Journal of Applied Phycology (2005) 17: 355–362 DOI: 10.1007/s10811-005-8005-y
C Springer 2005
Effects of potassium iodide on the growth and metabolite accumulation of two planktonic diatoms Weifa Zheng1,∗ , Caifa Chen1 , Yiqin Wang2 , Kangde Bao3 , Xuemei Wang1 & Chengcai Chu2 1
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, Xuzhou Normal University, Xuzhou, P.R. China; 2 State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P.R. China; 3 Department of Biology, Anhui Normal University, Wuhu, P.R. China ∗
Author for correspondence: e-mail:
[email protected]; fax: +86 516 3403179
Received 19 December 2004; accepted 9 May 2005
Key words: biomass accumulation, metabolite accumulation, Nitzschia closterium, Phaedactylum tricornutum, potassium iodide Abstract In this study the effects of potassium iodide on the growth and metabolite accumulation of Nitzschia closterium (Ehr.) W. Smith and Phaedactylum tricornutum Bolin were investigated to assess its possible application to the mass culture of the two diatoms in open environment, extensive systems. The results indicated that supplementation of potassium iodide at a concentration of 1000 mg L−1 resulted in a reduction of the induction phase in cultures of N. closterium and P. tricornutum and led to an increase in the accumulation of biomass and extracellular polymeric substances. Conversely, the addition of potassium iodide, at all concentrations tested, showed no obvious effect on the fatty acid profiles of the two diatoms, particularly in the content of eicosapentaenoic and decosahexaenoic acid. Potassium iodide was also found to inhibit the growth of Dunaliella salina, Cryptomonas sp. and Chlorella sp. at minimum inhibitory concentrations of 356.8, 475.9 and 696.2 mg L−1 , respectively. It also inhibited bacteria, including species isolated from the two diatom cultures, at a minimum concentration of 400 mg L−1 . These results suggest that potassium iodide is an effective agent for inhibiting the proliferation of certain flagellate and nonflagellate algae, and bacteria, thus forming a favorable environment for diatoms to proliferate and consequently improving accumulation of biomass and EPS. These properties of potassium iodide provide a possible solution for preventing contamination from flagellate and non-flagellate algae in mass culture of the two diatoms without causing significant changes in their fatty acid composition. Abbreviations: DHA, decosahexaenoic acid; DW, dry weight; EPA, eicosapentaenoic acid; EPS, extracellular polymeric substances; N., Nitzschia; P., Phaedactylum; PUFA, polyunsaturated fatty acids; ZBNC, the medium of Zheng and Bao for Nitzschia closterium; ZBPT: the medium of Zheng and Bao for Phaedactylum tricornutum
Introduction Planktonic diatoms are one of the major groups of primary producers in marine ecosystems and are an essential food source for aquaculture (St John et al., 2001). The species, Nitzschia closterium (Ehr.) W. Smith and Phaedactylum tricornutum Bolin are frequently chosen for feeding juvenile shellfish (Qu, 1995) and some fish species (Brown et al., 1997) because of their high
content of polyunsaturated fatty acids. However, the quantity of the two diatoms cannot satisfy the needs of a growing aquaculture sector because they are sparsely populated under natural conditions (Mischke et al., 2004), making it necessary to develop mass cultures of these two diatoms. Previous reports indicated that cultures of the two diatoms in open environment, extensive systems were easily contaminated by flagellate algae such as Dunaliella and Cryptomonas as well
356 as by non-flagellate Chlorella species (Wang, 1995), frequently leading to culture failure. In our previous studies, adequate supplementation of potassium iodide into the cultures of N. closterium and P. tricornutum generated higher yields of biomass with less contamination by flagellates and Chlorella sp. (Zheng & Bao, 2004a,b). However, it has not yet been clear how potassium iodide exerted its impact on the growth and metabolite accumulation of the two diatoms. During the growth of diatoms, most species, including planktonic diatoms, produce extracellular polymeric substances (EPS) predominantly comprised of acidic polysaccharides and small quantities of proteins and sulfides (Kelly, 1989; Hoagland et al., 1993; Poulet & Martin-Jezequel, 1983). The diatoms also accumulate ca. 10–39% polyunsaturated fatty acids (PUFA) as one of the major metabolites (Shin et al., 2003); variation in PUFA content directly affects feeding efficiency (Qu, 1995). In the present study, the effects of potassium iodide on growth and accumulation of biomass, EPS and PUFA in N. closterium and P. tricornutum were investigated to assess its possible application to mass cultures of the two diatoms in extensive systems.
Materials and methods Microalga and culture N. closterium and P. tricornutum were isolated from the beach of Lianyun Port, a coastline city of the Yellow China Sea. Voucher specimens (JKL003 for N. closterium and JKL004 for P. tricornutum) were identified by Prof Zhili Liu and preserved in the Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, P.R. China. N. closterium was maintained in ZBNC medium (KNO3 , 100 mg; K2 HPO4 , 80 mg; NaHCO3 , 600 mg; vitamin B12 , 1.5 × 10−4 mg; vitamin B1 , 1.5 × 10−3 mg; ferric citrate, 0.5 mg; EDTA, 2 mg; artificial seawater, 998 mL) supplemented with 100 mg of Na2 SiO3 ·H2 O and 4 mg of ureophil, and the pH of the medium was adjusted to 7.8 with 1 N NaOH. P. tricornutum was maintained in ZBPT medium (KNO3 , 200 mg; K2 HPO4 , 25.8 mg; NaHCO3 , 459 mg; Na2 SiO3 ·H2 O, 170 mg; artificial seawater, 998 mL) supplemented with 1 mL of a stock solution containing f/2 trace elements, 1.5 × 10−4 mg of vitamin B12 , and 1.5 × 10−3 mg of vitamin B1 . The algal cells were incubated in static culture at 25 ◦ C under light irradiation of 81.04 µmol m−2 s−1 and a 12 h/12 h
light/dark cycle in in 500-mL conical flasks containing 250 mL of medium. Air-agitated cultures supplemented with potassium iodide Air-agitated cultures of N. closterium and P. tricornutum were grown in 1000-mL conical flasks, each containing 800 mL of ZBNC or ZBPT medium under the same conditions as in non-agitated cultures. Agitation was provided by three turbine impellers (4.8 cm in diameter) at speeds varying between 500 and 700 rpm to maintain the dissolved oxygen level above 45% saturation. Compressed air (200 mL min−1 ) was supplied to the flasks through a circular stainless steel sparger. To start the cultures, 10 mL of the two algae at a density of 2 × 104 mL−1 was inoculated separately in optimized ZBNC or ZBPT medium, each containing potassium iodide at serial concentrations of 0, 800, 1000, and 1200 mg L−1 to assess the effects on biomass, EPS accumulation, and fatty acids composition. Bacterial growth inhibition test Nutrient agar plates containing various concentrations of potassium iodide were prepared in quintuplicate. The test bacteria, included Staphylococcus auerus (JKB068), Escherichia coli (JKE029) Bacillus subitilis (JKS007), as well as two Clostridium species that had been isolated from the cultures of N. closterium and P. tricornutum, were inoculated by standardized loops and incubated at 37 ◦ C. After 24-h-incubation, the plates were examined for bacterial growth and MIC100 values were determined as the minimum concentration of potassium iodide that inhibited their growth. Toxicity to flagellate algae and chlorella species Serial concentrations of potassium iodide were supplemented into the modified Johnson’s medium for the culture of Dunaliella salina, f/2 medium for Cryptomonas sp. and Chlorella sp., the ZBNC medium for N. closterium and the ZBPT medium for P. tricornutum in quintuplicate. The inocula, each containing 3 × 104 alga cells, were added to 10 mL of the corresponding media and incubated in an oscillator shaker at a rate of 150 rpm for 10 days under the same light and temperature conditions as for the cultures of the two diatoms. The growth of algae was determined by counting the number of cells using a haemacytometer. The minimum
357 concentration of potassium iodide that inhibited growth was designated as MIC100 . Analysis Cell density was determined by a haemacytometer. Biomass was determined as cell dry weight (DW) as described by Wen and Chen (2000). For analysis of EPS accumulation, the authrone-sulfuric acid method reported by Winder et al. (1999) was used for detecting polysaccharides; the protein content in EPS was determined as described by Tang et al. (1994) and sulfide was determined by the tube-colorimetric method (Liu & Qin, 1997). Lyophilized cells were used to measure fatty acids composition. Fatty acid methyl esters were prepared by transmethylation with the acetyl chloride-methanol method (Jiang & Chen, 1999) and analyzed by GC-MS as described by Zheng and Chen (2003). The data collected in the experiments were processed using SPSS 10.0 software. The assumptions of analysis of variance were considered to be statistically significant at p < 0.05. The results are expressed as mean ± S.E. Results Effect on cell growth and biomass accumulation In general, the two diatoms experienced a 2- or 3-day induction phase before initiating exponential growth
(Zheng & Bao, 2004a,b). The induction phase, however, was reduced to several hours after adequate supplementation of potassium iodide. In cultures of N. closterium and P. tricornutum, the induction phase is normally 48 and 25 h, respectively. The addition of potassium iodide at concentrations of 800, 1000, and 1200 mg L−1 drastically reduced the induction phase to 7.5, 2 and 1.4 h, respectively, for N. closterium and to 3.5, 2 and 1.4 h, respectively, for P. tricornutum (Figure 1). We then tested whether the presence of potassium iodide affected cell density and duration of the exponential growth phase of the two diatoms. The exponential growth of N. closterium in ZBNC medium was sustained for 4 days and reached its maximum cell density of 4.67×106 mL−1 . The addition of potassium iodide at concentrations of 800 and 1000 mg L−1 did not extend the exponential growth phase, but did increase the maximum cell density to 4.95 × 106 and 7.02 × 106 mL−1 , respectively. A further increase in concentration of potassium iodide to 1200 mg L−1 resulted in the reduction of both cell density (3.9 × 106 mL−1 ) and exponential phase (3 days) (Figure 2). In contrast, the exponential growth of P. tricornutum in ZBPT medium lasted for nearly 7 days with a maximum cell density of 4.65 × 106 mL−1 . In the culture containing 800 mg L−1 potassium iodide, exponential growth was reduced to 4.66 days, but the maximum cell density increased moderately to 5.65 × 106 mL−1 . In the culture containing 1000 mg L−1 of potassium iodide, the exponential
Figure 1. Effect of potassium iodide on induction phase of the two diatoms. Note. (a) P < 0.001 versus 0 mg L−1 in N. closterium; (a ) P < 0.001 versus 0 mg L−1 and (b ) P < 0.05 versus 800 mg L−1 in P. tricornutum (SSPS).
358
Figure 2. Effect of potassium iodide on growth of N. closterium in ZBNC medium.
Figure 3. Effect of potassium iodide on growth of P. tricornutum in ZBPT medium.
growth phase was restored back to 7 days and the maximum cell density was increased to 6.75 × 106 mL−1 . A further increase to 1200 mg L−1 reduced the exponential growth phase to 5 days while the cell density was slightly increased to 4.95 × 106 mL−1 (Figure 3). The biomass accumulation of the two diatoms was significantly affected by potassium iodide. In cultures without potassium iodide, the biomass of N. closterium and P. tricornutum was 1159.8 and 1312.9 mg L−1 ,
respectively, by the tenth day after inoculation. Supplementing potassium iodide to a concentration of 800 mg L−1 , increased the biomass of the two diatoms to 1289.7 and 1423.7 mg L−1 , respectively. Further increases to 1694.8 and 1735.7 mg L−1 , respectively, were obtained at 1000 mg L−1 potassium iodide. However, by the tenth day after inoculation a concentration of 1200 mg L−1 resulted in a decreased biomass of 987.26 and 1210.7 mg L−1 for N. closterium and
359
Figure 4. Effect of potassium iodide on biomass accumulation of N. closterium and P. tricornutum. Note. (a) P < 0.05 and (c) P < 0.01 versus 0 mg L−1 ; (d) P < 0.01 versus 800 mg L−1 in N. closterium; (a ) P < 0.05 and (c ) P < 0.01 versus 0 mg L−1 ; (d ) P < 0.05 versus 800 mg L−1 in P. tricornutum (SSPS).
P. tricornutum (Figure 4). These results indicate that potassium iodide at 1000 mg L−1 yielded the highest accumulation of biomass for both diatom species. Effect on EPS accumulation The results showed that the EPS released by the two diatoms contained only polysaccharides. In the culture without potassium iodide, N. closterium produced 1159.8 mg EPS L−1 compared with 450.1 mg L−1 produced by P. tricornutum. Addition of potassium iodide to the culture resulted in increased accumulation of EPS in both diatoms (Figure 5). At 800 mg L−1 , a slight increase to 149.4 mg L−1 of EPS in N. closterium and 115 mg L−1 in P. tricornutum, was observed. At 1000 mg L−1 potassium iodide, EPS increased to 409 mg L−1 in N. closterium and 257 mg L−1 in P. tricornutum, and at 1200 mg L−1 there was an increase of EPS in both diatoms similar to that in the culture containing 800 mg L−1 (Figure 5).
tively. In addition, 1.4–2.1% linolenic acid was also detected from P. tricornutum. The presence of potassium iodide at the concentrations tested resulted in no obvious changes in the profiles of fatty acids, particularly in the contents of EPA and DHA (Table 1). Inhibitory effect against bacteria, flagellate algae and chlorella species Potassium iodide showed different inhibitory effects against the test bacteria, flagellate algae and Chlorella, as well as the two diatoms. Addition of potassium iodide at concentrations higher than 400 mg L−1 completely inhibited the growth of S. auerus, E. coli and B. subitilis, two Clostridium species and Dunaliella salina. At concentrations >700 mg L−1 , the growth of Cryptomonas sp. and Chlorella sp. was inhibited. In contrast, the two diatoms demonstrated a high tolerance to potassium iodide, showing MIC100 of 2600.4 and 2590.7 mg L−1 , respectively (Table 2).
Effect on composition of fatty acids Discussion The composition of fatty acids was determined by the GC-MS method. In cultures without potassium iodide, 38.1 and 34.9% EPA, 3.4 and 33.9% DHA, 13.9 and 18.4% hexadecanoic acid were detected from the total fatty acids of N. closterium and P. tricornutum, respec-
Cultures of diatoms in open environment extensive systems are frequently contaminated by flagellate algae including Dunaliella sp. and Cryptomonas sp. as well as the non-flagellate Chlorella sp. (Wang, 1995).
360 Table 1. Effect of potassium iodide on fatty acid composition of N. closterium and P. tricornutum (%) PI/0 (mg L−1 )
PI/800 (mg L−1 )
PI/1000 (mg L−1 )
PI/1200 (mg L−1 )
Fatty acids
N.
P.
N.
P.
N.
P.
N.
P.
14:0 14:1 i-15:0 15:0 16:0 16:1 (n−7) i-17:0 16:2 (n−6) 16:2 16:2 (n−4) 16:3 (n−4) 16:4 (n−1) 18:0 18:1 (n−9) 18:1 (n−7) 18:2 (n−6) 18:3 (n−6) 20:3 (n−6) 20:4 (n−6) 20:4 (n−3) 20:3 (n−3) 20:5 (n−3) 22:5 (n−3) 22:6 (n−3)
–
1.4 0.3 0.5 13.3 18.4 1.2 0.5 2.3 – 2.2 1.7 0.4 0.5 1.8 4.7 – 1.9 0.8 3.8 0.7 0.7 34.9 1.4 3.9
–
1.2 0.6 0.9 12.8 20.1 0.9 0.3 2.9 – 1.5 1.4 0.2 0.8 1.7 3.9 – 2.1 0.6 3.7 0.2 1.1 35.1 1.2 4.1
–
0.5 0.5 1.1 11.4 19.3 1.5 0.4 2.2 – 2.7 1.3 0.5 0.7 1.9 3.5 – 2 0.7 3.1 0.3 1.5 36.7 0.5 4.5
–
0.1 2.7 0.9 16.3 17.1 3.9 0.7 1.9 – 2.1 0.8 0.4 1.2 2.2 3 – 1.4 0.9 4.2 0.1 0.9 34.8 0.1 4.4
0.2 0.9 10.4 13.9 0 4.7 0.1 8.1 – 4.7 0.5 3.1 0.8 2.1 0.9 – 0.3 – 1.5 – 38.1 – 3.4
0.5 1.4 12.5 16.3 2.3 1.4 3.5 5.6 – 1.7 0.9 0.7 1.9 1.2 0.7 – 0.2 – 1.5 – 38.9 – 3.3
3.8 1.3 12.6 12.7 2.4 1.2 5.2 7.4 – 0.8 1.8 1.3 1.3 2.1 0.4 – 0.5 – 1.1 – 38.48 – 3.4
3.3 1.5 13.1 15.7 1.9 1.5 3.8 8.5 – 0.8 1.7 1.2 1.5 2.7 0.6 – 0.8 – 0.8 – 36.9 – 3.7
Figure 5. Effect of potassium iodide on EPS accumulation of N. closterium and P. tricornutum. Note. (a) P < 0.05, (b) P < 0.01 versus 0 mg L−1 ; (d) P < 0.01 versus 800 mg L−1 in N. closterium; (a ) P < 0.05, (b ) P < 0.01 versus 0 mg L−1 ; (d ) P < 0.05 versus 800 mg L−1 in P. tricornutum (SSPS).
361 Table 2. Inhibitory effects against bacteria, flagellates and chlorella as well as the two diatoms. Species
MIC100 (mg L−1 )
Staphylococcus auerus (JKB068) Escherichia coli (JKB029) Bacillus subitilis (JKBS007) Clatridium sp.1 Clatridium sp.2 Dunaliella salina Cryptomonas sp. Chlorella sp. Nitzschia closterium Phaedactylum tricornutum
358.3 ± 17.1 219.4 ± 9.6 147.6 ± 10.2 231.5 ± 13.1 200.9 ± 14.7 356.8 ± 14.6 475.9 ± 10.3 696.2 ± 9.4 2600.4 ± 16.9 2590.7 ± 13.7
A successful diatom culture depends on its competitive growth against other algae (Davis & Fourqurean, 2001), which can be influenced by the length of the induction phase. A previous report indicated that the length of the induction phase was dependent on the concentration of hydroxyl acetic acid released by the alga itself (Zanjiang Aquaculture College, 1980). In our study, potassium iodide showed much higher toxicity to the test flagellates and Chlorella sp. than to the two diatoms. The addition of potassium iodide to the culture significantly reduced the induction phase of cell proliferation and resulted in early entry to the exponential growth, enabling the two diatoms to be more competitive. At the same time higher concentrations of potassium iodide (above 800 mg L−1 ) inhibited the growth of flagellates and Chlorella sp., leading to a favorable environment for cell proliferation and hence biomass accumulation of the two diatoms. EPS of diatoms is an important source of carbon circulation in marine micro-food chains consisting of algae and bacteria (Hoagland et al., 1993; Kelly, 1989; Poulet & Martin-Jezequel, 1983). Predominately consisting of polysaccharides, EPS of diatoms is readily degraded by bacteria (Naoshige et al., 2001). Therefore, the accumulation of EPS of a diatom largely depends on the amount of bacteria in the culture medium. Our results showed that at relatively low concentrations potassium iodide had bactericidal action against a number of bacteria including species present in the culture of the two diatoms as well as in seawater. The presence of potassium iodide in the culture invariably inhibited the proliferation of bacteria and hence the biodegradation of EPS, leading to an increased accumulation. We noted that the presence of potassium iodide did not affect the profile of fatty acids of the two diatoms
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