Spray Irrigation Culture of Macroalgae in CO2 Enriched Atmosphere using Recirculated Seawater

Amir Neori 1, Patricia Abelin 2, Sitti Raehanah M.Shaleh 2, Dominick Mendola 2, B. Greg Mitchell 2
1. National Center for Mariculture Eilat, Israel Oceanographic and Limnological Research 2. Scripps Photobiology Group, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA

ABSTRACT
HN 4+m M - o r ng m ni
NH4+ M - afternoon NH4+ M - morning
tn eirtu n x 2 + 2OC + riA
2 OC

Figure 4: Monitoring the residual of daily ammonia uptake at 10AM before nutrient addition and at 4PM. Each day at
10 am we added a very small volume of concentrated media to result in 300 M or 600 M (2x) final ammonium concentration. Adding 2x led to accumulation of excess ammonium.

pH - o rn ng m i

Figure 5: Variation of pH during the culture period in the morning

at 10AM before starting the CO2 injection (left panel) and at 4PM after 6 h of CO2 enrichment (right panel).

RESULTS
The specific growth rate of Ulva thalli (Figure 2) ranged between 3.7 and 6.4% / day. Two-way ANOVA test has shown that air enrichment with CO 2 did not significantly (P>0.05) affect growth of Ulva sp within treatments starting with the same biomass density. On the other hand, stocking density (in the range of 20 to 70g/m2 was found to have a significant effect (P<0.05) on the SGR. The SGR from treatment starting with the lowest biomass density (20g/m2) was significantly higher than the treatment starting with 40g/m2 when both biomass densities were tested in the same experiment. However, in a separate experiment with higher initial stocking density (70g/m2) the SGR was found to not be significantly different than the 20g/m 2 initial stocking density treatment. This result is perhaps due to differences between seaweed batches used for the experiments conducted at different times. Yield (stocking density x growth rate) increased with stocking density (Figure 3), i.e., optimal stocking density was not achieved under the low light levels of these experiments. Nutrients were added to saturation as shown in the results of ammonia uptake (Figure 4). The treatment receiving double the basal quantity of nutrients per day showed ammonia residual in the media not absorbed by the sample. Treatments exposed to the addition of CO2 ( six hours each day) had lower pH ~ 7.3 during this period of CO2 exposure (Figure 5 - right panel) and returned to ~8.2 by the time of the sample the next morning (Figure 5 - left panel).

CONCLUSIONS AND IMPLICATIONS

The novel experimental laboratory-scale spray system has been shown to be low cost, simple to operate and supplies reliable and consistent experimental results with multiple replicates. We are encouraged with the preliminary results using this system, and are continuing to improve various aspects of its design and operation for subsequent testing and experimentation with macroalgae. We intend to test growth-related parameters, such as yield, nutrient uptake, stocking densities, and chemical composition as a function of key operational parameters, including water exchange, light levels, temperature, CO2 enrichment and nutrient supply. Although the yields of seaweed biomass in spray cultures is still lower than that for submerged cultures reported in the literature. However, the potential for lower cost delivery of CO2 and harvesting may offset the lower productivity at larger scale.

SPRAY CULTURE SYSTEM

The culture set-up consisted of 15 experimental units. Each unit combines 3 separate, stacked, translucent plastic containers (Figure 1). In practice, a pre-weighed mass of seaweed thalli was placed onto the 144 cm2 porous plate located on the bottom the middle culture container. Seawater-based media (using ammonia nitrogen and phosphate salts) was continuously dripped onto the seaweed sample via a perforated distribution plate which received re-circulated media flow (~ 0.5 LPM) from a submersible pump in the reservoir beneath the seaweed culture container (Figure 1). Air, or air enriched with CO2 , flowed through the free volume of the culture container at various flow-rates depending upon treatment. Fresh seaweed biomass was collected from the natural populations in La Jolla, CA, and acclimated to laboratory culture conditions and culture media prior to testing. Light was provided by a bank of cool white fluorescent tubes (F54T5/850; 50 watt) which provided approximately 140 E/cm-2/s illumination at the level of the seaweed in each container. Culture temperature ranged from 2427oC.

CITATIONS
Haglund, K. and Pedersen, M. 1988. Aquaculture, 72:181-189. Hanisak, D.M. 1987. in: Developments in Aquaculture and Fisheries Science. No. 16, pp.191-218. Elsevier Science Publishers Indergaard, M., Ostgaard, K., Jensen, A. and Storen, O. 1986. J. Exp. Mar. Biol. Ecol., 98: 199-213. Lignell, A. and Pedersen, M. 1986. Bot.Mar., 29:509-516 Moeller, H.W., Garber, S.M. and Griffen, G.F. 1984. Hydrobiologia, 116/117:299-302. Msuya, F.E. and Neori, A. 2010. J.Phycol. 46:813-817. Neori, A., Msuya, F.E., Shauli,L.,Schuenhoff,A.,Kopel,F. and Shpigel,M. 2003. J.Appl.Phycol. 15:543-553. Pickering, T.D., Gordon, M.E. and Tong,L.J. 1995. Aquaculture, 130:43-49. Sahoo, D. and Yarish,C. 2005. Anderson R.A.(eds.) In: Algal Culturing Techniques, pp.219-238. Elsevier Academic Press. Schuenhoff,A., Shpigel,M., Lupatsch,I., Ashkenazi,A., Msuya, F.E. and Neori, A. 2003. Aquaculture 221;167-181.

G row t ra t % d ay ) h e ( /

Figure 2: Specific Growth Rate after five days of Ulva sp. culture stocked at three initial densities ( 20 40 70 grams DW/m2) cultivated in spray systems with injection of only air or injection of air plus CO2.
8

SGR =

loge DW2 - logeDW1 _______________________ x (t2 t1)

100

6 5 4 3 2 1 0 0 0 10 10 20 20 30 30 40 40 50 50 60 60 70 70 80 80 90 90 100 100

Where : SGR = specific growth rate (% day -1) DW1 = dry weight of the plant at start; DW of the initial biomass was calculated using wet weight and converting with a the ratio of wet to dry weight determined with the source biomass. DW2 = dry weight at a time t t = time (days) Dry weight content of harvested biomass was determined by oven drying the samples to a constant weight at 60oC;. A 2-way ANOVA test was used to determine the effect of the treatments while the Tukey test was applied to evaluate which variables had a significant effect on the specific growth rate. Data analysis was done using SYSTAT 13.

Initial weight of Ulva (g DW / m 2) Figure 3: Ulva yield in grams of dry weight /m2/day as a function of stocking density. Data from four experiments . For a comparison, Ulva yield in outdoor tanks (full sunlight) is on an annual average above 14 g DW / m2 / day (Msuya and Neori, 2010).

2 OC

riA

Experiments were conducted over a five-day period following two days acclimation under culture conditions. Culture variables tested were biomass density, media exchange and enrichment rates, effect of CO2 addition, and nutrient uptake. Residual ammonia left in the media before and after the daily introduction of nutrients was used as the indicator of nutrient uptake. Specific growth rate (% / day) was calculated using the following the formula (Hunt, 1978):

TREATMENTS AND MEASUREMENTS

Air

Air + CO2

ACKNOWLEDGEMENTS
This work was partially-supported by a sub-contract from the Gas Technology Institute, Des Plaines, Illinois, under a 2010 DOE/NETL funded project (DE-FE0002640) entitled Macroalgae for CO2 Capture and Renewable Energy A Pilot Project. Dr. A. Neori was supported in this research by his institution with supplemental funds from UCSD-SIO, as a visiting scientist on sabbatical leave in the laboratory of Dr. B. G. Mitchell at UCSD Scripps Institution of Oceanography, 2009-2010.

syad de spalE

2.7

0.7

syad despalE

sirT + 2OC + riA 2OC + riA riA

2.7

0.7

The plants were placed on a 12 x 12 cm perforated platform in the middle container. Water spray from the top container kept the algae moist and was drained through the perforated platform in the middle container to the bottom container media reservoir and pumped continuously to the top container.

6.7

4.7

4.7

Figure 1: Set up of 15 individual spray irrigation units

s ir T + 2OC + r iA

2 OC + riA

r iA

8.7

8.7

6.7

Marine macroalgae seaweeds are a fast growing renewable resource, mostly cultured in the sea for food, feed, and a wide range of chemicals. In recent years, large-scale seaweed culture is gaining more attention for generating biomass for fuel production and atmospheric carbon uptake, as well as for commodity alginates and for specialty pharmaceutical usage. The current yearly world production of all types of macrophytes is approximately 15 million tons, using low-cost, in-sea rope or latticestructure culture techniques (Sahoo & Yarish, 2005). Seaweed culture in ponds in Israel has been used for biofiltration of marine fish-farm effluents (Neori, et al., 2003), while in earlier years, seaweeds were cultivated in ponds in Florida for energy production (Hanisak, 1987). Seaweed cultivation using spray culture methods has been previously described (Haglund et al.,1988 ; Indergaard et al., 1986 ; Lignell & Pedersen, 1986) Mueller et al., 1984). In a recent study, (Msuya & Neori 2010), seaweed thalli (Ulva spp.) were cultured outdoors on horizontal plastic mesh beds, and nutrient rich seawater media plus waste nutrients was constantly sprayed over the supported thalli. Pickering et al., (1995), also conducted spray culture experiments with Gracilaria spp. on mesh platforms suspended in tanks The spray-culture methods employed here allow for controlled laboratory study of various macroalgal species and strains to test for their ability to grow and produce biomass under varying conditions of light, temperature, nutrient mixture, and CO2 gas enrichment. Information gained in these studies can be applied to design larger, outdoor-scale units.

2.8

0.8

2.8

0.8

INTRODUCTION

pH - a fte r oon n

syaD despalE

+ riA riA

4.8

006 005 004 003 002 001

syad de spalE

tneirt un x 2 + 2OC + riA 2OC + riA riA

006 005 004 003 002 001 0 4.8

Here we demonstrate a laboratory-scale closed spray-culture system for testing growth and environmental response of marine seaweed biomass. The system does not submerge the cultured thalli, but constantly sprays them with nutrient rich media while exposing them to CO2-enriched air captured within the closed culture chamber. To date, we have evaluated the performance of the system using Ulva spp., while a second set of tests are currently underway with Gracilaria spp. (both collected locally). Using the lab-system we are able to control for varying light levels, temperature, stocking densities, nutrient levels, media exchange rates, plus CO2 enrichment in the air phase. Growth results reported in this preliminary study are consistent with previously published data from the literature. The goal of this work is to develop a laboratory spray culture system to experimentally evaluate different macroalgae strains in spray culture with respect to the effect of CO2-enrichment on growth and productivity. Scaledup versions are envisioned that employ sealed greenhouse-type enclosures, wherein flue-gases from power plants or other CO2 generators, and nutrients from agriculture or domestic wastewaters, would be used to grow macroalgal crops for feed, fuel and energy.

NH 4+mM - a ft r oon en

2 m/g 02

2 m/g 04

2 m/g 07

8 6 4 2