Bioscience, Biotechnology, and Biochemistry

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Effect of Vitamin B12-Enriched Thraustochytrids

on the Population Growth of Rotifers

Masahiro HAYASHI, Tsugiyo YUKINO, Fumio WATANABE, Emi MIYAMOTO &

Yoshihisa NAKANO

To cite this article: Masahiro HAYASHI, Tsugiyo YUKINO, Fumio WATANABE, Emi MIYAMOTO
& Yoshihisa NAKANO (2007) Effect of Vitamin B12-Enriched Thraustochytrids on the Population
Growth of Rotifers, Bioscience, Biotechnology, and Biochemistry, 71:1, 222-225, DOI: 10.1271/
bbb.60308

To link to this article: https://doi.org/10.1271/bbb.60308

Published online: 22 May 2014.

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Biosci. Biotechnol. Biochem., 71 (1), 222–225, 2007

Note
Effect of Vitamin B12-Enriched Thraustochytrids
on the Population Growth of Rotifers
Masahiro H AYASHI,1; y Tsugiyo Y UKINO,2 Fumio WATANABE,3
Emi M IYAMOTO,4 and Yoshihisa N AKANO5
1
Department of Biological Production and Environmental Science, Faculty of Agriculture,
University of Miyazaki, 1-1 Gakuenkibanadainishi, Miyazaki 889-2192, Japan
2
Department of Nutritional Sciences, Faculty of Health and Welfare Science, Nayoro City University,
Kita 8-chome, Nishi 4-jo, Nayoro 096-8641, Japan
3
Department of Agricultural, Biological, and Environmental Sciences, Faculty of Agriculture,
Tottori University, 4-101 Koyamacho-minami, Tottori 680-8550, Japan
4
Department of Health and Nutrition, Nagasaki International University,
2825-7 Huis Ten Bosch-cho, Sasebo 859-3298, Japan
5
Division of Applied Biological Chemistry, Graduate School of Agriculture,
Osaka Prefecture University, 1-1 Gakuencho, Sakai 599-8531, Japan

Received June 5, 2006; Accepted October 17, 2006; Online Publication, January 7, 2007
[doi:10.1271/bbb.60308]

Newly isolated thraustochytrids showed uptake of
vitamin B12 from the culture into the cells. Cultivation
of thraustochytrids in a medium containing 1 g/ml of
vitamin B12 greatly increased the contents of vitamin
B12 in the cells. Similarly to Schizochytrium limacinum,
odd numbered fatty acids decreased in the cells of new
isolates cultivated with vitamin B12. Vitamin B12-
enriched thraustochytrids, strain mh0186, enhanced
the population growth of rotifers fed on the cells as sole
feed.
Key words: thraustochytrids; docosahexaenoic
(DHA); rotifer; vitamin B12
Industrial utilization of thraustochytrids as potential
sources of docosahexaenoic acid (DHA) and carotenoids
has been investigated.1–6) It is known that many strains
of thraustochytrids accumulate a large amount of DHA
in the cells. Schizochytrium limacinum SR-213) and
Thraustochytrium KH1055) show marked accumulation
of DHA and carotenoids respectively. Moreover, the
practical use of thraustochytrids as a feed for zooplank-
tons, such as rotifers and Artemia, has also been
reported.7,8) It has been shown that feeding certain
thraustochytrids to rotifers and Artemia improved the
nutritional value of the zooplanktons for marine fish
larvae, but no information about the population growth
of rotifers fed on thraustochytrids is available.
It is widely recognized that Chlorella is a profitable
feed for rotifer cultivation in hatcheries.9) Since vitamin
B12 (B12) is an essential nutrient for rotifers,10)
commercial condensed Chlorella is enriched with B12.
Enrichment of Chlorella cells with B12 greatly im-
proved the nutritional value of Chlorella cells for the
population growth of rotifers fed on it.11,12) In the
present study, the effects of B12-enriched thraustochy-
trids on rotifer cultivation were evaluated.
Sources for the isolation of thraustochytrids, such as
coastal water, sediments in the sea, dead leaves of
mangrove forests, and so on, were identified in the area
acid of the Yaeyama Islands in Okinawa. From these sources,
zoospores of thraustochytrids were collected by the pine
pollen method.13) The collected zoospores were spread
on potato-dextrose agar (PDA) plates prepared in half-
strength artificial sea water (Marine Art High, Senju
Seiyaku Co., Ltd., Osaka). After incubation of the PDA
plates at 28
C for 5–7 d, colonies of thraustochytrids
were identified, transferred, and purified on other PDA
plates.
Isolates of thraustochytrids were cultivated in 500 ml
shaking flasks at 28
C for 4 d. The culture medium
consisted of 3% (w/v) glucose and 1% (w/v) yeast
extract in half-strength artificial sea water (pH 6.0). For
enrichment of the cells with B12, the vitamin (1 mg/ml)
was added into the basal medium. After cultivation for
4 d, cells were harvested by centrifugation (1;500  g,
10 min) and washed 3 times with distilled water.
Total lipids of thraustochytrids and rotifers were

y
To whom correspondence should be addressed. Tel/Fax: +81-985-58-7225; E-mail: hayash-m@cc.miyazaki-u.ac.jp
Abbreviations: DHA, docosahexaenoic acid; PDA, potato dextrose agar; B12, vitamin B12
 Effect of B12-Enriched Thraustochytrids on Rotifers 223
14) Table 1. Contents of B12 in Thraustochytrids Cultivated with or
extracted by the Folch method after freeze drying of
the cells or rotifers. Fatty acid compositions of the lipids without B12
were determined by gas liquid chromatography after Contents of B12 (mg/100 g)
methanolysis with 10% (w/v) HCl–methanol solution. Strains
B12 +B12
The contents of B12 were determined according to the
microbiological method with Lactobacillus delbrueckii mh0186 1.1 420
mh0190 1.8 1160
subsp. Lactis ATCC7830 and a B12 assay medium mh0192 2.7 1440
(Nissui, Tokyo).15) mh0194 3.9 890
Rotifers, Brachionus rotundiformis, were cultured in mh0253 4.1 1070
1 liter flasks at 28
C. The initial density of rotifers was mh0254 1.3 1500
1,000 individuals/ml. The densities of rotifers in flasks mh0255 2.5 650
mh0247 1.5 1440
were determined daily by microscopic observation. For
rotifer cultivation, freeze-dried cells of isolate mh0186 B12, cells cultivated without B12;
+B12, cells cultivated with B12.
with or without B12 enrichment were given to rotifers.
The daily feeding rate of the cells was 200 mg/l for each
flask.
In 2000–2002, 380 strains were isolated from the area efficiency of its uptake into cells.
of the Yaeyama Islands. These strains had widely On the other hand, cultivation of thraustochytrids
different growth rates, lipid contents, and DHA contents with B12 caused great changes in their fatty acid com-
of the cells. Among these isolates, eight strains of positions (Table 2). Thraustochytrids usually contain a
thraustochytrids were selected as appropriate strains for marked amount of odd numbered saturated fatty acids,
feeding rotifers. They showed high growth (higher such as C15:0 and C17:0. The addition of B12 into the
OD660 than 5.0) after cultivation for 4 d in basal medium medium decreased the rate of odd numbered fatty acids
at 28
C, and had high (over 20% in dry cells) lipid in the total fatty acids of the cells. Recently, it was
contents and high (over 45% of total fatty acids) DHA suggested that B12 activated methylmalonyl-CoA mu-
contents. tase (EC5.4.99.2) in thraustochytrids cells. According
For estimation of the uptake of B12 by these strains, to Shirasaka et al., propionyl-CoA, a primer for odd
they were cultivated with or without supplementation of numbered fatty acid synthesis, was converted to succin-
the medium with B12. The cells cultivated without yl-CoA, and it was consumed in the TCA cycle in
addition of B12 contained a small amount of B12. thraustochytrids cultivated with B12.16) Our results in
However, the cultivation of each strain with 1 mg/ml of this study also support their suggestion.
cyanocobalamin resulted in a great increase in cellular It has been shown that the feeding of thraustochytrids
B12 contents. In particular, cells of mh0190, mh0192, to rotifers for several hours improved the DHA contents
mh0247, mh0253, mh0254 cultivated with B12 con- and the nutritional value of the rotifers for marine fish
tained up to 1,000 mg/100 g dry cells (Table 1). These larvae,7,8) whereas information about the cultivation of
results are the first findings of the incorporation and rotifers for several days by feeding of thraustochytrids as
accumulation of B12 in thraustochytrid cells, while sole feed were not available. In the present study, strain
Maruyama et al.11) reported that Chlorella strains also mh0186, which had the highest DHA content was given
incorporate B12 in their cells, resulting in Chlorella to rotifers as sole feed for population growth. Mh0186
vulgaris K-22 cells containing 1,100 mg/100 g of dry cells cultivated without B12 gave a poor growth of the
cells. However, further studies are required for the rotifer population after 4 d of cultivation. However,
evaluation of the optimum concentration of B12 and the mh0186 cells cultivated with B12 greatly improved in

Table 2. Fatty Acid Compositions of Thraustochytrids Cultivated with or without B12

mh0186 mh0190 mh0192 mh0194 mh0247 mh0253 mh0254 mh0255

B12 +B12 B12 +B12 B12 +B12 B12 +B12 B12 +B12 B12 +B12 B12 +B12 B12 +B12
C14:0 0.1 0.6 0.6 1.7 0.7 1.2 1.3 1.1 0.8 2.9 0.1 0.5 0.5 0.8 0.5 1.2
C15:0 11.7 2.1 19.9 3.8 7.8 1.2 9.3 4.1 32.1 4.3 14.6 2.9 9.4 1.5 13.2 2.1
C16:0 9.4 23.5 9.6 28.1 17.4 24.3 20.6 21.0 5.7 31.5 12.0 22.8 14.1 21.9 17.2 32.4
C17:0 5.3 1.0 5.6 1.3 2.9 0.5 3.1 1.5 5.2 1.2 6.6 1.9 3.3 0.6 8.7 1.6
C22:5 n-6 10.3 9.7 10.1 11.3 11.2 12.1 12.0 14.0 8.8 11.4 9.5 9.7 11.7 12.0 9.5 10.5
C22:6 n-3 58.0 57.1 47.7 47.5 56.1 56.4 47.5 51.7 36.7 40.5 51.9 54.7 55.5 56.0 45.7 47.0
Others 5.2 6.0 6.5 6.3 3.9 4.3 6.2 6.6 10.7 8.2 5.3 7.5 5.5 7.2 5.2 5.2 (%)
Total fatty acids 125.0 119.1 141.3 149.2 120.2 131.2 100.1 107.0 122.2 130.3 125.3 140.6 109.8 121.7 172.4 169.2
(mg/g of dry cells)
B12, cells cultivated without B12;
+B12, cells cultivated with B12.
224
7000
Rotifer density (indiv./ml)
6000
5000
4000
3000
2000
1000
0
0 1 2 3
Cultuivation period (d)
Fig. 1. Population Growth of Rotifers Fed on mh0186 Cells
Cultivated with or without B12.
Values are the mean  SD (n ¼ 5). , rotifers fed on mh0186
cells cultivated without B12; , rotifers fed on mh0186 cells
cultivated with B12.
population growth of rotifers over 4 d (Fig. 1). More-
over, the rotifers cultivated with B12-enriched thraus-
tochytrids contained 3.1% (w/w) of DHA. As for the
Chlorella, feeding cells enriched with B12 enhanced
the population growth of rotifers.12) Maruyama et al.
indicated that B12 was essential for the population
growth of rotifers.17) In this study, it was found that
enrichment of thraustochytrids with B12 improved the
population growth of rotifers. The population growth of
rotifers fed on mh0186 cells cultivated with B12 equaled
that of B12-enriched Chlorella.
Rotifers fed on B12-enriched thraustochytrids con-
tained a large enough amount of DHA as a feed for
appropriate growth of marine fish larvae. In general,
rotifers are nutritionally enriched with DHA by feeding
of a DHA-containing commercial diet for several hours
prior to feeding to fish larvae, while rotifers fed on B12-
enriched thraustochytrids richly contained DHA in this
study. These results indicate that the feeding of B12-
enriched thraustochytrids represents a novel procedure
for the production of marine fish larvae without nutri-
tional enrichment of rotifers.
In the present study, it was found that B12 is taken up
into thraustochytrids cells, and that the addition of B12
to the medium decreases the cellular contents of odd
numbered fatty acids. The feeding of B12-enriched
thraustochytrids to rotifers greatly improved the pop-
ulation growth of rotifers. These results indicate that
B12-enriched thraustochytrids should be a profitable
feed for rotifer cultivation. This feed yields efficient
production of larval marine fish, and may contribute to a
decrease in feed and labor costs in fisheries.
Acknowledgment
We are grateful to Mr. K. Ohta, Ms. M. Imaizumi, and
Ms. K. Hamamoto of Marine Service KATSU for their
M. HAYASHI et al.
help in collecting samples. We also thank Mr. K.
Tamashiro of Katsusuisan, Co., Ltd., for providing
important information during our stay in the Yaeyama
islands.
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