Arch. Microbiol. 112, 57-59 (1977) Archives of

Microbiology �9 by Springer-Verlag 1977

The Cell Content and Secretion of Water-Soluble Vitamins by Several Freshwater Algae*

S. A A R O N S O N * * , S. W. D H A W A L E , and N. J. P A T N I

Biology Department, Queens College, City University of New York, Flushing, New York 11367, U.S.A.

B. D E A N G E L I S , O. F R A N K , and H. B A K E R

Departments of Medicine and Preventive Medicine, New Jersey Medical School, East Orange, New Jersey 07018, U.S.A.

Abstract. Three green algae, Chlamydomonas rein-

hardii, Chlorella vulgaris and Scenedesmus obliquus,

and one b lue-green alga, Anabaena cyclindrica, were g rown in chemica l ly defined media . Al l the a lgae examined con ta ined folates, f l -carotene and v i tamins C and E; several o f the B-v i tamins and v i t amin A were found in va ry ing a m o u n t s in some bu t no t in all the a lgae examined . Al l the green a lgae secreted signif icant a m o u n t s o f fo la te and b io t in and all bu t Scenedesmus secreted p a n t o t h e n a t e in to thei r g rowth m e d i u m ; Anabaena secreted fola te and pan to thena te .

Key words." W a t e r soluble v i tamins (secret ion, cell conten t ) - Chlamydomonas - Chlorella - Scene- desmus - Anabaena.

Wate r - so lub l e v i tamins are k n o w n to be requi red by several a lgae (Droop , 1962; Provaso l i , 1958, 1963; P rovaso l i and Car lucci , 1974) as well as m a n y o ther m i c r o o r g a n i s m s (Sebrel l and Harr i s , 1 9 6 7 - 1 9 7 2 ; Burkho lde r , 1963). M u c h less is k n o w n of the v i t amin con ten t o f a lgae (Droop , 1962; P rovaso l i and Car lucci , 1974) u p o n which the food p y r a m i d rests and even less is k n o w n a b o u t v i t amin secre t ion (excret ion) in a lgae (Provaso l i and Car lucci , 1974). The meager in- f o r m a t i o n ava i lab le on v i t amin secre t ion is ma in ly qual i ta t ive . K n o w l e d g e o f the v i t amin con ten t o f a lgae is b e c o m i n g increas ingly i m p o r t a n t as they now are used or a re be ing cons idered for h u m a n or domes t i c an ima l nu t r i t i on (Mate les and Tannen- baum, 1968; Bha t tachar jee , 1970). I n f o r m a t i o n on v i t amin secret ion by a lgae m a y give us more in fo rma- t ion on the a lgal con t r ibu t ions to the fer t i l i ty o f na tu r a l waters . W e repor t here on the con ten t and

* This work was done with the support of grant BMS 74-08918 from the National Science Foundation ** To whom offprint requests should be sent

secre t ion o f water - so lub le and several l ip id-so luble v i t amins by four species o f f reshwater a lgae found in b l o o m s (Hawkins , 1972).

M A T E R I A L S A N D M E T H O D S

Organisms and Growth Conditions. Axenic cultures of the several algae (obtained through the kindness of Dr. L. Provasoli) were grown in the following media: Anabaena cylindrica in the medium of Healey (1973); Chlamydomonas reinhardii in the low salt medium of Sueoka (1960) ; Chlorella vulgaris in the medium of Sorokin and Krauss (1958) and Scenedesmus obliquus in the medium of Kessler et al. (1963). All cultures except Scenedesmus were grown in a refri- gerated incubator at 25~ with 150 ft. candle of white fluorescent light. Scenedesmus was grown similarly at 15~ Experimental cultures were grown in 150 ml volumes in 1 1, screw-cap Eden- meyer flasks.

Stationary-phase cells (11 days) were harvested at 4~ by centrifugation at 12100g for 20min in a Sorval RC-2B re- frigerated centrifuge.

Vitamin Analysis of Algal Cells and Medium. Centrifuged and washed algae and cell free media were suspended in known volume of distilled water and assayed for vitamins with the following modifica- tions of the methods as described and applied to biologic fluids and tissues (Baker and Frank, 1968). Unless otherwise indicated all dilutions were made in distilled water. All buffers were same as used earlier (Baker and Frank, 1968). 1. Folates: Cells and medium were diluted 1:20 with buffer containing 0.05 ~ ascorbate, autoclaved for 10 min and assayed at 10, 20, and 40 ml/100 ml. Lactobacillus casei (ATCC no.7469) was used for the assay of total folates. 2. Vitamin B12: i ml of sample was diluted with 2 ml of buffer containing 0.01 ~ sodium metabisulfate, autoclaved for 10 min, diluted to 5 ml and assayed at i0, 20, and 30 ml/100 ml with Oehromonas malharnensis. 3. Thiamin: 1 ml sample was diluted with 2ml of 0.5~ papain (crude) and incubated for 3days at 37 ~ C with volatile preservative (chlorobenzene : 1,2-dichloroethane : 1-chlorobutane, 1 : 1 : 2); after incubation 2 ml of buffer contain- ing 0.1 ~ Tween 80 was added, the mixture was autoclaved for 10 min to remove volatile preservative and assayed at 20, 30, and 40 ml/100 ml with Ochromonas danica. 4. Biotin: 1 ml sample was diluted with 4 ml of 0.2 ~ clarase (Diastase-Fisher Scientific Co.), incubated for 3 days at 37~ with volatile preservative. After incubation the suspension was autoclaved for 30 min, assayed at 20, 30, and 40 ml/100 ml with Ochromonas danica. 5. Pantothenate: Same procedure as for biotin was used, and the mixture was assayed with Tetrahymenapyriformis. 6. Vitamin B6:1 ml of sample

58 Arch. Microbiol., Vol. 112 (1977)

was diluted with 1 ml of 0.05 M citrate-phosphate buffer, pH 4.5, autoclaved for 15min cooled and 2.0ml of 0.4~ clarase was added and mixture incubated for 3 days at 37~ with volatile preservative. After incubation, the suspension was autoclaved for 15 rain, 6 ml of distilled water added, and the sample assayed at 10, 20, and 30 ml/100 ml with Tetrahymena pyriformis. 7. Riboflavin: Using the same procedure as for vitamin B6, riboflavin was

Table 1. Vitamin assay and sensitivity

Vitamin Assay method Sensitivity/ml

Folates Lactobacillus casei ATCC 7469 < 10 pg B12 Ochromonasmalhamensis < 0.3pg Thiamin Ochromonas danica < 0.1 ng Biotin Ochromonas danica < 3 pg Pantothenate Tetrahymena pyriformis < 3 ng B6 Tetrahymena pyriformis < 0.i ng Riboflavin Tetrahymena pyriformis < 1 ng Nicotinate Tetrahymena pyriformis < 3 ng A Colorimetric 250 ng B-carotene Colorimetric 400 ng E Colorimetric 1.0 ~tg C Colorimetric 1.0 Ixg

assayed at 20, 30, and 40 ml/100 ml with Tetrahymena pyriformis. 8. Nicotinate: 1 ml of sample was diluted with 4 ml of 1 ~ papain and incubated for 3 days at 37~ with volatile preservative, then autoclaved for 3'0 rain, and assayed at 5, 10, and 20 ml/100 ml with Tetrahymena pyriformis. Suitable enzyme controls were included, correction factors were applied for enzymic contribution of vitamins if more than a trace concentration was detected.

Table 1 lists the assay organism or procedure employed and its sensitivity.

RESULTS AND DISCUSSION

Autolysis was ruled out as the source of the B-vitamins in the medium because 1. only a few of the B-vitamins found in the cells were found in the medium, and 2. the cell-free medium of all the algae examined here con- tained protein, most contained carbohydrate and none contained lipids or nucleic acids. If autolysis were a significant source of molecules, the medium would have contained most if not all of the B-vitamins and lipids and macromolecules assayed.

In Table 2 it can be seen that algae analyzed here vary appreciably in their content of water-soluble

Table 2. Vitamin content of several freshwater algae

Vitamin (Conc./mg dry weight)

Cyanophyta Chlorophyta

A n a b a e n a Ch lamydomonas Chlorella Scenedesmus

Total folates" ng 15 9 11 6 B12 pg X nd nd nd Thiamin ng X nd nd nd Biotin pg X 260 448 nd Pantothenate ng 88 nd 79 nd B6 ng 7 nd 11 nd Riboflavin ng 55 nd 68 46 Nicotinate ng 78 nd 56 nd A ng nd 105 nd nd B-carotene ng 1468 1680 781 222 E ~tg 4 4 2 1 C Ixg 2 2 15 2 Cell yield/ml x 10 ~ filamentous

growth 1.0 1.5 0.6

a Total folates include reduced folates, N 5 methyltetrahydrofolate and oxidized monoglutamates X = added to nutrient medium; nd = not detected at assayable levels

Table 3. Water-soluble vitamins in the cell-free culture medium of several freshwater algae a

Vitamin (Conc./ml of A n a b a e n a Ch lamydomonas Chlorella Scenedesmus cell-free medium)

Total folate ng 3 9 4 2 Biotin pg X 996 214 173 Pantothenate ng 70 63 51 nd

a B12, B6, Nicotinate, Riboflavin, Thiamin, Vitamin A, Vitamin C, Vitamin E and B-carotene were not secreted into the culture media by these algae in detectible amounts X = added to nutrient medium; nd = not detected

S. Aaronson et al. : Content and Secretion of B-Vitamins by Algae 59

and lipid-soluble vitamins. It becomes important to know if algae contain adequate amounts of vitamins if they are to serve as a major source of animal nutrition and ultimately provide food material for man.

Vitamins found in natural waters are usually at- tributed to secretion by bacteria (Provasoli and Carlucci, 1974; Overbeck, 1974). Algae may, how- ever, secrete significant amounts of vitamins into their environment (Table 3). That these do not result from autolysis may be deduced from the limited number of vitamins found in the medium (Table 3) compared to those found in the cell (Table2). Provasoli and Carlucci (1974) have reviewed earlier work, most of which was qualitative. Among the vitamins secreted by algae were nicotinate (Nakamura and Gowans, 1964), biotin (Bednar and Holm- Hansen, 1964), B12 (Carlucci and Bowes, 1970), riboflavin (Schwarze, 1975) and ascorbate, B6 folates, nicotinate and pantothenate (Aaronson et al., 1971). Here we extend and supplement our earlier report with more evidence for algal secretion of vitamins. Unlike Schwarze (1975), we did not find riboflavin secreted by our strain of Chlorella vulgaris.

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Received July 2, 1976