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The influence of duckweed species diversity on biomass productivity and nutrient removal efficiency in swine wastewater
Accepted Manuscript The influence of duckweed species diversity on biomass productivity and nutrient removal efficiency in swine wastewater Zhao Zhao, Huijuan Shi, Yang Liu, Hai Zhao, Haifeng Su, Maolin Wang, Yun Zhao PII: DOI: Reference:
S0960-8524(14)00875-X http://dx.doi.org/10.1016/j.biortech.2014.06.031 BITE 13565
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
31 March 2014 6 June 2014 8 June 2014
Please cite this article as: Zhao, Z., Shi, H., Liu, Y., Zhao, H., Su, H., Wang, M., Zhao, Y., The influence of duckweed species diversity on biomass productivity and nutrient removal efficiency in swine wastewater, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.06.031
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The influence of duckweed species diversity on biomass productivity and nutrient removal efficiency in swine wastewater Zhao Zhaoa, Huijuan Shia, Yang Liub, Hai Zhao b, Haifeng Sua, Maolin Wanga, Yun Zhao a* a Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 610064 Chengdu, China b Chengdu Institute of Biology, Chinese Academy of Sciences, 610041 Chengdu, China
Abstract The effect of temperature, light intensity, nitrogen and phosphorus concentrations on the biomass and starch content of duckweed (Landoltia punctata OT, L. minor OT) in monoculture and mixture were assessed. Low light intensity promoted more starch accumulation in mixture than in monoculture. The duckweed in mixture had higher biomass and nutrient removal efficiency than those in monoculture in swine wastewater. Moreover, the ability of L. punctata C3, L. minor C2, Spirodela polyrhiza C1 and their mixtures to recovery nutrients and their biomass were analyzed. Results showed that L. minor C2 had the highest N and P content, while L. punctata C3 had the highest starch content, and the mixture of L. punctata C3 and L. minor C2 had the greatest nutrient removal rate and the highest biomass. Compared with L. punctata C3 and L. minor C2 in monoculture, their biomass in the mixture increased by 17.0 and 39.8%, respectively. Key words: Duckweed; Mixture; Swine wastewater; Biomass; Starch 1. Introduction *
Corresponding author address: Sichuan University, No. 24 South Section 1, Yihuan Road, 610064 Chengdu, China. Tel : +86 028 85418776. E-mail address: [email protected] (Y Zhao).
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Large amounts of nutrients are discharged annually from swine farms, greatly increasing the levels of eutrophication in local rivers and lakes. Developing a value way to purify water in a simple, cheap and energy-efficient method has been more and more important to swine industry. A number of technologies have been developed for this purpose (Chen et al., 2004; Kim et al., 2004; Bortone, 2009) , of which the conversion of nutrients into valuable plant biomass, in addition to making full use of the postharvest biomass in order to recycle the nutrients, has drawn an increasing amount of attention (Xu and Shen, 2011). For instance, the cultivation of wetland plant species (capable of utilizing the excess nutrients) in wastewater has been found to be a great purification method. Not only are these plants able to purify water in a simple, inexpensive and energy-efficient manner, but the biomass produced by the plants can also be used as fodder for cattle (Fang et al., 2007). The duckweed belonging to Lemnaceae family lives in standing and slowly flowing waters all over the world, except in arctic and antarctic regions. There are 37 species belonging to 4 genera (Lemna, Spirodela, Wolffia and Wolffiella) (Cheng and Stomp, 2009). Duckweed species primarily reproduce asexually, grow fast, and increase biomass rapidly. Many species of duckweed can double their biomass every 2 or 3 days (Rusoff et al., 1980). Once established, duckweed efficiently lowers the level of carbon dioxide in the air and reduces nitrogen and phosphor in the water (Stomp 2005). Since swine wastewaters are already rich in nitrogen and phosphorus, they offer a readily available and cost-effective growth medium for duckweed. Duckweed can assimilate nutrient in wastewater, thus integrate of wastewater purification and biomass production
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(Su et al., 2012). In addition, duckweed is also known to tolerate high ammonia nitrogen and has an excellent ability to uptake nitrogen with a preference for ammonium, the dominant nitrogen form in swine wastewaters (Cheng et al., 2002). Because of these characteristics, various duckweed species have already been used for the treatment of municipal, industrial, and swine wastewaters in many countries, including Bangladesh, Israel, and the U.S (Oron, 1994; Vander Steen et al., 1998; El-Shafai et al., 2007). Moreover, duckweed is considered as a potential bioenergy source for bioethanol production due to its excellent growth and starch accumulation capability (Cheng and Stomp, 2009; Xu et al., 2011; Chen et al., 2012). It can also produce high levels of protein and starch. The protein content of a number of duckweed species grown under varying conditions has been reported to range anywhere from 15 to 45% of the dry weight (Porath et al., 1979) and starch contents range from 3 to 75% (Reid and Bieleski, 1970). Previously, the production of valuable biomass through nutrient recovery was found to have huge differences among various duckweed species and geographical isolates (Bergmann et al., 2000). Therefore, selecting the best duckweed strain from a collection of local strains is a prerequisite for the establishment of an effective duckweed cropping system (Xu and Shen 2011). In addition, only a single species such as L. punctata, L. minor and Wolffia arrhiza was reported at a time as high-potential candidates for domestic wastewater treatment and biofuel production (Cheng et al., 2002; Suppadit 2011; Ge et al., 2012). However, it is difficult to maintain a single species throughout an industrial operation because of the constant contamination of
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other species into the system (Su et al., 2012). Nonetheless, little attention has been paid to a mixture of different duckweed species in treating wastewater and creating biomass. Although a mixed culture was previously found to increase the overall methionine content, as compared to monoculture (Ismail 1998), biomass and starch contents were not discussed. Some researchers also presumed that mixed cultures of certain favorable microalgae candidates may increase biomass production because of their own advantage that can optimize the system performance (Olguin 2003), but this has not been properly investigated. As such, it is still necessary to evaluate whether mixed cultures of duckweed can enhance nutrient recovery and biomass production, as compared to a monoculture of duckweed, in swine wastewater. In the current paper, the ability of 2 local strains (L. minor and L. punctata) to increase their biomass and starch content either in mixture or in monocultures was investigated in response to light intensity, temperature, and nutrient concentration. The efficiency of 5 local strains isolated from different sources (S. polyrhiza, L. minor, and L. punctata) were further evaluated in nutrient recovery and biomass production either in mixture or monoculture. 2. Materials and Methods 2.1 Plant material and culture conditions The strains L. punctata OT and L. minor OT were isolated from the same pond in Huilong, Chengdu, China. In addition, S. polyrhiza C1, L. minor C2 and L. punctata C3 were obtained from a sewage treatment facility in Chengdu, Sichuan province. Following collection, the plants were rinsed gently with distilled water to remove
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debris and the healthy fronds were placed in plastic aquaria containing Hoagland E+ solution (macronutrients: 1 mM KH2PO4, 10 mM KNO3, 2 mM Ca(NO3)2, and 2 mM MgSO4; micronutrients: 46 mM H3BO3, 9 mM MnCl2.4H2O, 0.76 mM MnSO4.7H2O, 0.32 mM CuSO4, and 0.55 mM H2MoO4; iron source: 78 mM Fe-EDTA. Following 7 days of adaptation in the Hoagland E+ solution, the plants were transferred to 5% swine lagoon liquid to generate new and young rapid growing fronds acclimatized to the experimental conditions. The swine lagoon water was provided by a swine farm from Leshan city in Sichuan province. The concentrations of NH4-N, and PO4-P in the undiluted swine water were 1020mg/L and 224 mg/L, respectively. 2.2 Experimental protocol Duckweed was cultured in 1000ml-plastic container (12cm×18cm×5cm) at 25±1 for 12 days under a 16:8 light: dark cycle, with a light intensity of 7000 lux. The nutrient conditions were diluted one tenth of the full-strength Hoagland E+ solution concentration. Mixed duckweed cultures were obtained by combining either two or three of the species of duckweed in ratios of 1:1 or 1:1:1. A total of 3 initial grams of fresh materials were inoculated to cover the entire water surface with a single layer of fronds. The influence of temperature, light intensity, and N and P concentrations on biomass and starch content in L. punctata OT , L. minor OT and their mixture were analyzed in Hoagland E+ solution condition. Temperatures were set at 20, 25, or 30 , light intensities were set to 2000, 5000, or 10000 lux, while nutrient conditions were regulated according to the method of Josphine (Josphine et al., 2011), where the
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full-strength concentrations(340 mg N L−1, 150 mg P L-1) were chosen as the high-nutrient availability treatment and then diluted. The final concentration of N and P were therefore as follows: (340 mg N L−1, 150 mg P L-1), (34 mg N L−1 15 mg P L-1), (3.4 mg N L−1 1.5 mg P L-1), (0.34 mg N L−1 0.15 mg P L−1) and (0 mg N L−1 0 mg P L−1). In order to reduce algae growth and to keep nutrient levels constant, we renewed the growth solution every two days. Each treatment was replicated three times. For the swine wastewater studies, L. punctata OT, L. minor OT and their mixture were cultured in swine wastewater with 51mg/L of NH4-N and 12 mg/L of PO4-P for nutrient recovery and biomass production. Moreover, S. polyrhiza C1, L. minor C2 and L. punctata C3 and their different combinations were investigated under similar conditions, but with slightly modified concentrations of NH4-N (31.6mg/L) and (PO4-P 2.4 mg/L). In order to retain an optimal growth environment, the pH was adjusted to approximately 7.0 throughout the experiment. Any water lost to evaporation was replaced with tap water every day throughout the experiments. To measure the fresh weight (FW), duckweed were centrifuged in a washing machine to remove the surplus water and subsequently measured with a balance (Bergmann et al., 2000). To measure the dry weight (DW), the samples were dried at 60
until the weight was constant. The growth rate was measured as follows: Growth
rate = increased dry weight/area/time (g/m2/day). Furthermore, micro-Kjeldahl N-digestion was used to determine total-N content of biomass samples (Markus et al. 1985), while the P content was determined by the Vanado-molybdo-phosphoric acid colorimetric method (Kuo, 1996) and crude protein
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content was estimated by N × 6.25 (Xiao et al., 2013). To measure the starch content, dry duckweed powder was homogenized in 3mL 6M HCl, heated in boiling water for 2h in water bath, pH-adjusted to 7.0±0.5 with HCl (6M) or KOH (6M), and then added to 200uL Pb(COOH)2 and centrifuged at 5000g for 5min. Sugars in the supernatant were analyzed by HPLC (Thermo 2795, Thermo Corp., USA) with an Evaporative Light-scattering Detector (All-Tech ELSD 2000, All-tech., Corp., USA) and the starch content was determined using the total sugar content (starch content = glucose content × 0.909) (Zhang et al. 2011). Finally, the NH4-N and PO4-P levels in the swine water were measured by a Spectroquant testing kit (MERCK Corp., Germany) using PhotoLab 6100 (WTW Corp., Germany). 2.3 Calculations and statistics Each data point represents the results of three samples per experiment. All results are expressed as means ± standard error in the figures and significance (P < 0.5) was assessed using Student’s t-test. 3 Results and discussion 3.1 The effect of temperature on the duckweed growth Changes in temperature had substantial effects on both growth rate and starch content in duckweed with the two strains investigated exhibiting distinct growth characteristics in different temperatures. The results showed that the optimal temperature for the growth rates of L. punctata OT and L. minor OT was 25 g/m2/d) and 20
(4.2
(3.9 g/m2/d), respectively (Fig 1 a). Interestingly, the growth rate of the
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mixture was between those of L. punctata OT and L. minor OT at higher temperatures (30
and 25 ), but it was almost the same as that of the L. punctata OT at 20 .
Alternatively, the starch content of L. punctata OT, L. minor OT, and the mixture decreased by 28.6, 34.9 and 39.7 %, respectively, as the temperature increased from 20 to 30
(Fig 1 a). Overall, however, mixed cultures did not have a significant advantage
over the monoculture in terms of the biomass and the starch content. 3.2 The effect of light intensity on duckweed growth Light intensity significantly affected growth rate of duckweed. Under low light (2000 lux) the growth rate of L. punctata OT, L. minor OT, and the mixture were 1.31, 0.71, and 1.28 g/m2/d, respectively. As the light intensity increased to 5000 lux, the growth rate increased by 1.3, 2.5, and 1.1 fold, respectively, but did not increase further at 10000 lux (Fig 2a). The growth rate of the mixture was between L. minor OT and L. punctata OT in monoculture in light intensity of 5000 and 10000 lux. Interestingly, the mixture reached the highest growth rate (1.28 g/m2/d) almost as well as L. punctata OT (1.31 g/m2/d) (P>0.05) at low irradiance (2000 lux). The result showed that the mixture promoted the population to grow in low light intensity. It was observed that the roots of both L. punatata OT and L. minor OT in mixture were considerably longer than those in monoculture in the low light intensity (unpublished data). In this regards, there is a long-standing controversy about root involvement in nutrient uptake in Lemnaceae species (Cedergreen and Madsen, 2002), but recent studies have shown that L. punctata can uptake nitrogen by both the roots and fronds (Fang et al. 2007).
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Moreover, L. minor can regulate NO3- uptake via fronds or roots depending on light intensity (Cedergreen and Madsen 2004). The above results show that the competition between L. punatata OT and L. minor OT in low light promotes longer roots, increasing NO3 - uptake to promote growth. The starch content of the duckweed tended to be higher in high light intensity (Fig 2b). The starch contents of L. punctata OT and L. minor OT in monoculture increased by 1.6 and 2.2 fold, respectively, as the light intensity increased from 2000 lux to 5000 lux. But as the light intensity increased from 5000 lux to 10000 lux, the starch content of L. minor OT did not increase obviously, however, the starch content of L. punctata OT increased by 1.53 fold. When L. punctata OT and L.minor OT were mixed culture, the starch content increased by 1.5 fold as the light intensity increased from 2000 lux to 5000 lux, but decreased by 3% at 10000lux. The starch content of the mixture (21.8% DW) was between those of L. punctata OT (24.6% DW) and L. minor OT (17.6% DW) at 10000 lux irradiance, but the starch contents of the mixture (22.3% and 8.8%) were significantly higher than L. punctata OT (18.3% and 7.0%) and L. minor OT (17.0% and 5.4%) in monoculture at 5000 and 2000 lux irradiance (Fig 2b). Higher starch content in duckweed is favorable to species competition at low irradiance, so duckweed in the mixed culture tends to accumulate more starch, as compared to that in monoculture. There is a particular climate in the Sichuan Basin. Cloudy and rainy days occur often in Chengdu, resulting in low light intensity year round with a lowest recorded value at noon of approximately 1000 lux (Xiao et al.,
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2013). Therefore, the ideal method for producing starch at low irradiance areas such as Sichuan Basin is via a mixed culture of duckweed. 3.3 The effect of N and P content on duckweed growth It was observed that the duckweed grown in high N and P concentration had large, thin and dark green fronds and short, thin roots. By contrast, plants grown in low N and P concentration were small, thick and pale with long thick roots. Results revealed that the growth rate of L. punctata OT, L. minor OT and their mixture changed significantly depending on N and P concentrations (Fig. 3 a). The higher N and P concentrations in A and B medium were clearly favorable for the growth of L. punctata OT alone. Moreover, the growth rate of L. punctata OT significantly decreased at levels below that of solution B (34.5mg N L-1 and 15 mg P L-1). Alternatively, the growth rate of L. minor OT did not change obviously until they were grown in D and E medium, at which point the growth rate began to decrease at a rapid rate. Interestingly, the growth rate of the mixture rose initially and then fell as the N and P concentration decreased from A to D. The highest growth rate of the mixture was observed in C medium (Fig. 3 a). The growth rate of L. punctata OT was significantly higher than that of L. minor OT at every concentration combination of N and P. When higher concentration of N and P were used in the A or B media, the growth rate of the mixture of duckweed was between those of L. punctata OT and L. minor OT. When N and P concentration decreased and was lower than that in C medium, the growth rates of the mixture were almost equal to L. punctata OT in monoculture (Fig. 3a). These results indicated that the
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competition of the duckweed in the mixture promoted the growth of either one or both of the species when N and P were in low concentration. It was observed that the roots of L. minor OT and L. punctata OT in mixed cultures were separately longer than those in monocultures when N and P concentrations were low (data not shown), indicating that the competition of the two species resulted in the extension of plant root system. The extended root system can increase the absorption of the nutrients, especially phosphorus (Smith and Read, 1997). In addition, the duckweed had the capacity to take up NO3– through both roots and fronds, the over-all contribution of root and frond to whole-plant uptake varied depending on plant N status and the root contribution increased from 32 to 73% for N-satiated and N-depleted plants, respectively (Cedergreen and Madsen 2002). In low N concentration, the root had more contribution for NO3–. The longer roots of the L. minor OT and L. punctata OT in mixture would have more NO3 – absorption capacity to promote the growth. So in low N and P concentration, the competition of different duckweed species would promote each other for the use of nitrogen, resulting in higher growth rate. The concentration of N and P also strongly affected the starch content and the starch content in the duckweed tended to be accumulated in lower N and P concentration. The starch content of L. punctata OT, L. minor OT and their mixture increased by about 5.6, 9.9 and 7.1 fold as N and P concentration decreased from A (345mg N L-1 and 150 mg P L-1) to E (0 mg N L-1 and 0 mg P L-1). At higher concentrations (A or B), the starch content of the mixture was between L. punctata OT and L. minor OT in monoculture. As the N and P concentration decreased (C, D or E
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media), however, the starch content of the mixture rose to become equivalent with that of L. punctata OT (Fig 3b). These results revealed that the lack of nutrition prompted duckweed to rapidly accumulate starch, and starch accumulation was in favor of duckweed competition and surviving in adverse environments. 3.4 The removal of nutrient in swine wastewater The change of nutrient in the swine lagoon liquid covered with L. minor OT, L. punctata OT and the mixture of the two species, respectively, was analyzed every three days. Results indicated that the duckweed could efficiently remove nitrogen and phosphorus from the swine wastewater. The vast majority of NH4-N and PO4-P was removed from the swine wastewater in 6 or 9 days (Table 1, 2). During the first 3 days, the NH4-N was removed 60.00-67.84% from the swine wastewater and there were no advantages in the remove effect of NH4-N with the mixed culture of L. punctata OT and L. minor OT over monoculture (Table 1). From 3 to 6d, however, the highest remove rate of NH4-N (6.65 mg/l/d) was obtained from the mixed culture and the remove rate of NH4-N of L. minor OT and L. punctata OT in monoculture was 4.81 and 5.65 mg/l/d, respectively. Compared with monoculture, the mixed culture of the two duckweed species enhanced the NH4-N removal capacity obviously during 3 to 6 cultured days. The uptake rate of PO4-P was different in different species and different culture phase. During the first 3 days, the highest remove rate of PO4-P was observed in L. minor OT. Following 6 days of culture, however, the highest remove rate of PO4-P was found in the mixed culture, with a PO4-P value of 1.2 mg/L, significantly lower
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than those in monoculture (3.5 mg/L and 4.8 mg/L). Finally, after 9 days of culture, all PO4-P content in the swine wastewater were below 0.1 mg/L and the minimum PO4-P content was also found in the mixed culture (Table 2). These results indicated that the mixed culture of different duckweed species could enhance the PO4-P removal capacity. It was also observed that the roots of L. punctata OT and L. minor OT in mixed culture in swine water were longer than those in monocultures. Additional 3 strains (S. polyrhiza C1, L. minor C2 and L. punctata C3) isolated from a sewage treatment facilities were also mono- and mixed culture in swine wastewater in order to verify further the advantages that the mixed culture of different duckweed species could enhance the nutrient removal capacity. During the first 2 days, the duckweeds were in a lag period and had very little uptake of NH4-N. Following 2 days of culture, the duckweeds were in the growth period and began to uptake NH4-N at a rapid rate. After 12 days of culture, the overall NH4-N content in any mixed culture of any two strains was significantly lower than those in monoculture, but the mixed culture of all three species could not enhance the NH4-N removal capacity (Fig 4a). The remove rate by NH4-N of S. polyrhiza C1, L. minor C2 and L. punctata C3 in monoculture was 2.72, 2.53 and 2.80 mg/l/d, respectively. Meanwhile, the remove rate of NH4-N of the different mixture was 2.90 (mixture of S. polyrhiza C1 and L. minor C2), 2.94 (mixture of S. polyrhiza C1 and L. punctata C3), 3.01 (L. minor C2 and L. punctata C3), and 2.65 (mixture of S. polyrhiza C1, L. minor C2 and L. punctata C3) mg/l/d, respectively. These results indicated that effect of the NH4-N removal of the mixed culture of two species was obviously better than those of
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mono or mixed culture of three species. Furthermore, the PO4-P content of swine wastewater rapidly decreased when it was treated with duckweed regardless of which culture was used (Fig 4b). Following 12 day of culture, the PO4-P content in any mixed culture of any two or three species was significantly lower than those in monoculture (Fig 4b). The overall remove rate of PO4-P by S. polyrhiza C1, L. minor C2, and L. punctata C3 in monoculture was 0.187, 0.181 and 0.184 mg/l/d, respectively, while in the mixed cultures, the remove rate was 0.197 (mixture of S. polyrhiza C1 and L. minor C2), 0.199 (mixture of S. polyrhiza C1 and L. punctata C3), 0.196 (L. minor C2 and L. punctata C3) and 0.195 (mixture of S. polyrhiza C1, L. minor C2 and L. punctata C3) mg/l/d, respectively. These results indicated that effect of the PO4-P removal of the mixed culture of two or three species was obviously better than those of mono species culture. A number of nutrient removal rates between the separate populations of duckweed (Fig 4 and Tables 1 and 2) seem to be inconsistent, especially during the first few days. For instance, the process of phosphate removal occurred a few days later than that of NH4-N removal (Table 1, 2), but phosphate removal was much faster in sewage water (Fig4 a, b). These results are similar to a previous report looking at the ability of a mixture of algae to process swine wastewater with different N/P ratios (Su et al., 2012), indicating the importance of the N/P ratio for nutrient removal. As such, it was calculated that the N/P ratio (4.5:1) in swine wastewaters in table 1 and 2 was much lower than that (15:1) in the fig 4. Therefore, the inconsistent results should be due to the different N/P ratio in the wastewater.
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The results of nutrient removal rates of duckweed were similar to the results from those of algae. The nutrient removal rates by using mixed algal culture are higher than those of three algae species in monoculture (Chlamydomonas reinhardtii, Scenedesmus rubescens and Chlorella vulgaris), further substantiating the similar results revealed in this paper (Su et al., 2012). 3.5 Nutrient content and biomass of duckweed in swine wastewater The growth rate and biomass were analyzed when the duckweed strains were mono-/ mixed culture in the swine lagoon liquid. The results showed that the growth rate of duckweed in mixed culture was obviously higher than that of monoculture. After 12 days of culture, the growth rate of L. minor OT, L. punctata OT and the mixture of the two were 16.20, 21.72 and 27.75 g/m2/d in fresh weight, respectively (data not shown). Furthermore, the biomass of each culture at harvest was 31.6, 37.5 and 41.4 g/m2 in dry weight (Table 3), respectively. The biomass of the mixture increased by 31.1 and 10.4% over that of L. minor OT and L. punctata OT in monoculture, respectively. The other 3 strains (S. polyrhiza C1, L. minor C2 and L. punctata C3) were also mono-/ mixed culture in swine wastewater and the similar results were obtained. The harvest biomass of S. polyrhiza C1, L. minor C2 and L. punctata C3 in monoculture were 48.39, 55.69 and 66.55 g/m2, respectively, while the biomass of the mixture in different combinations of duckweed strains were 77.87 (mixture of L. punctata C3 and L. minor C2), 71.36 (mixture of L. punctata C3 and S. polyrhiza C1), 73.00 (mixture of L. minor C2 and S. polyrhiza C1) and 76.00 (mixture of S. polyrhiza C1, L. minor C2 and L. punctata C3) g/m2, respectively (Table 4). The biomass of duckweed in the
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mixed culture increased by 7.2-57.1% than that in monoculture. The nutrient of duckweed harvested from the swine wastewater was analyzed. L. minor OT, L. punctata OT and their mixture were first assessed. L. minor OT had the highest crude protein, N and P content, while L. punctata OT was found to have the highest starch content (Table 3). The crude protein, N, and starch content in the mixed culture was in between that of L. minor OT and L. punctata OT in monoculture, however, the P content was the lowest (Table 3), indicating that the mixture consumed a greater amount of P in order to maintain its growth rate. In addition, the nutrient content of S. polyrhiza C1, L. minor C2 and L. punctata C3 was analyzed . The results showed the similar results that a higher N and P content was assessed in the L. minor C2, while a higher starch content was observed in L. punctata C3 (Table 4). The P content in the mixture of different species was always lower than that in mono-culture except the mixture of L. minor C2 and S. polyrhiza C1. Only some of the mixed cultures had starch content values that fell between those in monoculture. Interestingly, the starch content in the mixture of S. polyrhiza C1 and L. punctata C3 was significantly higher than that of S. polyrhiza C1 and L. punctata C3 in monoculture (Table 4). Taking all the data into consideration, the mixture of L. punctata C3 and S. polyrhiza C1 produced the most starch, while the mixture of L. punctata C3 and L. minor C2 produced the greatest amount of crude protein (Table 4). Therefore it was a good method to mix duckweed species in culture to increase the production of biomass, the starch and crude protein. In addition, we must protect the duckweed species
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diversity. 4. Conclusion The duckweed in the mixture had higher growth rate, total biomass, starch, crude protein and nutrient removal efficiency than those in monoculture in swine wastewater. L. minor OT had higher crude protein content, while L. punctata OT had the higher starch content in monoculture. In different mixed cultures with L. punctata C3, L. minor C2 and S. polyrhiza C1, the increase biomass was different. The species diversity significantly affects the biomass productivity and nutrient removal efficiency of duckweed in swine wastewater, and the proper mixture of duckweed significantly abrogates the eutrophication of swine wastewater. Acknowledgements We would like to thank Dr. Q.S. Liu from Sichuan University for reviewing the manuscript. This work was supported in part by grants from the Ministry of Science and Technology of PR China (2011BAD22B03) and the Chinese Academy of Science (KSCX2-EW-J-22 and Y2C5021100).
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Cedergreen, N., Madsen T.V., 2002. Nitrogen uptake by the floating macrophyte Lemna minor. New Phytol. 155(2), 285-292. Cedergreen, N., Madsen, T.V., 2004. Light regulation of root and leaf NO3- uptake and reduction in the floating macrophyte Lemna minor. New Phytol. 161(2), 449-457. Chen, M., Kim J.H., Kishida, N., Nishimura, O., Sudo, R., 2004. Enhanced nitrogen removal using C/N load adjustment and real-time control strategy in sequencing batch reactors for swine wastewater treatment. Water Sci. and Technol. 49(5-6),309-314. Chen, Q., Jin, Y.L., Zhang,G.H., Fang, Y., Xiao, Y., Zhao, H., 2012. Improving Production of Bioethanol from Duckweed (Landoltia punctata) by Pectinase Pretreatment. Energies. 5 (8),3019-3032. Cheng, J.J., Stomp A.M., 2009. Growing Duckweed to Recover Nutrients from Wastewaters and for Production of Fuel Ethanol and Animal Feed. Clean-Soil, Air, Water. 37(1),17-26. Cheng, J., Bergmann, B.A., Classen, J. J., Howard, J.W., 2002. Nutrient recovery from swine lagoon water by Spirodela punctata. Biresour. technol. 81,81-85. El-Shafai, S.A., El-Gohary, F.A., Nasr, F.A., vander Steen, N.P., Gijzen, H.J., 2007. Nutrient recovery from domestic wastewater using a UASB-duckweed ponds system. Bioresour. Technol. 98, 798–807. Fang, Y. Y., Babourina, O., Rengel, Z., Yang, X.E., Pu, P. M. , 2007. Ammonium and nitrate uptake by the floating plant Landoltia punctata. Ann. Bot,99(2):
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365-370. Garrett, K.A., Mundt C.C., 1999. Epidemiology in mixed host populations. Phytopathol. 89(11), 984-990. Ge, X.M., Zhang, N.N., Phillips, G.C., Xu, J. F., 2012. Growing Lemna minor in agricultural wastewater and converting the duckweed biomass to ethanol. Bioresour. Technol. 124,485-488. Ismail, M., 1998. Chemical Characterization of Protein Contentrates of Duckweed. Pertanika J. Sci. & Techno.6(1),7-21. Josphine N., Iris S., Ludwig T., 2011. Competition between Lemna minuta and Lemna minor at different nutrient. Aquat Bot. 94,158-164. Kim, J. H., Chen, M.X., Kishida, N., Sudo, R., 2004. Integrated real-time control strategy for nitrogen removal in swine wastewater treatment using sequencing batch reactors. Water Res.38(14-15), 3340-3348. Kuo, S., 1996. Phosphorus. In: Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabai, M.A., Johnston, C.T., Sumner, M.E. (Eds.), Methods of Chemical Analysis, Part 3. Chemical Methods. SSSA (Soil Science Society of America), Madison,WI, pp. 869–919. Markus, D. K., McKinnon, J.P., Buccafuri, A.F., 1985. Automated analysis of nitrite, nitrate, and ammonium nitrogen in soils. Soil Sci Soc Am J. 49(5), 1208-1215. Olguin, E.J., 2003. Phycoremediation: key issues for cost-effective nutrient removal processes. Biotechnol. Adv. 22(1-2), 81-91. Oron, G.., 1994. Duckweed culture for wastewater renovation and biomass production.
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Agric. Water Manag. 26, 27–40. Porath, D., Hepher, B., Koton, A., 1979. Duckweed as an aquatic crop: evaluation of clones for aquaculture. Aquat. Bot, 7, 273 – 278. Reid, M.S., Bieleski, RL., 1970. Response of Spirodela oligorrhiza to phosphorus deficiency. Plant Physiol. 46(4), 609-613. Rusoff, L.L., Blakeney, E.W., Culley, D.D., 1980. Duckweeds (Lemnaceae family): a potential source of protein and amino acids. J Agr Food Chem. 28(4), 848-850. Stomp, A.M. 2005. The duckweeds: a valuable plant for biomanufacturing. Biotechnology. Annu Review. 11, 69-99. Su, Y., Mennerich, A., Brigitte, U., 2012. Coupled nutrient removal and biomass production with mixed algal culture: Impact of biotic and abiotic factors. Bioresour. Technol. 118, 469-476. Suppadit, T., 2011. Nutrient removal of effluent from quail farm through cultivation of Wolffia arrhiza. Bioresour. Technol. 102(16), 7388-7392. Smith, S. E., Read, D. J., Mycorrhizal Symbiosis 2nd edn (Academic, San Diego, 1997) Vander Steen, P., Brenner, A., Oron, G., 1998. An integrated duckweed and algae pond system for nitrogen removal and renovation. Water Sci. Technol., 38, 335–343 Xiao, Y., Fang Y., Jin, Y.L., Zhang, G.H., Zhao, H., 2013. Culturing duckweed in the field for starch accumulation. Ind Crops Prod. 48, 183-190. Xu, J., Shen G., 2011. Growing duckweed in swine wastewater for nutrient recovery and biomass production. Bioresour. Technol. 102(2), 848-853. Xu, J., Cui, W., Cheng, J.J., Anne-M, S., 2011. Production of high-starch duckweed and
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its conversion to bioethanol. Biosyst Eng, 110:67-72. Zhang, L., Zhao, H., Gan, M.Z., Jin, Y.L., Gao, X.F., Chen, Q., 2011. Application of simultaneous saccharification and fermentation (SSF) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales. Bioresour. Technol. 102(6), 4573-4579.
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Fig. 1. Change of growth rate (a) and starch content (b) of the duckweed in the mixture and monoculture under the influence of temperature. DW=Dry weight. Values represent mean± S.E. (n=3). The growth rate and starch content were measured at 12 days after inoculation. The asterisk indicated that the growth rate/ starch content of L. punctata / L. minor in uni-cultures that were significantly different from those in mixture with p<0.05 in an unpaired Student’ s t test. Fig. 2. Change of growth rate (a)and starch content (b) of the duckweed in the mixture and monoculture under the influence of light intensity. DW=Dry weight. Values represent mean± S.E. (n=3). The growth rate and starch content were measured at 12 days after inoculation. The asterisk indicated that the growth rate/ starch content of L. punctata/L. minor in uni-cultures that were significantly different from those in mixture with p<0.05 in an unpaired Student’ s t test. Fig. 3. Change of growth rate (a) and starch content (b) the duckweed in the mixture and monoculture in media with different concentration of N and P. DW=Dry weight. Values represent mean± S.E. (n=3). A= 345 mg N L-1 150 mg P L-1; B=34.5 mg N L-1 15 mg P L-1; C=3.45 mg N L-1 1.5 mg P L-1; D=0.345 mg N L-1 0.15 mg P L-1; E=0 mg N L-1 0 mg P L-1. The growth rate and starch content were measured at 12 days after inoculation. The asterisk indicated that the growth rate/ starch content of L. punctata / L. minor in uni-cultures that were significantly different from those in the mixture with p<0.05 in an unpaired Student’s t test. Fig. 4. The NH4-N (a) and PO4-P (b) content in the swine wastewater covered with duckweed in different days. Values represent mean ± S.E. (n=3).
22
Table 1. The NH4-N content (mg/L) in the swine wastewater covered with duckweed in different days. Time (day) 0 3 6 9 12
L. punctata OT
The mixture
L. minor OT
51±1.37
51±1.37
51±1.37
17.5±0.24
20.1±0.53
18.8±1.06
0.54±0.06
0.16±0.05
4.37±1.16
0
0
0
0
23
0.35±0.13 0
Table 2 The PO4-P content (mg/L) in the swine wastewater covered with duckweed in different days. Time (day) 0 3 6 9 12
L. punctata OT
The mixture
L. minor OT
11.2±0.74
11.2±0.74
11.2±0.74
10.3±1.51
9.6±1.35
5.6±0.15
3.5±0.66
1.2±0.25
4.8±0.15
0.05±0.02
0.04±0.01
0.09±0.01
0.05±0.01
0.03±0.02
0.06±0.01
24
Table 3 The nutrient content of duckweed in swine wastewater on 12d. Strains
N(%)
P(%)
Starch
Crude
content
protein
(%)
content
Biomass 2
(g/m )
Starch 2
(g/m )
Crude protein 2
(g/m )
(%) L. punctata OT
4.81b
0.46b
16.74a
30.06b
37.5b
6.28a
11.3c
L. minor OT
5.15a
0.81a
9.37c
32.19a
31.6c
2.95b
10.2b
Mixture (L. punctata OT+ L. minor OT)
4.61b
0.39b
15.52b
28.81b
41.4a
6.42a
11.9a
The concentration of NH4-N and PO4_P in the swine waste water were 51mg/L and 12 mg/L. Different lower-case letters in the same column denote significant differences according to Tukey test (P<0.05).
25
Table 4
The nutrient content of duckweed in swine water on 12d.
Strains
N(%)
P(%)
Starch
Crude
content
protein
(%)
content
Biomass 2
(g/m )
Starch 2
(g/m )
Crude protein (g/m2)
(%) L. punctata C3
4.04b
0.53b
28.60a
25.25b
66.55c
19.03b
16.80bc
L. minor C2
4.35a
1.25a
13.42c
27.19a
55.69d
7.47f
15.14c
S. polyrhiza C1
3.47c
0.49b
22.92b
21.69c
48.39e
11.09d
10.50d
Mixture (L. Punctata C3+L. minor C2)
4.19a
0.23c
24.80b
26.19a
77.87a
19.31b
20.40a
Mixture (L. punctata C3+S. polyrhiza C1)
3.51c
0.43b
29.67a
21.94c
71.36b
21.17a
15.66c
Mixture (L. minor C2 + S. polyrhiza C1)
4.25a
1.02a
13.35c
25.56a
73.00b
9.75e
18.66b
Mixture
4.01b
0.45b
23.33b
25.06b
76.00a
17.73c
19.05a
(L.punctata C3+L.minor C2+S.polyrhiza C1)
The concentration of NH4-N and PO4_P in the swine waste water were 31.6mg/L and 2.4 mg/L. Different lower-case letters in the same column denote significant differences according to Tukey test (P<0.05).
26
27
28
29
30
Highlights ●
The duckweed in mixture produced more starch in low light intensity.
●
The mixture promoted the growth and nutrient removal in swine wastewaters.
●
The duckweed in the mixture had lower P content than those in the monoculture.
●
The proper combinations of duckweed significantly increased biomass.
31
S0960-8524(14)00875-X http://dx.doi.org/10.1016/j.biortech.2014.06.031 BITE 13565
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
31 March 2014 6 June 2014 8 June 2014
Please cite this article as: Zhao, Z., Shi, H., Liu, Y., Zhao, H., Su, H., Wang, M., Zhao, Y., The influence of duckweed species diversity on biomass productivity and nutrient removal efficiency in swine wastewater, Bioresource Technology (2014), doi: http://dx.doi.org/10.1016/j.biortech.2014.06.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The influence of duckweed species diversity on biomass productivity and nutrient removal efficiency in swine wastewater Zhao Zhaoa, Huijuan Shia, Yang Liub, Hai Zhao b, Haifeng Sua, Maolin Wanga, Yun Zhao a* a Key Laboratory of Bio-Resources and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 610064 Chengdu, China b Chengdu Institute of Biology, Chinese Academy of Sciences, 610041 Chengdu, China
Abstract The effect of temperature, light intensity, nitrogen and phosphorus concentrations on the biomass and starch content of duckweed (Landoltia punctata OT, L. minor OT) in monoculture and mixture were assessed. Low light intensity promoted more starch accumulation in mixture than in monoculture. The duckweed in mixture had higher biomass and nutrient removal efficiency than those in monoculture in swine wastewater. Moreover, the ability of L. punctata C3, L. minor C2, Spirodela polyrhiza C1 and their mixtures to recovery nutrients and their biomass were analyzed. Results showed that L. minor C2 had the highest N and P content, while L. punctata C3 had the highest starch content, and the mixture of L. punctata C3 and L. minor C2 had the greatest nutrient removal rate and the highest biomass. Compared with L. punctata C3 and L. minor C2 in monoculture, their biomass in the mixture increased by 17.0 and 39.8%, respectively. Key words: Duckweed; Mixture; Swine wastewater; Biomass; Starch 1. Introduction *
Corresponding author address: Sichuan University, No. 24 South Section 1, Yihuan Road, 610064 Chengdu, China. Tel : +86 028 85418776. E-mail address: [email protected] (Y Zhao).
1
Large amounts of nutrients are discharged annually from swine farms, greatly increasing the levels of eutrophication in local rivers and lakes. Developing a value way to purify water in a simple, cheap and energy-efficient method has been more and more important to swine industry. A number of technologies have been developed for this purpose (Chen et al., 2004; Kim et al., 2004; Bortone, 2009) , of which the conversion of nutrients into valuable plant biomass, in addition to making full use of the postharvest biomass in order to recycle the nutrients, has drawn an increasing amount of attention (Xu and Shen, 2011). For instance, the cultivation of wetland plant species (capable of utilizing the excess nutrients) in wastewater has been found to be a great purification method. Not only are these plants able to purify water in a simple, inexpensive and energy-efficient manner, but the biomass produced by the plants can also be used as fodder for cattle (Fang et al., 2007). The duckweed belonging to Lemnaceae family lives in standing and slowly flowing waters all over the world, except in arctic and antarctic regions. There are 37 species belonging to 4 genera (Lemna, Spirodela, Wolffia and Wolffiella) (Cheng and Stomp, 2009). Duckweed species primarily reproduce asexually, grow fast, and increase biomass rapidly. Many species of duckweed can double their biomass every 2 or 3 days (Rusoff et al., 1980). Once established, duckweed efficiently lowers the level of carbon dioxide in the air and reduces nitrogen and phosphor in the water (Stomp 2005). Since swine wastewaters are already rich in nitrogen and phosphorus, they offer a readily available and cost-effective growth medium for duckweed. Duckweed can assimilate nutrient in wastewater, thus integrate of wastewater purification and biomass production
2
(Su et al., 2012). In addition, duckweed is also known to tolerate high ammonia nitrogen and has an excellent ability to uptake nitrogen with a preference for ammonium, the dominant nitrogen form in swine wastewaters (Cheng et al., 2002). Because of these characteristics, various duckweed species have already been used for the treatment of municipal, industrial, and swine wastewaters in many countries, including Bangladesh, Israel, and the U.S (Oron, 1994; Vander Steen et al., 1998; El-Shafai et al., 2007). Moreover, duckweed is considered as a potential bioenergy source for bioethanol production due to its excellent growth and starch accumulation capability (Cheng and Stomp, 2009; Xu et al., 2011; Chen et al., 2012). It can also produce high levels of protein and starch. The protein content of a number of duckweed species grown under varying conditions has been reported to range anywhere from 15 to 45% of the dry weight (Porath et al., 1979) and starch contents range from 3 to 75% (Reid and Bieleski, 1970). Previously, the production of valuable biomass through nutrient recovery was found to have huge differences among various duckweed species and geographical isolates (Bergmann et al., 2000). Therefore, selecting the best duckweed strain from a collection of local strains is a prerequisite for the establishment of an effective duckweed cropping system (Xu and Shen 2011). In addition, only a single species such as L. punctata, L. minor and Wolffia arrhiza was reported at a time as high-potential candidates for domestic wastewater treatment and biofuel production (Cheng et al., 2002; Suppadit 2011; Ge et al., 2012). However, it is difficult to maintain a single species throughout an industrial operation because of the constant contamination of
3
other species into the system (Su et al., 2012). Nonetheless, little attention has been paid to a mixture of different duckweed species in treating wastewater and creating biomass. Although a mixed culture was previously found to increase the overall methionine content, as compared to monoculture (Ismail 1998), biomass and starch contents were not discussed. Some researchers also presumed that mixed cultures of certain favorable microalgae candidates may increase biomass production because of their own advantage that can optimize the system performance (Olguin 2003), but this has not been properly investigated. As such, it is still necessary to evaluate whether mixed cultures of duckweed can enhance nutrient recovery and biomass production, as compared to a monoculture of duckweed, in swine wastewater. In the current paper, the ability of 2 local strains (L. minor and L. punctata) to increase their biomass and starch content either in mixture or in monocultures was investigated in response to light intensity, temperature, and nutrient concentration. The efficiency of 5 local strains isolated from different sources (S. polyrhiza, L. minor, and L. punctata) were further evaluated in nutrient recovery and biomass production either in mixture or monoculture. 2. Materials and Methods 2.1 Plant material and culture conditions The strains L. punctata OT and L. minor OT were isolated from the same pond in Huilong, Chengdu, China. In addition, S. polyrhiza C1, L. minor C2 and L. punctata C3 were obtained from a sewage treatment facility in Chengdu, Sichuan province. Following collection, the plants were rinsed gently with distilled water to remove
4
debris and the healthy fronds were placed in plastic aquaria containing Hoagland E+ solution (macronutrients: 1 mM KH2PO4, 10 mM KNO3, 2 mM Ca(NO3)2, and 2 mM MgSO4; micronutrients: 46 mM H3BO3, 9 mM MnCl2.4H2O, 0.76 mM MnSO4.7H2O, 0.32 mM CuSO4, and 0.55 mM H2MoO4; iron source: 78 mM Fe-EDTA. Following 7 days of adaptation in the Hoagland E+ solution, the plants were transferred to 5% swine lagoon liquid to generate new and young rapid growing fronds acclimatized to the experimental conditions. The swine lagoon water was provided by a swine farm from Leshan city in Sichuan province. The concentrations of NH4-N, and PO4-P in the undiluted swine water were 1020mg/L and 224 mg/L, respectively. 2.2 Experimental protocol Duckweed was cultured in 1000ml-plastic container (12cm×18cm×5cm) at 25±1 for 12 days under a 16:8 light: dark cycle, with a light intensity of 7000 lux. The nutrient conditions were diluted one tenth of the full-strength Hoagland E+ solution concentration. Mixed duckweed cultures were obtained by combining either two or three of the species of duckweed in ratios of 1:1 or 1:1:1. A total of 3 initial grams of fresh materials were inoculated to cover the entire water surface with a single layer of fronds. The influence of temperature, light intensity, and N and P concentrations on biomass and starch content in L. punctata OT , L. minor OT and their mixture were analyzed in Hoagland E+ solution condition. Temperatures were set at 20, 25, or 30 , light intensities were set to 2000, 5000, or 10000 lux, while nutrient conditions were regulated according to the method of Josphine (Josphine et al., 2011), where the
5
full-strength concentrations(340 mg N L−1, 150 mg P L-1) were chosen as the high-nutrient availability treatment and then diluted. The final concentration of N and P were therefore as follows: (340 mg N L−1, 150 mg P L-1), (34 mg N L−1 15 mg P L-1), (3.4 mg N L−1 1.5 mg P L-1), (0.34 mg N L−1 0.15 mg P L−1) and (0 mg N L−1 0 mg P L−1). In order to reduce algae growth and to keep nutrient levels constant, we renewed the growth solution every two days. Each treatment was replicated three times. For the swine wastewater studies, L. punctata OT, L. minor OT and their mixture were cultured in swine wastewater with 51mg/L of NH4-N and 12 mg/L of PO4-P for nutrient recovery and biomass production. Moreover, S. polyrhiza C1, L. minor C2 and L. punctata C3 and their different combinations were investigated under similar conditions, but with slightly modified concentrations of NH4-N (31.6mg/L) and (PO4-P 2.4 mg/L). In order to retain an optimal growth environment, the pH was adjusted to approximately 7.0 throughout the experiment. Any water lost to evaporation was replaced with tap water every day throughout the experiments. To measure the fresh weight (FW), duckweed were centrifuged in a washing machine to remove the surplus water and subsequently measured with a balance (Bergmann et al., 2000). To measure the dry weight (DW), the samples were dried at 60
until the weight was constant. The growth rate was measured as follows: Growth
rate = increased dry weight/area/time (g/m2/day). Furthermore, micro-Kjeldahl N-digestion was used to determine total-N content of biomass samples (Markus et al. 1985), while the P content was determined by the Vanado-molybdo-phosphoric acid colorimetric method (Kuo, 1996) and crude protein
6
content was estimated by N × 6.25 (Xiao et al., 2013). To measure the starch content, dry duckweed powder was homogenized in 3mL 6M HCl, heated in boiling water for 2h in water bath, pH-adjusted to 7.0±0.5 with HCl (6M) or KOH (6M), and then added to 200uL Pb(COOH)2 and centrifuged at 5000g for 5min. Sugars in the supernatant were analyzed by HPLC (Thermo 2795, Thermo Corp., USA) with an Evaporative Light-scattering Detector (All-Tech ELSD 2000, All-tech., Corp., USA) and the starch content was determined using the total sugar content (starch content = glucose content × 0.909) (Zhang et al. 2011). Finally, the NH4-N and PO4-P levels in the swine water were measured by a Spectroquant testing kit (MERCK Corp., Germany) using PhotoLab 6100 (WTW Corp., Germany). 2.3 Calculations and statistics Each data point represents the results of three samples per experiment. All results are expressed as means ± standard error in the figures and significance (P < 0.5) was assessed using Student’s t-test. 3 Results and discussion 3.1 The effect of temperature on the duckweed growth Changes in temperature had substantial effects on both growth rate and starch content in duckweed with the two strains investigated exhibiting distinct growth characteristics in different temperatures. The results showed that the optimal temperature for the growth rates of L. punctata OT and L. minor OT was 25 g/m2/d) and 20
(4.2
(3.9 g/m2/d), respectively (Fig 1 a). Interestingly, the growth rate of the
7
mixture was between those of L. punctata OT and L. minor OT at higher temperatures (30
and 25 ), but it was almost the same as that of the L. punctata OT at 20 .
Alternatively, the starch content of L. punctata OT, L. minor OT, and the mixture decreased by 28.6, 34.9 and 39.7 %, respectively, as the temperature increased from 20 to 30
(Fig 1 a). Overall, however, mixed cultures did not have a significant advantage
over the monoculture in terms of the biomass and the starch content. 3.2 The effect of light intensity on duckweed growth Light intensity significantly affected growth rate of duckweed. Under low light (2000 lux) the growth rate of L. punctata OT, L. minor OT, and the mixture were 1.31, 0.71, and 1.28 g/m2/d, respectively. As the light intensity increased to 5000 lux, the growth rate increased by 1.3, 2.5, and 1.1 fold, respectively, but did not increase further at 10000 lux (Fig 2a). The growth rate of the mixture was between L. minor OT and L. punctata OT in monoculture in light intensity of 5000 and 10000 lux. Interestingly, the mixture reached the highest growth rate (1.28 g/m2/d) almost as well as L. punctata OT (1.31 g/m2/d) (P>0.05) at low irradiance (2000 lux). The result showed that the mixture promoted the population to grow in low light intensity. It was observed that the roots of both L. punatata OT and L. minor OT in mixture were considerably longer than those in monoculture in the low light intensity (unpublished data). In this regards, there is a long-standing controversy about root involvement in nutrient uptake in Lemnaceae species (Cedergreen and Madsen, 2002), but recent studies have shown that L. punctata can uptake nitrogen by both the roots and fronds (Fang et al. 2007).
8
Moreover, L. minor can regulate NO3- uptake via fronds or roots depending on light intensity (Cedergreen and Madsen 2004). The above results show that the competition between L. punatata OT and L. minor OT in low light promotes longer roots, increasing NO3 - uptake to promote growth. The starch content of the duckweed tended to be higher in high light intensity (Fig 2b). The starch contents of L. punctata OT and L. minor OT in monoculture increased by 1.6 and 2.2 fold, respectively, as the light intensity increased from 2000 lux to 5000 lux. But as the light intensity increased from 5000 lux to 10000 lux, the starch content of L. minor OT did not increase obviously, however, the starch content of L. punctata OT increased by 1.53 fold. When L. punctata OT and L.minor OT were mixed culture, the starch content increased by 1.5 fold as the light intensity increased from 2000 lux to 5000 lux, but decreased by 3% at 10000lux. The starch content of the mixture (21.8% DW) was between those of L. punctata OT (24.6% DW) and L. minor OT (17.6% DW) at 10000 lux irradiance, but the starch contents of the mixture (22.3% and 8.8%) were significantly higher than L. punctata OT (18.3% and 7.0%) and L. minor OT (17.0% and 5.4%) in monoculture at 5000 and 2000 lux irradiance (Fig 2b). Higher starch content in duckweed is favorable to species competition at low irradiance, so duckweed in the mixed culture tends to accumulate more starch, as compared to that in monoculture. There is a particular climate in the Sichuan Basin. Cloudy and rainy days occur often in Chengdu, resulting in low light intensity year round with a lowest recorded value at noon of approximately 1000 lux (Xiao et al.,
9
2013). Therefore, the ideal method for producing starch at low irradiance areas such as Sichuan Basin is via a mixed culture of duckweed. 3.3 The effect of N and P content on duckweed growth It was observed that the duckweed grown in high N and P concentration had large, thin and dark green fronds and short, thin roots. By contrast, plants grown in low N and P concentration were small, thick and pale with long thick roots. Results revealed that the growth rate of L. punctata OT, L. minor OT and their mixture changed significantly depending on N and P concentrations (Fig. 3 a). The higher N and P concentrations in A and B medium were clearly favorable for the growth of L. punctata OT alone. Moreover, the growth rate of L. punctata OT significantly decreased at levels below that of solution B (34.5mg N L-1 and 15 mg P L-1). Alternatively, the growth rate of L. minor OT did not change obviously until they were grown in D and E medium, at which point the growth rate began to decrease at a rapid rate. Interestingly, the growth rate of the mixture rose initially and then fell as the N and P concentration decreased from A to D. The highest growth rate of the mixture was observed in C medium (Fig. 3 a). The growth rate of L. punctata OT was significantly higher than that of L. minor OT at every concentration combination of N and P. When higher concentration of N and P were used in the A or B media, the growth rate of the mixture of duckweed was between those of L. punctata OT and L. minor OT. When N and P concentration decreased and was lower than that in C medium, the growth rates of the mixture were almost equal to L. punctata OT in monoculture (Fig. 3a). These results indicated that the
10
competition of the duckweed in the mixture promoted the growth of either one or both of the species when N and P were in low concentration. It was observed that the roots of L. minor OT and L. punctata OT in mixed cultures were separately longer than those in monocultures when N and P concentrations were low (data not shown), indicating that the competition of the two species resulted in the extension of plant root system. The extended root system can increase the absorption of the nutrients, especially phosphorus (Smith and Read, 1997). In addition, the duckweed had the capacity to take up NO3– through both roots and fronds, the over-all contribution of root and frond to whole-plant uptake varied depending on plant N status and the root contribution increased from 32 to 73% for N-satiated and N-depleted plants, respectively (Cedergreen and Madsen 2002). In low N concentration, the root had more contribution for NO3–. The longer roots of the L. minor OT and L. punctata OT in mixture would have more NO3 – absorption capacity to promote the growth. So in low N and P concentration, the competition of different duckweed species would promote each other for the use of nitrogen, resulting in higher growth rate. The concentration of N and P also strongly affected the starch content and the starch content in the duckweed tended to be accumulated in lower N and P concentration. The starch content of L. punctata OT, L. minor OT and their mixture increased by about 5.6, 9.9 and 7.1 fold as N and P concentration decreased from A (345mg N L-1 and 150 mg P L-1) to E (0 mg N L-1 and 0 mg P L-1). At higher concentrations (A or B), the starch content of the mixture was between L. punctata OT and L. minor OT in monoculture. As the N and P concentration decreased (C, D or E
11
media), however, the starch content of the mixture rose to become equivalent with that of L. punctata OT (Fig 3b). These results revealed that the lack of nutrition prompted duckweed to rapidly accumulate starch, and starch accumulation was in favor of duckweed competition and surviving in adverse environments. 3.4 The removal of nutrient in swine wastewater The change of nutrient in the swine lagoon liquid covered with L. minor OT, L. punctata OT and the mixture of the two species, respectively, was analyzed every three days. Results indicated that the duckweed could efficiently remove nitrogen and phosphorus from the swine wastewater. The vast majority of NH4-N and PO4-P was removed from the swine wastewater in 6 or 9 days (Table 1, 2). During the first 3 days, the NH4-N was removed 60.00-67.84% from the swine wastewater and there were no advantages in the remove effect of NH4-N with the mixed culture of L. punctata OT and L. minor OT over monoculture (Table 1). From 3 to 6d, however, the highest remove rate of NH4-N (6.65 mg/l/d) was obtained from the mixed culture and the remove rate of NH4-N of L. minor OT and L. punctata OT in monoculture was 4.81 and 5.65 mg/l/d, respectively. Compared with monoculture, the mixed culture of the two duckweed species enhanced the NH4-N removal capacity obviously during 3 to 6 cultured days. The uptake rate of PO4-P was different in different species and different culture phase. During the first 3 days, the highest remove rate of PO4-P was observed in L. minor OT. Following 6 days of culture, however, the highest remove rate of PO4-P was found in the mixed culture, with a PO4-P value of 1.2 mg/L, significantly lower
12
than those in monoculture (3.5 mg/L and 4.8 mg/L). Finally, after 9 days of culture, all PO4-P content in the swine wastewater were below 0.1 mg/L and the minimum PO4-P content was also found in the mixed culture (Table 2). These results indicated that the mixed culture of different duckweed species could enhance the PO4-P removal capacity. It was also observed that the roots of L. punctata OT and L. minor OT in mixed culture in swine water were longer than those in monocultures. Additional 3 strains (S. polyrhiza C1, L. minor C2 and L. punctata C3) isolated from a sewage treatment facilities were also mono- and mixed culture in swine wastewater in order to verify further the advantages that the mixed culture of different duckweed species could enhance the nutrient removal capacity. During the first 2 days, the duckweeds were in a lag period and had very little uptake of NH4-N. Following 2 days of culture, the duckweeds were in the growth period and began to uptake NH4-N at a rapid rate. After 12 days of culture, the overall NH4-N content in any mixed culture of any two strains was significantly lower than those in monoculture, but the mixed culture of all three species could not enhance the NH4-N removal capacity (Fig 4a). The remove rate by NH4-N of S. polyrhiza C1, L. minor C2 and L. punctata C3 in monoculture was 2.72, 2.53 and 2.80 mg/l/d, respectively. Meanwhile, the remove rate of NH4-N of the different mixture was 2.90 (mixture of S. polyrhiza C1 and L. minor C2), 2.94 (mixture of S. polyrhiza C1 and L. punctata C3), 3.01 (L. minor C2 and L. punctata C3), and 2.65 (mixture of S. polyrhiza C1, L. minor C2 and L. punctata C3) mg/l/d, respectively. These results indicated that effect of the NH4-N removal of the mixed culture of two species was obviously better than those of
13
mono or mixed culture of three species. Furthermore, the PO4-P content of swine wastewater rapidly decreased when it was treated with duckweed regardless of which culture was used (Fig 4b). Following 12 day of culture, the PO4-P content in any mixed culture of any two or three species was significantly lower than those in monoculture (Fig 4b). The overall remove rate of PO4-P by S. polyrhiza C1, L. minor C2, and L. punctata C3 in monoculture was 0.187, 0.181 and 0.184 mg/l/d, respectively, while in the mixed cultures, the remove rate was 0.197 (mixture of S. polyrhiza C1 and L. minor C2), 0.199 (mixture of S. polyrhiza C1 and L. punctata C3), 0.196 (L. minor C2 and L. punctata C3) and 0.195 (mixture of S. polyrhiza C1, L. minor C2 and L. punctata C3) mg/l/d, respectively. These results indicated that effect of the PO4-P removal of the mixed culture of two or three species was obviously better than those of mono species culture. A number of nutrient removal rates between the separate populations of duckweed (Fig 4 and Tables 1 and 2) seem to be inconsistent, especially during the first few days. For instance, the process of phosphate removal occurred a few days later than that of NH4-N removal (Table 1, 2), but phosphate removal was much faster in sewage water (Fig4 a, b). These results are similar to a previous report looking at the ability of a mixture of algae to process swine wastewater with different N/P ratios (Su et al., 2012), indicating the importance of the N/P ratio for nutrient removal. As such, it was calculated that the N/P ratio (4.5:1) in swine wastewaters in table 1 and 2 was much lower than that (15:1) in the fig 4. Therefore, the inconsistent results should be due to the different N/P ratio in the wastewater.
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The results of nutrient removal rates of duckweed were similar to the results from those of algae. The nutrient removal rates by using mixed algal culture are higher than those of three algae species in monoculture (Chlamydomonas reinhardtii, Scenedesmus rubescens and Chlorella vulgaris), further substantiating the similar results revealed in this paper (Su et al., 2012). 3.5 Nutrient content and biomass of duckweed in swine wastewater The growth rate and biomass were analyzed when the duckweed strains were mono-/ mixed culture in the swine lagoon liquid. The results showed that the growth rate of duckweed in mixed culture was obviously higher than that of monoculture. After 12 days of culture, the growth rate of L. minor OT, L. punctata OT and the mixture of the two were 16.20, 21.72 and 27.75 g/m2/d in fresh weight, respectively (data not shown). Furthermore, the biomass of each culture at harvest was 31.6, 37.5 and 41.4 g/m2 in dry weight (Table 3), respectively. The biomass of the mixture increased by 31.1 and 10.4% over that of L. minor OT and L. punctata OT in monoculture, respectively. The other 3 strains (S. polyrhiza C1, L. minor C2 and L. punctata C3) were also mono-/ mixed culture in swine wastewater and the similar results were obtained. The harvest biomass of S. polyrhiza C1, L. minor C2 and L. punctata C3 in monoculture were 48.39, 55.69 and 66.55 g/m2, respectively, while the biomass of the mixture in different combinations of duckweed strains were 77.87 (mixture of L. punctata C3 and L. minor C2), 71.36 (mixture of L. punctata C3 and S. polyrhiza C1), 73.00 (mixture of L. minor C2 and S. polyrhiza C1) and 76.00 (mixture of S. polyrhiza C1, L. minor C2 and L. punctata C3) g/m2, respectively (Table 4). The biomass of duckweed in the
15
mixed culture increased by 7.2-57.1% than that in monoculture. The nutrient of duckweed harvested from the swine wastewater was analyzed. L. minor OT, L. punctata OT and their mixture were first assessed. L. minor OT had the highest crude protein, N and P content, while L. punctata OT was found to have the highest starch content (Table 3). The crude protein, N, and starch content in the mixed culture was in between that of L. minor OT and L. punctata OT in monoculture, however, the P content was the lowest (Table 3), indicating that the mixture consumed a greater amount of P in order to maintain its growth rate. In addition, the nutrient content of S. polyrhiza C1, L. minor C2 and L. punctata C3 was analyzed . The results showed the similar results that a higher N and P content was assessed in the L. minor C2, while a higher starch content was observed in L. punctata C3 (Table 4). The P content in the mixture of different species was always lower than that in mono-culture except the mixture of L. minor C2 and S. polyrhiza C1. Only some of the mixed cultures had starch content values that fell between those in monoculture. Interestingly, the starch content in the mixture of S. polyrhiza C1 and L. punctata C3 was significantly higher than that of S. polyrhiza C1 and L. punctata C3 in monoculture (Table 4). Taking all the data into consideration, the mixture of L. punctata C3 and S. polyrhiza C1 produced the most starch, while the mixture of L. punctata C3 and L. minor C2 produced the greatest amount of crude protein (Table 4). Therefore it was a good method to mix duckweed species in culture to increase the production of biomass, the starch and crude protein. In addition, we must protect the duckweed species
16
diversity. 4. Conclusion The duckweed in the mixture had higher growth rate, total biomass, starch, crude protein and nutrient removal efficiency than those in monoculture in swine wastewater. L. minor OT had higher crude protein content, while L. punctata OT had the higher starch content in monoculture. In different mixed cultures with L. punctata C3, L. minor C2 and S. polyrhiza C1, the increase biomass was different. The species diversity significantly affects the biomass productivity and nutrient removal efficiency of duckweed in swine wastewater, and the proper mixture of duckweed significantly abrogates the eutrophication of swine wastewater. Acknowledgements We would like to thank Dr. Q.S. Liu from Sichuan University for reviewing the manuscript. This work was supported in part by grants from the Ministry of Science and Technology of PR China (2011BAD22B03) and the Chinese Academy of Science (KSCX2-EW-J-22 and Y2C5021100).
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Cedergreen, N., Madsen T.V., 2002. Nitrogen uptake by the floating macrophyte Lemna minor. New Phytol. 155(2), 285-292. Cedergreen, N., Madsen, T.V., 2004. Light regulation of root and leaf NO3- uptake and reduction in the floating macrophyte Lemna minor. New Phytol. 161(2), 449-457. Chen, M., Kim J.H., Kishida, N., Nishimura, O., Sudo, R., 2004. Enhanced nitrogen removal using C/N load adjustment and real-time control strategy in sequencing batch reactors for swine wastewater treatment. Water Sci. and Technol. 49(5-6),309-314. Chen, Q., Jin, Y.L., Zhang,G.H., Fang, Y., Xiao, Y., Zhao, H., 2012. Improving Production of Bioethanol from Duckweed (Landoltia punctata) by Pectinase Pretreatment. Energies. 5 (8),3019-3032. Cheng, J.J., Stomp A.M., 2009. Growing Duckweed to Recover Nutrients from Wastewaters and for Production of Fuel Ethanol and Animal Feed. Clean-Soil, Air, Water. 37(1),17-26. Cheng, J., Bergmann, B.A., Classen, J. J., Howard, J.W., 2002. Nutrient recovery from swine lagoon water by Spirodela punctata. Biresour. technol. 81,81-85. El-Shafai, S.A., El-Gohary, F.A., Nasr, F.A., vander Steen, N.P., Gijzen, H.J., 2007. Nutrient recovery from domestic wastewater using a UASB-duckweed ponds system. Bioresour. Technol. 98, 798–807. Fang, Y. Y., Babourina, O., Rengel, Z., Yang, X.E., Pu, P. M. , 2007. Ammonium and nitrate uptake by the floating plant Landoltia punctata. Ann. Bot,99(2):
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365-370. Garrett, K.A., Mundt C.C., 1999. Epidemiology in mixed host populations. Phytopathol. 89(11), 984-990. Ge, X.M., Zhang, N.N., Phillips, G.C., Xu, J. F., 2012. Growing Lemna minor in agricultural wastewater and converting the duckweed biomass to ethanol. Bioresour. Technol. 124,485-488. Ismail, M., 1998. Chemical Characterization of Protein Contentrates of Duckweed. Pertanika J. Sci. & Techno.6(1),7-21. Josphine N., Iris S., Ludwig T., 2011. Competition between Lemna minuta and Lemna minor at different nutrient. Aquat Bot. 94,158-164. Kim, J. H., Chen, M.X., Kishida, N., Sudo, R., 2004. Integrated real-time control strategy for nitrogen removal in swine wastewater treatment using sequencing batch reactors. Water Res.38(14-15), 3340-3348. Kuo, S., 1996. Phosphorus. In: Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabai, M.A., Johnston, C.T., Sumner, M.E. (Eds.), Methods of Chemical Analysis, Part 3. Chemical Methods. SSSA (Soil Science Society of America), Madison,WI, pp. 869–919. Markus, D. K., McKinnon, J.P., Buccafuri, A.F., 1985. Automated analysis of nitrite, nitrate, and ammonium nitrogen in soils. Soil Sci Soc Am J. 49(5), 1208-1215. Olguin, E.J., 2003. Phycoremediation: key issues for cost-effective nutrient removal processes. Biotechnol. Adv. 22(1-2), 81-91. Oron, G.., 1994. Duckweed culture for wastewater renovation and biomass production.
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Agric. Water Manag. 26, 27–40. Porath, D., Hepher, B., Koton, A., 1979. Duckweed as an aquatic crop: evaluation of clones for aquaculture. Aquat. Bot, 7, 273 – 278. Reid, M.S., Bieleski, RL., 1970. Response of Spirodela oligorrhiza to phosphorus deficiency. Plant Physiol. 46(4), 609-613. Rusoff, L.L., Blakeney, E.W., Culley, D.D., 1980. Duckweeds (Lemnaceae family): a potential source of protein and amino acids. J Agr Food Chem. 28(4), 848-850. Stomp, A.M. 2005. The duckweeds: a valuable plant for biomanufacturing. Biotechnology. Annu Review. 11, 69-99. Su, Y., Mennerich, A., Brigitte, U., 2012. Coupled nutrient removal and biomass production with mixed algal culture: Impact of biotic and abiotic factors. Bioresour. Technol. 118, 469-476. Suppadit, T., 2011. Nutrient removal of effluent from quail farm through cultivation of Wolffia arrhiza. Bioresour. Technol. 102(16), 7388-7392. Smith, S. E., Read, D. J., Mycorrhizal Symbiosis 2nd edn (Academic, San Diego, 1997) Vander Steen, P., Brenner, A., Oron, G., 1998. An integrated duckweed and algae pond system for nitrogen removal and renovation. Water Sci. Technol., 38, 335–343 Xiao, Y., Fang Y., Jin, Y.L., Zhang, G.H., Zhao, H., 2013. Culturing duckweed in the field for starch accumulation. Ind Crops Prod. 48, 183-190. Xu, J., Shen G., 2011. Growing duckweed in swine wastewater for nutrient recovery and biomass production. Bioresour. Technol. 102(2), 848-853. Xu, J., Cui, W., Cheng, J.J., Anne-M, S., 2011. Production of high-starch duckweed and
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its conversion to bioethanol. Biosyst Eng, 110:67-72. Zhang, L., Zhao, H., Gan, M.Z., Jin, Y.L., Gao, X.F., Chen, Q., 2011. Application of simultaneous saccharification and fermentation (SSF) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales. Bioresour. Technol. 102(6), 4573-4579.
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Fig. 1. Change of growth rate (a) and starch content (b) of the duckweed in the mixture and monoculture under the influence of temperature. DW=Dry weight. Values represent mean± S.E. (n=3). The growth rate and starch content were measured at 12 days after inoculation. The asterisk indicated that the growth rate/ starch content of L. punctata / L. minor in uni-cultures that were significantly different from those in mixture with p<0.05 in an unpaired Student’ s t test. Fig. 2. Change of growth rate (a)and starch content (b) of the duckweed in the mixture and monoculture under the influence of light intensity. DW=Dry weight. Values represent mean± S.E. (n=3). The growth rate and starch content were measured at 12 days after inoculation. The asterisk indicated that the growth rate/ starch content of L. punctata/L. minor in uni-cultures that were significantly different from those in mixture with p<0.05 in an unpaired Student’ s t test. Fig. 3. Change of growth rate (a) and starch content (b) the duckweed in the mixture and monoculture in media with different concentration of N and P. DW=Dry weight. Values represent mean± S.E. (n=3). A= 345 mg N L-1 150 mg P L-1; B=34.5 mg N L-1 15 mg P L-1; C=3.45 mg N L-1 1.5 mg P L-1; D=0.345 mg N L-1 0.15 mg P L-1; E=0 mg N L-1 0 mg P L-1. The growth rate and starch content were measured at 12 days after inoculation. The asterisk indicated that the growth rate/ starch content of L. punctata / L. minor in uni-cultures that were significantly different from those in the mixture with p<0.05 in an unpaired Student’s t test. Fig. 4. The NH4-N (a) and PO4-P (b) content in the swine wastewater covered with duckweed in different days. Values represent mean ± S.E. (n=3).
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Table 1. The NH4-N content (mg/L) in the swine wastewater covered with duckweed in different days. Time (day) 0 3 6 9 12
L. punctata OT
The mixture
L. minor OT
51±1.37
51±1.37
51±1.37
17.5±0.24
20.1±0.53
18.8±1.06
0.54±0.06
0.16±0.05
4.37±1.16
0
0
0
0
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0.35±0.13 0
Table 2 The PO4-P content (mg/L) in the swine wastewater covered with duckweed in different days. Time (day) 0 3 6 9 12
L. punctata OT
The mixture
L. minor OT
11.2±0.74
11.2±0.74
11.2±0.74
10.3±1.51
9.6±1.35
5.6±0.15
3.5±0.66
1.2±0.25
4.8±0.15
0.05±0.02
0.04±0.01
0.09±0.01
0.05±0.01
0.03±0.02
0.06±0.01
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Table 3 The nutrient content of duckweed in swine wastewater on 12d. Strains
N(%)
P(%)
Starch
Crude
content
protein
(%)
content
Biomass 2
(g/m )
Starch 2
(g/m )
Crude protein 2
(g/m )
(%) L. punctata OT
4.81b
0.46b
16.74a
30.06b
37.5b
6.28a
11.3c
L. minor OT
5.15a
0.81a
9.37c
32.19a
31.6c
2.95b
10.2b
Mixture (L. punctata OT+ L. minor OT)
4.61b
0.39b
15.52b
28.81b
41.4a
6.42a
11.9a
The concentration of NH4-N and PO4_P in the swine waste water were 51mg/L and 12 mg/L. Different lower-case letters in the same column denote significant differences according to Tukey test (P<0.05).
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Table 4
The nutrient content of duckweed in swine water on 12d.
Strains
N(%)
P(%)
Starch
Crude
content
protein
(%)
content
Biomass 2
(g/m )
Starch 2
(g/m )
Crude protein (g/m2)
(%) L. punctata C3
4.04b
0.53b
28.60a
25.25b
66.55c
19.03b
16.80bc
L. minor C2
4.35a
1.25a
13.42c
27.19a
55.69d
7.47f
15.14c
S. polyrhiza C1
3.47c
0.49b
22.92b
21.69c
48.39e
11.09d
10.50d
Mixture (L. Punctata C3+L. minor C2)
4.19a
0.23c
24.80b
26.19a
77.87a
19.31b
20.40a
Mixture (L. punctata C3+S. polyrhiza C1)
3.51c
0.43b
29.67a
21.94c
71.36b
21.17a
15.66c
Mixture (L. minor C2 + S. polyrhiza C1)
4.25a
1.02a
13.35c
25.56a
73.00b
9.75e
18.66b
Mixture
4.01b
0.45b
23.33b
25.06b
76.00a
17.73c
19.05a
(L.punctata C3+L.minor C2+S.polyrhiza C1)
The concentration of NH4-N and PO4_P in the swine waste water were 31.6mg/L and 2.4 mg/L. Different lower-case letters in the same column denote significant differences according to Tukey test (P<0.05).
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27
28
29
30
Highlights ●
The duckweed in mixture produced more starch in low light intensity.
●
The mixture promoted the growth and nutrient removal in swine wastewaters.
●
The duckweed in the mixture had lower P content than those in the monoculture.
●
The proper combinations of duckweed significantly increased biomass.
31