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Feb 15, 2014 - the potential of the red macroalgae, Chondrus crispus. Stackhouse and Palmaria palmata (Linnaeus) Weber & Mohr, to remediate effluent from ...
J Appl Phycol (2014) 26:2173–2181 DOI 10.1007/s10811-014-0247-0

Intermittent aeration affects the bioremediation potential of two red algae cultured in finfish effluent Scott Caines & Juan A. Manríquez-Hernández & Jim Duston & Peter Corey & David J. Garbary

Received: 25 September 2013 / Revised and accepted: 22 January 2014 / Published online: 15 February 2014 # Springer Science+Business Media Dordrecht 2014

Abstract The high cost of aeration needed to tumble culture macroalgae is a limiting factor for integration with land-based finfish culture. Toward reducing this electricity cost, we compared intermittent aeration (16 h on:8 h off) with continuous aeration (24 h on) on the productivity of two strains of Chondrus crispus (Basin Head and Charlesville) and Palmaria palmata from Atlantic Canada between May and June 2011. Algal fronds were cultured under a 16:8-h light/ dark photoperiod in 50-L tanks supplied with finfish effluent (49 μmol L−1 of ammonium and 11 μmol L−1 of phosphate) at a mean water flow rate of 0.4 L min−1. Nitrogen (N) influx was 1.8 gN m−2 day−1, and phosphorus (P) influx was 0.9 gP m−2 day−1, with uptake rates ranging from 0.02 to 2.4 gN m−2 day−1 and −0.2 to 0.4 gP m−2 day−1. On average, the macroalgae culture system (algae and biofilms) removed 1.0 gN m−2 day−1 (51.9 %). The growth of macroalgae (pooled across treatment and strain) ranged from 0.5 to 1.6 % day−1, which accounted for a yield of 2.2 to 5.4 g DW m−2 day−1. Switching off aeration at night improved the growth rate of Basin Head Chondrus by 146 % and had no effect on growth rate or nitrogen and carbon removal by P. palmata and Charlesville Chondrus. Growth and yield of Basin Head Chondrus under intermittent aeration were over two times greater than both Charlesville Chondrus treatments.

S. Caines : J. A. Manríquez-Hernández : J. Duston Department of Plant and Animal Sciences, Faculty of Agriculture, Dalhousie University, Truro, NS, Canada P. Corey Scotian Halibut Ltd., Wood’s Harbour, NS, Canada D. J. Garbary (*) Department of Biology, St. Francis Xavier University, Antigonish, NS, Canada e-mail: [email protected]

Keywords Chondrus crispus . Dulse . Effluent treatment . Irish moss . Palmaria palmata . Sustainable aquaculture

Introduction The integration of macroalgae into land-based marine finfish culture is an attractive combination: producing a second crop and reducing the risk of eutrophication from farm effluent. Typical land-based closed systems effectively remove feces and waste food but fail to deal with the dissolved nutrients (Crab et al. 2007). Nitrogen (N)- and phosphorus (P)-rich effluent discharged into coastal waters can result in damaging eutrophication (Kaartvedt et al. 1991; Troell et al. 1999), although the effects can sometimes be beneficial (White et al. 2011). Culturing macroalgae can help purify the finfish effluent (Neori et al. 2003; Matos et al. 2006; RobertsonAndersson et al. 2008). Our contribution has been to study the potential of the red macroalgae, Chondrus crispus Stackhouse and Palmaria palmata (Linnaeus) Weber & Mohr, to remediate effluent from a land-based Atlantic halibut farm (Hippoglossus hippoglossus; Corey et al. 2012, 2013; Kim et al. 2013). The problem is that tank-cultured red macroalgae must be “tumbled” by vigorous aeration to optimize growth and maintain health (Bidwell et al. 1985; Craigie and Correa 1996; Lüning and Pang 2003; Msuya and Neori 2008). The cost of electricity needed to operate air pumps can exceed the value of the macroalgae in our region, hence our interest in intermittent aeration. Intermittent aeration received attention during the 1980s and 1990s but has been mostly ignored in recent years (DeBusk et al. 1986; Friedlander and Ben-Amotz 1991; Ugarte and Santelices 1992; Msuya and Neori 2008). C. crispus grew best when agitated continuously by paddle wheels but achieved 82 % of maximum growth when agitated for 12 h (50 % reduction in power usage) during daylight

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hours from February to May (Shacklock et al. 1975). Bubbling air proved better than paddle wheels to keep C. crispus tumbling and could be turned off at night with no loss of production, although no data were presented (Bidwell et al. 1985). Macroalgae with different morphologies, a syntagmatic red macroalga, Gracilaria, and a bilayer green macroalga, Ulva, showed similar productivity for 12 and 24 h aeration cycles (Guerin and Bird 1987; Vandermeulen and Gordin 1990). Studies with natural or nutrient supplemented seawater suggest that 4 to 10 hours of intermittent aeration a day is required to maintain high growth of Gracilaria spp. (Ryther et al. 1984; Ugarte and Santelices 1992). Short intermittent aeration cycles of 1 minute to 2 hours have been successfully used, but cost savings with these intermittent regimes can be offset by spikes in electricity consumption during frequent starting of air compressors (Ryther et al. 1984; DeBusk et al. 1986; Friedlander and Ben-Amotz 1991). Some observations with C. crispus and Gracilaria conferta in seawater alone suggest that aeration during the night is not needed to achieve high yields, while the green macroalga, Ulva lactuca, can be cultured without aeration when sufficient agitation is provided by water circulation (Shacklock et al. 1975; Bidwell et al. 1985; Friedlander and Ben-Amotz 1991; Msuya and Neori 2008). To date, no study has investigated the effects of major reductions in aeration or intermittent aeration as a means of cultivating red macroalgae for the bioremediation of finfish effluent. This study investigates the effects of intermittent aeration on growth, N and carbon (C) removal by algal fronds, nitrogen and P removal by culture units, and ammonium and phosphate uptake of P. palmata and C. crispus supplied with finfish effluent from a land-based farm. P. palmata and C. crispus were chosen as they are common to Atlantic Canada and have been successfully integrated into landbased finfish farms (Matos et al. 2006; Kim et al. 2013). Two populations of C. crispus, a population with vegetative propagation from Basin Head, Prince Edward Island, Canada (see Sharp et al. 2010 for history and status) and a sporeforming population newly isolated from the Atlantic coast of NS, Canada, were cultured to determine if one is more suited for integration with finfish.

Materials and methods Palmaria palmata was collected in June 2009 from a natural population at Point Prim, NS, Canada (44°41′N, 65°46′W) where commercial harvesting occurs (Garbary et al. 2012). A population of Chondrus crispus originally from Basin Head (herein Basin Head Chondrus), Prince Edward Island, Canada was obtained from the Sandy Cove Aquaculture Research Station of the National Research Council, Halifax, NS in June 2009. The derived population at Wood’s Harbour forms large

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foliose blades in situ and is gametophytic (Garbary et al. 2011). A population of C. crispus (herein Charlesville Chondrus) was collected from the intertidal zone at Charlesville, NS (43°34′N, 65°47′W) in May 2011. Basin Head Chondrus and P. palmata were cultured in the effluent of a land-based Atlantic halibut farm (H. hippoglossus), Scotian Halibut Ltd., Wood’s Harbour, NS (43°31′N, 65°44′ W), from June 2009 to experiment acclimation. Over the year, the effluent temperature ranged from 1 to 18 °C and the nitrogen concentration between 6 and 564 μM N (Corey 2012). Charlesville Chondrus was maintained in effluent for 17 days prior to acclimation. Culture facility Macroalgae were cultured during May–June 2011 in 18 white tanks (50-L laundry tub: 50 L×50 W×20 H cm) arranged in three rows of six within a black plastic enclosure inside a large barn. Temperature ranged from 11.5 to 16.2 °C (Table 1). The tanks were illuminated by 32 W T8 fluorescent lamps (Philips), with16:8-h light/dark photoperiod, with the irradiance at the water surface at a photosynthetic photon flux rate of 112±5 μmol photons m−2 s−1 (LI-192SA, Li-Cor, USA). The mean (±SE) concentration of ammonium and phosphate in the inflowing fish effluent was 49.4 ± 6.6 and 10.8 ± 0.6 μmol L−1, respectively. Effluent was supplied at a rate of 2,558.8 L m−2 day−1, which accounted for a nutrient influx of 0.9±0.04 gP m−2 day−1 and 1.8±0.1 gN m−2 day−1. Salinity was 30 psu. Vigorous agitation of macroalgae was achieved by supplying air (4.5 hp regenerative compressor: Fuji Electric, USA) from 1 mm holes drilled every 2 cm in a 1.3 cm (inside diameter) vinyl tubing lining the bottom of each tank in a 30-cm-diameter circle. Fronds were cut between 4 and 6 cm in length, and 500 g was stocked into each tank at 2 kg m−2 (10 g L−1) fresh weight (FW), an optimum density for productivity and nutrient removal (Kim et al. 2013). Following a 7-day acclimation period, the biomass of each tank was restocked to 2 kg m−2 by removing excess material. Each strain was grown under two aeration treatments: (1) 24 h continuous aeration and (2) a 16-h on:8-h off (16 h herein) aeration, achieved by manually turning the air supply on and off. The biomass in each tank was measured and readjusted to 2 kg m−2 each week for a total of 3 weeks. Measurements Temperature and pH (YSI 60) were monitored twice daily (0800 and 1600 h), and water flow was measured daily and adjusted to maintain a flow rate between 0.4 and 0.6 L min−1. The total FW at each sampling period was obtained by spinning the macroalgae for 1 to 2 min at ca. 500 rpm in a residential washing machine (drum diameter 53 cm and depth

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Table 1 Physical parameters in May to June 2011 during testing the effect of intermittent aeration on the growth and bioremediation capacity of three strains of red macroalgae, Basin Head and Charlesville C. crispus and P. palmata, grown in land-based finfish effluent Parameter

Mean

Range

Temperature (°C) 24 h aeration 16 h aeration pH 24 h aeration 16 h aeration Water flow (L min−1) 24 h aeration 16 h aeration Turnover (day−1) 24 h aeration 16 h aeration Mean daily light dose (mol photons m−2 day−1) 16:8-h light/dark 24 h aeration 16 h aeration

13.4±0.04 13.6±0.05 13.3±0.05 7.5±0.02 7.7±0.01 7.3±0.03 0.4±0.01 0.5±0.02 0.4±0.02 12.8±0.4 14.1±0.7 11.6±0.6 6.4±0.3

11.5–16.2 11.6–16.2 11.5–16.0 6.6–8.0 6.6–8.0 6.6–7.9 0.05–0.9 0.1–0.9 0.05–0.9 1.5–26.5 3.5–26.5 1.5–25.3 4.1–8.9

6.3±0.3 6.6±0.5

4.6–7.7 4.1–8.9

Values are mean±SE

35 cm; Maytag, USA) and weighing within 2 min of spinning. Dry weight (DW) was calculated from 1 to 10 g samples dried to constant weight at 60 °C. Dried tissues were ground to powder with either a ball mill (MM301, Retsch, USA) or a roller-grinder custom built for D. Lynch (DAL-AC). Total tissue N and C were determined using either a CNS-1000 (Leco, USA) or a VarioMAX (Elementar, Germany). Specific growth rate (SGR; % day−1) was calculated as follows: SGR; % day−1 ¼

ln M 2 −ln M 1  100 T 2 −T 1

Removal; gN day−1 m−2

Nitrogen removed (gN m−2 day−1) by culture unit was calculated as follows:  ð F  ðN 1 −N 0 ÞÞ  10−6  Mm Removal gN m−2 day−1 ¼ SA ð5Þ

ð1Þ

Nitrogen removed (gN m−2 day−1) by macroalgae was based on the equation in Kim et al. (2007): M 2 −M 1 DW  Tissue N  T −T FW ¼ 2 1 SA

removal was calculated with the same equation, except tissue C was substituted for tissue N. Removal is a direct measurement of nitrogen and carbon mitigation by cultured macroalgae, while uptake efficiency represents the bioremediation potential of the culture system, including cultured species and microalgae and bacterial films on the surfaces of macroalgae and tanks (Dvir et al. 1999; Hernández et al. 2006; Corey et al. 2012). Water samples were collected from the inlet of a single tank and from the drains of each tank after each weekly biomass determination at three times over a 24-h period (0800, 1600, and 0800 h). Replicate water samples from each treatment were combined to form a single composite sample for each sampling time. A mean inlet and outlet concentration (μmol L−1) was calculated for ammonium and phosphate over two periods (0800 to 1600 and 1600 to 0800 h). Ammonium was analyzed using a Technicon Auto Analyzer II (SEAL Analytical, USA) following the Technicon industrial method no. 155-71W. Phosphate was determined following Parsons et al. (1984). Uptake efficiencies were calculated following a modified version of the equation used by Evans and Langdon (2000):   N 1 −N 0 Uptake efficiency ðU Þ ¼ F   100 ð4Þ N1

ð2Þ

where U is the percent of ammonium or phosphate removed by the system; F is the mean flow over a 24-h period (L day−1); N1 and N0 are the initial and final mean concentrations (μmol L−1) over a sampling period, respectively; and Mm is the molar mass of nitrogen or phosphorus. Statistical analysis

Yield (g DW m−2 day−1) was based on the equation in Mata et al. (2010):

Yield; g DW m

−2

day

−1

M 2 −M 1 DW  FW T −T ¼ 2 1 SA

ð3Þ

where M2 and M1 are biomass (g) at day T2 (final) and day T1 (initial), respectively; DW is dry weight; FW is fresh weight; and SA is the surface area (m2) of the culturing unit. Carbon

To account for correlation between measurements on the same tank over time (3 weeks), specific growth rate, N and C removal and tissue N and C were analyzed as a repeated measures mixed model design. The factors were strain (three levels: P. palmata, Basin Head Chondrus and Charlesville Chondrus) and treatment (two levels: 24 h and 16 h aeration). Different types of covariance structure were tested, and those with a lower Akaike’s information criterion (AIC) that met the normality requirement were selected to run the Mixed procedure (Elliot and Woodward 2010). Reporting models with low

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AIC values ensures the most appropriate covariance structures are used, which decreases the divergence between the predicted and actual densities, reduces the amount of information loss, and improves the strength of data interpretation. The Anderson–Darling normality test (Anderson and Darling 1952) was conducted in Minitab 16 (Minitab Inc., USA), using the residuals of the Mixed procedure of SAS statistical software (SAS 9.3, Institute Inc., USA). Tissue C was natural log-transformed to meet the normality criterion. Least-squares means analysis (p=0.05) was used as a post hoc test to determine pairwise relationships among all combinations. Mean ammonium and phosphate uptake efficiency data were calculated by strain and treatment; a one-way ANOVA was determined in Minitab 16.

Results Intermittent aeration significantly improved the growth of Basin Head C. crispus by 145.6 %, but did not have a significant effect on the growth of Charlesville Chondrus or P. palmata (Fig. 1). The mean SGR of Charlesville Chondrus supplied with intermittent aeration was 8.4 % higher than continuous aeration, while growth of P. palmata supplied with intermittent aeration achieved 83.6 % of continuously aerated fronds. Overall, P. palmata achieved the highest SGR (1.6±

0.30

A

a

24 h 16 h 1.5

ab b

1.0 c

c

c 0.5

Nitrogen removal (gN m-2 d-1)

Specific growth rate (% d-1)

2.0

0.1 % day−1, mean±SE; pooled across treatments and weeks), which was 3.5-fold higher than 24 h aerated Basin Head Chondrus and up to 3.6-fold higher than the Charlesville Chondrus under both aeration treatments (Fig. 1). Growth (pooled across algal strains and treatments) decreased from 1.2 ± 0.2 to 0.8 ± 0.1 % day−1 (ANOVA: Fdf = 9.33(2,22), p=0.001) between weeks 1 and 3 (Table 2). The two-way and three-way interaction terms for week were not significant (p>0.05), so data were pooled over strain and treatment to represent variation in specific growth rate, removal of N and C by algal tissues, and ammonium and phosphate uptake over time (Table 3). Tissue N and C and the removal of N and C by algal tissues were not affected by intermittent aeration (Table 4; Fig. 1). Overall, tissue N for each strain differed by as little as 0.1 % between aeration treatments and by as much as 0.2 % between strains (Table 4). Tissue C differed between strains and was the highest in P. palmata (32.8±0.5 %; ANOVA: Fdf =0.48(2, 12), p=0.024). Mean removal of N and C by Basin Head Chondrus was up to 2.2-fold greater under intermittent aeration compared to continuous aeration. P. palmata (pooled across treatments) removed the most N (0.22±0.01 g N m−2 day−1), which was 2.2-fold higher than Charlesville Chondrus (LSD p=0.009; Fig. 1). The removal of C was 2.3-fold higher for P. palmata compared to Charlesville Chondrus (Fig. 1). Nitrogen and carbon removal by algal tissues

B

a

a

0.25

ab

0.20 b 0.15

b b

0.10 0.05 0.00

0.0

BH

CV

PP

Strain 2.0

a

C

80

1.5

1.0

b

b

D

Ammonium Phosphate

a

Uptake (% removed)

Carbon removal (gC m-2 d-1)

a

b

0.5

0.0

60

40

20

0

BH

CV

PP

Strain

Fig. 1 Mean (SE) specific growth rate (A), nitrogen (B) and carbon removal by algal tissues (C), and ammonium and phosphate uptake (D) of Basin Head (BH) and Charlesville (CV) C. crispus and P. palmata (PP)

BH(24h) BH(16h) CV(24h) CV(16h) PP(24h) PP(16h) Strain (treatment)

grown with 24 h and 16 h aeration between 30 May and 20 June 2011. Values sharing the same letters are not significantly different (α=0.05). Values in D are not significantly different with each nutrient

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Table 2 Temporal variation in specific growth rate, nitrogen and carbon removal, and ammonium and phosphate uptake pooled across strain (P. palmata, and Basin Head and Charlesville C. crispus) and aeration treatments (24 and 16 h) Week Parameter

1

2

3

0.8±0.1b 0.8±0.1b Specific growth rate (% day−1) 1.2±0.1a N removal (g m−2 day−1) 0.2±0.02a 0.1±0.02b 0.1±0.02b C removal (g m−2 day−1) 1.5±0.1a 1.0±0.1b 1.0±0.1b Ammonium uptake % 34.5±7.3b 60.1±4.0a 61.1±3.5a Phosphate uptake % 9.5±4.0b 25.9±2.3a 25.0±3.3a Values are the mean±SE. Values sharing the same letters are not significantly different (α=0.05)

decreased by up to 35 % between weeks 1 and 3 (Table 2). The ratio of C to N was on average 7.1 and ranged from 6.9 to 7.4 (Table 4). Nitrogen influx, in the form of ammonium, in effluent across all treatments was on average 1.8±0.1g m−2 day−1, while P influx, in the form of phosphate, was 0.9±0.04 g m−2 day−1. Nitrogen removal by the culture system (algal tissues and biofilms) was as high as 1.2±0.3 g m−2 day−1 in 16 h aerated Basin Head Chondrus and was on average 1.0 ± 0.1gN m−2 day−1 (51.9 %) across all treatments and strains (Table 5). The removal of N by algal tissues was on average 0.2±0.01 gN m−2 day−1, which accounted for 16.5 % of the total N removed by the culture system. The removal of P by the culture system was on average 0.2±0.02 g m−2 day−1, which was 20.1 % of the total phosphorus supplied in effluent (Table 5). Table 3 Results from repeated measures mixed model analyses showing degrees of freedom (df), F values, and p values for individual factors and interaction terms for specific growth rate, carbon and nitrogen removal by algal tissues, carbon to nitrogen ratio, and tissue carbon and nitrogen of C. crispus (Basin Head and Charlesville) and P. palmata, grown at two aeration treatments (24 and 16 h aeration) in land-based finfish effluent

Factor (s) Treatment Strain Treatment×strain Week Treatment×week Strain×week Treatment×strain×week Factor (s) Treatment Strain Treatment×strain Week Treatment×week Strain×week Treatment×strain×week

The uptake of ammonium and phosphate was independent of aeration treatment and algal strain but varied significantly between sampling period (Fig. 1; Table 2). Among the three strains of macroalgae, the uptake of ammonium and phosphate (pooled across treatments) was the highest for Charlesville Chondrus (53.9±4.4 % and 21.9±3.1 %, respectively, Fig. 1). There was a 2.6-fold increase in the uptake of phosphate (pooled across treatments and strains) between weeks 1 and 3 (Table 2).

Discussion A limiting factor for the integration of macroalgae into aquaculture is offsetting operating costs (electricity, labor, and pumping water) relative to the income generated from algal sales (Bidwell et al. 1985; McLachlan 1991). An 8-hour period of no aeration during the night had no adverse effects on the growth or bioremediation capabilities of red macroalgae cultured in finfish effluent. Culturing red algae with no aeration during the night can reduce air compressor electricity costs by 33 to 50 % and increase the viability of integrated aquaculture. Overall, 52 % of nitrogen, in the form of ammonium, was removed from the culture system, with approximately 17 % of this being incorporated into algal tissues. Circulating macroalgae with bottom aerated tanks is thought to improve growth by minimizing the diffusion boundary layer and increasing exposure of fronds to light (Friedlander and Ben-Amotz 1991; Gonen et al. 1993, 1995; Msuya and Neori 2008). Adequate aeration is essential with increasing tank depth or highly stocked tanks, where light and

Specific growth rate df F p 1 1.49 0.2456

Carbon removal df F p 1 0.89 0.3646

Nitrogen removal df F p 1 1.09 0.3172

2 22.56 2 4.61 2 9.33 2 0.20 4 0.48 4 0.28 C/N ratio df F 1 0.00 2 0.47 2 0.09 2 3.52 2 0.71 4 3.71 4 0.81

2 8.22 2 2.78 2 5.98 2 0.07 4 0.76 4 0.71 Tissue carbon df F 1 1.35 2 5.19 2 0.48 2 5.18 2 0.59 4 0.37 4 0.81

2 5.16 2 2.59 2 4.55 2 0.21 4 0.80 4 0.90 Tissue nitrogen df F 1 0.18 2 0.03 2 0.03 2 4.30 2 0.65 4 3.36 4 1.48