Thesis for the Degree of Master of Science

Thesis for the Degree of Master of Science Mass culture of a freshwater green alga Chlorella sorokiniana HS1 under mild salinity and induction of neutral lipids, chlorophyll a and b, and total carotenoids by salinity change after enrichment by Geun Soo Kim Department of Microbiology The Graduate School Pukyong National University February 2017

Mass culture of a freshwater green alga Chlorella sorokiniana HS1 under mild salinity and induction of neutral lipids, chlorophyll a and b, and total carotenoids by salinity change after enrichment ( 약한염도조건에서담수녹조류인 C. sorokiniana HS1 의대량배양과농축후염도변화에의한중성지방, 클로로필, 카로티노이드의유도 ) Advisor: Prof. Tae-Jin Choi by Geun Soo Kim A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Depart of Microbiology, The Graduate School, Pukyong National University February 2017

Mass culture of a freshwater green alga Chlorella sorokiniana HS1 under mild salinity and induction of neutral lipids, chlorophyll a and b, and total carotenoids by salinity change after enrichment A dissertation by Geun Soo Kim Approved by: (Chairman) Myung-Suk Lee (Member) Young-Tae Kim (Member) Tae-Jin Choi February 2017

Contents ABSTRACT... 1 INTRODUCTION... 3 MATERIALS AND METHODS... 7 CELL STRAIN AND CULTURE CONDITIONS... 7 CULTIVATION OF C. SOROKINIANA HS1 IN DIFFERENT SODIUM CHLORIDE CONCENTRATION... 8 AFTER THE MASS CULTURE OF C. SOROKINIANA HS1 AND ESTABLISH STORAGE CONDITIONS UNDER SALT STRESS... 8 NILE RED STAINING AND NEUTRAL LIPID DETERMINATION... 9 SPECTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLL A AND B AND TOTAL CAROTENOID CONTENTS... 11 RESULTS AND DISCUSSION... 13 CULTIVATION OF C. SOROKINIANA HS1 IN VARIOUS NACL CONCENTRATIONS... 13 AFTER THE MASS CULTURE OF C. SOROKINIANA HS1 AND ESTABLISH STORAGE CONDITIONS UNDER SALT STRESS... 18

NEUTRAL LIPID CONTENTS IN RESPONSE TO CHANGES IN SALINITY... 21 CONTENTS OF CHLOROPHYLL A AND B, AND TOTAL CAROTENOIDS IN RESPONSE TO CHANGES IN SALINITY... 25 CONCLUSION... 30 국문초록... 33 REFERENCES... 35

Lists of figures Figure 1. Growth curve of C. sorokiniana HS1 in different concentration of NaCl...16 Figure 2. Neutral lipid contents of C. sorokiniana HS1 in various NaCl concentrations...17 Figure 3. Growth curve of concentrated C. sorokiniana HS1 in different concentration of NaCl...20 Figure 4. Neutral lipid contents by change of sodium chloride concentration and times...24 Figure 5. Contents of chlorophyll a in response to changes in salinity and culture time...27

Figure 6. Contents of chlorophyll b in response to changes in salinity and culture time...28 Figure 7. Total carotenoid contents in response to changes in salinity and culture times...29

Lists of tables Table 1. The formulas that used in the calculation of chlorophyll - a, b and total carotenoid levels...12

Mass culture of a freshwater green alga Chlorella sorokiniana HS1 under mild salinity and induction of neutral lipids, chlorophyll a and b, and total carotenoids by salinity change after enrichment Geun Soo Kim Department of Microbiology, The Graduate School, Pukyong National University Abstract Salinity is a major abiotic stress for terrestrial plants and freshwater microalgae. Dunaliella microalgae display decreased photosynthesis and carotenoid production under salinity stress. However, Chlorella sorokiniana HS1 is a novel freshwater microalga strain that can adapt to seawater salinity. This report determined the optimal growth conditions for C. sorokiniana HS1 in response to changes in salinity (10, 20, 30 and 40 g L -1 NaCl). Also, the neutral lipids, chlorophyll and carotenoid contents were compared using Nile red staining and spectrophotometric analysis after enrichment by membrane 1

filtration. The comparative growth rates of C. sorokiniana HS1 under freshwater and four different salinity conditions showed the chlorella grew fastest at 10 g L -1 NaCl. Neutral lipid, chlorophyll a and b, and total carotenoid characterization of enriched C. sorokiniana HS1 in response to changes in salinity (10, 20, 30 and 40 g L -1 NaCl) and culture times (24~72 hours), showed the chlorella grew well at 10 g L -1 NaCl. Under this condition, it produced high contents of neutral lipids, chlorophyll and carotenoids. Thus, the present study proposed that a mass culture system using brine groundwater may be an efficient approach for large-scale chlorophyll and lipid production. 2

Introduction The US consumes over 50 billion gallons of diesel fuel per year for transportation purposes. In 2007, the US Government Accountability Office reported the need to develop a strategy for addressing a peak and decline in oil production. Declining oil production will cause oil and diesel prices to rise sharply creating a strong market for replacement fuels. Biodiesel is an alternative liquid fuel that can substantially replace conventional diesel and reduce exhaust pollution and engine maintenance costs (Gallagher, 2011). This renewable fuel can be produced from different feedstocks such as soybeans, sunflowers, canola, jathropha, waste cooking oil, and microalgae. Among these various feedstocks, microalgae recently come into the spotlight. Microalgae are the fastest-growing plants in the world. Industrial reactors for algal culture are open ponds, photobioreactors and closed systems. Microalgae are very important as a biomass source. Microalgae will someday 3

be competitive as a source for biofuel. Microalgae can be a replacement for oil based fuels, one that is more effective and has few disadvantages. Microalgae are among the fastest-growing plants in the world, and about 50% of their weight is oil. This lipid oil can be used to make biodiesel for cars, trucks, and airplanes (Demirbas & Demirbas, 2011). However, algae-to-biodiesel production have obvious disadvantage. It is apparent that an economically viable microalgae-to-biodiesel commercialization will initially depend on government subsidies and the future price of oil, in addition to optimized biomass yields (Gallagher, 2011). To overcome these advantages, there are many studies have been carried out (Scott et al., 2010; Hossain et al., 2008). Microalgae provide several advantages for biodiesel production compared to oil crops as they are not dependent on fertile land, offer higher productivities and can use wastewater (Chisti, 2007). However, microalgae also have apparent disadvantages, such as high water usage for culture, high nutrient input and contamination by competing species. In particular, the accumulation of useful substances, like lipids, can be enhanced under stress conditions (Christenson & Sims, 2011; Smith, Sturm, & Billings, 2010; 4

Wijffels, Barbosa, & Eppink, 2010). Among the stressed environments, nutrient starvation is particularly difficult to establish and involves high cost. Consequently, continuous mass cultivation in a nutrient starvation environment has many limitations and other methods are required to accumulate lipids using microalgae (Hu, Sommerfeld, Jarvis, Ghirardi, Posewitz, Seibert, & Darzins, 2008). Salinity stress is one of the easiest ways to create a stress environment. Therefore, mass cultivation of microalgae in combination with salinity may be a simple approach to enhance biodiesel production (Perrineau, Zelzion, Gross, Price, Boyd, & Bhattacharya, 2014). However, the economic feasibility must be considered when using salinity stress. In general, microalgae are cultivated in at least a few thousand tons for the production of biodiesel, which requires an enormous amount of salt to raise the salinity of 10 ppt. From this perspective, it is inefficient to apply salinity stress to microalgae during general mass culture. Therefore, to secure economic efficiency in producing biodiesel using microalgae, it is necessary to enrich the microalgae. Chlorella is a green microalga with a spherical single cell, sized 2~10 μm 5

in diameter. It proliferates four times every 20 hours and is the fastest growing food crop known (Barclay, Meager, & Abril, 1994; Kang & Sim, 2004). Chlorella grows through the photosynthesis process and accumulates useful substances, such as intracellular pigments (chlorophyll and carotenoids) and lipids (Kim, Won, & In, 2014). It contains about 14% lipid along with a large amount of protein (Han, Kang, Kim, & Kim, 2002; Illman, Scragg, & Shales, 2000). Chlorella species are suitable for the production of biodiesel due to their ability to outcompete other species and resist environmental stress. The green alga Chlorella sorokiniana is the most promising of the Chlorella strains for biodiesel applications. It is common in freshwater and is known to be inhibited at 0.035 g L -1 NaCl, but is tolerant to heat, ph and light (de-bashan, Trejo, Huss, Hernandez, & Bashan, 2008; Moronta, Mora, & Morales, 2006). In this study, the growth rate of a novel freshwater green alga Chlorella sorokiniana HS1 was compared during incubation in freshwater and various salinity conditions. Then, lipid, chlorophyll and carotenoid production of the enriched culture broth in response to changes in salinity and culture time were determined. 6

Materials and methods Cell strain and culture conditions C. sorokiniana HS1 strain was obtained from Dr. Hee-Sik Kim in Sustainable Bioresource Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Republic of Korea, and was incubated in BG11 medium (Castenholz, 1988) at 24±1 under 20,000 lux light intensity. C. sorokiniana HS1 was able to grow in all the tested concentrations of sodium chloride (0~40 g L -1 ). The salinity of the culture media used 0, 10, 20, 30 and 40 g L -1 Sodium chloride. For salt shock condition, C. sorokiniana HS1 was grown in BG11 media until early exponential phase. 7

Cultivation of C. sorokiniana HS1 in different sodium chloride concentration To research C. sorokiniana HS1 s resistance to salinity, C. sorokiniana HS1 was grown in BG11 medium with different Sodium chloride concentrations (0, 10, 20, 30 and 40 g L -1 ). Cell numbers was counted using a hemocytometer (Gimzo Supply Co., USA) every day till 10 days. After the mass culture of C. sorokiniana HS1 and establish storage conditions under salt stress The neutral lipid, chlorophyll a and b, and total carotenoid contents of C. sorokiniana HS1 cultured in BG11 medium with various NaCl concentrations (0, 10, 20, 30 and 40 g L -1 NaCl) were determined. When the number of microalgae cells reaches a certain threshold, it no longer increases (Richmond, 1992). Therefore, to efficiently obtain the most useful materials produced by the microalgae in the predetermined space, it is necessary to concentrate the microalgae culture fluid. 8

C. sorokiniana HS1 was cultured in BG11 medium with 10 g L -1 NaCl and concentrated using a membrane filtration method. After enrichment, NaCl was added to the concentrated culture inoculum to achieve various NaCl concentrations (10, 20, 30 and 40 g L -1 ) and then kept for 3 days. The cell numbers were counted every 24 hours using a hemocytometer (Gizmo Supply Co., USA). Nile red staining and neutral lipid determination Nile red (9-(diethylamino)-5H-benzo[a]phenoxazin-5-one) (Sigma Aldrich, USA) was prepared as a stock solution at 250 mg L -1 in acetone. The modified Nile red staining procedure was performed as described by Chen, Zhang, Song, Sommerfeld, and Hu (2009). For effective staining of the microalgal cells, the neutral lipid contents were measured after adjusting the absorbance of the microalga to 0.5 (OD 750 nm ). The microalga culture was suspended in 25% dimethyl sulfoxide (DMSO, Sigma Aldrich), stained with 0.5 μg ml -1 Nile red and incubated in a 96-well plate in the dark at 40 for 10 min. The fluorescence was then measured using a spectrofluorophotometer (Infinite 9

F200 Pro, Tecan, Austria) at excitation and emission wavelength of 495 and 620 nm, respectively. The fluorescence intensity was calculated by subtracting the background fluorescence intensity of the microalga from the measured value. For lipid quantification, standard quantitative curves were constructed using triolein (Sigma Aldrich, USA) (Bertozzini, Galluzzi, Penna, & Magnani, 2011), which showed good linearity (R 2 = 0.99935). 10

Spectrophotometric determination of chlorophyll a and b and total carotenoid contents Aliquots (1 ml) of the microalga were incubated in BG11 medium at 10, 20, 30 and 40 g L -1 NaCl for 3 days, then centrifuged at 13,000 rpm (Centrifuge 5424, Eppendorf, Hamburg, Germany) for 2 min. The supernatant was discarded and the pellets were resuspended in 1 ml methanol, incubated at 60 for 30 min and then cooled at 0 for 5 min. The cooled solutions were centrifuged at 13,000 rpm for 2 min and the supernatant transferred to a fresh tube before the absorbance was measured using a UV/Vis spectrophotometer (Biochrom, MA, USA) at 666, 653 and 470 nm. The contents of chlorophyll a and b, and total carotenoids were calculated as shown in Table 1 (Lichtenthaler & Wellburn, 1983; Şükran, Güneş, & Sivaci, 1998).. 11

Table 1. Formulae used to calculate chlorophyll a and b, and total carotenoid contents Chlorophyll a (mg/l) = 15.65 A 666 nm 7.340 A 653 nm Chlorophyll b (mg/l) = 27.05 A 653 nm 11.21 A 666 nm Total carotenoids (mg/l) = 1000 A 470 nm 2860 C a 129.2 C b / 245 C a = Chlorophyll a, C b = Chlorophyll b, A = absorbance 12

Results and Discussion Cultivation of C. sorokiniana HS1 in various NaCl concentrations First of all, C. sorokiniana HS1 showed the highest growth rate in BG11 medium with 10 and 20 g L -1 NaCl than with freshwater (no NaCl supplementation) (Fig. 1). It was also confirmed that C. sorokiniana did not grow well in BG11 medium with salt concentrations above 40 g L -1. According to previous research, Chlorella sp. can grow both in freshwater and mild saline conditions (< 10 g L -1 ) (de-bashan et al., 2008; Moronta et al., 2006). C. sorokiniana HS1 was isolated from swine wastewater, which has a relatively high salinity level. Thus, it can tolerate salt concentrations over 10 g L -1 and can even tolerate seawater salinity (Kim, Ramanan, Kang, Cho, Oh, & Kim, 2016). In this study, however, there was a slight difference in the growth rate of C. sorokiniana HS1 in freshwater and saline conditions. When cultured at a salinity of 10 g L -1 NaCl, it grew about 2.2 2.3 times more rapidly than 13

without NaCl. Also, a previous study showed that compared to the growth rate at 10 g L -1 NaCl, the cell numbers decreased gradually at a salinity over 30 g L -1 NaCl (Kim et al., 2016). However, in this study, there was no significant difference between culturing at 30 and 40 g L -1 NaCl relative to freshwater. These results demonstrated C. sorokiniana HS1 can tolerate a certain level of salinity, in agreement with previous studies. Moreover, because the growth rate was drastically reduced at NaCl of 30 g L -1 or more, it is considered that C. sorokiniana HS1 can be cultured efficiently if an environment of mild salinity (10 g L -1 ) is provided. Secondly, C. sorokiniana HS1 showed the highest neutral lipid in BG11 medium with 30 g L -1 NaCl than with freshwater (no NaCl supplementation) (Fig. 2). It was also confirmed that neutral lipid was dramatically decressed in BG11 medium with salt concentrations above 40 g L -1. This results are similar to previous research s results. When cultured at a salinity of 30 g L -1 NaCl, it contained about 2.8 times more rapidly than without NaCl. Also, a previous study showed that compared to the growth rate at 30 g L -1 NaCl, the cell numbers decreased gradually at a salinity over 40 g L -1 NaCl (Kim et al., 2016). In considering the growth rate in 30 g L -1 NaCl concentration, it is also 14

inefficient way to get neutral lipid at culture medium with 30 g/l NaCl concentration. 15

Fig. 1. Growth curve of C. sorokiniana HS1 in various NaCl concentrations 16

Fig. 2. Neutral lipid contents of C. sorokiniana HS1 in various NaCl concentrations 17

After the mass culture of C. sorokiniana HS1 and establish storage conditions under salt stress Based on the above-mentioned preliminary findings for C. sorokiniana HS1 growth rate in the presence of NaCl, the strain was cultured at 10 g L -1 NaCl. Before enrichment, C. sorokiniana HS1 showed 5.00 ⅹ 10 6 cells/ml, and after enrichment, the cell numbers were 2.97 ⅹ 10 9 cells/ml. In particular, in the presence of NaCl, the cell numbers of C. sorokiniana HS1 decreased dramatically within the first 24 hours (Fig. 3). After that time, there was no notable change in the cell number. It is known that microalgae are difficult to cultivate over a certain density (Richmond, 1992). In commercial scale operations, cell concentrations are typically less than 1 g L -1 (Benemann, 1989). Similar to previous research, this study also confirmed that the number of cells in the concentrated culture decreased with time. Despite the decrease in biomass, the concentrated microalgal culture fluid was required to efficiently obtain useful constituents from the strain in a 18

predetermined space. Therefore, to be commercially viable, the most efficient interval is found by calculating the amount of useful materials that can be recovered from microalgae relative to the biomass within certain time intervals. 19

Fig.3. Growth curve of concentrated C. sorokiniana HS1 in various NaCl concentrations 20

Neutral lipid contents in response to changes in salinity The neutral lipid contents of microalgae are influenced by the growth conditions, such as culture temperature, light exposure and nutrients in the media (Kim, 1999). In this study, the highest lipid contents were detected after culture in 20 g L -1 NaCl (Fig. 4). Also, the most effective culturing time, regarding the neutral lipid contents, was at 48 hours (Fig. 4). Generally, it is known that the lower the cell growth, the higher the lipid content (Kim, Park, Kim, Joo, & Lee, 2013). Lipid content in algae biomass increases at high salt concentrations due to the accumulation of small molecules (e.g., glycerol) responding to osmotic pressure (Cuaresma, Janssen, Vílchez, & Wijffels, 2009; Wijffels et al., 2010). For instance, the freshwater microalga Chlamydomonas mexicana has been shown to grow in a high salinity medium with an increase in glycerol accumulation (Salama et al., 2014). It is known that Dunaliella species also have an increased lipid content with increasing salt concentration (Sharma, Schuhmann, & Schenk, 2012). In contrast, Botryococcus braunii is a green alga that has both high lipid and 21

carotenoid contents at a mild salinity (Rao, Dayananda, Sarada, Shamala, & Ravishankar, 2007). In this study, increasing the concentration of NaCl also increased the neutral lipid contents. However, excessive salinity stress (over 30 g L -1 NaCl and 72 hours, Fig. 4) sharply decreased both the cell numbers and the neutral lipid contents. These results suggest that the lipid synthesis in microalgae cells occurs actively in response to an external stress factor. Moreover, these findings concur with previous studies, showing that the accumulation of useful constituents is generally increased at mild salinity. However, unlike a previous study (Kim et al., 2016), in which the lipid content of C. sorokiniana HS1 was the highest at a salt concentration of 30 g L -1, this study revealed the highest lipid content at 10 g L -1 NaCl. In the instance of Chlorella vulgaris cultured in a nitrogen-limited medium, it is reported that the lipid contents increased about 40% compared to the control group (Illman et al., 2000). Also, exposure to stress factors, causes the microalgae to alter their lipid metabolism or lipid accumulation as a means of self-protection (Choi & Lee, 2015). Although the growth of microalgae under 22

environmental stress is reported to contribute to increased lipid accumulation, it is also reported to slow the growth of single cells. The current results highlight the efficient lipid production from C. sorokiniana HS1 using salinity as a simple environmental stress factor. 23

Fig 4. Neutral lipid contents in response to changes in salinity and culture time. 24

Contents of chlorophyll a and b, and total carotenoids in response to changes in salinity Among the salinity conditions evaluated, the contents of chlorophyll and carotenoids were most abundant at 40 g L -1 NaCl (Fig. 5, 6, 7). However, in comparison to 10 g L -1 NaCl, culturing at 40 g L -1 NaCl did not result in a notable difference. Regarding the culturing time, the chlorophyll a and b contents were highest at 48 hours, while the carotenoid contents were maximal at 24 hours. Commonly, when microalgae are exposed to stressed environments, the cells attempt to accumulate useful materials, such as lipids and chlorophyll (Choi, Kim, Kim, & Lee, 2013). Indeed, in this research, high chlorophyll and carotenoid contents were confirmed at 40 g L -1 NaCl. Conversely, however, the contents of chlorophyll and carotenoids measured after culturing at 20~30 g L -1 NaCl were lower than at 10 g L -1. Similar to these results, Botryococcus braunii microalga also had high chlorophyll and carotenoid contents at low salt concentrations but the 25

differences were not noticeable (Rao et al., 2007). In the instance of Thalassiosira, Cyclotella, Syracosphaera, Cryptomonas, and Monochrysis, there was little difference in chlorophyll content due to salinity change (McLachlan, 1961). Based on these results, it is considered that the content of pigments, such as chlorophyll and carotenoids, according to salinity change is influenced by the nature of the microalgae. Besides salinity, light intensity is also used as an external stress factor but it has not shown a marked difference in chlorophyll content (Cuaresma et al., 2009). 26

Fig 5. Contents of chlorophyll a in response to changes in salinity and culture time. 27

Fig 6. Contents of chlorophyll b in response to changes in salinity and culture time. 28

Fig 7. Total carotenoid contents in response to changes in salinity and culture times. 29

Conclusion This study compared the production of neutral lipids, chlorophyll and carotenoids by C. sorokiniana HS1 exposed to salinity stress. Comparison of the growth rate in response to the salinity change, revealed C. sorokiniana HS1 cultured well in 10 g L -1 NaCl, growing two-fold faster than in freshwater. This finding was not observed in a previous study (Kim et al., 2016). However, in a mass culture environment, the enriched culture medium showed a dramatic decrease in cell number after 24 hours, under all salinity conditions evaluated (10 40 g L -1 ). After 24 hours, there was no noticeable change in cell number. Nile red staining and spectrophotometric analyses revealed the lipid content was maximal in 20 g L -1 NaCl. This result corresponded to approximately a twofold increase in lipid content compared to the culture in 30

freshwater. Similar changes in lipid content, in response to salinity stress, have been published in the literature (Kim et al., 2016; Rao et al., 2007; Sharma et al., 2012). Conversely, the highest contents of chlorophyll and carotenoids were achieved by culturing in 40 and 10 g L -1 NaCl, respectively. However, these results were comparable to the control group. In conclusion, it is considered that mass cultivation of C. sorokiniana HS1 is most effective in 10 g L -1 NaCl to produce useful materials, like lipids, chlorophyll and carotenoids. In considering the relatively costly use of NaCl (Moon, Jung, Kim, & Shin, 2004) in mass cultivation of microalgae, brine groundwater may be considered as an economically efficient alternative stress inducer that can be readily prepared by simple water treatment. Providing there is no specific contaminants present, brine groundwater might be suitable for C. sorokiniana HS1 cultivation due to its low salinity compared to sea water. The high lipid and chlorophyll contents acquired from the mass cultivation of C. sorokiniana HS1 in 10 g L -1 NaCl are expected to also be acquired when using brine groundwater at similar salinity. The results of the present study 31

propose that this approach is used as the basis for the production of useful substances, such as lipids and chlorophyll, through the mass culture of C. sorokiniana HS1. 32

국문초록 약한염도조건에서담수녹조류인 C. sorokiniana HS1 의대량배양과농축 후염도변화에의한중성지방, 클로로필, 카로티노이드의유도 김근수 부경대학교대학원미생물학과 요약 염분은육상식물과미세조류담수종모두에게주요한비생물적스트레스 원으로작용한다. 일반적으로, 일부 Dunaliella 종의경우, 염도스트레스에 광합성과카로티노이드합성이감소한다. 그러나, 새롭게알려진녹조류인 C. sorokiniana HS1 은담수종임에도불구하고해수의염도에도적응한다고알려져 있다. 본연구에서, 염도변화에따른 C. sorokiniana HS1 의최적의성장조건을 33

알아보고농축후, 중성지질과클로로필그리고카로티노이드함량을비교하였다. 그과정으로 C. sorokiniana HS1 을담수와 4 개의서로다른염도구간에서배양 한후, 성장속도를비교하였다. 그결과 C. sorokiniana HS1 은 10 g L -1 의 염도에서가장빠르게성장했다. 이후, 막여과방식을이용하여 C. sorokiniana HS1 을농축하였다. 농축후, 4 개의서로다른염도구간에서 24 ~ 72 시간동안 보관하였다. 그후, 염도변화와시간에따른중성지질과클로로필 - a, b 그리고 카로티노이드함량의변화를 Nile red 염색법과분광분석법을이용하여 비교하였다. 결론적으로, C. sorokiniana HS1 은 10 g L -1 의염도조건에서중성지질과 클로로필그리고카로티노이드함량이가장높았다. 이연구의결과는지하염수 이용한미세조류의대량배양을통한클로로필의생산과지방추출에유용하게 사용될것이라제안한다. 34

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