Characterization of nutrient status of Halamphora luciae (Bacillariophyceae) using matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Figures & data

Table 1. Monosaccharide composition (mol%) of the different extracts from Halamphora luciae cells cultured in f/2 medium. Data are expressed as media ± SD.

Monosaccharide^a	RTW	W100
Rha	tr	3.7 ± 0.5
Fuc	8.5 ± 0.5	25.4 ± 2.1
Ara	2.9 ± 1.2	nd
Xyl	4.1 ± 0.9	3.2 ± 0.5
Man	5.9 ± 2.2	8.7 ± 2.4
Gal	13.9 ± 1.5	20.5 ± 0.6
Glc	64.7 ± 3.1	38.6 ± 0.3

^aRha=Rhamnose; Fuc=Fucose; Ara=arabinose; Xyl=Xylose; Man=Mannose; Gal=Galactose; Glc=Glucose; tr=trace (< 1 mol %); nd=not determined.

Table 2. Average molecular weights obtained for MALDI mass spectrometry of Halamphora luciae extracts in f/2 medium.

			DPmax^a	DPn^b	DPrange
RTW	1769	1893	9	13	7–19
W100	2255	2120	11	14	7–20

^a Degree of polymerization (DP) corresponding to the most abundant ion. ^b Number-average degree of polymerization calculated considering all the observed ions.

Fig. 1. MALDI mass spectra of the (a) room temperature (RTW) and (b) hot water (W 100) extraction from Halamphora luciae cells grown in f/2 medium. Matrix: DHBA-SA mixture.

Table 3. Composition (mol %) of monosaccharide produced by methylation and hydrolysis of the RTW extract from Halamphora luciae. Data are expressed as media ± SD.

Monosaccharide^a	Deduced glycosidic linkage	RTW-m
2,3,4,6-Me4Glc	Terminal	20.8 ± 1.4
2,4,6-Me3Glc	3-linked	47.4 ± 3.9
4,6-Me2Glc	3-linked 2-substituted	10.6 ± 1.7
2,4-Me2Glc	3-linked 6-substituted	12.7 ± 2.3
Glc		8.5 ± 1.9

^a2,4-Me₂Glc=1,3,5,6-tetra-O-acetyl-2,4-di-O-methylglucitol; 4,6-Me₂Glc=1,2,3,5-tetra-O-acetyl-4,6-di-O-methylglucitol; 2,4,6-Me₃Glc=1,3,5-tri-O-acetyl-2,4,6-tri-O-methylglucitol; 2,3,4,6-Me₄Glc=1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol.

Table 4. Constituent monosaccharide (mol%) of extracts obtained from the hydrolysates of entire cells of Halamphora luciae cultured in f/2, f/2-N and f/2-P media. Data are expressed as media ± SD.

Monosaccharide^a	f/210d^b	f/220d^c	f/230d^d	f/2-N^e	f/2-P^e
Rha	2.1 ± 0.4	3.4 ± 0.4	4.1 ± 0.4	tr	2.1 ± 1.4
Fuc	15.2 ± 0.3	11.5 ± 1.5	12.5 ± 1.6	2.2 ± 0.1	2.5 ± 0.9
Ara	tr	nd	nd	tr	nd
Xyl	tr	1.9 ± 1.2	nd	tr	17.5 ± 2.3
Man	3.2 ± 0.9	2.4 ± 0.8	4.8 ± 1.5	tr	8.9 ± 4.1
Gal	34.6 ± 1.1	21.5 ± 0.4	16.1 ± 0.1	2.9 ± 0.9	6.2 ± 0.5
Glc	43.9 ± 0.8	58.9 ± 0.9	62.5 ± 0.5	94.9 ± 0.3	62.8 ± 1.3

^a Rha=Rhamnose; Fuc=Fucose; Ara=arabinose; Xyl=Xylose; Man=Mannose; Gal=Galactose; Glc=Glucose; tr=trace (< 1 mol %), nd=not determined. ^b Cells were grown in f/2 medium and harvested at day 10. ^c Cells were grown in f/2 medium and harvested at day 20. ^d Cells were grown in f/2 medium and harvested at day 30. ^e Cells harvested at day 10.

Table 5. Average molecular weights obtained for MALDI mass spectrometry of Halamphora luciae cells in different culture conditions and culture ages.

	n	w	DPmax^c	DPn^d	DPrange
Growth medium^a
f/2 medium	5192	5243	32	32	26–38
N depleted medium	4921	5062	34	30	15–43
P depleted medium	5983	6073	38	38	27–47
Culture age^b
5-day culture	3936	4223	19	27	14–38
10-day culture	4666	4931	32	29	15–40
15-day culture	4703	5104	32	29	13–42
25-day culture	5187	5267	32	32	23–41
35-day culture	4973	5221	32	30	14–44

^a The cells of H. luciae harvested at day 15. ^b Cells were grown in f/2 medium. ^c Degree of polymerization (DP) corresponding to the most abundant ion. ^d Number-average degree of polymerization calculated considering all the observed ions.

Fig. 2. MALDI mass spectra of Halamphora luciae cells under different culture media harvested at day 15. (a) f/2 (control), (b) f/2-N and (c) f/2-P. Matrix: CHCA.

Fig. 3. (a) Distribution of the polymerization of the polymers from Halamphora luciae cells grown in f/2 medium (black circles), f/2-N (white circles) and f/2-P (white triangles), (b) Cells grown in f/2 medium and harvested at days 5 (black circles), 10 (white circles), 15 (black triangles), 25 (white triangles) and 35 (black squares). The relative abundance was based on the polymer with the most abundant ion, which was represented as one unit in the curve.

Fig. 4. MALDI mass spectra of Halamphora luciae cells growth in f/2 medium harvested at days (a) 5, (b) 10, (c) 15, (d) 25 and (e) 35. Matrix: SA.

Figs 5–7. Halamphora luciae cells cultured in f/2 medium and stained with aniline blue. Fig. 5. Arrows in the light micrographs indicate the chloroplast. Figs 6-7. Fluorescence micrographs with (Fig. 6) blue filter (Ex 450–490 nm, 510 nm FT, and Em 520 nm) showing chlorophyll autofluorescence (black arrow) and (Fig. 7) with UV filter (Ex 330–380 nm, FT 400 nm, Em 420 nm) without chlorophyll autofluorescence. In Figs 6-7 the brighter fluorescence at the girdle bands indicates the presence of a β-1,3 glucan (white arrows). Scale = 10 μm.