. and the green filamentous algae Chromatium sp. can be utilised to reduce the dissolved concentrations of Mn and Fe in mine drainage using small pond systems (40–46 m2). Although the main focus of these studies was metals removal by algae, none reported on sulphur reduction in the ponds. The only study that showed reduction of sulphate is a bench-scale experiment by Sheoran and Bhandari (2005) using algae-microbial mats with the cyanobacteria Oscillatoria as the dominant sp. In the latter study, substantial quantities of Fe, Cu, Zn, Co, Pb, Ni, Mn and SO4 were removed in a short period. O’Halloran et al. (2008) reported that metal concentrations most likely play a secondary role to pH in impacting on macroalgae. These authors identify Al and Fe as metals of particularly secondary impact, as they precipitate out at a pH > 3.5. Therefore, it was important in the current study to take into account the different pH ranges in relationship with possible metal toxicity to determine the different responses by algae after the 192 h of exposure. Nevertheless, whether algal species can grow at neutral pH values or not, defines them as an acidtolerant and acidophilic species (Gross 2000). Previous studies (e.g. Gimmler and Weis 1992) have shown that such species are able to maintain a constant, neutral cytosolic pH over a wide range of external pH values. For maintaining a neutral intracellar pH, acidophilic algae have developed several strategies to survive under low pH conditions. These include (a) a positive membrane potential and a positive charge outside the cytosolic membrane, and (b) a decreased permeability of the cytosolic membrane for protons or for active proton pumping activity (Remis et al. 1994; Gimmler and Weis 1992). Although several previous studies showed metal bioaccumulation in filamentous macroalgae (Das et al. 2009a, b; Oberholster et al. 2014), little is known about bioaccumulation of certain growth elements (e.g. P, Ca, Mg and S) in such algae at various pH levels. Sulphur is an essential element for autotrophs and heterotrophs. In its reduced oxidation state, sulphur plays an important part in the structure and function of proteins (Davidian and Kopriva 2010). Three amino acids found in almost all proteins, namely cysteine, cystine and methionine, contain carbon-bounded sulphur. Sulphur is also found in sulfolipids, some vitamins, sulphate esters and a variety of other compounds. Sulphate is the predominant form of sulphur in oxic waters. Uptake of sulphate into algal cells is through an active transport mechanism, and inserted into an energetically activated molecule, APS (adenosine-5′-phosphosulfate), which can be further activated at the expense of one ATP molecule to PAPS (3′-phosphoadenosine-5′-phosphosulfate). It is then transferred to a thiol carrier (a molecule with a -SH group) and reduced to the 22 oxidation state (Davidian and Kopriva 2010). In contrast to nitrate assimilation, where the various intermediates are freely present in the cytoplasm, sulphur remains attached to a carrier during the reduction sequence
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Fig. 4 Ordination plot of principal component analysis showing distinct clustering of M. tumidula with Ca and Mg, and O. crassum with S, P and chl-a, at pH values of 5, 6.14 and 7
(Schmidt 1986). It is evident from the study reported in this paper that M. tumidula has a much greater affinity for sulphur than K. klebsii and O. crassum, rendering this species an ideal candidate in the treatment of AMD containing high concentrations of sulphur. Magnesium, unlike toxic metals, is essential for the growth and development of algae (Ayed et al. 2016). It occupies a central position in the chlorophyll molecule and influences the activity of the photosynthetic enzymes. In addition, Mg acts with other chlorophyll compounds as antennae to capture light energy necessary for photosynthetic reaction (Carvalho et al. 2011). The greater bioaccumulation of Mg by O. crassum at the pH of 6.14 and 7 compared to the other macroalgae at lower pH values might be related to growth. A previous study by Finkle and Appleman (1952) showed that chlorophyll synthesis in the microalgae Chlorella vulgaris revealed striking differences in the growth patterns of this alga when cultured in nutrient solutions containing different amounts of Mg. Munir et al. (2015) report that Oedogonium sp. showed low growth in terms of fresh weight at a pH of 6.5, while at a pH of 7.5 and 8 the fresh weight increased significantly. The latter observation was concurrent with the current study. Oedogonium crassum showed very little increase in chl-a (mg g−1 fw) and mg g−1
AFDW after exposure to mine water after 192 h at all four pH ranges. However, the species M. tumidula show significant increases in chl-a (mg g−1 fw) at pH values 5, 6.14 and 7. Magnesium is also involved in the aggregation of ribosomes in functional units and in the formation of catalase (Encarnaçao et al. 2012). The higher concentrations of Mg uptake by the filamentous algae K. klebsii under the low pH condition of 3 can possibly be related to the formation of catalase (Fig. 2). Although catalase was not tested in the current study, a previous study by Soto et al. (2011) showed that catalase activity increased significantly after exposure of the green alga Pseudokirchneriella subcapitata to Cu and Zn, causing the formation of reactive oxygen species inside cells. According to Gorain and Bagchi (2013), both Ca and Mg were found to be critical for biomass yield and lipid accumulation in tested microalgae. Previous studies have shown that P, rather than N, is the primary limiting nutrient for algae in many natural environments, and that it is an important component required for normal growth and development of alga cells (Larned 1998). Phosphorus typically constitutes 1% of the dry weight of algae, but it may require in significant excess since not all added phosphorus is bioavailable due to formation of complexes with metal ions such as Al (Das et al. 2009a, b; Parent and
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Campbell 1994). The immediate effects of P limitation on algae include a reduction in the synthesis and regeneration of substrates in the Calvin-Benson cycle, and a consequential reduction in the rate of light utilisation required for carbon fixation. The current study shows that the highest uptake of P occurred at a pH of 3 by the algae K. klebsii. According to Nishikawa et al. (2003), metal-tolerant algae would accumulate polyphosphate in higher concentrations than non-tolerant algae, leading to a higher phosphate demand as observed in the current study. Furthermore, phosphates form an integral part of essential molecules such as ATP that are needed to maintain a neutral intracellular pH in an acidic pH external environment, by pumping protons out of the cell. However, the lower uptake of P by M. tumidula at a pH of 3 might be related to the ability of this species to adjust its internal pH in response to external pH fluctuations, thereby maintaining an energy advantage over other species that are more acid intolerant (Juneja et al. 2013). From a previous report by Regel et al. (2002), it was evident that Al interferes with basic cellular functions such as the phosphoinositide and the intracellular Ca signalling pathways, which are involved in a myriad of cellular metabolic functions. The results from the current study show that of the subject macroalgae, only M. tumidula and O. crassum bioaccumulate Ca after 192 h at pH levels of 5 and 7. Calcium is a crucial regulator of growth and development in algae, and it is well known that Ca plays an important role in controlling membrane structure and function. From a physiological perspective, Ca controls membrane permeability— cells cultured in solutions of low Ca, especially in the presence of EDTA, leakage ions and metabolites. The bioaccumulation of Ca at a pH of 5, 6.14 and 7 by O. crassum might be ascribed to the prevention of leakage of ions from the cells, or increased growth. A laboratory study by Singh and Chaudhary (1988) showed that the formation of oogonium as generative organs in O. hatei was observed in the pH range 7–9, and that no oogonial development was observed at a pH of 4. A previous laboratory study by Oberholster et al. (2014) showed that after 192-h exposure to AMD at different selected pH values, the most efficient macroalgae to sequestrate the metals Al, Fe, Mg, Mn and Zn were O. crassum > K. klebsii > M. tumidula. A study conducted by Roberts et al. (2013) demonstrated that the freshwater macroalgae Oedogonium is an ideal candidate for bioremediation of metal-contaminated waste streams, which concurred with previous findings by Oberholster et al. (2014). Roberts et al. (2013) showed in their study that algal cultures delivered significant improvement in ash dam water quality during winter, reducing the elements Al, As, Cd, Ni and Zn to meet guideline values within 2 to 4 weeks. The authors showed in a later study (Roberts et al. 2015) that slow pyrolysis of the cultivated algae in the ash dam water immobilised the accumulated metals in a recalcitrant C-rich biochar that can be used as an
ameliorant for low-fertility soils. In the current study, it was evident that M. tumidula was a much better bioaccumulator of sulphur than the species O. crassum or K. klebsii, indicating that a consortium of macroalgae, rather than one single species, must be used to treat AMD from coal mines or constructed wetlands during winter periods.
Conclusion The macroalga M. tumidula has a much greater affinity for S than K. klebsii and O. crassum. The highest bioaccumulation of S occurred at a pH of 5, making this species an ideal candidate for use in the treatment of sulphur-rich water from coal mines. Furthermore, M. tumidula also accumulated the highest P at a pH of 7. The species O. crassum shows the greatest bioaccumulation of Mg and Ca at a pH of 7. It was evident from the study that the low pH value of 3 may reduce the uptake of Mg in comparison to a pH value of 7. However, it must be taken into account that coal mines in different regions have different natural geology and different AMD metal concentrations that may affect the uptake of S, P, Mg and Ca in the tested algae differently. Acknowledgements The authors express their gratitude to the CSIR for funding the project as well as to colleagues for helping with the water and algae sampling. The authors also thank the unknown referees for their critical review of and constructive suggestions toward improving the manuscript.
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