Nutrient recovery from swine lagoon water by Spirodela punctata

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1 Bioresource Technology ) 81±85 Short communication Nutrient recovery from swine lagoon water by Spirodela punctata Jiayang Cheng a, *, Ben A. Bergmann b, John J. Classen a, Anne M. Stomp b, James W. Howard a a Department of Biological and Agricultural Engineering, North Carolina State University, Campus Box 7625, Raleigh, NC , USA b Department of Forestry, North Carolina State University, Campus Box 8002, Raleigh, NC , USA Received 22 May 2000; received in revised form 11 June 2001; accepted 13 June 2001 Abstract Spirodela punctata 7776, the best duckweed strain in total protein production selected from in vitro screening experiments with synthetic swine lagoon water medium was examined for N and P recovery. It has shown a capability to grow in and to remove N and P from synthetic swine lagoon water with high N 240 mg NH 4 ±N=l) and P 31.0 mg PO 4 ±P=l) levels. A lag period of approximately 96 h was observed before the duckweed started to grow. During the lag period, utilization of N and P by the duckweed was very slow. The rates of N and P uptake, and duckweed growth increased with the increase of the initial N and P concentrations in the medium. The highest rates of N and P uptakes, and duckweed growth observed in this study were 0.955, mg/l-h, and 1:33 g=m 2 -h or 31:92 g=m 2 -day), respectively. The N:P ratio in swine lagoon water is adequate for growing the duckweed. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Duckweed; Lemnaceae; Nitrogen; Nutrient recovery; Phosphorus; Spirodela punctata; Swine lagoon water; Wastewater 1. Introduction * Corresponding author. Tel.: ; fax: address: jay_cheng@ncsu.edu J. Cheng). Swine production has experienced a tremendous growth in the southeastern US in recent years. This dramatic increase in swine production has resulted in a large amount of waste and nutrients that require management. Swine farms in the region usually use anaerobic lagoons for treatment and temporary storage of the animal wastewater. The e uent of the lagoons is used for irrigating cropland and recycling to ush manure out of the swine houses. However, most swine farms in the southeastern region are specialized in hog production and have limited cropland available solely for removing nutrients from swine wastewater. With the growth of swine production, there are growing concerns of potential environmental contamination of excess nutrients, including ammonia emissions, nitrates in water supplies, and eutrophication in rivers and lakes. In coastal areas of North Carolina, recent outbreaks of sh kills have been linked to the stimulation of the dino agellate P esteria caused by nutrient runo Burkholder, 1999). There is great interest in developing alternative nutrient management systems for swine wastewater treatment to resolve environmental concerns. A potential option for nutrient management is to recover nutrients in aquatic plants that may have economic value as a byproduct, and could be removed from the farm. Duckweed is a free- oating aquatic plant that proliferates through vegetative budding of new fronds and accumulates biomass at rates greater than most other plants including eld crops Landolt, 1986). Duckweed also has a longer growing period than most other plants. In some areas with warm climates, duckweed can grow in all seasons. Duckweed has been used for tertiary treatment of municipal and industrial wastewaters for more than a decade Alaerts et al., 1996; Oron, 1994; Oron et al., 1988). Oron et al. 1988) reported using duckweed for domestic wastewater treatment to recycle the nutrients. E uent quality was at a secondary level and met criteria for reuse for agricultural irrigation. Dry yield of the duckweed approached 15 g=m 2 -day with a protein content of about 30%. Alaerts et al. 1996) demonstrated nutrient removal by duckweed in a sewage lagoon. Total Kjeldahl nitrogen TKN) and total phosphorus TP) were removed by 74±77%. A relatively clean e uent with 2.7 mg TKN/l and 0.4 mg TP/l was achieved /02/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S )

2 82 J. Cheng et al. / Bioresource Technology ) 81±85 Duckweed has a greater rate of nutrient uptake when compared to other aquatic macrophytes and has the advantage of preferential uptake of ammonium Oron et al., 1988). Ammonium uptake is critically important to the treatment of swine wastewater because ammonium is the primary form of nitrogen in the wastewater and can accelerate eutrophication in open ponds or can result in nitrate formation if moved into ground water Oron et al., 1988). Culley et al. 1981) found that a mixture of duckweed species could take up to 1378 kg of N predominately as ammonium), 347 kg of P, and 441 kg of potassium from 1 ha of water area in a year under the climatic conditions of Louisiana, USA. Duckweed can also tolerate high nutrient levels with reports of Spirodela polyrrhiza being capable of growth in 1 g/l N and 1.5 g/l P Landolt, 1986). The ability to tolerate high nutrient concentrations is valuable because swine wastewater usually contains a high level of nutrients 200±800 mg/l N and 30±100 mg/l P). Duckweed also has a very high content of proteins, ranging between 15% and 45% dry weight Landolt, 1986). Protein content is an important characteristic for several potential end uses of duckweed. Many researchers reported that duckweed could be utilized as human nutrition and animal, poultry, and sh feed Bhanthumnavin and McGarry, 1971; Edwards, 1974; Hillman and Culley, 1978; Ruso et al., 1978; Shireman et al., 1978; Truax et al., 1972). Duckweed cell walls lack lignin and are easy to digest, making duckweed a valuable source of livestock feed protein. Duckweed protein has an amino acid composition that varies by genotype and species but is generally of high quality Porath et al., 1979). The objectives of the research presented here were to investigate the rates of ammonia±n and phosphate±p uptakes from synthetic swine lagoon water by S. punctata 7776, the best duckweed strain in total protein production selected from in vitro screening experiments Bergmann et al., 2000), to study the relationship between nutrient uptake and duckweed growth, and to obtain information for determining appropriate ammonium and phosphate levels to start a continuous duckweed based nutrient recovery system for swine wastewater treatment. 2. Methods 2.1. Batch test Batch tests on the rate of nutrient NH 4 ±N and PO 4 ±P) uptake from synthetic swine lagoon water by S. punctata 7776 were conducted in 300 ml 5:08 cm 5:08 cm 11:62 cm or 2 in: 2in: 4:58 in.) polypropylene boxes Magenta, Chicago, IL) in a constant temperature growth chamber. Each box contained 150 ml 5.81 cm deep in the box) of culture medium, SAM, which was formulated to closely resemble the nutrient pro le, total ionic strength, ph, and buffering capacity of typical swine lagoon water in North Carolina Bergmann et al., 2000). The medium was sterilized in an autoclave for 30 min at 121 C before the test started. The carbon source in SAM was 3.0% sucrose with 1.15 g/l citric acid used as a bu ering agent. The phafter autoclaving was approximately 7. Fortyfour boxes including 11 control boxes without duckweed and 3 11 triplicate sample boxes with duckweed were used in each batch test. Each sample box was initially seeded with the same amount of S. punctata to cover the whole surface area of 5:08 cm 5:08 cm. Prior to culture initiation, the duckweed had been pre-cultured for two weeks on Schenk and Hildebrandt 1972) medium supplemented with 3.0% sucrose. The boxes were then placed into a 23 C growth chamber with 16-h photoperiod and a photosynthetic photon ux density of 40 lmol=m 2 -s provided by wide spectrum uorescent tubes Bergmann et al., 2000). The medium and the duckweed in each box were mixed every day. Destructive sampling was used to monitor the nutrient level and duckweed growth by taking a whole box as a sample for analysis. Triplicate samples and a control sample were taken every 48 h in each batch test. During the process of an experiment, phdecreased as the duckweed grew, so 10 M NaOHsolution was added to the boxes to keep the pharound 7.0. To better understand the relationship between the nutrient uptake rate and the duckweed growth rate, di erent dilutions of N and P concentrations in SAM were used as the initial media in the batch tests. To investigate the e ect of N concentration in the medium on the N and P uptake by S. punctata 7776 and duckweed growth, batch tests were conducted using di erent initial NH 4 ±N concentrations 240, 125, and 63 mg/l) while maintaining the same initial PO 4 ±P concentration around 30 mg/l) in the media. Similarly, to study the e ect of P concentration in the medium on the N and P uptake by S. punctata 7776 and duckweed growth, batch tests were conducted using di erent initial PO 4 ±P concentrations 31.0, 14.2, and 5.9 mg/l) while maintaining the same initial NH 4 ±N concentration around 240 mg/l) Table 1 Combinations of initial N and P concentrations in the media for batch tests on nutrient removal from synthetic swine lagoon e uent by S. punctata 7776 Initial NH 4 ±N concentration mg/l) Batch test Batch test Batch test Batch test Batch test Initial PO 4 ±P concentration mg/l)

3 J. Cheng et al. / Bioresource Technology ) 81±85 83 in the media. The combinations of initial N and P in these batch tests are illustrated in Table Chemical analysis Medium samples were analyzed for total nitrogen TN), NH 4 ±N, total phosphorus TP), ortho-phosphatephosphorus o-po 4 ±P, and ph. The wet and dry weights of the duckweed in each sample were also measured immediately after sampling. All the chemical analyses were conducted in the Environmental Analysis Laboratory of the Biological and Agricultural Engineering Department at North Carolina State University. EPA Methods 1983) and and Standard Methods 1995) 4500-P C, 4500-P F, and 4500-H were used for TN, NH 4 ±N, TP, o-po 4 ±P, and phanalyses, respectively. The wet weight of the duckweed was measured immediately after sampling with a balance. Free water on the duckweed was removed with a paper towel before the measurement. The wet duckweed was then dried in an oven at 105 C for about 12 h to obtain the dry weight of the duckweed. 1. Spirodela punctata 7776, 2. Lemna gibba 8678, 3. Lemna minor 7501, 4. Spirodela punctata 7488, 5. Lemna obscura 7720, and 6. Lemna aequinoctialis 8715 Bergmann et al., 2000). These duckweed strains were further studied for their nutrient uptake rates and their potential in nutrient recovery from swine lagoon e uent. Duckweed takes up nutrients from a medium to support its growth as it proliferates. In uence of replicate was not signi cant p > 0:15 for any variables in all the batch tests in this study except the duckweed biomass in batch tests 3 and 5 p ˆ 0:03 and p ˆ 0:02, respectively). Removal of N and P from the medium by growing duckweed, S. punctata 7776, in batch test 1 is illustrated in Fig. 1. NH 4 ±NandPO 4 ±P concentrations in the medium did not change in the control boxes, which had no duckweed, during the 480 h of the test Fig. 1 a) and b)). TN and TP concentrations did not change in those boxes, either data not presented). This 2.3. Statistical Analysis Statistical analyses on the data obtained from three replicates in each batch test were performed using SAS SAS Institute, Cary, NC). General linear models were used for the analysis of variance with replicate and time as independent variables and N, P, and duckweed biomass dry) as dependent variables. 3. Results and discussion Previous applications of duckweed in wastewater treatment generally dealt with low nutrient concentrations Alaerts et al., 1996; Oron, 1994; Oron et al., 1988). In our study, duckweed has been used to recover nutrients from swine lagoon water with high N and P contents. A systematic screening of duckweed geographic isolates to determine promising candidates for the treatment of wastewater was undertaken with swine arti cial medium SAM) that approximated typical swine lagoon water in North Carolina, USA. An in vitro screening test started with a collection of approximately 1000 geographic isolates belonging to 36 species of the four genera in Lemnaceae: Lemna, Spirodela, Wol a, and Wol ella. Forty-one geographic isolates representing 12 species from the four genera were selected from the collection based on the observation that they exhibited rapid growth during routine culture maintenance. The selected duckweed geographic isolates were grown in vitro on bu ered SAM for 11 days and compared for their performance. The top ranking duckweed geographic isolates in total protein production were: Fig. 1. Ammonium and phosphate removal from a synthetic swine lagoon water SAM) initial NH 4 ±N, 240 mg/l; initial PO 4 ±P, 31.0 mg/ l) by growing S. punctata 7776: medium volume ˆ 150 ml, surface area ˆ 25:81 cm 2, temperature ˆ 23 C, photon ux density ˆ 40 lmol=m 2 -s, and photoperiod ˆ 16 h/day. a) Ammonium±N removal; b) phosphate±p removal; c) duckweed biomass dry) accumulation.

4 84 J. Cheng et al. / Bioresource Technology ) 81±85 indicates that biological activity other than duckweed growth can be neglected in the batch test. The averages and standard deviations of NH 4 ±Nand PO 4 ±P concentrations and duckweed biomass dry basis) in the triplicate samples with initial duckweed seed were plotted against experimental time in Fig. 1. There was a lag period of approximately 96 h while the duckweed became acclimated to the new environment in the medium. During this period, there was slight N and P reduction in the medium, but the duckweed biomass remained constant. This phenomenon indicates that the duckweed accumulated N and P in its cells during the lag period. Rapid N and P removal from the medium occurred when the duckweed started to grow and the removal rate remained until NH 4 ±N disappeared at around 300 h. After the depletion of NH 4 ±N, the uptake of P by the duckweed decreased to a much lower rate. There were clearly two phases of NH 4 ±N uptake by the duckweed: a low NH 4 ±N uptake rate mg/l-h) during the lag period of duckweed growth, and a high NH 4 ±N uptake rate mg/l-h) during the fastgrowing period Fig. 1 a)). Three phases of PO 4 ±P utilization by the duckweed were observed in this test: a slow PO 4 ±P uptake mg/l-h) during the lag period of duckweed growth, a rapid uptake mg/l-h) during the fast-growing period, and a slow uptake mg/l-h) again after the depletion of NH 4 ±N Fig. 1 b)). Duckweed grew at an almost constant rate 1:33 g=m 2 -h after the lag period Fig. 1 c)). This growth rate remained even after NH 4 ±N and PO 4 ±P were completely consumed in the medium. At that time the duckweed probably grew new fronds which received N and P from their parent fronds, resulting in low N and P concentrations in the duckweed. Landolt 1986) indicated that N and P concentrations of duckweed increase with the increase of N and P concentrations in the medium. Based on our experimental results it appears that duckweed growth rate was almost constant while N and P concentrations in the medium decreased, which is in agreement with Landolt's observation Landolt, 1986). Similar experimental results were obtained from batch tests 2 and 3. In these tests, lower initial N concentrations were used while the same initial P concentration was used as in batch test 1. As in batch test 1, N and P concentrations in the media did not change in the control boxes, which had no duckweed, during the batch tests. The lag period for the duckweed in both tests was approximately 96 h, which was the same as in batch test 1. Linear N removal was observed in both batch tests, and higher NH 4 ±N uptake rate occurred in the test with higher initial N concentration in the medium. The P removal from the media in these two tests followed the same pattern as in batch test 1. Almost no P uptake by the duckweed was observed during the lag period and linear P uptake occurred after the duckweed started to grow. The initial P concentrations in batch tests 1±3 were about the same, but the PO 4 ±P removal rate in these tests decreased with the decrease of the initial N concentration. Duckweed growth rate also decreased with the decrease of initial N concentration in the batch tests. Based on the experimental results obtained from batch tests 1±3, the rates of NH 4 ±N uptake, PO 4 ±P uptake, and the duckweed growth decreased with the decrease of the initial N concentration in the medium. Similar tests batch tests 4 and 5) were conducted to determine the e ect of P concentration in the medium on the N and P uptake by the duckweed and the duckweed growth using media with di erent initial P concentrations. When compared to the results obtained from batch test 1, NH 4 ±N was utilized by the duckweed in a similar way with a slow initial reduction and a rapid removal afterwards. The slow initial N reduction occurred during the lag period of duckweed growth. The overall NH 4 ±N uptake rates from these media decreased with the decrease of initial P concentration in the media. No signi cant PO 4 ±P removal from the media was observed during the lag period of duckweed growth. Linear P utilization occurred with the growth of the duckweed. However, the overall P removal and duckweed growth rates decreased with the decrease of initial P concentration. The results obtained from the batch tests indicate that S. punctata 7776 grew well in the synthetic swine lagoon water. The highest nutrient N and P) uptake rates and the duckweed growth rate occurred in batch test 1 with initial NH 4 ±N and PO 4 ±P concentrations of 240 and 31.0 mg/l, respectively. The duckweed growth rate was about twice of that reported by Oron et al. 1988). In this batch test, the ratio of initial N:P was about the same as that in typical swine lagoon water, indicating that the N:P ratio in swine lagoon water is adequate to grow the duckweed. These results are important information for starting up a nutrient recovery system with growing S. punctata for swine wastewater treatment because an optimal duckweed growth is desired in the start-up. 4. Conclusions Spirodela punctata 7776, the best duckweed strain in total protein production selected from in vitro screening experiments with synthetic swine lagoon water medium, has shown the capacity to grow in and to remove N and P from the swine lagoon water with high N and P levels. The N:P ratio in swine lagoon water is adequate for growing the duckweed. The utilization of N and P by the duckweed was very slow during the lag period of the duckweed growth. The N and P utilization rates increased with the increase of the initial N and P concentrations during the growth period of the duckweed. The duckweed growth rate also increased with the increase of N and P concentrations. In the growth period of duck-

5 J. Cheng et al. / Bioresource Technology ) 81±85 85 weed, the NH 4 ±N and PO 4 ±P utilization and duckweed growth were linear over time. In our study, the highest NH 4 ±N uptake mg/l-h), PO 4 ±P uptake mg/ l-h), and duckweed growth 1:33 g=m 2 -h or 31:92 g=m 2 -day were observed in the medium with the initial NH 4 ±N and PO 4 ±P concentrations of 240 and 31.0 mg/l, respectively. This growth rate of S. punctata 7776 was approximately twice that reported by Oron et al. 1988) in a eld test for domestic wastewater treatment. Acknowledgements The authors would like to thank Rachel Huie, Chris Hayes, and Indira Thillai for their analytical service, Nirmala Rajbhandari and Xiaohong Lin for their help in maintaining the duckweed cultures, and Ye Sun for her assistance in statistical analyses. This is paper no of the Journal Series of the Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC , USA. Funding for this work was provided by North Carolina Agricultural Research Service. The views expressed are entirely those of the authors and do not necessarily re ect the views of the agency. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned. References Alaerts, G.J., Rahman Mahbubar, M.D., Kelderman, P., Performance analysis of a full-scale duckweed-covered sewage lagoon. Wat. Res. 30, 843±852. Bergmann, B.A., Cheng, J., Classen, J.J., Stomp, A.-M., In vitro selection of duckweed geographical isolates for potential use in swine lagoon e uent renovation. Bioresource Technol. 73 1), 13± 20. Bhanthumnavin, K., McGarry, M.G., Wol a arrhiza as a possible source of inexpensive protein. Nature ), 495. Burkholder, J., The lurking perils of P steria. Sci. Am. August), 42±49. Culley Jr., D.D., Rejmankova, E., Kvet, J., Frye, J.B., Production, chemical quality, and use of duckweed Lemnaceae) in aquaculture waste management, and animal feeds. J. World Maric. Soc. 12, 27±49. Edwards, D.J., Weed preference and growth of young grass carp in New Zealand. N. Z. J. Mar. Freshwater Res. 8 2), 341±350. Hillman, W.S., Culley Jr., D.D., The use of duckweed. Am. Sci. 66, 442±451. Landolt, E., Biosystematic investigation on the family of duckweeds: the family of Lemnaceae ± a monograph study. Geobotanischen Institute ETH, Stiftung Rubel, Zurichbergstrasse 38. Methods for Chemical Analysis of Water and Waste, United States Environmental Protection Agency EPA), Cincinnati, OH. Oron, G., Duckweed culture for wastewater renovation and biomass production. Agric. Wat. Manage. 26, 27±40. Oron, G., de-vegt, A., Porath, D., Nitrogen removal and conversion by duckweed grown on wastewater. Wat. Res. 22, 179± 184. Porath, D., Hepher, B., Koton, A., Duckweed as an aquatic crop: evaluation of clones for aquaculture. Aquat. Bot. 7, 273±278. Ruso, L.L., Zeringgue, S.P., Achacoso, A.S., Culley, D.D., Feeding value of duckweed an aquatic plant, family Lemnaceae) for ruminants. J. Dairy Sci. 61 Suppl. 1), 186. Schenk, R.U., Hildebrandt, A.C., Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199±204. Shireman, J.V., Colle, D.E., Rottmann, R.W., Growth of grass carp fed natural and prepared diets under intensive culture. J. Fish. Biol. 12 5), 457±464. Standard Methods for the Examination of Water and Wastewater, 19th Ed., American Public Health Association, American Water Works Association, and Water Environment Federation, Washington, DC. Truax, R.E., Culley, D.D., Gri th, M., Johnson, W.A., Wood, J.P., Duckweed for chick feed? L. Agric. 16 1), 8±9.