Growth and metal bioconcentration by conspecific freshwater macroalgae cultured in industrial waste water

Biomass and effluent collection

Three species of filamentous algae from the genus Oedogonium were used in the cultivation experiments. One species was collected from the Ash Dam water (ADW) storage at Tarong power station in October 2012 (‘Tarong Oedogonium’), while the other two species were collected from irrigation ditches in the Brandon sugarcane region. Attempts at species level identification of the three species were made using taxonomic keys (Entwisle et al., 2007), however, the lack of clear morphological characteristics meant that the species could not be identified beyond genus level. The species were therefore assessed using molecular techniques, arguably the most effective approach to identifying cryptic species, and each was determined to be a unique genotype, supporting its assignment as a unique species (Lawton et al., 2014). As the three isolates could not be matched to extant species of Oedogonium on the basis of classical taxonomic features they are hereafter referred to by their GenBank accession numbers. The Tarong Oedogonium isolate (26.76°S, 151.92°E; temperate) has the accession number KC606974, while the two strains isolated from the Brandon region (19.56°S, 147.36°E; tropical) in far north Queensland, have the accession numbers KC701473 and KF606977. The strains from the Brandon region were first collected in 2010 and have been maintained in culture at James Cook University (JCU) for more than 2 years. All three species have been maintained in stock cultures at the Marine and Aquaculture Research Faculties Unit (MARFU), JCU, Douglas campus (19.33°S, 146.76°E), in dechlorinated town water (DTW) with f/2 media addition since collection.

The ADW used in this study is an effluent created during ash disposal practices at a coal-fired power station. ADW was sourced directly from the Tarong coal-fired power station in south-east Queensland (26.76°S, 151.92°E) and transported to James Cook University (JCU), Townsville in clean plastic 1000 L Intermediate Bulk Containers (IBCs) in November 2012. The ADW was then stored at ambient temperature in 12,000 L storage tanks. A sub-sample of this water was taken from the storage tank for this study in December 2012 and subject to elemental analysis to quantify initial conditions in the effluent (Table 1). The ADW was provided by Stanwell Energy Corporation.

Experimental design

A cultivation study was conducted to assess the growth of the three species of Oedogonium, as well as the bioconcentration of metals and metalloids from the ADW. The experiments were conducted within a climate controlled room (28°C, 100 µmol photons m⁻² s⁻¹ and a 12 light: 12 dark photoperiod). The experimental temperature was chosen to reflect the median daytime temperature at the time of running the experiments. Stock cultures of each species were maintained for five weeks in DTW with f/2 nutrient addition (0.1 g Aquasonic^® f/2 growth media L⁻¹) to acclimate them to the experimental conditions. The aim of the acclimation period was to attain ‘steady state’ productivity, defined here as <5% change in Specific Growth Rate (SGR) for two consecutive weeks prior to initiating the experiment. This was achieved on day 35 (Fig. S1) , and from this time point onwards the three species were cultivated in either Ash Dam water or DTW with f/2 addition as described below, for three consecutive growth periods of seven days. The experimental period therefore spanned 21 days and consisted of three cultivation and harvest cycles of equal duration as described below.

The three species of Oedogonium were cultured in 1 L Schott bottles at an initial stocking density 0.5 g fresh weight (FW) L⁻¹. The FW was measured by gently blotting surface moisture from the biomass and weighing it. The Schott bottles were stocked with biomass from one of the three species and filled with either ADW or DTW amended with f/2 media, and then placed randomly in the climate controlled room. The bottles were aerated with compressed air delivered via a Pasteur pipette (at a flow rate of 0.2 L min⁻¹) to each replicate bottle, and they were randomly redistributed on a daily basis to avoid possible light bias in the experimental area. Each replicate bottle was harvested every seven days by pouring the contents through 20 µm filter mesh. A sample of the water was filtered (0.45 µm) and retained for metal analysis. The biomass was blot-dried with paper towel and weighed to the nearest 0.1 g. A sub-sample of this biomass was returned to the bottle with new water and growth media to reset the stocking density at 0.5 g FW L⁻¹. Any remaining biomass from each harvest was then dried in a dehydrator for 48 h at 60 °C.

Growth rates for each of the treatments were calculated based upon the FW determined for eight consecutive growth periods of seven days (t = Day 7). The Specific Growth Rate, SGR (% FW d⁻¹), was calculated using the equation: ${\displaystyle \mathrm{SGR}=Ln\left(W_f/W_i\right)/T\ast 100}$ where W_f, is the final weight (g FW) of biomass, W_i, is the initial weight (g FW) of biomass, and T, is the number of days in culture (7).

Elemental analysis

The algal biomass and the two water sources (ADW and DTW) were each analysed for the same 17 elements at the start of the experiment (Table 1). Water samples were collected using a 60 mL syringe and filtered (0.45 µm) to remove particulates. The concentrations of the 17 elements (Table 1) were also determined for all three species of Oedogonium grown in the Ash Dam water treatments before exposure to Ash Dam water, and at each of the three time points during the 21 day experimental period when Oedogonium was cultivated in Ash Dam water. All biomass was prepared for the analysis by drying in a dehydrator for 48 h at 60 °C, then digesting 100 milligrams (mg) of the dried algae in a Teflon digestion vessel with 3.0 milliliters (ml) double distilled HNO₃ and 1.0 ml analytical grade H₂O₂. The solution was digested for 2 h then heated in a microwave oven to 180 °C for 10 min, then diluted with Milli-Q water. The concentrations of Al, As, Ba, Cd, Co, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Pb, Se, Sr, V and Zn were measured with a Bruker 820-MS Inductively Coupled Plasma Mass Spectrometer (ICP-MS). An external calibration strategy was used for both instruments with a series of multi-element standard solutions containing all of the elements of interest (three standard solutions were tested containing 10, 25 and 50 µg L⁻¹ of each element) and the results were reported after subtracting the procedure blanks. The multi-element standards were obtained from Choice Analytical (Sydney, Australia). Collisional Reaction Interface (CRI) was used for As (H₂) and V (He), while ⁸²Se isotope was used for Se quantification, to eliminate polyatomic interferences for these elements. A 1% HCl solution was spiked with 1 ppb As, Se and V and measured three times for quality control; recovery between 98.5 and 110% indicated no significant interferences. All analyses were conducted at the Advanced Analytical Centre at JCU, Townsville.

Statistical analysis

As the aim of the experiment was to compare long-term growth rates for the three species of Oedogonium in Ash Dam water with DTW, the mean growth rate was determined for each replicate over the course of the three week experimental period containing the ADW treatments. The mean growth rate was then analysed by two-factor analysis of variance (ANOVA) for the main experimental factors of water source (Ash Dam water and DTW) and species. Where necessary the growth data were log transformed (Quinn & Keough, 2002). Residual histograms and scatter plots of residuals vs. estimates were assessed to determine normality and homogeneity of variance respectively (Quinn & Keough, 2002). Post-hoc comparisons for main effects and their interactions were made using Tukey’s HSD multiple comparisons.

The concentrations of metals in the algal biomass were first subject to multivariate ordination with non-metric Multi-dimensional Scaling (nMDS) to assess multivariate patterns in the temporal profile of metal bioconcentration from Ash Dam water by the three species. The mean data for the metal concentration within the biomass (mg kg⁻¹) were reassembled in a Bray-Curtis similarity matrix, focusing specifically on elements for which there are existing Australian and New Zealand Environmental Control Council (ANZECC) water quality criteria (see Table 1 for ANZECC water quality criteria). Mean metal contents of the three species were then contrasted for individual metals with a one-way ANOVA with the factor ‘species’. The elemental concentration of the algal biomass was also converted to a bioconcentration factor (BCF) for elements that have stipulated ANZECC trigger values. The BCF is the ratio of the element concentration in the water, to the final concentration in the biomass (DeForest, Brix & Adams, 2007). The bioconcentration factor (BCF) was calculated using the equation: ${\displaystyle \mathrm{BCF}=C_b/C_w}$ where, C_b is the concentration of the element in the dry macroalgal biomass (mg kg⁻¹) and C_w is the initial concentration of the element in the water phase (mg L⁻¹). The BCF is therefore a dimensionless ratio that expresses uptake of elements relative to availability in the water sample.

Elemental profile of Ash Dam water and DTW

The elemental composition of the Ash Dam water was complex with several metals and metalloids at concentrations significantly higher than the relevant ANZECC water quality criteria (Table 1). The concentrations of Al, As, Cd, Mg, Mn, Mo, Ni, Se, Sr, V and Zn were all significantly higher in Ash Dam water than DTW (two-tailed t test, P < 0.05), with the concentration of Al, As, Cd, Ni, Se and Zn exceeding the ANZECC trigger values designated for the protection of aquatic life at the 95% level (Table 1). The Ash Dam water is therefore a complex industrial effluent with multiple metal (Al, Cd, Ni and Zn) and metalloid (As and Se) targets for bioremediation. The remainder of the results section will focus on those elements that exceed ANZECC water quality criteria as the aim of the study is to ascertain interspecific variation in the potential for bioremediation.

Between-species variation in growth rates

‘Steady state’ productivity (defined here as <5% change in SGR for two consecutive weeks) was attained after 5 weeks of acclimation for all three species (Fig. S1). Acclimation to ‘steady state’ productivity was conducted to minimise any anomalous growth rates during the experiments as a result of changing photo-regime (µmol photons m⁻² s⁻¹) and photoperiod associated with the biomass being sourced from stock cultures and subsequently exposed to controlled experimental conditions. During this period, one of the Oedogonium species (KC701473) had a significantly higher mean productivity (23% SGR) than the other two species (∼20% SGR for both KF606977 and KC606974; Fig. S1).

After acclimation, all three species were exposed to either Ash Dam water or DTW with nutrient addition. There was a significant ‘species x water source’ interaction for the mean growth rates of the three species of Oedogonium (“Species x Water Source”: F_2,12 = 4.95, P = 0.027). The growth rate of one species of Oedogonium (KC701473) was significantly higher than the growth rates of the two other species in DTW (Fig. 1). KC701473 had a mean SGR of 26% in DTW, in comparison to the other species that had a mean SGR of 20% (KF606977) and 16% (KC606974) over the three week experimental period (Fig. 1).

The overall growth rates for each species were reduced in Ash Dam water relative to those in DTW, with the greatest reduction for KC701473 (decreasing from 26% in DTW to 7.5% in Ash Dam water) (Fig. 1). However, in contrast to the significant difference observed in growth between the three species when grown in DTW, there was no difference in the growth rates between the three species of Oedogonium in Ash Dam water (Fig. 1). There was an apparent trend towards higher SGR for the Oedogonium from the Brandon region (KF606977) and Tarong (KC606974) than KC701473 in Ash Dam water (Fig. 1), but these differences were not statistically significant (Fig. 1).

Patterns in metal and metalloid bioconcentration

All three species had low concentrations of most elements at the end of the acclimation period in DTW, although KC701473 had a significantly higher K content than the other two Oedogonium species (Table S1). There were no significant differences between the three species of Oedogonium with respect to mean bioconcentration of metals or metalloids over the course of the experiment (Fig. 2). The only elements for which there was a significant difference in bioconcentration between species of Oedogonium were Cd and As. Cd was bioconcentrated to a significantly higher concentration in KF606977 and KC606974 than KC701743 (Fig. 3B, Table 2), while As was bioconcentrated to a significantly higher concentration in KC701743 than the other two species (Fig. 3E, Table 2). The remaining elements showed no significant differences in their bioconcentration between the three species of Oedogonium (Fig. 3, Table 2). The majority of elements that did not initially exceed ANZECC criteria also had similar concentrations in the three species of Oedogonium at the conclusion of the experiment (Fig. S2 and Table S2).

While the mean bioconcentration of metals and metalloids did not differ between the three species by the end of the experiment, there were minor differences in the temporal pattern of metal bioconcentration. The clustering of the three species with respect to the vector orientation in the MDS supports a temporal sequence in metal uptake and that this differs between the three species. In the first week following the transfer of the cultures to Ash Dam water, all three species bioconcentrated a subset of elements from Ash Dam water (Mn, Pb and Fe, Fig. 4), however, KC606974 and KF606977 bioconcentrated the metals Cd, Ni and Zn much more rapidly in the first week than KC701473 (Fig. 4). By week two, KC701473 had a similar element profile to the other species having bioconcentrated the trace elements Al, Cd, Ni, Se and Zn, while KC606974 clustered with the group of element vectors of Se, Ni, Zn, Cd, Al and B and had the highest internal concentrations of these elements in week 2 (Fig. 4). In the third week of cultivation, the elemental profile of the Oedogonium KC606974 was similar to its initial (post-acclimation) composition, while the other two Oedogonium species (KC701473 and KC606974) continued to have higher concentrations of elements (Fig. 4).

These patterns are also clearly reflected in the temporal sequence of bioconcentration of metals (Al, Cd, Ni and Zn) and metalloids (As, Se) that exceeded ANZECC criteria by the three species of Oedogonium. The four metals (Al, Cd, Ni and Zn) were bioconcentrated more rapidly by Oedogonium isolate KC606974 and KF606977 than KC701473. There were significantly higher internal concentrations of Al, Cd, Ni and Zn in these two species after one week of exposure to Ash Dam water (Figs. 5A–5D). However, the internal concentrations of Al, Cd and Ni in the three species converged and were not different by the end of the experiment, while Zn remained higher in KF606977 than the other two species (Fig. 5D). In contrast, bioconcentration of As differed between the three species, with significantly higher As contents in KC701473 than KF606977 and KC606974 by the end of the experiment (Figs. 5E) . There were no significant differences in the rate or extent of bioconcentration of Se by the three Oedogonium species (Fig. 5F).

Metal and metalloid bioconcentration factors

The element bioconcentration factors were very consistent between the three species of Oedogonium. For all species, the highest BCF was attained for Cu, Mn, Zn and Ni with values ranging from 709–1418 for Zn, 889–1256 for Ni, 6938–8943 for Cu, and 5892–16719 for Mn (Tables 3 and 4). Conversely, Al, Se and Mo all had the lowest BCF with values of 3–4 for Mo, 79–93 for Se and 259–351 for Al (Tables 3 and 4). The ranking of the BCF amongst elements was, however, relatively consistent with the same four elements (Cu, Mn, Zn and Ni) having the highest BCF for all three species, while the same three elements (Al, Se and Mo) had the lowest BCF for all species (Table 4).