Culture and Use of Algae in Southeast Asia Compressed

CULTURE AND USE OF ALGAE
IN SOUTHEAST ASIA
Proceedings of the Symposium
on Culture and Utilization of Algae
in Southeast Asia
Editors
I.J. Dogma, Jr.
G.C. Trono, Jr.
R.A. Tabbada
CULTURE AND USE OF ALGAE
IN SOUTHEAST ASIA
Proceedings of the Symposium on

Culture and Utilization of Algae
in Southeast Asia
8-11 December 1981
Tigbauan, Iloilo
Philippines
AQUACULTURE DEPARTMENT
SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER
Tigbauan, Iloilo, Philippines
1990
CULTURE AND USE OF ALGAE IN SOUTHEAST ASIA
Culture and Utilization of Algae in
Southeast Asia, 8-11 December 1981
ISBN 971-8511-11-3
Published and Printed by

Aquaculture Department
Southeast Asian Fisheries
Development Center (SEAFDEC)
Copyright 1990
All Rights Reserved
No part of this publication may be
reproduced or transmitted in any form
or by any means, electronic or mechanical,
including photocopy, recording, or any
information storage and retrieval system,
without permission in writing from the
publisher.
CULTURE A N D USE OF ALGAE IN SOUTHEAST ASIA
in Southeast Asia
CONTENTS
Foreword v
Seaweed resources in the developing countries of Asia:

Production and socioeconomic implications 1
G.C. TRONO, JR.
Utilization and farming of seaweeds in Indonesia 9

A. SOEGIARTO & SULUSTIJO
Present status of seaweed culture in Korea 21

J.W. KANG
Utilization of seaweeds in Thailand 27

K. LEWMANOMONT
Life history of Acrothrix pacifica and Sphaerotrichia divaricata

in laboratory cultures 31
I. UMEZAKI
Antithamnion sparsum, its life history and hybridization

with A. defectum in culture 39
I.K. LEE & S.M. BOO
Water quality assessment of the Langat River, Selangor, Malaysia

using the natural algal periphyton community and laboratory bioassays
of two Chlorella species 51
A. ANTON
Growth and development of Trentepohlia odorata in culture 55

C.T. LEE, Y.C. WEE & K.K. HO
Agroindustrial waste products as sources of cheap substrates for

algal single-cell protein production 67
M.T. ZAFARALLA, L.R. VIDAL & L.E. TRAVINA
Utilization of seaweed resources 77

G.J.B. CAJIPE
Culture and utilization of freshwater algae as protein source 81
B.R. RODULFO
Philippine algal taxonomy: Past, present, and future 89

P.A. CORDERO, JR.
Algal production and utilization relevant to aquaculture

in the Philippines 99
J.B. PANTASTICO, J.P. BALDIA,
C.C. ESPEGADERA & D.M. REYES, JR.
FOREWORD
The idea of holding this Symposium was brought about by the growing
interest in the role of algae in the economic life of the peoples of the
developing countries in Southeast Asia. The use of algae as food, as raw
materials in the production of industrial phycocolloids, and as natural feed
for economically important aquaculture species has been highlighted re-
cently in developed countries in Asia and other parts of the world. The need
for information on recent developments in this area of concern was the
primary consideration in the holding of this Symposium.
Efforts to organize the Symposium started in early 1981 under the

auspices of UNESCO through its Regional Headquarters of Network for
Microbiology in Southeast Asia in cooperation with the National Institute of
Science and Technology Authority. The Aquaculture Department of the
Southeast Asian Fisheries Development Center (SEAFDEC) in Tigbauan,
Iloilo, hosted the Symposium and provided the secretariat services and
funds to support a number of local participants.
Attendance in the Symposium included fifteen full participants from

different institutions from seven countries, students and faculty of the
University of the Philippines in the Visayas, and other observers.
Thirteen papers were presented covering a wide variety of topics from

culture of micro and macroalgae to processing of algal products and their
utilization in industry as natural feed for aquaculture animals, status of
seaweed resources and their production, and the biology and use of algal
population as indicator of the environmental state of the aquatic environ-
ment.
This late the Aquaculture Department of SEAFDEC decided to publish

the proceedings of the Symposium because the information therein continue
to be relevant to aquaculture.
F.J. Lacanilao
Chief
Aquaculture Department
SEAFDEC
February 1990
Participants from Indonesia, Japan, Korea, Malaysia, Philippines, Singapore and Thailand in the Symposium on Culture and
Use of Algae in Southeast Asia, 8-11 December 1981, Tigbauan Research Station, SEAFDEC Aquaculture Department,
Tigbauan, Iloilo, Philippines.
SEAWEED RESOURCES IN THE DEVELOPING
COUNTRIES OF ASIA: PRODUCTION AND SOCIO-
ECONOMIC IMPLICATIONS
Gavino C. Trono, Jr.

Marine Sciences Institute, College of Science
University of the Philippines
Quezon City, Philippines
ABSTRACT
The bulk of world seaweed production today comes from devel-

oped countries in the temperate region, including Japan, China,
and Korea. The seaweed production potentials in the developing
countries of Asia will have to be explored to meet the increasing
world demand. Extensive shallow and farmable reef areas as well
as cheap labor highly favor seaweed production. Harvesting from
natural stocks, usually the practice in most developing countries,
is unreliable. Efforts should thus be directed toward actual farming
of seaweeds.
In the Philippines, development of the farming technology on

Eucheuma alvarezii and E. denticulatum significantly increased
production by the middle of the 70's. Harvests of Eucheuma from
farms and other seaweeds from natural stocks now rank third
among the fishery exports of the country.
The socioeconomic implications of the development of the sea-

weed resources in the developing countries of Asia are discussed.
The Philippine experience is cited specifically to show the benefits
derived from seaweed farming technology.
INTRODUCTION
Although no accurate data are available, world production of seaweeds

at present seems quite sizeable. For instance, Naylor (1976) estimates a
world production of 1 170 000 and 2 400 000 metric tons (mt) of wet weight
for 1963 and 1973, respectively. The 1973 production was valued at approxi-
mately US$765 million at primary source. In 1974, production was esti-
mated at 2 664 000 mt. An examination of the data, however, shows that the
bulk of production was contributed by developed countries in the temperate
2 Culture and Use of Algae
regions including Japan, China, and Korea which produced 697 800 mt
(1974), 700 000 mt (1973), and 335 700 mt (1975), respectively. These
countries are the major producers of seaweeds in Asia.
In contrast, developing countries contributed very little to the total sea-

weed production in the world. For instance, among the Asian countries, the
Philippines produced only 7 000, Indonesia 15 000 and India 6 000 mt, or an
aggregate of only 28 000 mt in 1974. Productions in the other countries of
Asia were negligible. Hence the 1974 production of these countries repre-
sents a very small portion of the total world production. Naylor's (1976)
estimate for the Philippines, however, is incorrect. The 7 000 mt produced in
1974 was on dry weight, not wet weight, basis.
Present estimates (Naylor 1976) show that seaweed production in devel-

oped countries cannot cope with the highly increasing demands for food,
pharmaceutical, and industrial purposes. It is estimated that the annual
increase in the demand for seaweeds and seaweed products is 8-10% (Naylor
1976). Hence, countries such as the United States, Canada, Denmark,
Japan, France, Australia, and Germany are importing large amounts of
dried seaweeds from developing countries in Southeast Asia.
STATUS OF SEAWEED PRODUCTION IN ASIA
Except for Japan, China, and Korea which have a well-established

seaweed industry, most Asian countries are still dependent on the wild or
natural seaweed crops, i.e., fishermen directly gather seaweeds from natural
stocks. Actual farming of seaweeds in the Philippines is limited only to three
commercial species, e.g., Eucheuma alvarezii (commercially known as the
"cottonii type"), E. denticulatum (the "spinosum type") and Caulerpa lentil-
lifera. The first two species are produced through mariculture while C.
lentillifera is cultivated in ponds.
The harvesting of natural seaweed stocks is very unreliable. Production

is highly dependent on the growth of the species which in turn is highly
influenced by monsoons and other environmental stresses. In addition, the
absence of a management program for the naturally produced species often
results in the depletion and/or destruction of natural stocks due to over-
harvesting. In contrast, production through farming is not only reliable but
is also a very efficient way of conserving local stocks. The principal seaweed
genera, their uses, and present status of production in Asia are listed in
Table 1.
The potentials of seaweed production in the developing countries of Asia

are very high. Many Asian countries possess well developed coral reefs which
could support high seaweed production. These shallow and farmable reef
Seaweed Resources in Asia 3
areas, however, are presently utilized mainly for small-scale fishing. In the
Philippines, the increased harvests of Eucheuma from farms reflect the
development of such potentials and have made the country the major
supplier of this species in the international market. Due to low production
costs, seaweeds and seaweed products from developing countries in Asia can
be highly competitive in the international market.
Table 1. Principal seaweed genera of economic potential in Asia
Country Genera Uses Status of

production
Philippines Caulerpa food pond culture &

wild crops
Codium food wild crops
Sargassum alginate, feeds wild crops
Porphyra food wild crops
Gelidiella agar wild crops
Gracilaria agar, food wild crops
Eucheuma carrageenan, food mariculture
Indonesia Gracilaria agar, food wild crops

Eucheuma carrageenan wild crops,
mariculture
Gelidiella agar, food wild crops
Hypnea carrageenan, food wild crops
Caulerpa ' food wild crops
Acanthophora food wild crops
Singapore Eucheuma carrageenan wild crops

Gracilaria agar wild crops
Sargassum alginate wild crops
Brunei Gracilaria agar wild crops
East Malaysia Porphyra food wild crops

Eucheuma carrageenan wild crops
Caulerpa food wild crops
Gracilaria agar wild crops
West Malaysia Gracilaria agar wild crops
Thailand Gracilaria agar wild crops

Vietnam Gracilaria agar wild crops

Hong Kong Sargassum alginate wild crops

Table 1 continued..
Taiwan Gracilaria agar pond culture
Porphyra food wild crops;
culture
Sri-Lanka Gracilaria agar wild crops

India Gracilaria agar wild crops;

mariculture
Gelidiella agar wild crops
Hypnea carrageenan wild crops
Burma Gracilaria agar wild crops

Gelidium agar wild crops
Pakistan Gracilaria agar wild crops

Gelidium agar wild crops
Hypnea carrageenan wild crops
SEAWEED PRODUCTION IN THE PHILIPPINES
The Philippine seaweed export profile is reflected in Table 2. No official

records of local seaweed production are available before 1967 when the
Philippines started exporting dried seaweeds to other countries. Production
from 1967 to about 1972 was mostly harvests from natural stocks by
fishermen. During this period, the unregulated harvests in response to the
high demand of the dried produce in the world market led to the depletion
of the natural stocks. Toward the end of the 1960's and during the early
1970's, production was maintained mainly by the discovery of new seaweed
beds in very far and hardly accessible reef areas.
The development of farming technology in the early 70's made its full
impact on production toward the middle 70's when production came mainly
from the farming of two species of Eucheuma, namely, E.alvarezii and E.
denticulatum. The farming of the second species contributed significantly to
production during the latter half of the decade. A small portion of the total
seaweed production was derived from natural stocks of other seaweeds such
as Gracilaria, Gelidiella, Caulerpa, and Sargassum. Except for Caulerpa,
their production is dependent up to now on natural stocks.
Pond culture of Caulerpa is presently done in Mactan, Cebu. Although

the produce is locally sold in open markets of Metro Manila, Cebu City,
Cagayan de Oro City, and Zamboanga City, recently a significant portion is
being exported to Okinawa in a partially dehydrated (salted) state.
Table 2. Philippine seaweed export, 1967-1980
Year Metric Ton Value (Pesos)
1967 674.5 351 989

1968 263.9 221 056
1969 427.6 447 908
1970 318.1 527 321
1971 339.8 675 504
1972 483.9 1 414 051
1973 1 432.7 4 062 086
1974 5 039.6 14 973 151
1975 4 514.8 13 292 226
1976 3 950.1 12 366 568
1977 6 094.1 14 666 768
1978 13 575.3 42 480 674
1979 16 495.8 58 521 274
1980 13 191.3 55 667 616
Source: Exports of fish and fishery products cleared by the Bureau of Fisheries
and Aquatic Resources, by kind, quantity, and value, Fisheries Statistics, 1967-1980.
All Sargassum productions in Central Visayas are being exported to

Japan whereas those in Northern Mindanao are utilized in the local
manufacture of feeds. A significant amount of Gracilaria and Gelidiella is
exported while the rest is locally processed into crude agar bars. Production
of other genera such as Codium, Hypnea, and Porphyra is dependent on
natural stocks and the produce is only locally consumed (BFAR Statistics
1980).
The data on Philippine seaweed exports indicate an almost twenty-fold

increase in production from 1967 to 1980 (BFAR Statistics 1980). From a
minor product, seaweeds now rank third, behind tuna and shrimps, among
Philippine fishery exports.
There are many other seaweed species which still remain to be tapped
and/or developed. The rational development of these resources, however, is
hampered by the lack of appreciation on the part of the policy makers of the
importance and economic potentials of seaweeds as a fishery resource. This
attitude is reflected in the priorities and the funding support allocated to
seaweed research and development compared to other natural resources
such as energy, forestry, minerals, etc. Notwithstanding the many reasons
for this negative attitude, the developing countries of Asia should take a hard
look at the history of the seaweed industry of Japan, China, and Korea where
seaweeds as a resource touch on the everyday life of the people. Once this
attitude is resolved, the solution to the other major problems such as lack
of expertise, facilities, and funds will naturally follow. Solving these prob-
lems may take some time but surely time will be a minor constraint. And the
resolution of the foremost problem of lack of appreciation for seaweeds may
be hastened through the influence of international agencies such as the

UNESCO, FAO, UNDP, and others.
SOCIOECONOMIC IMPLICATIONS OF
SEAWEED RESOURCE DEVELOPMENT
The socioeconomic impact of the development of the seaweed resources

in the developing countries can be readily appreciated by first looking at the
present status of the quality of life, resources, and livelihood of the people
living in coastal areas in relation to the present emphasis on industrial
development and urbanization.
A large portion of the Asian population lives along coastal areas and is
intimately associated with the sea and its resources. Most of these coastal
populations are located in far-flung areas which are hardly benefited by
modern industrial development and urbanization. Being in the tropics, the
coastal areas of Asian countries are characterized by well-developed coral
reefs, shallow bays and coves which used to abound with fishery resources.
Through intensive fishing, these coastal areas have been and will continue
to be depleted of resources on which the very lives of the coastal population
directly or indirectly depend. The grave concern for the consequences of the
depletion of the fisheries in shallow coastal areas is best dramatized by the
closure of traditional fishing grounds to big fishing operations. The Philip-
pines, Indonesia and Thailand have enacted measures to prevent the over-
exploitation of the fishery stocks in some fishing grounds in consideration
of the plight of the small-scale or artisanal fishermen who can hardly afford
to have more sophisticated fishing crafts to fish in far areas which still
support good fisheries. The decline in coastal productivity coupled with
population increase consequently would lead to the lowering of living
standards in the coastal areas. The development of seaweeds as a resource
is an alternative activity which should rank high in government programs
if the idea of a more equitable distribution of wealth and benefit is to
accommodate the poor fishermen. In the Philippines, for instance, more than
600 000 of the fishing force are small-scale fishermen.
A Philippine experience, as exemplified by the development of the

Danajon Reef in northern Bohol in Central Visayas, exquisitely demon-
strates the socioeconomic benefits that can be derived from seaweed farm-
ing. The development of Eucheuma farming in this area is better docu-
mented than that in the Sulu Archipelago. During the peak production of
E. denticulatum (Lim and Porse 1980) in nothern Bohol in 1979 there were:
200 farm houses and drying platforms constructed, 500 hectares of the reef
planted with seaweeds, 2 000 people working daily on the farms, and 1 000
families or approximately 8 500 people fully or partially dependent on
seaweed farming.
The above data do not include the people who were in some way benefited
by the farming activities, e.g., the middlemen, the small store owners, and
suppliers of farm materials, among others. The secondary impact of the
farming activities is of course very hard to measure. However, favorable
effect which the seaweeds brought to the people in terms of improvement of
their life styles was best illustrated by their acquisition of simple luxury
items such as radios, gas stoves, clothes, and motorized bancas. But the best
proof that seaweed farming is a productive form of livelihood is the shift from
fishing into seaweed farming. Results of interviews show that local fisher-
men were earning a net average of P12 per day. A hectare of seaweed farm
netted a farmer an average of P1 200 per month excluding the capital
investment.
The production in northern Bohol in 1979 was, however, small compared

to that in Sulu in the southern Philippines which still accounts for the bulk
of farmed seaweed.
As an entrepreneur, the seaweed farmer is subject to both favorable and

adverse effects of the demand and price fluctuations in the international
market.
Since seaweed farming is labor-intensive, the industry can employ the

otherwise idle or underutilized labor force in the coastal areas.
LITERATURE CITED
Bureau of Fisheries and Aquatic Resources Statistics. 1980. Export of fish and
fishery products cleared by BFAR, by kind, quality, and value. 1967-1980.
Lim, J.R. and H. Porse. 1980. Breakthrough in the commercial culture of Eucheuma
spinosum in northern Bohol, Philippines. Paper presented at the Xth Interna-
tional Seaweed Symposium. Goteborg, Sweden, August 1980.
Naylor, J. 1976. Production, trade and utilization of seaweeds and seaweed prod-
ucts. FAO Fish. Tech. Paper No. 159. Rome. 73pp.
UTILIZATION AND FARMING OF SEAWEEDS
IN INDONESIA
Aprilani Soegiarto
and
Sulustijo
National Institute of Oceanology
Jakarta, Indonesia
ABSTRACT
A great variety of seaweeds grow abundantly along the 81 000-

km coastline of the 13 000 islands comprising the Indonesian
archipelago. However, it is only recently that the economic impor-
tance of seaweeds has really been appreciated.
At present, seaweeds collected in Indonesia are mainly used

for food supplement, domestic agar manufacture, and for export.
Because of the increasing demands for the carrageenan-containing
seaweed, mass cultures have been undertaken in both experimen-
tal and production sites established in many parts of the country.
These efforts are expected to increase the annual volume of exports
from 2 000 to 6 000 mt.
The paper reviews the state and problems of seaweed utiliza-

tion, development, and farming efforts in Indonesia.
INTRODUCTION
The Indonesian archipelago is situated between the Asian and the

Australian continents and between the Pacific and the Indian Oceans.
Geographically, it is located between 94°-141°E and 6°N-11°S. Roughly, it
consists of 13 667 islands with more than 81 000 km of coastline. Marine
waters constituting two-thirds of the country's territory are an ample
resource for maritime opportunities. Hence, a considerable amount of scien-
tific survey and research on the marine environment had been undertaken.
Early marine research in Indonesia started when Amboina was a

headquarter for the Dutch East Indies Company. However, modern marine
research stemmed from the Siboga Expedition of 1899-1900 (Tydeman 1903)
which focused on the marine flora and fauna and their biogeography.
Physical oceanography was brought to the fore in the 1930's by the Snellius
(Riehl 1952) and the Dana Expeditions and more recently by the world-
renowned Albatross and Galathea Expeditions in 1948 and 1951, respec-
tively.
The earliest report on seaweeds in Indonesian waters probably was that

of Rumphius (1750). Through the untiring effort of Weber van Bosse during
the Siboga Expedition, phycology flourished in Indonesia. Unfortunately,
however, it is only recently that the economic potential of seaweeds has been
appreciated. This paper describes the state of research on algae and their
significance in Indonesia.
SEAWEED PRODUCTION
A number of surveys indicate that the distribution and density of marine

algae in different areas vary according to the type of bottom, season,
hydrographic conditions, and species composition at a given time. Table 1
presents the great variations in standing crop from one region to another and
from one species to another. It is, thus, imperative to consider these
differences in the utilization of seaweed resources.
Table 1. Variations in standing crops of seaweed in Indonesia and other countries
Location Species Standing crop (tons/ha)
INDONESIA
Wawarada Bay Eucheuma spinosum 4-18**
Moluccas and E. spinosum 0.6-3.4**
East Nusa
Tenggara
Seribu Islands E. spinosum 0.11**
Tenjung Benoa E. serra 0.46**
Gracilaria lichenoides 0.96**
Hypnea spp. 1.52**
Ulva spp. 1.63**
Southeast Moluccas E. spinosum 2.27**
Central Moluccas E. edule 5.02**
Gracilaria spp. 2.13**
OTHERS
Philippines E. spinosum 9**
La Jolla, California Laminaria sp. 62.5-100**
Scotland Laminaria cloustonii 19.5-45**
Hanauma Bay, Sargassum obtusifolium 5.7-10.2*
Hawaii S. echinocarpum 4.7-6.5**
Kaneohe Bay, Dictyosphaeria cavernosa 0.07-7.27*
Hawaii
* Dry weight; ** Wet weight.

Farming of Seaweeds in Indonesia 11
The identification of factors responsible for such variations must then be

one major area for seaweed research. Fortunately, this has been recognized
in Indonesia recently. For example, Soegiarto (1963) observed that the
standing crop of seaweeds in Wawarada Bay, Sumbawa, ranged from 4 to 18
tons wet weight per hectare and about half (52.6%) of the mixed populations
consisted of Eucheuma spinosum. In 1974, a large-scale survey was carried
out to determine the location and production potentials of the seaweed
Eucheuma in the Moluccas and the East Nusa Tenggara (Lesser Sunda)
waters (Mubarak 1974). Based on this survey, it was estimated that
production of Eucheuma from the Moluccas is about 1 750 mt/yr and from
the East Nusa Tenggara 200 mt/yr. It seems that these estimates were
rather low since figures from the Directorate General of Fisheries showed
that the Moluccas produced 2 277 mt in 1973, 2 636 mt in 1974 and 7 160 mt
in 1975 (Table 2). This discrepancy only indicates that further surveys and
research are needed in order to arrive at reasonable estimates on seaweed
production in each region. Other surveys were carried out by Sulustijo and
Atmadja (1980) in the Seribu Islands, Jakarta Bay; Sulustijo and Yusuf in
Southeast Moluccas; and Sahupala et al. (1977) in Central Moluccas.
UTILIZATION OF SEAWEEDS
At present, the seaweeds collected in Indonesia are mainly used for food,
agar extraction, and export.
Table 2. Seaweed production from selected coastal waters of Indonesia*
Place Production (metric tons)

1973 1974 1975
Bengkulu, West Sumatra 5 5 3

South Sumatra 39 13 645
Riau Archipelago (Strait
of Malacca) ** ** 195
Northcoast of East Java 56 234 **
Yogyakarta, Southcoast
of Java ** ** 9
Bali 60 74 61
Lesser Sunda Islands
(Nusa Tenggara) 329 154 150
Southeast Celebes 387 126 203
Moluccas 2 277 2 636 7 160
West Irian 3 990 **
*Source: The Indonesian Directorate General of Fisheries.

**No available data.
Food
The widespread use of seaweeds as human food in Indonesia was

recorded as early as 1292 when the first European ships sailed through
Indonesian waters. For centuries, the islanders utilized marine algae as food
supplement, especially as "sayur" or vegetable (Zaneveld 1955). However,
algal consumption was limited to fishermen and normally these edible
species did not reach local markets. But some species used in making
sweetened jellies were transported farther inland.
Hogue (1922) mentioned some twenty-one species of useful seaweeds in

Indonesia. The list was later expanded by Zaneveld (1955) to include also the
economically important algae of the Southeast Asian waters. This fact
indicates that Indonesians eat seaweeds in various forms: raw as salads,
boiled as vegetable, mixed with various spices, pickled, cooked with coconut
milk, for soup thickening, pudding, and sweetened jellies. Some species even
serve as medicine.
The organic composition of seaweeds varies from one division to another

and from one species to another. For any one species, it can also vary
geographically, according to depth, and even from one part of a thallus to
another. Therefore, it is hardly surprising that the published data on their
organic contents are highly variable.
Table 3 shows the chemical analyses of six red algae collected from
Sumbawa Island (Nusa Tenggara). Generally, proteins are present in small
quantities and are hardly assimilated by human beings. However, animals
may utilize certain proteins better. This is probably one of the reasons for
using some seaweeds for stock feed instead of for human consumption.
Carbohydrates, occurring mainly as cell wall and intracellular storage

materials, constitute the bulk of algal organic matter. However, the high
carbohydrate content is of low value as a source of energy since most of these
substances are hardly digested in man. It has been suggested, however, that
people who eat seaweeds from childhood may acquire a specialized bacterial
flora in the intestines to help digest these algal carbohydrates. Thus,
islanders might obtain more food value from seaweeds than others and,
possibly also, animals not used to a diet which includes algae.
Even if all the carbohydrates and proteins in seaweeds were fully

digestible, these could not significantly supplement the nutrients derived
from present food or dietary sources. Rather, the nutritive value of seaweeds
is based on their mineral and vitamin contents. Biochemists have definitely
established the presence of carotene, and vitamins B1, B2, B12, C, D, and E in
marine algae. Again, the vitamin concentration varies from one species to
another. Intraspecific differences also occur geographically and even with
depth.
Table 3. Proximate analysis of some Indonesian seaweeds (After Soegiarto 1968)
Proximate analysis Eucheuma Eucheuma Gracilaria Gracilaria Gelidiopsis Hypnea

(% dry weight) spinosum sp. sp. confervoides sp. sp.
Moisture 27.50 16.99 19.01 24.91 12.95 25.15
Protein 5.40 2.48 4.17 3.14 9.98 1.59
Carbohydrates 33.22 63.19 42.59 37.52 54.43 32.25
Fats 8.62 4.30 9.54 5.52 11.09 5.81
Crude fiber 3.01 - 10.51 9.14 - 11.43
Ash 22.25 23.04 14.18 15.77 11.75 23.77

Organic Substances
Seaweeds are important sources of chemical substances for industrial or

medical purposes, ranking them among the important resources of the sea.
The agar industry in Indonesia is of recent development. Incidentally, the
term 'agar' is of Indonesian origin. An attempt to manufacture agar commer¬
cially was first reported in 1910. Hofstede(1921a, b, 1923) surveyed the agar
content of various seaweeds to determine the possibility of establishing
large-scale agar production in Indonesia. But due to rather crude and
inadequate analytical methods, he concluded that (1) among the edible
seaweeds in Indonesia, only a few contain agar, (2) the agar-containing
seaweeds are not abundant, and (3) the seaweeds exported in large quanti-
ties do not contain agar. Later, the use of improved methods of analysis,
however, showed that the seaweeds occurring in Indonesia contain suffi-
cient agar to justify large-scale agar manufacture for export, an opinion
shared also by Zaneveld (1955).
The Indonesian government had been giving full support to the estab-
lishment of agar factories. Between 1947 and 1952, the Laboratory for
Chemical Research in Bogor analyzed the agar content of practically all
species of commercial seaweeds collected from Indonesian waters. This work
later showed that Eucheuma spp. did not contain agar but another sub-
stance known as the iota-carrageen in extract from E. spinosum which is
becoming more important as an additive in various industrial products,
especially in America and Europe.
The first large-scale agar factory in Indonesia was set up in 1930 in

Kudus along the north coast of Central Java. More factories were soon
established in other cities. Unfortunately, the industry suffered a severe
setback during the Second World War. But the high price of agar and full
government support after the war stimulated the emergence of smaller
factories in Jakarta, Surabaya, Padang, Bandung, and other cities. Table 4
shows the present locations of agar factories in Java.
At present, Gracilaria lichenoides is the most important raw material for

agar manufacture in Indonesia (Table 4). Two other species also commonly
used by the industry are Gracilaria blodgettii and Gelidium latifolium. On
the other hand, Gracilaria confervoides is not used in agar processing in
Indonesia but is exported from Ujungpandang (Macassar) in rather great
quantities. There is, however, a need to improve the quality of the agar
product For example, by using a combination of G. latifolium and G. liche-
noides, an agar of better quality can be obtained. Research in this field is
badly needed.
Export
For many years, Indonesian export of seaweeds has been an important

Table 4. Some agar factories in Java (After Soegiarto et al., 1975)
Raw materials
No. of Species Production
Name needed
workers used (kg/month)
(kg/month)
Universal
Surabaya 4 Gracilaria spp. 710 42
Sinar Kencana 32 Gracilaria spp. 13 000 800

Wonocolo, Surabaya and Gelidium spp.
Sriti
Surabaya - - 250 15
Sari Jaya
Surabaya - - 2278 166
Oen Brothers 7 Gracilaria spp. 417 25

Surabaya and Gelidium spp.
Sumber Laut 7 Gracilaria spp. 1000 60

Surabaya and Gelidium spp.
Hasalin 60 Gracilaria spp. 20 000- 2000-

Jakarta and Gelidium spp. 30 000 3000
Djawa
Jakarta 25 Gracilaria spp. 15 000 1500
economic activity. Early records show that for over a century, seaweeds had
been exported to China. Before the Second World War, the volume of export
was more than 1000 mt/yr but it decreased immediately after the war. In the
last few years, the demand has increased considerably. With attractive
export regulations on soft products, including many marine resources, the
volume of seaweed export reached an all-time high of 5 923 mt in 1966 (Table
5) but it later declined due to changes in export regulations, the government's
tight money policy, and the decrease in seaweed price.
Table 5. Annual export of seaweeds (dry weight, metric tons) from major Indonesian
ports in 1960-1976
Tanjung Priok, Ujungpandang

Year Surabaya Ambon Others
Jakarta (Macassar)
1960 104.50 197.87 305.20 - 20.00

1961 286.05 72.61 562.99
1962 76.50 40.76 300.99 -
1963 146.00 - - - 12.00
1964 147.00 - 110.00 - 28.00
1965 335.00 - 770.00 - 27.00
1966 329.00 137.00 5 246.00 - 211.00
1967 244.70 335.00 265.80 - 72.70
1968 380.80 182.70 1 571.40 89.00 170.80
1969 136.00 227.50 1 040.30 490.20 289.30
1970 888.30 5.50 1 076.50 1 373.20 463.90
1971 45.90 24.30 1 125.80 2 283.50 254.00
1972 80.20 137.40 1 480.40 1 804.50 219.40
1973 52.60 64.20 1 587.10 1 296.60 250.70
1974 8.60 53.20 1 446.20 1 605.60 187.70
1975 9.50 23.00 764.70 745.70 59.70
1976 10.00 43.20 1 313.60 505.50 115.70
Source: The Indonesian Directorate General of Fisheries.
By far, Ujungpandang has been the major market and shipping harbor
for seaweeds harvested mainly from the south coast of Celebes and the
adjacent islands. In recent years, harvests from Nusa Tenggara (Sumbawa,
Flores, Sumba) and the Moluccas region have also been transported to
Ujungpandang. The seaweed production from the Moluccas now is rather
substantial (over 2 000 mt in 1971) to make Ambon another center of
seaweed trade.
Between 1920 and 1930, Indonesian seaweeds exported almost exclu-

sively to China consisted chiefly of E. spinosum. The export statistics for
1930-1940 also indicates that 20-30% of the exported seaweeds landed in
Japan and consisted mainly of G. blodgettii, G. lichenoides, G. amansii and
G. rigidum. Singapore and Hong Kong are important transitory ports of
seaweed exports destined for European, American, or even Japanese mar-

kets. However, in the last few years, direct exportation has increased
steadily.
SEAWEED FARMING
One of the problems of the seaweed export industry is the lack of a

constant supply of seaweeds of good quality. Shipments containing some
low-grade seaweeds are unacceptable in the international market
and are usually sold at low prices. Steps are being taken to remedy this
problem.
Some commercial seaweed buyers are sponsoring programs on the

cultivation of E. spinosum in tropical Southeast Asian countries such as
Singapore, Philippines, and Indonesia. Successful field cultivation will
ensure yields of predictable quantity and quality and possibly also reduce
transport costs. So far, the results have been very encouraging and the
Philippine experience has become a model of such efforts (Doty 1973).
Indonesian seaweed culture in the laboratory started as early as 1966

(Soerjodinoto 1968, Soegiarto 1968, Ismail 1971) but the study progressed
only slowly due, in part, to political and economic instability in the country
during that time. In 1972 the study was resumed with strong government
support, especially by the Indonesian National Institute of Oceanology
(NIO) and the Marine Fisheries Research Institute (MFRI). In the last few
years, a number of seaweed farms have been set up by NIO at Pari Islands,
Jakarta Bay (Soegiarto et al. 1975) and the Tanjung Benoa, Bali (Sulistijo
and Atmadja 1980); by MFRI and Copenhagen Pectin Company in Samar¬
inga Islands, Central Celebes (Mubarak 1975); by fishermen in North
Tanimbar Island and in Maumere (Mubarak 1974); by MFRI and Marine
Colloids in Riau (Mubarak 1976); by Directorate General Cooperative and
USAID in Aru Islands, Moluccas (Mubarak 1978); by fishermen in coopera-
tion with the Moluccas Government and NIO Ambon Station in Geser,
Ceram Island, Moluccas; and by fishermen with the assistance of Copen-
hagen Pectin Company in Terora-Tanjung Benoa, Bali (Sulustijo and At-
madja 1980).
The E. spinosum farm in Terora-Tanjung Benoa, Bali, so far has

produced the best results, with a monthly production of 15-20 mt dry weight
in 1979. However, due to the decreased prices, the monthly production
declined to around 10 mt after one year. Furthermore, experimental results
have shown that the growth rate on floating rafts is slightly better than that
of the bottom method. For example, in the farms in Tanjung Benoa, Bali, the
average growth rate was 4-5%/day on the floating rafts and less than 3%/day
in the bottom method.
CONCLUSIONS
The economic value of seaweeds in Indonesia is not yet fully appreciated.

The full potential of the country's seaweed resources remains to be tapped.
More extensive surveys and research are urgently needed to determine

the potential production and the factors which govern the productivity of the
economically important species.
Only a few species have so far been investigated for their nutritional
value and chemical composition which considerably vary from one species to
another and with geographical distribution and seasonal changes.
The expansion and upgrading of agar factories in Indonesia will depend

on government protection against imported products, capital investments,
improved management, continuous supplies of good quality raw materials,
and the application of more modern technology.
Seaweed exports can be increased through attractive export regulations

and bonuses in combination with strict quality control.
Seaweed farming should be encouraged to increase production, control

the quantity and the quality of seaweeds, and reduce interinsular transport
costs and risks. Technically, seaweed farming is easy and does not require
much capital investment. It will also provide new job opportunities for
fishermen.
Appropriate marketing and pricing systems should be developed in

order to protect the seaweed farmers from unscrupulous dealers or buyers.
LITERATURE CITED
Doty, M.S. 1973. Eucheuma farming for carrageenin. Sea Grant Advisory Report.
UNIHAW Seagrant A-273-02.
Hogue, K. 1922. Algen. In de Nuttige Planten van Nederlandsch Indie. Dept. Landb.
Nijverh. & Hand., Buitenzorg, vol. 1:33-38.
Hofstede, H.W. 1921a. Is een agar-agar industri in Nederlandsch. Indie mogelijk?

Alg. Landbouweekbl. Ned. Indie. 6(15):671-673. 6(17) 1921b:738-740. 8(9)
1923:319-324.
Ismail, W. 1971. Metode kultur dari beberapa jenis rumput laut. MFRI Unpublished
Report. 16 pp.
Mubarak, H. 1974. Laporan survey Eucheuma di perairan Maluku dan Nusa

Tenggara Timur, Juli-Nopember 1974. Lap. Penel. Perik. Laut 1:1-29.
Mubarak, H. 1975. Percobaan penanaman rumput laut Eucheuma spinosum

(Rhodophyta, Gigartinales) di P. Samaringa, Kecamatan Menur Kepulauan,
Sulawesi Tengah, akhir Juli-awal Oktober 1975. LPPL 1/75 - PL.053/75:78-
101.
Mubarak, H. 1976. Laporan percobaan budidaya rumput laut Eucheuma spinosum

de Pulau Teland, Riau. LPPL, Jakarta.
Mubarak, H. 1978. Rumput laut (Algae), Manfaat, Potensi dan Usaha Budidayanya.
Lembaga Oseanologi Nasional - LIPI, Jakarta. 61 pp.
Riehl, P.M. van 1952. The Snellius Expedition. J. Cons. Inst. Expl. Mer. 7:213-217.
Rumphius, G.E. 1750. Het Amboinsche Kruidbook, Herbarium Amboinense. Amste-

laedami. 6. 256 pp.
Sahupala, I., O.K. Sumadiharga, D. Sapulete, L.P. Wenno, and Sulustijo. 1977. Hasil
penelitian rumput laut dari perairan pulau-pulao Nasalaut, Kefing dan Geser,
Maluku Tengah. Proyek Penelitian Kultivasi Rumput Laut Pemda DT I Ma-
luku, Ambon: 27 pp.
Soegiarto, A. 1963. Primary productivity of benthic algae in a tropical bay. M.S.

thesis, University of Hawaii. 53 pp.
Soegiarto, A. 1968. Seaweed resources from Indonesian waters. NAS-LIPI Workshop

on Food, Indonesian Professional Paper, Working Group IV, paper 5. 21 pp.
Soegiarto, A., K. A. Soegiarto, and S. Soemodihardjo. 1975. Bibliografi Beranotasi III

Algae yang berpotensi Ekonomi. LON-LIPI. 42 pp.
Soegiarto, A., Sulustijo, and W.S.Atmadja. 1975. Pertumbuhan Alga Laut Eucheuma
spinosum pada berbagai kedalaman. Fourth Biological Seminar-Second Na-
tional Biological Congress, Yogyakarta. 13 pp.
Soerjodinoto. 1968. Is the cultivation of seaweed (.Eucheuma spinosum and Eu-

cheuma edule) in Indonesia technically possible and economically justified?
IPFC/C 68 Tech. 21. 4 p.
Sulustijo and W.S. Atmadja. 1980. Laporan Tahunan LON-LIPI, Jakarta.
Tydeman, G.F. 1903. Hydrographic results of the Siboga Expedition. Siboga Exp. 3.
93 pp.
Zaneveld, J.S. 1955. Economic marine algae of tropical South and East Asian and
their utilization. IPFC Special Publication No. 3. 35 pp.
PRESENT STATUS OF SEAWEED CULTURE
IN KOREA
Jae Wong Kang

National Fisheries University of Pusan
Pusan, Korea
ABSTRACT
Seaweeds from natural stocks as well as from aquaculture

have been widely utilized in Korea for a long time. Porphyra was
first cultivated 360 years ago. The culture of Undaria pinnatifida
was introduced more than 10 years ago. Laminaria spp. were also
introduced by employing an artificial culture method. L. religiosa
was grown in natural beds along the middle part of the eastern
coast after the initiation of farming of this species. At present, pro-
duction of U. pinnatifida from farms is much greater than the
natural harvest.
The most widely cultured species of Porphyra is P. yezoensis,

but P. tenera is also farmed in some areas. Recently, some varieties
that were known to grow faster and to be more resistant to diseases
were introduced from Japan. After Conchocelis was successfully
grown, the artificial seeding method became very popular.
The production of cultured Porphyra was 34 025 mt in 1980; U.

pinnatifida, 153 333 mt in 1979; Laminaria, 5192 mt in 1979.
INTRODUCTION
Seaweeds from natural stocks and aquaculture have been widely

utilized in Korea for a long time now. Chung (1814) described thirty-four
species of seaweeds with particular emphasis on their use as human food.
Porphyra was first cultivated on tidal flats of Kwang-Yang Bay near the
estuary of the Seom-Jin River 360 years ago. Later it was grown on shrub
branches standing on tidal flats. Since then more efficient culture methods
have been developed. Split-bamboo blinds replaced the shrub branches as
culture substrates. Initially, one lateral margin of the split-bamboo blind
was fixed to the sea bottom and the other side was left free in the water at
certain level within the tidal range. In the 1930's, this method was modified
by setting the split-bamboo blind substrate in a horizontal position.
Undaria pinnatifida has been harvested for a long time from natural
rocks by using a method that eliminates 'pest' weeds such as Sargassum
Phyllospadix, and articulated coralline. The cultivation of the species
started in 1970 and at present the production from cultures is much higher
than natural harvests.
Laminaria spp. are distributed along the eastern coast of northern

Korea. In the south, where it is too warm for the natural growth of the
species, L. japonica and L. religiosa have been introduced employing an
artificial culture method. L. religiosa later started to grow on natural rocks
along the middle part of the eastern coast sometime after the initial farming
of this species.
As these species of sea vegetables are now grown extensively by a

number of sea farmers, seedling production which is undertaken by highly
skilled farmers is a major concern of the seaweed industry in South Korea.
PORPHYRA CULTURE
Among Porphyra species, P. yezoensis is at present most commonly

cultivated although P. tenera, which used to be more popular, is still grown
in some farms (Kang 1970). Recently, some varieties that grow faster and
are more resistant to diseases were introduced from Japan. The limited
success in the cultivation of these varieties may be due to differences in en-
vironmental conditions and the lack of experience of farmers in culturing
new cultivars.
About 4 to 5 years ago, Conchocelis was cultured by the free-growing

method before the technique of substrate culture in shells. However,
farmers generally do not utilize this method. They obtain their seeds from
the National Fisheries Research and Development Agency of Korea or
import them from Japan to get new strains.
Before the technique of artificial seeding from Conchocelis cultures was

developed about 10 years ago, farmers obtained their seeds from natural
spores settling in the field. Now, the old method of natural spore-settling is
no longer practiced. Artificial seeding is usually carried out in large plastic
envelopes. The cultured Conchocelis and nets for spore-settling are placed
inside the envelope containing sea water. The envelope is then set under
water in the field to simulate or approximate natural conditions. Since
seeding requires exact timing of spore release, farmers usually prefer
artificial seeding under natural conditions than under fully controlled
conditions in tanks installed on land.
Seaweed Culture in Korea 23
The Porphyra culture method is as follows:
Culture of Conchocelis
(control of light intensity, water temperature,
salinity, and diseases)
Artificial seeding (spore-settling on net material) (under

natural or controlled conditions)
Nursery rearing
Cold storage of
nursery nets
Transplant to farming site
Growing (control of diseases,

management of water level)
Harvest
The nets are then suspended in seawater during the nursery growing
period of about 55 days. The grow-out nursery nets are almost directly
transplanted to the final growing ground. Only very few farmers keep some
of the nursery nets refrigerated.
The old practice of using split-bamboo blinds as culture substrate has

been substituted by the net-culture method which is more convenient and
efficient for spore-settling. The seeded nets (1.8 × 4m) are usually placed and
maintained at a certain level within the tidal range by attaching them to
supporting poles. The nets float near the water surface and are exposed to
the air twice a day during the spring low-tide period. The average time of
each exposure is 3 to 4 hours.
If the exposure time is short during the early stages of the culture period,
Porphyra grows fast but is more vulnerable to diseases. On the other hand,
with a long exposure period it grows slowly but is more resistant to diseases.
Morever, when warm temperatures accompany the calm weather during
December, it is very difficult to grow healthy Porphyra by adjusting the
exposure time alone. However, farmers can grow Porphyra fast and increase
yields by reducing the exposure time in December. But the next crop usually
suffers from diseases.
The nets in the drift system are always floating on the surface of the
water and maintained in place by anchors. This culture system is beginning
to be practiced toward the open sen because of the pollution of inland
seawaters. Recently, production of Porphyra has expanded to meet increas-
ing demands. However, there is the need to improve its quality.
UNDARIA CULTURE
Korea has two species of Undaria: U. pinnatifida and U. peterseniana.

Because the distribution of the latter is restricted to Cheju Island and its
vicinity, it is not as popular as the widely distributed U. pinnatifida which
is preferred by the Korean people. The thalli of another species, Costaria
costata, is also utilized as food.
The culture technique of Undaria was introduced more than ten years
ago in the vicinity of Pusan (Saito 1964) which is located along the country's
southeastern coastline. About 70% of the total production of the species in
Korea today, however, is produced along the southwestern coastal areas.
The culture and production of Undaria involve:
Artificial seeding (spore-settling)
Indoor culture of nursery stage
Preliminary growing (rearing of

the young sporophytes at sea)
Rearing of thalli
Harvest
Raw Drying Salting
Marketing
Artificial seeding is usually carried out from late May to early June when the
seawater temperature increases to around 17°C. When shade-dried sporophylls are
immersed in seawater, the zoospores are discharged. The zoospores germinate into
gametophytes which grow very fast up to the young sporophyte stage when the water
temperature is below 20°C until July before the onset of high water temperature. If
light intensity is reduced, gametophytes grow only into a few cells. Growth of
gametophytes is slow until August since water temperature thereafter begins to
increase.
The success of Undaria culture basically depends on the development of the

young sporophytes. Factors like high water temperature and transpar-
Seaweed Culture in Korea 25
ency as well as unstable sea conditions owing to prevailing strong winds and
fluctuation of water temperatures apparently influence the growth of young
sporophytes. Recently, certain diseases caused by bacterial pathogens and
parasites, such as Thalestris sp. and other copepods, have been observed in
cultures.
The market price of the dried product fluctuates considerably. At the

very start of the harvest season in December, it is about ten times as much
as in February to April. Therefore, some farmers start to grow young
sporophytes early in September, in spite of the adverse effects of warm
temperatures, so as to harvest in December when prices are high. However,
when fully grown sporophytes experience high water temperatures during
the hot season, survival of the sporophytes is low.
LAMINARIA CULTURE
Although Laminaria generally thrives in cold temperate waters, it is

now possible to farm it in warmer temperate waters through artificial seed
production (Li 1969). In this method, spores are collected on strings from
blades of mature Laminaria in May and then kept in the dark and at low
temperature indoors during summer in Japan. They are moved to the sea
in autumn and then grown to marketable size by July in the following year.
When cultured from spores, the seaweed still behaves as a biennial and
develops the same thickness and taste as naturally grown individuals.
Laminaria is now cultured in South Korea in areas other than its natural
habitats.
The culture of Laminaria in Korea consists of the following:
Artificial seeding (October)
Indoor culture of seeds

(gametophytes to young sporophytes)
Preliminary growing
(rearing of the young sporophytes at sea)
Transplant to growing ground (November)
Rearing
Partly preserved for seed Harvest (late June to

production early August next year)
In Korea, seedling culture begins in October and the rearing of young

sporophytes is carried out from late October to November when the water
temperature is below 18°C. It is harvested by late July before the water
temperature rises to 20°C. In summer, typhoons may damage seaweeds
farms, and the cultures may be affected by the growth of bryozoans.
PRODUCTION OF CULTURED SEAWEEDS
The data on the production of cultured Porphyra, Undaria, and Laminaria

in South Korea in 1975-1980 are given in Table 1. Annual total harvests for
each seaweed during this period varied greatly, but Undaria clearly consti-
tuted the bulk, followed by Porphyra and Laminaria. Porphyra production
Table 1. The production of cultured seaweeds in South Korea
Production (metric tons)

Species
1975 1976 1977 1978 1979 1980
Porphyra - 29 090 11750 23 610 29 240 34 025

Undaria 132 328 161 770 208 620 158 342 153 333 -
Laminaria 2 759 8 342 2 122 5 516 5 192 -
was a high 34 025 mt in 1980. Undaria and Laminaria accounted respec-

tively for 153 333 and 5 192 mt in 1979; no data are available for both
seaweeds for 1980.
LITERATURE CITED
Chung, Y.S. 1814. The Fishes Ja-San (Ja-San Eco-Bo). (In Chinese.)
Kang, J.W. 1970. Species of cultivated Porphyra in Korea. Bull. Korean Fish. Soc.
3(2):77-92.
Li, A. 1969. Laminaria culture: a success in warm waters. Yoshoku 4(11):12-18. (In
Japanese.)
Saito, Y. 1964. Cultivation of Undaria pinnatifida (Harv.) Sur. Japan Fishery

Resources: Conservation Association, Tokyo. (In Japanese.)
UTILIZATION OF SEAWEEDS IN THAILAND
Khanjanapaj Lewmanomont
Faculty of Fisheries
Kasetsart University
Bangkok 9, Thailand
ABSTRACT
Different seaweeds harvested from natural stocks are utilized

in Thailand as human food and animal feed and for medicinal
purpose and extraction of crude agar. Gracilaria and Porphyra are
the most exploited commercially. Commercial cultivation through
seaweed farming is recommended.
INTRODUCTION
Thailand, which lies between Latitudes 5° to 21°N and Longitudes 97° to

160°E is one of the countries in Asia favorable for the growth of seaweeds.
Being an entirely tropical monsoon country, pronounced wet and dry
seasons characterize the climate of the land. The rainy season at the upper
part of the country is from May to October, while in the south the rainy period
is from October to February. The dry season begins in November and lasts
until April. Thailand has 2 527 kilometers of coastline which abounds with
seaweeds. The southern coastlines border the Gulf of Thailand on the east
and Andaman Sea on the west and consist of sandy-rocky shores, coves, and
mangroves areas. However, not much Phycological work has been done
along the sea coast.
SEAWEED UTILIZATION
The Thai people use seaweeds as food, as animal feed, for medicinal
purposes, and for extracting agar. Seaweeds are eaten only in particular
areas, especially along the coast of the Gulf of Thailand and Andaman Sea.
The majority of edible seaweeds belong to the genera Gracilaria, Porphyra,
Caulerpa, Sargassum, Hypnea, Laurencia, Acanthophora, Padina, Dictyota,
Hydroclathrus, and Chaetomorpha. These seaweeds are consumed fresh or
blanched as salad vegetables, mixed with some ingredients, or used in soup
preparations (Lewmanomont 1978).
Gracilaria is the only genus used for agar extraction. To extract agar,
local people boil in water the bleached, dried Gracilaria, filter the mixture
through muslin, and let the filtrate set into a gel. Agar can be made into
different desserts (Boon-nag 1935).
Seaweeds used for medicinal purpose are Sargassum and Laurencia.

Both are used in the treatment of goiter. Dried Sargassum is also boiled and
taken as tea to lower body temperature when one has fever.
For animal feed, only the green seaweed Ulva reticulata is used in the
diet of pigs.
Among the useful seaweeds, Gracilaria and Porphyra are more popular
than the other genera. Both are exploited commercially, but are harvested
only from natural stocks.
Gracilaria
This genus occurs in many areas in Thailand. More than ten species had
been reported (Lewmanomont 1978). The most common species, G. ver-
rucosa, is widely distributed in the Gulf of Thailand and Andaman Sea. The
other common species are G. blodgettii and G. crassa.
Based on the report of the Department of Customs (1956-1980), Thai-

land exported Gracilaria to many countries for agar extraction in 1956 to
1961 and again from 1975 to 1980. In 1980, Thailand exported more than 200
tons of dried Gracilaria to Japan, Federal Republic of Germany, Italy, and
Hong Kong. Only a small volume was utilized locally as food and for
extracting agar. During the same period, Thailand imported agar from
Japan, Hong Kong, Korea, Argentina, United States, United Kingdom, and
Federal Republic of Germany. It seems ridiculous to export seaweed raw
material abroad and then import the final product, agar. In 1966, Thailand
imported only 66 tons of agar. Since then, imports have increased dramati-
cally every year. In 1979, 225 tons of agar worth 67 million baht was
imported. Therefore, if the cultivation of Gracilaria in Thailand becomes
successful, it will increase the income of the Thai people living along
coastlines and also minimize agar imports once an agar-extracting factory
in Thailand is established.
Porphyra
This genus is an expensive red seaweed used as food in Thailand. The

common species is P. vietnamensis. This species occurs only in the south at
Seaweed Utilization in Thailand 29
Songkhla, Pattani, and Narathiwas during November to February when the

salinity and temperature of seawater are low. It grows on exposed rocks
constantly splashed by waves. The local people collect Porphyra by hand and
sell it fresh in the market or dry it into sheets. The annual production is
variable and depends on environmental conditions. It is only at Songkhla
where the alga is commercially exploited. The annual yield is about 500 kg
fresh weight (Lewmanomont and Ogawa 1979; Prommanond and Saha¬
watcharin 1968; Thiemmedh 1960). Since P. vietnamensis is a tropical
species that can tolerate high temperatures, its commercial cultivation in
Thailand is possible.
PROSPECTS OF SEAWEED FARMING
Seaweed farming can provide a steady supply of raw material to a

seaweed industry. The potential of seaweed farming in Thailand is rather
high since favorable environmental conditions therein such as high light
intensity and temperature throughout the year support good seaweed
growth. The productivity of seaweed farming is higher in warmer areas than
in cold regions. This may be due to faster seaweed growth rates and longer
growing seasons in warm areas. Moreover, seaweeds of commercial impor-
tance occur in Thailand which are easier to culture than introduced species.
Gracilaria and Porphyra offer the best prospect for seaweed farming in
Thailand. A well-planned project is seriously needed. Cooperation with
other countries and aid from foreign specialists are also required.
LITERATURE CITED
Boon-nag, K. 1935. On the agar-agar obtained from the marine algae of Siam.
Department of Science, Bangkok, Popular Bulletin No. 5. 12 p.
Department of Customs. 1956-1980. Annual Statement of Foreign Trade of

Thailand.
Lewmanomont, K. 1978. Some edible algae of Thailand. Kasetsart Journal

12(2):119-129.
Lewmanomont, K. and H. Ogawa. 1979. Study on the life history of Porphyra of

Thailand. A report to the Faculty of Fisheries, Kasetsart University, Bangkok,
Thailand. 27 p.
Prommanond, P. and S. Sahawatcharin. 1968. Sarai Bai. Annual Report of the

Songkhla Marine Station. Department of Fisheries, Ministry of Agriculture. p.
144-156.
Thiemmedh, J. 1960. A report on the Sarai Bai (Porphyra sp.) of' Songkhla. Thai
Fisheries Gazette 13(4):311-339.
LIFE HISTORY OF ACROTHRIX PACIFICA AND
SPHAEROTRICHIA DIVARICATA IN LABORATORY
CULTURES
Isamu Umezaki
Laboratory of Fishery Resources
Division of Tropical Agriculture
Graduate School of Agriculture
Kyoto University, Kyoto 606, Japan
ABSTRACT
The life histories of Acrothrix pacifica Okamura & Yamada

(Acrothricaceae) and Sphaerotrichia divaricata (Ag.) Kylin (Chord-
ariaceae) in the Chordariales, Phaeophyceae were studied in the
laboratory. Both species showed an alternation of macroscopic
sporophyte (2n) and microscopic gametophyte (n).
In A. pacifica, unfused gametes developed into haploid sporo-

phytes under cooler conditions or into gametophytes under warmer
conditions. In Sphaerotrichia pacifica, unfused gametes developed
into gametophytes under warmer conditions.
INTRODUCTION
The Chordariales in Japan contains four families and seventeen genera:

Leathesiaceae (Petrospongium, Cylindrocarpus, Leathesia), Chordariaceae
(Chordaria, Cladosiphon, Eudesme, Haplogloia, Heterosaudersella, Myriog-
loia, Analipus, Papenfusiella, Pseudochorda, Sauvageaugloia, Saundersella,
Sphaerotrichia, Tinocladia), Spermatochnaceae (Nemacystis), and Acrothri-
caceae (Acrothrix). Most of the genera and species in this order are distrib-
uted in the northern part of Japan, except for Cladosiphon okamuranus, a
locally edible species which grows in the subtropical waters of Okinawa and
Amami Islands. Other edible species such as Nemacystis decipiens, Tinocla-
dia crassa, Sphaerotrichia divaricata, and Acrothrix pacifica are distrib-
uted along the Pacific Ocean and Japan Sea coasts in Honshu and western
and northern Kyushu. These five species have been used as "sea vegetables"
in Japan.
The life histories of N. decipiens and T. crassa have been studied by

Yotsui (1975, 1980), Yotsui and Migita (1974) and Yotsui (1978, 1979a, b),
respectively. Shinmura (1974, 1975, 1976, 1977a, b) and Shinmura and
Yamanaka (1974a, b) studied the life history and ecology of C. okamuranus.
Based on these studies, N. decipiens and C. okamuranus are now artificially
cultivated in the sea to increase production.
Since an understanding of the life history is fundamental to the cultiva-

tion of algal species in the sea, a study was conducted on the life history of
A. pacifica and S. divaricata in the laboratory. In laboratory cultures, the
life cycles of these two species were previously found to be heteromorphic,
with an alternating macroscopic sporophyte and microscopic gametophyte
(Ajisaka 1979; Ajisaka and Umezaki 1978). Furthermore, under given
conditions, the sporophytic and gametophytic stages were alternately re-
peated without producing any further generations.
MATERIALS AND METHODS
The sporophytes of A. pacifica and S. divaricata were collected at

Takahama in Wakasa Bay, which is in the middle part of Honshu facing
Japan Sea. The fertile sporophytes bearing unilocular sporangia were
collected during the early summer months of June and July. The isolation of
zoospores discharged from the sporophyte was done by the micropipette
method. The culture medium used in this study was Provasoli's ES medium.
Cultures were illuminated with cool white flourescent lamps (ca. 1500-3000
lux) and were incubated under the following temperature-photoperiod
regimes: 20°C: 18-6 hr (set l); 20°C: 10-14 hr (set 2); 15°C: 14-10 hr (set 3);
15°C: 10-14 hr (set 4); 10°C:14-10hr(set5). For the A. pacifica cultures, there
were two additional regimes: 10°C: 10-14 hr (set 6); 5°C: 10-14 hr (set 7).
RESULTS AND DISCUSSION
Acrothrix pacifica
The unilocular sporangia of A. pacifica are elongated obovoid 44-66 x 25-

41 (32 × 55 average) urn in size. Zoospores discharged from the unilocular
sporangium are pear-shaped, with a single chromatophore and an eyespot,
and laterally biflagellated. Soon after settling on the substratum (glass
slide), zoospores became spherical with a diameter of 4.4-7.6 (5.6 average)
urn.
The settled spores germinated and developed into creeping uniseriate

filaments on which hyaline hairs were produced. Under warmer conditions
Life History of A. pacifica & S. divaricata 33
(sets 1 and 2, 20C), the creeping filaments developed into prostrate and
upright systems forming dense tufts. Under cooler conditions (sets 5 and
6, 10"C), the creeping filaments developed into comparatively smaller tufts
and produced many larger erect filaments from the center. Each erect
filament profusely branched off on the opposite side or on all sides. At 10°C,
most of the cells of the branches and branchlets of erect filaments were
transformed into uni- or biseriate plurilocular gametangia. The gameto-
phytes mature within 2-3 months under set 5 (10°C: 14-10 hr) and within 3
months under set 6 (10°C: 10-14 hr).
The gametes from the gametangium are pear-shaped, measuring 5.7-

10.2 x 3.0-5.3 mm. Under cooler conditions (sets 4-7, 10-15°C), gametic
conjugations (usually isogamous, rarely anisogamous) were observed. The
fused gametes settled on the substratum (glass slide) and soon became
spherical. The settled zygote germinated to produce filament that later
developed into a monosiphonous central axis. Each cell of the central axis
divided giving rise to a primary assimilating filament. Then some basal cells
of the primary assimilating filaments divided to produce a medullary layer
which grew trichothallically into a cylindrical plant. Under cooler conditions
(5-10°C), the cylindrical plant branched off laterally and after 3 months, it
matured producing many unilocular sporangia which released zoospores.
The branched fertile plants are similar in habit to sporophytes from the sea.
The gametophytes derived from zoospores of sporophytes carry 8-14

chromosomes while the sporophytes from zygotes of cultured gametophytes
have 14-19 chromosomes (Table 1). This indicates that the sporophytes are
diploid and the gametophytes are haploid. Unfused gametes germinated into
sporophytes under cooler conditions (10°C). The sporophytes have 8-14 (of
which 50% has 9) chromosomes and, therefore, seemed to be haploid. The
haploid sporophytes bore unilocular sporangia and released zoospores. The
zoospores directly germinated and gave rise to haploid gametophytes. On the
other hand, under warmer conditions (sets 1 and 2, 20°C), most of the
unfused gametes germinated into haploid gametophytes, repeating the
same generation. Under moderate conditions (set 3 and 4, 15°C), the unfused
gametes germinated into sporophytes (more in number than gametophytes).
When one-celled germlings of unfused gametes were cultured under cooler
conditions (10-15°C), they germinated into sporophytes which, however,
grew smaller than normal diploid sporophytes and died within a month. On
the other hand, when the unfused gametes under warmer conditions (10-
15°C) were transferred into cooler conditions (5-10°C), they again germi-
nated into gametophytes.
This study indicates that the life history of Acrothrix pacifica is morpho-
logically an alternation of macroscopic sporophytes with microscopic game-
tophytes or, karyologically, a diploid sporophyte alternates with a haploid
gametophyte. Furthermore, unfused gametes developed into haploid sporo-
phytes under cooler conditions and into gametophytes under warmer condi-
Table 1. Chromosome number of Acrothrix pacifica
Karyology: diploid haploid haploid
sporophyte gametophyte sporophyte

Plant: from zygote from zoospore from unfused
in culture in culture gamete in
culture
Chromosome 14-19 8-14 8-14

number: (50% is 9)
tions. This culture study also suggests that the sporophytes prefer the cooler
waters of winter and spring. On the other hand, gametophytes, which have
not yet been found in the sea, prefer the warmer waters of summer and
autumn (Fig. 1).
WINTER SPRING SUMMER
Macroscopic unilocular zoospore microscopic

sporophyte sporangium gametophyte
R! unfused
gamete
+
plurilocular
zygote gamete gametangium
WINTER AUTUMN SUMMER
Fig. 1. Life history of Acrothrix pacifica ( haploid, diploid, R! meoisis).
Sphaerotrichia divaricate
S. divaricata grows epiphytic on Sargassum confusum and S. pilulif-

erum or on rocks l-2 m below low tide mark. The fertile sporophytes have
unilocular sporangia. The sporangia are elongated obovoid measuring 61-
75 x 26-41 urn. Zoospores discharged from the sporangium are pear-shaped
with a single chromatophore and an eyespot 4.7-6.6 x 2.8-3.8 μm in size, and
laterally biflagellated. Soon after settling on the substratum (glass slide)
they became spherical with a diameter of 2.8-4.8 mm. Later, they germinated
and developed into creeping filaments. They branched laterally, forming
dense tufts composed of prostrate and upright systems. Under warmer and
long-day conditions (set 1, 12°C: 16-18 hr), the upper parts of some erect
filament were transformed into uniseriate plurilocular gametangia within
13 days. However, under set 2 conditions of 20°C: 10-14 hr, the tufts were
larger (over 1 cm dia.) and never bore plurilocular gametangia even after 2
months. Under cooler (10°C) and long-day (14-10 hr) conditions, 8-day old
filaments which are usually provided with hairs, developed into simple tufts
composed of small prostrate and sparsely branched erect systems. Within
20 days they matured and farmed plurilocular gametangia. Under moderate
conditions (set 3, 15°C: 14-10hr; set 4, 15°C: 10-14 hr) two types of tufts were
formed: dense tufts at 20°C (sets 1 and 2), and simple ones at 10°C (set 5).
These two types were formed at nearly equal rates and matured and started
liberating gametes within 18 days.
The gametes discharged from the gametangium were morphologically

similar to zoospores, laterally biflagellated, and measured 4.7-6.2 x 3.3-5.2
urn in size. The discharge of gametes was induced when cultures were
transferred from dark to light. The conjugation between gametes was
isogamous or rarely anisogamous. While the rate of the gametic conjugation
was 70-80% under cooler conditions (set 5, 10°C), the process scarcely oc-
cured under warmer conditions (sets 1 and 2, 20°C) and most of the gametes
germinated without fusion and each developed into a gametophyte, repeat-
ing the same generation.
The zygotes became spherical and soon germinated to develop into

cylindrical plants consisting of assimilating filaments, cortical and medul-
lary layers. Under cooler conditions (set 5, 10°C: 14-10 hr), the erect
cylindrical plants branched off and grew larger, with a habit similar to that
of sporophytes in the sea.
Unfused gametes germinated and developed into gametophytes. The

gametophytes grew faster and became fertile earlier under warmer and
long-day conditions (set 1, 20°C: 16-18 hr) than under cooler and short-day
conditions (set 6, 10°C:10-14 hr; set 7, 5°C: 10-14 hr.).
The chromosome number of sporophytes from the sea was 23-30 and that
of gametophytes derived from zoospores was 7-17. Eighty percent of the
gametophytes, however, had a chromosome number of 9-12 (Table 2).
Although the sporophytes derived from conjugated gametes in laboratory
culture had 15-27 chromosomes, 90% of them had 18-24.
This culture study has demonstrated that cooler conditions favor sporo-
phyte growth while warmer temperatures induce sporophytes to produce
unilocular sporangia earlier. Moreover, the study has confirmed that the life
history of S. divaricata is heteromorphic, an alternation of diploid macro-
Table 2. Chromosome number of Sphaerotrichia diuaricata
Karyology: diploid haploid diploid
Plant: sporophyte gametophyte sporophyte

from sea from zoospore from conjugated
in culture gametes in culture
Chromosome 23-30 7-17 15-27

number: (80% is 9-12) (90% is 18-24)
scopic sporophytes and haploid microscopic gametophytes. Meiosis may

occur during the formation of zoospores in the unilocular sporangium on the
diploid sporophyte (Fig. 2).
The results of culture experiments also suggest that although gameto-

phytes have not yet been found in the sea, both gametophytes and sporo-
phytes are present. In summer, gametophytes grow well and mature faster.
Most of the gametes germinated without conjugation, repeating the same
gametophytic generation. While the rate of sexual conjugation increases
from autumn to winter when seawater temperature drops, the sporophytes
derived from zygotes develop well into branched macroscopic fronds during
WINTER SPRING SUMMER
R!
macroscopic unilocular microscopic

zoospore
sporophyte sporangium gameotphyte
unfused
gamete
zygote + gamete plurilocular

gametangium
-
WINTER AUTUMN SUMMER
Fig. 2. Life history of Sphaerotrichia divaricata ( haploid, diploid, R!

meiosis).
winter and spring. The macroscopic sporophytes bear unilocular sporangia

in early summer when seawater temperature rises.
LITERATURE CITED
Ajisaka, T. 1979. The life history of Acrothrix pacifica Okamura et Yamada

(Phaeophyta, Chordariales) in culture. Jap. J. Phycol. 27:7-81.
Ajisaka, T. and I. Umezaki. 1978. The life history of Sphaerotrichia divaricata (Ag.)
Kylin (Phaeophyta, Chordariales) in culture. Jap. J. Phycol. 26:53-56.
Shinmura, I. 1974. Studies on the cultivation of an edible brown alga, Cladosiphon

okamuranus-III Development of zoospores from plurilocular sporangium.
Bull. Jap. Soc. Sci. Fish. 40:1213-1222.

okamuranus-lV. Development of zoospores from unilocular sporangium. Bull.
Jap. Soc. Sci. Fish. 41:1229-1235.

okamuranus-V. Conjugation of gamete and development of zygote. Bull. Jap.
Soc. Sci. Fish. 42:21-28.
Shinmura, I. 1977a. Life history of Cladosiphon okamuranus Tokida from southern

Japan. Bull. Jap. Soc. Phycol. 25 (suppl.):333-340.
Shinmura, I. 1977b. Fundamental studies on the cultivation of an edible alga,

Cladosiphon okamuranus. Jour. Kagoshima Pref. Fish. Sta. II:1-64.
Shinmura, I. and I. Yamanaka. 1974a. Studies on the cultivation of an edible brown

alga, Cladosiphon okamuranus-I. The season for seeding of zoospores and its
growth. Bull. Jap. Soc. Sci. 40:891-902.
Shinmura, I. and I. Yamanaka. 1974b. Studies on the cultivation of an edible brown

alga, Cladosiphon okamuranus-II. Field culture experiments with a culture-
net. Bull. Jap. Soc. Sci. Fish. 40:1133-1138.
Yotsui, T. 1975. Ecological studies on development of gametes of Nemacystis

decipiens. Bull. Nagasaki Pref. Fish. Sta. 1:1-6.
Yotsui, T. 1978. On the life cycle of an edible brown alga, Tinocladia crassa. Bull.
Jap. Soc. Sci. Fish. 44:861-868.
Yotsui, T. 1979a. Influences of environmental conditions on the gametophyte

maturity and gametic fusion of a brown alga, Tinocladia crassa. Bull. Nagasaki
Pref. Fish. Sta. 5:33-38.
Yotsui, T. 1979b. Influences of environmental conditions on the growth and
plurilocular sporangium formation in juvenile sporophytes of a brown alga,
Tinocladia crassa. Bull. Nagasaki Pref. Fish. Sta. 5:39-43.
Yotsui, T. 1980. Studies on the life cycle and artificial propagation of a brown alga
Nemacystis decipiens (Phaeophyta, Chordariales). Spec. Kept. Nagasaki Pref.
Fish. Sta. 7:1-48.
Yotsui, T. and S. Migita. 1974. Experiments in the culture of Nemacytis decipiens

(Phaeophyta, Chordariales). Bull. Jap. Soc. Fish. 40:1223-1228.
ANTITHAMNION SPARSUM,
ITS LIFE HISTORY AND HYBRIDIZATION WITH
A. DEFECTUM IN CULTURE
In Kyu Lee
and
Sung Min Boo
Department of Botany
Seoul National University
Seoul 151, Korea
ABSTRACT
Antithamnion sparsum Tokida isolated from the southern

and western coasts of Korea showed a basically Polysiphonia-type
life history. However, it sometimes exhibited a monoecious repro-
duction and the carpospores released from the cystocarp by self-
fertilization unexpectedly developed into plants bearing sperma-
tangia alone. These male plants were not functional up to 60 days
in culture.
The results of intraspeciflc crosses between populations of A.

sparsum were successful and the hybrid carpospores gave rise to
normal tetrasporophytes. On the other hand, the interspecific
crosses between A sparsum and A defectum were only partly
successful, as evidenced by gonimoblast development and the
release of carpospores in case of A. sparsum (male) × A defectum
(female),but not in A. sparsum (female)×A. defectum (male). These
results seem to suggest that both species are still undergoing
speciation.
INTRODUCTION
Several species of Antithamnion have been cultured in the laboratory.

These are considered to have a regular life history characterized by a
tetrasporophyte, dioecious gametophyte, and carposporophyte (Drew 1955,
Sundene 1959, Lee and West 1980). Some of them, however, show irregular
reproductive cycles in addition to a typical Polysiphonia-type of life history
(Sundene 1964, West and Norris 1966, Rueness and Rueness 1973). Still,
others in culture repeat the tetrasporic generation (Sundene 1962) or exhibit

vegetative growth alone (Whittick and Hooper 1976).
The life history of A. sparsum has not been confirmed in laboratory

culture. The species is expected to show a typical Polysiphonia-type
although cystocarpic plants have not been reported in the field (Tokida 1932,
1954, Kang 1966, Noda 1970, Lee and Kim 1977). The plants are distributed
from Saghalien to Korea (Tokida 1932, Kang 1966) and are closely related
to A. defectum occurring in the Pacific North America (Tokida 1932). The two
species have been distinguished by their cell dimensions, position of tetraspo-
rangia, and gross morphology (Tokida 1932) which, however, can be modified
by environmental factors (Sundene 1962). Thus, Wollaston (1971) suggested
that A. sparsum may be con specific with A. defectum, and Yoshida (1981)
recently proposed the former as a synonym of the latter.
In this paper the life history and reproduction of A. sparsum from Korea
were investigated in laboratory culture, and the assessment of the species
was considered by interspecific cross with A. defectum from the Pacific North
America.
Two isolates of A. sparsum were used in the study. One (#138 was
obtained from the southern coast of Korea at the intertidal zone of Jamdo,
Jinhae Bay (35°03'N, 128°40'E) on November 17, 1979, and the other (#238)
from the western coast of Korea at Gopado, Garolim Bay (36°24'N, 126°21'E)
on May 15, 1980. They were placed in cooler and transferred to the
laboratory for culture. On the other hand, for interspecific cross experi-
ments, culture strain of A. defectum (JAW #240, 241: Lee and West 1980)
from California coast was obtained through the courtesy of Dr. J.A. West,
University of California, Berkeley on October 8, 1980.
Unialgal cultures were set up using the methods of Lee and West (1980).
Preculture of all isolates was maintained in 1/2 PES medium under cool
white fluorescent light at an intensity below 300 lux. After 3-7 days, they
were transferred to the incubation condition in full strength PES media,
under 16-19°C, 800-1300 lux, 16:8 LD, using 7 x 7 cm glasswares. In order
to eliminate diatoms, GeO 2 solution was added to the culture medium for a
while (West 1970). The medium was usually changed every fortnight.
Tetraspores were obtained from fully mature tetrasporangia. After 24

h, the sporelings were transferred to a culture dish. Carpospores were
cultured in the same manner as the tetraspores. To observe fertilization, the
plants bearing cystocarps were isolated individually and cultured for a while
to make sure that unfertilized young branches were newly grown. In
Life History of A. sparsum and cross with A. defectum 41
addition, a few excised apices of a female plant were kept singly in a glass
container for observation of possible parthenogenesis.
RESULTS
The vegetative development of all isolates of A. sparsum was identical in

the laboratory. There was also no detectable difference in the vegetative
morphology of the tetrasporophyte from plants described from the field
(Tokida 1932, 1954).
Vegetative Morphology
Erect thallus with prostrate base is 3-5 cm high and is attached to the
glassware by means of rhizoidal filaments arising from the spherical basal
cells of the determinate branchlets. The rhizoidal filaments with blunt tips
are 4-8-celled. These also arise from the upper portion of the thallus. The
cells of the main axis are 59 urn broad and 348 urn long, about six times as
long as broad at maximum compared to 2-5.5 times in the field (Tokida 1932).
Determinate branchlets on the main axis are opposite, usually 12-16-celled
and semi-pinnately pectinate on the upper side. Indeterminate branches
arise from every 3-7 segments of main branch and basically produce no
branchlets at the opposite side.
Adventitious indeterminate branches sometimes arise from the basal

cell of determinate branchlets. Hairs, which have not been recorded in the
field, occur frequently on the terminal cell of determinate branchlets in the
apical portion of the thallus associated with sexual reproductive structures.
Gland cells are usually located on 2-3 cells of a pinnule and on the average
measure 24 mm long and 19 mm broad.
Reproduction in Culture
The germination pattern of both tetraspores and carpospores isolated

from the field for laboratory culture is basically identical. After attachment,
the spores synchronously develop two opposite primordia. One later forms
a rhizoid and the other develops into an apical cell from which the erect frond
appears (Figs. 1-5). However, there is no definite sequence in the appearance
of the rhizoid or the frond. In some sporelings, the rhizoidal cell, and in
others, the frond, develops much later. Determinate branchlets at first arise
alternately (Figs. 6, 8), or sometimes secondly (Fig. 7), with 16-18 days after
germination. Later, after full growth, they are situated opposite one another.
The tetraspores grow into gametophytes within 20-30 days after germi-
nation. Spermatangial ramuli are observed early, and carposporangial
plants appear about 10 days later. Gametophytes are basically dioecious in
culture. Spermatangia develop in all parts of the pinnules in determinate
branchlets. Each cell of a spermatangial ramulus cuts off a few sperma-
tangial parent cells, which divide once or a few times, forming 2 to 4 sperma-
tangia (Fig. 9).
Carpogonial branches are common in the upper to apical portion of the

thallus, occurring singly or very rarely in pairs successively on the basal cell
of determinate branchlets along the main axis and laterals. The small basal
cell bearing the carpogonial branch grows larger than others and becomes
the supporting cell. A mature carpogonial branch develops a long trichogyne
(Figs. 11, 17).
It is known that a single carpogonial branch on each fertile apex usually

matures into the cystocarp while the rest of the carpogonial branches
degenerate (Wollaston 1968). However, in our culture, two to three carpo-
gonial branches were not fertilized and grew into mature cystocarps at the
same time. Such an occurrence has not been observed previously among the
species of Antithamnion.
The development of the carposporophyte is basically similar to that

preciously described by Wollaston (1968) and Lee and West (1980). The
enlarged supporting cell, after fertilization, cuts off a characteristic
dome-shaped auxiliary cell and becomes acetabuliform. The carpogonium,
cutting off the trichogyne and leaving a cap cell at the top, produces a
connecting cell that fuses with the auxiliary cell (Fig. 12) and through
which the presumed diploid nucleus is moved into the auxiliary cell.
After fusion, the auxiliary cell divides transversely to form the lower foot
cell and upper central cell, which gives rise to the gonimoblast initials (Fig.
15). The gonimoblast cell is produced terminally on the auxiliary cell
and at this stage the axial, supporting, and the foot cells are fused (Fig. 14).
No special involucre is formed but the pinnae of axial cells below the
cystocarp grow upwards, partially surrounding the mature cystocarp. The
carpospores are released in thirty days after the formation of the auxiliary
cell.
Carpospores grow to tetrasporophytes that produce tetrasporangia in

thirty days after germination. Mature tetrasporangia are ovoid to ellipsoidal
and measure 41 x 59 urn on the average. Cruciate tetrasporangia are
pedicellate in one or two cells or sessile on the upper part of the pinnae of
determinate branchlets (Fig. 10) in contrast to the description by Tokida
(1954) that they are pedicellate or sessile. Tetrasporangia release tetraspores
in two weeks.
Thus, A. sparsum in culture completes its life cycle in four months.

Fig. 1-10. Development of vegetative thallus, tetrasporangia and spermatangia

of Antithamnion sparsum in culture. Fig.l. Released tetraspore. Figs. 2-3. One-day
tetrasporelings. Figs. 4-5. 4-celled stage with bipolar apices. Figs. 6-8. Branching
types of young plant. Fig. 9. Development of spermatangia. Fig. 10. Development
of tetrasporangia (ac: apical cell, g: gland cell, r: rhizoidal cell, s: spermatangial
parent cell, t: tetrasporangium).
Fig. 11-15. Development of female reproductive structure of Antithamnion

spars urn Tokida in culture. Fig. 11. Procarps in apical portion of main axis. Fig. 12
Auxiliary cell and connecting cell. Fig. 13. Development of early gonimoblast cells.
Fig. 14. A young cystocarp with secondary gonimoblast initial. Fig. 15. A mature
cystocarp (ac: auxiliary cell, ap: branch apex, c: carpogonium, cc: connecting cell, cn:
central cell, fc: foot cell, fu: fusion cell, gi: gonimoblast initial, pb: protein body, s:
sterile cell, su: supporting cell, t: trichogyne).
A typical Polysiphonia-type life history is repeated three times during

the culture period.
Unusual Life Histories
On the other hand, several monoecious gametophytes (#138-522) de-

rived from male gametophytes were observed in Jamdo isolates during
culture. Each monoecious gametophyte, isolated individually, developed
cystocarps, indicating self-fertilization. However, all the released carpo-
spores germinated and unexpectedly grew into plants bearing spermatangia
quite similar to a common male gametophyte. The fertility of the sperma-
tangia was, however, not confirmed completely. Even though these carpo-
spore-derived spermatangial plants were placed in culture together with
normal female plants for sixty days, no cystocarps were developed.
Intra- and Interspecific Crosses
The cross between the Jamdo (# 138) and the Garolim Bay (#238) isolates
of A. sparsum produced viable carpospores. Crosses between male A.
sparsum and female A. defectum were also successful; normal cytocarps and
viable carpospores were formed (Table 1). However, crosses between female
A. sparsum and male A. defectum produced no mature cytocarps and viable
carpospores. The gonimoblast stopped growing during early development
(Figs. 16-17).
Table 1. Cross experiments among populations of Antithamnion sparsum from

Korea and A. defectum from Pacific North America
Carpospore
Female Male Fertilization
release
A. sparsum #138 × A. sparsurn #138 + +

A. defectum #240 × A. defectum #241* + +
A. sparsum #138 × A. sparsum #238 + +
A. defectum #240 × A sparsum #138 + +
A. sparsum #138 × A defectum #241 + -
* After Lee and West (1980).
Fig. 16-17. Unsuccessful development of gonimoblast in interspecific cross of An-

tithamnion sparsum (female) and A. defectum (male) in culture. Fig. 16. Develop-
ment of connecting cell after fertilization. Fig. 17. Development of early gonimoblast
cell (a: auxiliary cell, ac: axial cell, c: carpogonium, cb: carpogonial branch, cc:
connecting cell, pb: protein body, su: supporting cell, t: trichogyne).
DISCUSSION
As summarized in Fig. 18, A. sparsum basically shows a typical Polysi-

phonia-type of life history (Drew 1955, Sundene 1959, Lee and West 1980).
Some unusual appearances of reproductive structures were also reported
among Antithamnion species not only in culture (Sundene 1962, 1964, West
and Norris 1966, Rueness and Rueness 1973), but also in the field (L'Hardy-
Halos 1968, Knaggs 1969).
Monoecious reproduction is another unusual phenomenon in the life

history of A. sparsum. Hence, the female reproductive structures were
developed on the male gametophyte, and the resulting carpospores, missing
the tetrasporic phase, developed exclusively male gametophytes whose
spermatia were not functional. On the contrary, no female plant developed
monoecious male branches in culture.
There are two previous reports on the monoecism of Antithamnion in

culture (Drew 1955, West and Norris 1966). Thus, monoecism may not be a
rare occurrence in this genus as it is also apparently common in Callitham-
nion, a related genus. However, it is peculiar that such monoecious plants
in the present experiment miss the tetrasporophyte. Whittick and West
(1979) demonstrated in the life history of a monoecious species of
Callithamnion that the carpospores from the cystocarp developed into
tetrasporophytes as seen in regular dioecious plants. Polanshek and West
(1977) also reported the repetition of cystocarpic generations in the life
history of Gigartina papillata. However, the lack of tetrasporic generation
would differentiate A. sparsum from these species.
While Sundene (1962, 1964) and West and Norris (1966) reported that
apomeiotic tetraspores in the gametophyte of Antithamnion developed only
gametophytes of the same sex as the parent, Rueness and Rueness (1973)
noted that the tetraspores in the male gametophytes developed into both
male and female plants. A similar phenomenon also occurs in Dasysiphonia
chejuensis (Lee and West, unpublished data). Light conditions apparently
play an important role in inducing sexual reproductive structures in An-
tithamnion (Rueness and Rueness 1973).
Van de Meer and Todd (1977) reported mixed phase reproduction in the
life history of Gracilaria sp., and suggested that the sexuality is controlled
by the genetic recombination of a pair of alleles rather than a pair of chro-
mosomes. But this was in case of diploid tetrasporophytes. They did not
explain the mixed reproduction in the gametophytes observed by West and
Norris (1966) and Rueness and Rueness (1973).
The monoecism of A. sparsum seems to be genetically stable since the

female branches that develop on the male thallus are sexually irreversible
and produce cystocarps successively, as in the case of tetrasporangium
meiosis
Tetrasporophyte (2n) Tetraspore (n)
♂ Gametophyte .. ♂ Gametophyte (n) ♀ Gametophyte (n)

?
Carpospore (2n) Carpospore
♂
♀
Cystocarp
Cystocarp (2n)
Fig. 18. Life h istory of Antithamnion sparsum in culture.
formation on cultured gametoph ytes of Symphyocladia pennata and D.

chejuensis (Lee and West 1979). However, th e presence of non-functional
spermatia from such male thalli suggests that these gametophytes would be
diploid rath er than haploid.
In reporting A. sparsum as a new species, Tokida (1932) mentioned that

this species showed more affinity to A. defectum Kylin and that both species
were basically distinguish ed by differences in cell dimension. Wollaston
(1971), therefore, doubted that both species might be conspecific and Yoshida
(1981) treated the former as a synonym of the latter. In fact, such morpho
logical characters used in separating the two species are easily subject to en
vironmental influence (Sundene 1962, Norris and West 1967), considered
vague (Wollaston 1968), and are also of little value in th is study. A
comparison of some significant taxonomic characters between A. sparsum
according to Tokida and A. defectum Kylin is shown in Table 2.
Table 2. A comparison of some significant taxonomic characters between Antitham-

nion sparsum and A. defectum Kylin
Characters A. defectum* A. sparsum** A. sparsum***
Attachment rhizoidal rhizoidal rhizoidal

Branching pattern opposite opposite opposite
Cell dimension 2-3 times 2-5.5 times 5-6 times
Cell tip tapering blunt blunt & tapering
Gland cell on 2-5 cells on 2-3 cells on 2-3 cells
Tetrasporangia 1-2-pedicellate 1-pedicellate 1-2-pedicellate
ovoid /80 µm sessile /ovoid sessile / ovoid
long 59 × 78 µm 41 × 59 µm
Spermatangia adaxial adaxial adaxial
*After Kylin (1925), Wollaston (1971).

**After Tokida (1932, 1954), Lee and Kim (1977).
***After present study.
The formation of carposporophyte and production of viable carpospores

in the cross between A. sparsum (male) × A. defectum (female) could indicate
that both species are partially interfertile. However, the reciprocal cross be-
tween the female A. sparsum and the male A. defectum was not successful.
They produced an auxiliary cell after fertilization, but failed to develop
gonimoblast cells (Figs. 16,17) which could suggest that both species are still
undergoing the speciation process.
LITERATURE CITED
Drew, K.M. 1955. Sequences of sexual and asexual phases in Antithamnion

spirographidia. Nature (London) 175:813.
Kang, J.W. 1966. On the geographic distribution of marine algae in Korea. Bull.
Pusan Fish. Coll. 7:1-125.
Knaggs, F.W. 1969. A review of Florideophycidean life histories and of the culture
techniques employed in their investigation. Nova Hedwigia 18:293-330.
Kylin, H. 1925. The marine algae in the vicinity of the Biological Station of Friday
Harbor Wash., Acta Univ. Lund 21:1-87.
Lee, I.K. and Y.H. Kim. 1977. A study on the marine algae in the Kwang Yang Bay.
3. The marine algal flora. Proc. Coll. Natur. Sci., S.N.U. 2:113-153.
Lee, I.K. and J.A. West. 1979. Dasysiphonia chejuensis gen. et sp. nov. (Rhodophyta,
Dasyaceae) from Korea. Syst. Bot. 4:115-129.
Lee, I.K. and J.A. West. 1980. Antithamnion nipponicum Yamada et Inagaki
(Rhodophyta, Ceramiales) in culture. Jap. J. Phycol. 28:19-27.
L'Hardy-Halos, M.-Th. 1968. Les Ceramiaceae (Rhodophyceae, Florideae)des cotes

Bretagne. 1. Le genre Antithamnion Nageli. Rev. Algol. 9:152-183.
Noda, N. 1970. On the marine algae of Awashima island in the Japan Sea. Bull. Jap.
Phycol. 18:147-153.
Norris, N.E. and J.A. West. 1967. Notes on the marine algae of Washington and
southern British Columbia. II. Madrono 19:111-116.
Polanshek, A.R. and J.A. West. 1977. Culture and hybridization studies on
Gigartina papillata (Rhodophyta). J. Phycol. 13:141-149.
Rueness, J. and M. Rueness. 1973. Life history and nuclear phases of Antithamnion
tenuissimum, with special reference to plants bearing both tetrasporangia and
spermatangia. Norw. J. Bot. 20:205-210.
Sudene, O. 1959. Form variation in Antithamnion plumula. Experiments on

Plymoth and Oslofjord. Nytt. Mag.Bot. 7:181-187.
Sundene, O. 1962. Reproduction and morphology in strains of Antithamnion boreale

originating from Spitsbergen and Scandinavia. Skr. Norke Vidensk. A-kad.
Oslo I. Mat.-Nat. Kl., N.S. 5:1-19.
Sundene, O. 1964. Antithamnion tenuissimum (Hauck) Schiffner in culture. Nytt

Mag.Bot. 11:5-10.
Tokida, J. 1932. On the two new species of Antithamnion from Japan. Bull. School
Fish., Hokkaido Univ. 2:1-34.
Tokida, J. 1954. The marine algae of the southern Saghalien. Mem. Pish. Hokkaido
Univ. 2:1-264, 15 pl.
van der Meer, J.P. and E.R. Todd. 1977. Genetics of Gracilaria sp. (Rhodophyceae,
Gigartinales). IV. Mitotic recombination and its relationship to mixed phases in
the life history. Can. J. Bot. 55:2810-2817.
West,J.A. 1970. The life history of Rhodochorton concrescens in culture. Bry. Phycol.
J. 5:179-186.
West, J.A. and R.E. Norris. 1966. Unusual phenomenon in the life histories of
Florideae in culture. J. Phycol. 2:54-57.
Whittick, A. and R.G. Hooper. 1976. The reproduction and phenology of Antitham-
nion cruciatum (Rhodophyta, Ceramiaceae) in insular Newfoundland. Can. J.
Bot. 55:520-524.
Whittick, A. and J.A. West. 1979. The life history of a monoecious species of
Callithamnion (Rhodophyta, Ceramiaceae) in culture. Phycologia 18:30-37.
Wollaston, E.M. 1968. Morphology and taxonomy of southern Australian genera of

Crouaniae Schmitz (Ceramiaceae, Rhodophyta). Aust. J. Bot. 16:217-417.
Wollaston, E.M. 1971. Antithamnion and related genera occurring on the Pacific
coast of North America. Syesi. 4:73-92.
Yoshida, T. 1981. Notes on Antithamnion sparsum Tokida (Rhodophyta, Cer-

amiaceae). Jap. J. Phycol. 29:47-50.
WATER QUALITY ASSESSMENT OF THE LANGAT
RIVER, SELANGOR, MALAYSIA USING
THE NATURAL ALGAL PERIPHYTON COMMUNITY
AND LABORATORY BIOASSAYS OF TWO
CHLORELLA SPECIES
Ann Anton
Department of Biology
Faculty of Science and Environmental Studies
University of Agriculture
Serdang, Selangor, Malaysia
ABSTRACT
The physico-chemical conditions in ten sampling stations off

the headwaters of the Langat River, Selangor, Malaysia were
studied. Monitoring was done twice a month from June to Decem-
ber 1980. Changes in water quality were observed downstream. A
total of 35 taxa of periphyton in four main divisions of algae were
identified. The decrease in the number of species in downstream
stations could be due to changes in the river rather than to chemical
pollution. Two species of Chlorella, namely, C. pyrenoidosa and C.
vulgaris, were grown in filtered river water obtained from the
different sampling stations to assess their growth responses. Results
suggest that pollution in the Langat River was caused mainly by
heavy siltation rather than chemical pollutants.
INTRODUCTION
It has been documented by the Environmental Protection Society Malay-

sia that 42 of the country's rivers are polluted and another 23 threatened by
human activities. However, only a few studies have been conducted on these
rivers. Most of the water pollution programs in Malaysian rivers have been
undertaken to assess the toxicity of pollutants.
This work is part of a series of surveys to study the effect of impoundment

on a river ecosystem. The study assessed the quality of the waters of Langat
River and determined the major cause of water pollution. Changes in the
periphyton community as well as the growth of two Chlorella species
cultured under laboratory conditions in water obtained from the river were
used as indicators of water pollution. The area chosen for the study was the
headwaters of the Langat River in the state of Selangor, flowing in the
geographical region of Latitudes 3°05'N to 3°13'N and Longitudes 101°47'E

to 101°53'E. The river flows along the valleys of hills about 1 000 ft above sea
level. The area was chosen because of: (1) the construction of the dam at the
upper reaches of the river that has resulted in a change in the physical
characteristics of the river; (2) an increase in human activities along the
river, such as establishment of towns and a rubber-processing factory, tin
mining, and paddy cultivations; (3) the utilization of water from the dam as
a source of water supply going through two treatment plants, 18 miles and
30 miles away, and (4) the increase of human settlers along the river,
creating domestic "tips" at various points along the river.
MATERIAL.S AND METHODS
Ten stations were established along the course of the river, each
approximately set 4.44 km apart, except for Stations 2, 3, and 4 which
were located in one area. Station 4 receives water from a mountain
stream, Station 2 from the reservoir, and Station 3 from waters used to
turn the turbines of a power plant operated by the National Electricity
Board.
Water samples were collected at each station twice a month from June
to December 1980. Other physical parameters that were measured included
pH using a pH meter, air and water temperatures, current velocities,
conductivity using a YSI 33-meter, and water depth and transparency. The
concentrations of sodium and potassium ions in the water samples were de-
termined using the flame photometer, nitrate-nitrogen (NO3-N) by the
Brucine method, and phosphate-phosphorus (PO4-P) by level II of the calo-
rimetric method. A slight modification in the phosphate determination was
the use of butan-1-10 instead of hexanol. The amount of total suspended and
dissolved solids was also determined at each station.
Samples of periphyton were collected from each station by scrapping

stones and rocks covered with algae. River water used in the culture of
Chlorella was collected from each station in 5-liter plastic bottles. On
reaching the laboratory, 25 ml of the river water was coursed through a 0.45
μm millipore filter paper into sterile flasks. One ml each of Chlorella
vulgaris and C. pyrenoidosa pure cultures was washed in 5% sodium
bicarbonate and then innoculated aseptically into separate flasks. There
were two replicates for each water sample. The initial density of cells in the
inoculum was also determined. The cultures were incubated under white
light provided by two fluorescent tubes 120 cm above the flasks and a 12-hour
light and 12-hour dark cycle. Growth of the cultures was quantified by
counting the number of cells in each flask up to a period of seven days after
innoculation, using a haemacytometer. Growth of the Chlorella cultures
was measured using the intrinsic growth rate, r:
Water Quality of Langat River, Malaysia 53
ln Nt - ln No
r = —————————
t
where: Nt = number of cells at time t;
N = number of cells at time 0;
t = maximum time when Nt occurs.
RESULTS
Analyses of the data on the physico-chemical properties of the river

water showed that the pH was generally lower at the downstream stations.
The highest value was recorded at Station 1 (pH = 7.6) and the lowest at
Station 10 (pH = 6.2). Water temperatures were constant at the different
stations except in Station 3 (21.9°C) which receives water from the power
station. The water used to turn the turbines originates from streams in the
mountain and is piped to the power station resulting in lower temperature.
In contrast, downstream stations were characterized by higher temperature
(28.5"C). Data on dissolved oxygen and carbon dioxide showed a drop in D.O.
downstream after Station 8, with Station 10 recording the lowest value of
7.05 mg l-1 and a corresponding increase in dissolved carbon dioxide (6.05 mg
l-1)• The level of phosphate-phosphorus in the river water was generally low,
varying from a low of 29.25 μg l-1 at Station 3 to a maximum of 72 μg l-1 at
Station 10. Nitrate-nitrogen values fluctuated from station to station. The
highest (45.17 mg l-1) was recorded at Station 1; downstream stations
recorded lower values.
The amount of both suspended and dissolved solids increased at the

downstream stations with a corresponding decrease in the water transpar-
ency. The suspended solids increased at an average of 20 mg l-1 at each
station from a minimum value of 30.6 mg l-1 at Station 3 to a maximum value
of 193.3 mg l-1 at Station 10. The amount of dissolved solids at Station 9 (91.3
mg l-1) was almost double that recorded upstream at Station 3 (55 mg l-1).
A qualitative survey of the periphyton flora recorded 23 species of algae

belonging to four major divisions. The diatoms were the most abundant.
Bioassay studies conducted utilizing two species of Chlorella, C. pyre-

noidosa and C. vulgaris, showed the maximum r for both species was
recorded in cultures utilizing river water from downstream stations, e.g.,
the highest r value was recorded at Station 10 while lower values were
recorded at the upstream stations.
DISCUSSION
The physical and chemical data from the ten stations indicated a change
in water quality further downstream. In general, stations further down-
stream showed low D.O. and pH levels and high concentrations of total
suspended and dissolved solids, nitrate-N2, and phosphate-phosphorus.
These observations suggest that the Langat River may already be polluted.
The more acidic conditions downstream could be attributed to wastes dis-
charged from the rubber factory as well as from domestic sources. Phos-
phates came mainly from detergents especially in Station 6 where the water
is intensively used for daily human activities. Nitrates originated mainly
from the dam as shown by the increase in its value at Station 1 as a result
of increased degradation of organic matter, viz, vegetation in the newly
impounded dam.
Construction work at the dam as well as road-building near Station 8

could account for the high levels of both suspended and dissolved solids that
are brought downstream.
The distribution of periphyton at all 10 stations showed a general

decrease; in the total number of species toward the downstream stations.
However, as the environmental conditions at each station change when the
river becomes deeper further downstream, the decrease in the number of
species could be due to the physical change in the depth of the river rather
than a result of pollution. The influence of physical changes is a common
problem encountered in the use of biotic indicators of pollution in natural
communities. The major parameters that determined the composition of the
periphyton community in Langat River appeared to be the nature of the river
beds and the velocity of the water currents.
It was hoped that the use of the laboratory culture of Chlorella to

determine the effect of any pollutant on the test organism would minimize
the problem posed by differences in environmental conditions. The higher
cell numbers of Chlorella grown in water obtained from stations further
downstream could indicate that growth of the alga was not determined by
any specific chemical substance present in the water medium since there
was a decrease in pH and a rise in PO4-P and NO3-N downstream. In
preparing the medium for the culture of Chlorella, the river water was
previously filtered. Filtration removed most of the solid particles. The
change in the quality of water further downstream is apparently caused by
the high levels of total suspended and dissolved solids. Pollution of Langat
River, on the whole, then is due to heavy siltation as a result of dam and road
constructions.
GROWTH AND DEVELOPMENT OF TRENTEPOHLIA
ODORATA IN CULTURE
C.T. Lee
Y.C. Wee
and
K.K. Ho
National University of Singapore
Kent Ridge, Singapore
ABSTRACT
Trentepohlia odorata, a filamentous green alga, grows pro-

fusely and imparts an orange color on walls of many high-rise
apartment buildings in Singapore. Since bulk cultures are needed
in the screening of possible chemicals to control infestation, vari-
ations in the growth and development of the alga in the laboratory
under different physico-chemical conditions were investigated.
Bold's medium either as a liquid or solidified with agar was

suitable substrate for the culture of the alga. An acidic growth
medium (pH 4.5-6.5) favored the formation of sporangia that
developed into yellowish-green colonies, whereas a basic medium
(pH 7.5-9.5) produced green colonies and no sporangia. An agar
concentration of 0.7% as well as the addition of thiamine to the
liquid basal medium enhanced the production of sporangia devel-
oping into numerous daughter colonies after two months. High
relative humidities increased colony growth and promoted the
formation of yellow colonies of cylindrical cells.
On walls of buildings, the cells are elliptical to barrel-shaped,

bright orange, and possess few sessile sporangia. In cultures where
moisture and nutrients are not limiting, cells are narrow and
elongated, with the filaments radiating from a central mass, and
the sporangia are of the sessile and pedicellate types.
INTRODUCTION
Trentepohlia is a filamentous green alga easily recognized by its charac-

teristic orange color due to an abundance of carotene in oil globules in its
cells. The alga is common in the tropics, with a few species even in the
temperate and sub-arctic areas. Five species have been recorded in Sin-
gapore (Johnson 1978), the more conspicuous being T. aurea (L.) Martius
and T. odorata (Wigg.) Wittr. The former is commonly found on tree trunks
and surfaces of rocks, forming short streaks of orange to green filaments. In
the shade, the filaments may be green while under full sunlight they may
turn orange. The latter forms a crustose layer on concrete surfaces and
building walls, especially those of the high-rise apartment buildings, giving
the surface a distinct tinge of orange (Wee and Lee 1981). T. odorata is of
particular interest in Singapore as its proliferation on surfaces of building
hastens their repainting. Hence the use of paints containing an effective
anti-algal chemical may reduce infestation. Unfortunately, anti-algal paints
are not as commonly available as those with anti-fungal additives. Faint
manufacturers may claim that their anti-fungal products are as effective on
algae as on fungi, but this has yet to be proven. Screening tests for potential
algicides, undertaken locally (Ngiam et al. 1973, Ngiam and Yong 1975) as
well as elsewhere (Drioko and Crylly 1974, Fitzgerald 1964) utilized blue-
green algae rather than T. odorata, the major organism colonizing on bare
walls in Singapore. Natural populations of the alga are, however, insuffi-
cient if large-scale screenings are to be undertaken. It was therefore
necessary to mass-produce the alga in the laboratory for screening purposes.
The following report gives an account of the influence of the pH and nutrient
strength of the culture medium, vitamin supplements, and relative humid-
ity on the growth and development of T. odorata under laboratory conditions.
The study, hopefully, will also provide basic information necessary for
controlling infestation.
Plant Materials
Samples of 7'. odorata were scraped from walls of buildings around the
former Bukit Timah Campus of the University of Singapore. A portion of the
scrapings was examined under the microscope and the morphological fea-
tures were noted. The rest was inoculated in Bold's basal medium contained
in 125 ml Erlenmeyer flasks (Nicholas and Bold 1965). These flasks were
placed under continuous lighting for three to four weeks to allow the
Trentepohlia, together with any other algae, to proliferate. The Trentepohlia
colonies were then isolated and reinoculated a number of times in the same
medium until pure cultures were obtained. In all experiments, an inoculum
of two colonies of equal diameter from the stock culture was introduced into
each flask.
pH of Culture Medium
Bold's medium after autoclaving had a pH of 6.5 which served as the

Trentepohlia odorata in Culture 57
control. Media of pH 4.5, 5.5, 7.5, 8.5 and 9.5 were prepared by adding dilute
sodium hydroxide or hydrochloric acid to autoclaved Bold's medium.
Agar Concentration
T. odorata was also cultured in Bold's medium with various agar concen-
trations of 0, 0.1, 0.3, 0.5, 0.7, 1.0, 1.3, and 1.5%. To vary the agar
concentration, the procedure of Hunter et al. (1966) was followed, i.e., the
agar and the mineral medium of double strength were prepared separately,
autoclaved, cooled to 50°C, and then mixed.
Strength of Culture Medium
Bold's basic medium of 0, 25, 50, 75, 100, and 200% strength were
prepared by varying the amounts of distilled water added to the basic
nutrients. The 100% or full strength medium served as the control and 0%
was plain distilled water.
Vitamins, namely thiamine hydrochloride (Vitamin B1), d-biotin (Vita¬

min H), and Vitamin B12 were used to supplement the culture medium. In
all cases, the vitamins were first dissolved in distilled water as stock
solutions, autoclaved, and then added to make concentrations of 0.1, 0.2, 0.3,
0.4, 0.5, and 0.6 mg/1 in the case of thiamine, and 0.2, 0.5, 1.0, 1.5, 3.0, and
6.0 mg/1 for the other two vitamins.
Saturated solutions of different chemicals were used to vary relative

humidities (Clayton 1967). These solutions were placed in petri dishes and
a glass slide with the alga was placed above, supported by a watch glass
inside. The petri dish was sealed with paraffin to maintain a constant
relative humidity within the dish. The dish was placed on the laboratory
bench at ambient temperature and standard lighting for 12 hours followed
by 12 hours of darkness. Each was replicated thrice, using algal colonies of
more or less similar size. Readings of length and breadth of cell as well as
length of sporangia were made after a three-week culture period.
RESULTS
T. odorata consists of a series of subspherical to barrel-shaped to

elliptical cells joined end to end in short chains. The cells are 9-17 urn long
and 7-15 μm wide with smooth walls of about 1 μm thick. The prostrate
filaments are more developed than the erect, and in many cases only the
former are found. Sporangia are sessile, terminal or intercalary, spherical
to oblong in shape, and are 10-25 μm long.
When grown in either liquid or solid culture, the filaments became

cylindrical and elongated. These filaments radiated from the central core of
original subspherical to elliptical cells and branched profusely. In liquid

medium, the colonies were circular and compact, showing a heterotrichous
condition. The typical cells developing in culture were longer than broad,
measuring 29-58 μm long and 30-5 μm wide. Under the light microscope, the
cells contained less carotene than those collected from walls. The chloro-
plasts appeared as parietal bands although discoid forms were also observed.
In culture, sessile and pedicellate sporangia developed laterally and are

generally bigger than those observed in the natural habitat. The mature
sporangia possessed a characteristic beak-like protrusion at the apical end.
pH of Culture Medium
The optimum pH was 7.5 in liquid culture, based on colony size, relative
abundance of new colonies formed, and dimensions of apical cells (Table 1).
Generally, growth was better with increasing pH. Sessile sporangia were
noted between pH 4.5 and 6.5, these being terminal at pH 4.5 and 5.5, but
lateral and confined to the older cells toward the center of the colony at pH
6.5 (Table 2). At the normal pH of 6.5, the cells were cylindrical and the
colony profusely branched. The few small oil globules containing carotene
were clustered at the center of the cells. With decreasing pH, the cells
became barrel-shaped, then elliptical, and accumulation of carotene in the
cells increased. On the other hand, increasing pH reduced the carotene
content and the chloroplasts became prominent bands.
Table 1. Growth of T. odorata in liquid medium at different pH after 6 weeks
Mean increase in
Mean size of apical cells (μm)*
pH diameter of colony
(mm) Width Length
4.5 0.05 9 29 (20-35)

5.5 0.75 4 35(17-46)
6.5 1.10 3 46 (32-58)
7.5 1.20 3 55 (40-70)
8.5 1.15 5 60 (46-70)
9.5 1.15 5 60 (46-70)
*n = 50.
Strength of Culture Medium
Dilution of Bold's medium depressed the growth of the alga, increased its
carotene content, and caused a reversion of the characteristic cylindrical
forms of actively growing cells to the elliptical shape of cells found naturally
on concrete walls (Table 3). At nutrient strengths of 75, 100, and 200%,
growth appeared normal, the cells remained green, and their shape was
characteristically cylindrical. When the normal concentration (100%) of the
Table 2. Morphological characteristics of T. odorata grown in liquid medium at

different pH after 6 weeks
Morphological characteristics*
pH Cell shape
Color of filament Sporangia Carotene
4.5 elliptical (++) yellow-green sessile and (+++)

cylindrical (+) terminal (++)
5.5 elliptical (+) yellow-green sessile; terminal (+++)

barrel-shaped (++) and lateral (++)
cylindrical (+)
6.5 cylindrical (+++) yellowish-green sessile, usually (++)

(profuse lateral (+)
branching)
7.5 cylindrical (+++) light-green (-) (++)
8.5 cylindrical (+++) green (-) (+)
9.5 cylindrical (+++) dark-green (-) (-)
*(-) absent; (+) sparse; (++) common/intense; (+++) very common/very intense.
Table 3. Cell shape and colony color of T. odorata in different strengths of Bold's
liquid medium after 6 weeks
Concentration
Cell shape* Colony color
of Bold's medium
0% elliptical (++) amber

sub-spherical (+++)
25% elliptical (++) yellow
cylindrical (+)
50% barrel-shaped (+) yellow
cylindrical (++)
75% barrel-shaped (+) yellow-green
cylindrical (++)
100% cylindrical (+++) green
200% cylindrical (+++) green
*(+) sparse; (++) common; (+++) very common.
medium was doubled, the green colonies consisted of cells packed with
chloroplasts, and the presence of carotene was hardly visible. Transferring
the alga from the diluted 25 and 50% culture media to normal strength
caused the cells to become green within four days and newly formed cells
were cylindrical.
Agar Concentration
The mean number of colonies after eight weeks increased from 33 in the
liquid medium (0%) to 390 in the medium with 0.7% agar (Table 4).
Similarly, the mean number of sporangia formed per colony increased with
higher agar concentrations up to 0.7%. At 1-1.5% agar concentrations, when
the medium was solid, the mean number of colonies decreased. The mean
number of sporangia per colony similarly showed a decline in the 1.3 and
1.5% agar media. At concentrations of 0.1-0.5%, the sporangia were sessile
and confined to the central region of the colony while sessile and pedicellate
sporangia developed all over the colony at agar concentrations of 0.7%; and
above.
Growth characteristics also varied with different agar concentrations.

In liquid and 0.1% agar media, the colonies were spherical and grew
suspended in the medium. At a higher concentration of 0.3%, the colonies
ranged from spherical to circular and flat, with the former suspended in the
medium and the latter growing on the surface. At concentrations of 0.5% and
above, the alga grew only on the surface of the medium with the circular
colonies close to one another and the filaments radiating delicately from the
center of each colony. In solid media of 1.3 and 1.5%, the circular colonies
developed a heterotrichous habit with erect filaments growing from the
center.
Table 4. Effects of agar concentrations on the growth of T. odorata after 8 weeks
Sporangial
State of Mean no. Colony shape
% Agar no. per
substratum of colonies and formation
colony
0 Liquid 33 8 Spherical, suspended in medium

0.1 Liquid 119 2 Spherical, suspended in medium
0.3 Gel 220 18 Spherical, suspended in medium
and circular on surface of
medium
0.5 Semi-solid 303 28 Circular and compact, on surface
of medium
0.7 Semi-solid 390 87 Circular and compact, on surface
of medium
1.0 Firm 14 70 Circular and compact, on surface
of medium
1.3 Hard 10 12 Circular and compact, with
aerial filaments
1.5 Hard 8 19 Circular and compact, with
aerial filaments
Vitamins
The addition of thiamine hydrochloride to liquid medium increased algal

wet weight through the enhancement of sporangia] formation which pro-
duced more colonies (Table 5). Most of the sporangia were intercalary or
terminal in position, d-biotin and vitamin B12 did not affect the growth of the
alga. The number of daughter colonies did not increase with the presence of
either vitamin in the medium.
The addition of any of the three vitamins did not apparently affect cell
size and shape. However, it was noted that d-biotin and Vitamin B12 at the
higher concentrations enhanced carotenogenesis since the culture appeared
more yellowish than those at lower concentrations. In the absence of
vitamins, cultures were green.
Table 5. Effects of thiamine on growth of T. odorata after 6 weeks
Thiamine Mean wet weight Cell

No. of Mean no.
conc. of colonies length range
sporangia of colonies
(mg/1) (mg) (μ)*
0 0 25 30.0 26-49(35)
0.1 3 26 43.4 29-46(41)
0.2 10 34 122.2 38-46 (35)
0.3 12 42 140.6 32-46 (38)
0.4 24 76 234.5 41-49(44)
0.5 39 72 154.0 26-41 (35)
0.6 26 48 142.0 38-46(41)
*Figures in parentheses are means of 50 cells measured from the first to fifth
cell after the apical cell.
Relative Humidity (RH)
Growth of the colony increased with increasing relative humidities

(Table 6). In the absence of moisture in the ambient air or at 0% RH, colonies
failed to form new cells nor increase in size, and turned uniformly green after
three weeks. Branching of the filaments was sparse and the cells of the
peripheral filaments were cylindrically elongated, a shape typical of the alga
under liquid culture.
At 35% RH, branches developed from the peripheral filaments. The

elliptical cells of the branches contained chloroplasts and numerous
small oil globules filled with carotene to impart a light color to the colony
(Table 6). At 55% RH, more profuse and longer branches were formed whose
cells were longer and cylindrical but subspherical to oval at terminals. At
higher humidities, the new cells were cylindrical and much elongated. The
yellowish colonies had older central cells packed with carotene-containing
Table 6. Effects of relative humidity (RH) on the growth and morphology of T. odorata
after 3 weeks
Mean increase in Shape

diameter of Color of colony of new Sporangial
RH(%)
origin
colony (µ) cells
0 0 Green
32 30 Light yellow Elliptical
55 50 Pale green Elliptical and
cylindrical
81 200 Yellow Cylindrical Intercalary
95 250 Yellow Cylindrical Intercalary
and apical
100 1100 Yellow Cylindrical Lateral and
apical
oil globules and younger peripheral cells with smaller globules and a less
intense carotenoid color. At 95 and 100% RH, most of the apical cells of the
peripheral filaments rounded off into sporangia. Many of these apical
sporangia were empty, indicating that the zoospores had been liberated as
confirmed by the presence of ten new colonies under an RH of 100%.
DISCUSSION
T. odorata is commonly found growing on walls of high-rise buildings in

Singapore, especially in areas where there is an excessive runoff of rain
water. The presence of the alga imparts an orange tinge to infested surfaces
which turn blackish with the presence of blue-green algae. Although T.
odorata is a green alga it appears orange due to the abundance of carotene
which masks the chlorophyll. Carotene is regarded by Senn (1911) as a food
reserve since its concentration declines when nutrients are readily available
and growth is active. When moisture and nutrients are limiting, the alga
accumulates carotene. Also, under shade conditions, the pigment may be
completely lacking and the alga appears green (Fritsch 1971).
it has also been suggested that carotene protects the chlorophyll against
phyto-oxidation under conditions of high light intensity (Griffiths et al.
1955). Thus, the accumulation of carotene by T. odorata growing on walls of
buildings enables the alga to survive such an exposed habitat. The ability
of the alga to withstand long dry periods also helps in its adaptation to a
habitat where moisture is only available during rainy months. The presence
of numerous, highly refractile fat granules of various shapes and sizes within
the cells would also contribute to the alga's ability to survive drought (Fritsch
1916, Piercy 1917). The complete absence or paucity of large vacuoles in the
protoplasm may likewise enable the cell to withstand dessication (Fritsch
1922). The ability of the cell walls to rapidly absorb atmospheric moisture,
as observed in T. aurea (Howland 1929), is also another adaptation to dry
conditions.
Geitler (1923) earlier pointed out that the appearance of cells of Trente-
pohlia under moisture stress and when nutrients are limiting closely
resembles the resting stages of many other algae. This observation is
supported by the morphological changes in the cells of T. odorata when
cultured in mineral nutrients. The shortly stringed elliptical cells give rise
to branched filaments of elongated cells, giving the impression of germinat-
ing resting cells. The newly formed colony, ball-like in liquid medium and
circular in solid, takes a yellow-green appearance as neither carotene nor
chlorophyll is the predominant pigment. In liquid culture and under
decreasing pH, the cells revert to the natural elliptical shape and have a high
carotene content. A reduction in relative humidity resulted in a similar
reversion.
In its natural habitat where growth is extremely slow, T. odorata is

usually sterile. The alga, with its short chain of elliptical cells reproduces
vegetatively by cells which readily separate. During periods of rain,
detached cells are washed down the surface as indicated by the characteris-
tic vertical orange striations on walls infested with the alga. During dry
periods, the detached cells are dispersed by wind as fine dust. Sporangia are
rarely formed in nature but, when present, are sessile, terminal, or interca-
lary in position. In laboratory cultures, sessile and pedicellate sporangia are
commonly formed and occur laterally. At pH 5.5 and 4.5 when cells revert
to the elliptical shape, the sporangia formed are of the terminal sessile type
rarely observed in nature.
The addition of thiamine to the liquid medium also enhances sporangia]

production, hence, the formation of daughter colonies. However, the produc-
tion of sporangia and daughter colonies is less than that in a medium with
0.7% agar. The necessity of vitamins for the growth of various groups of algae
has been reported. A few members of Cyanophyta are auxotrophs with an
absolute requirement for vitamin B12 (Van Baalen 1961). In Ochromonas, a
Chrysophyta, auxotrophy for various vitamins (thiamine, biotin, B12) is
common (Provasoli 1958). Members of the Pyrrophyta, especially the marine
dinoflagellates are similarly known to require vitamin B12 (Bold and Wynne
1978). Among the Chaetophorales (Chlorophyta), the first report of a
member requiring an external source of vitamin is Draparnaldiopsis
(Johnstone 1977). The positive response of T. odorata to thiamine (but not
to biotin and B12) is another record for the Chaetophorales.
The great increase in the number of daughter colonies in a semi-solid

medium (with 0.7% agar) can be attributed to the zoospores requiring
something solid to attach to prior to germination. These zoospores have been
observed to swarm for some time after liberation from the sporangium. They
then come to rest, attach their anterior end to the surface of the glass slide,
and rotate for a while before they withdraw their flagella and then germi-
nate. The substratum partly solidified with 0.7% agar is presumably hard
enough for the zoospores to attach to and soft enough for them to disperse
around, thus providing a larger area for the zoospores to swarm and
subsequently get attached prior to germination. When the medium is solid
(with 1% agar), the zoospores would not be able to penetrate the surface for
swarming.
T. odorata can be cultured easily in the laboratory using Bold's medium

either in a liquid or solidified with agar. The alga prefers slightly alkaline
(pH 7.5) environment although it can grow within a wide range of pH This
preference for an alkaline environment is not surprising. In nature it
colonizes whitewashed building walls as well as painted surfaces where the
pH is distinctly alkaline.
The addition of 0.4 ppm thiamine enhanced growth and stimulated the
production of sporangia which developed into numerous daughter colonies.
Culturing the alga in a semi-soil medium (with 0.7% agar) also promoted
the formation of daughter colonies.
LITERATURE CITED
Bold, H.C. and M.J. Wynne. 1978. Introduction to the algae. New Delhi: Prentice-
Hall.
Clayton, C.N. 1967. (termination of spores of powdery mildew fungi at various

relative humidities. In: A. Kelman (ed). Source book of laboratory exercises in
plant pathology. San Francisco & London: H.H. Freeman, p. 231.
Drioko, R.W., and J.B. Crylly. 1974. Control of algal growth on paints at tropical
locations. J. Paint Technol. 46:48-55.
Fitzgerald, G.P. 1964. Factors in the testing and application of algicides. Appl.
Microbiol. 12:247-253.
Fritsch. F.E. 1916. The algal ancestry of the higher plants. New Phycol. 15:233-250.
Fritsch,. F.E. 1922. The moisture relations of terrestrial algae. Ann. Bot. 36:1-19.
Fritsch. F.E. 1971. The structure and reproduction of the algae, vol. I. Cambridge:
University Press. Reprint.
Geitler, L. 1923. Studieu uber das hematochrome und die chromatophoren von
Trentepohlia. Osterr. Bot. Etschr. 73:76-83.
Griffiths, M.W., W.R. Sistrom, G.J. Cohen-Basaire, and R.Y. Stanier. 1955. Function
of carotenoids in photosynthesis. Nature 1976:1211-1215.
Howland, L.J. 1929. The moisture relations of terrestrial algae IV. Ann. Bot. 43:173-
202.
Hunter, S.H., A.C. Zahalsky, S. Aaronson, and H. Baker. 1966. Methods in cell
physiology 2:217-228. New York: Academic Press.
Johnson A. 1978. The Trentepholiaceae of Singapore Island. Gdns' Bull. Singapore

31:229-237.
Johnstone, L.M. 1977. Draparnaldiopsis: a filamentous green alga (Chlorophyta,

Chaetophorales) requiring vitamin B12. Phycologia 16:183-187.
Ngiam, T.L., F.M. Yong, H.A. Wong, and A.C. Lam. 1973. Inhibition of algal growth
on paints. J.S' pore Inst. Archet. 58:3-5.
Ngiam, T.L. and F.M., Yong. 1975. Inhibition of algal growth in paints (Part II). J.
S' pore Inst. Archet. 68:25-26.
Nicholas, H.W. and H.C. Bold. 1965. Trichosarcina polymorpha gen. et sp. nov. J.
Phycol. 1:34-38.
Piercy, A. 1917. The structure and mode of life of a form of Hormidium flaccidum
A. Braun. Ann. Bot. 31:513.
Provasoli, L. 1958. Nutrition and ecology of protozoa and algae. Annu. Rev.
Microbiol. 12:279-308.
Senn, B. 1911. Physiologische undersuchugen an Trentepohlia. Soc. Sci. Nat
Strasbourg Mem. 6:37-50.
Van Baalen, C. 1961. Vitamin B12 requirement of a marine blue-green alga. Science
133:1922.
Wee, Y.C. and K.B. Lee. 1981. Proliferation of algae on surfaces of buildings in
Singapore. Intn. Biodeterio. Bull. 16:113-117.
AGROINDUSTRIAL WASTE PRODUCTS AS
SOURCES OF CHEAP SUBSTRATES FOR ALGAL
SINGLE-CELL PROTEIN PRODUCTION
Macrina T. Zafaralla
Lina R. Vidal
and
Leonor Elizabeth Travina
College of Sciences and Humanities
University of the Philippines at Los Banos
College, Laguna, Philippines
ABSTRACT
Four types of agroindustrial waste products were tested for

their suitability as substrates for Chlorella single-cell protein
production. Based on cell density on day 7, unsterilized rice straw
filtrate, Tris-buffered inorganic medium (control), unsterilized
slop (1%) and unsterilized rice hull filtrate were suitable culture
media. Dry weight yield after two weeks did not vary among media.
Gross protein content of algae was highest in rice straw with or
without sterilization.
Substrates for algal SCP production are assessed on the basis

of their nutrient content, pH, and hygienic acceptability.
INTRODUCTION
Mateles & Tannenbaun (1967) in an overview of the status of algal

single-cell protein (SCP) production, proposed the establishment of national
centers in the United States for studies in the production and utilization of
algae. They conceptualized two types of centers operating in tandem, one to
work mainly with algal biomass production using inorganic media and
hygienic wastes, the other, to serve as model integrated agroindustrial
complex that will produce human- and animal-consumable algae and also
regenerate and recycle matter for resource and energy conservation. In the
Philippines, with the biotechnological thrusts spearheaded by the National
Institutes of Biotechnology and Microbiology (BIOTECH) in 1980, a signifi-
cant boost to algal SCP production was realized. Recently, the United
Nations Interim Fund for Science and Technology for Development ap-
proved a Philippine proposal to undertake research on microbial SCP
production, utilization, nutrition, and marketing. The organisms under

study are the bacteria, yeasts, filamentous fungi, and algae. The combined
financial and technological backing provided in the research grants places
applied algology, at least at the University of the Philippines at Los Banos
(UPLB), on a sustained basis for a minimum of three years. But this is not
to say that algal SCP production in the Philippines is in its primordial stage.
Noticeable strides during the early seventies include the establishment of
cultural requirements, production in synthetic media and animal manure,
and feeding experiments involving fish, poultry, and swine (Pantastico &
Sulabo 1974; Martinez 1976; Rigor et al. 1980). These past efforts have gone
up to the pond production scale.
Chlorella, whose potential as protein feed for swine (Rigor et al. 1980)
and as an unconventional protein food for man (Lee et al. 1967) is firmly
established, consists of from 50 to 65% protein (Endo & Shirota 1972). Mass
cultures of the alga yield from 50 to 65 g (dry weight) per litre of medium
under heterotrophic or mixotrophicconditions(Tamiya 1968). Local endeav-
ors, as computed from reports, have realized yields of 0.02 g/l/da in produc-
tion ponds, reflecting a maximum photosynthetic efficiency of 3.2% (Rigor et
al. 1980; Martinez 1980). Evidently, much still awaits algal SCP research
in terms of increasing dry matter yield under Philippine conditions.
The presently recognized primary concerns in algal SCP research in the

Philippines are the following:
To increase the kinds of test materials as substrates for SCP production

with emphasis on those whose product is fit for human consumption;
To establish the optimum cultural conditions with selected media

necessary for a pilot-plant production scale;
To expand utilization research after a thorough testing and evaluation

of reported processes;
To promote toxicological and epidemiological researches on identified

SCP; and
To appraise the marketability of the algal product.
Recognizing the importance of these inextricably linked objectives, we

have addressed some of our efforts on the first goal which is to widen the
range of test materials for substrates. The preliminary findings here
reported involved the use of rice straw, rice hull, molasses, slop, inorganic
medium, and Tris-buffered inorganic medium. The general objective is to
evaluate the suitability of these materials as sources of algal culture media
based on their nutrient content, pH, amount of algal biomass produced, and
protein yield of the algal product.
Chlorella Single-cell Protein Production 69
METHODOLOGY
Rice hull and rice straw filtrates were prepared from ashes of the
corresponding plant materials. Rice straw, as it is burned by farmers in the
field, is a mixture of rice straw and leaves and rice panicle residues. A 10%
(w/v) suspension of the ash was prepared with distilled water and passed
through coarse filter paper. The pH of the media was adjusted to 9 by
addition of 1N KOH. No attempt was made to remove the color of the
filtrates, orange-brown in rice hull and bright yellow in rice straw.
Slop and molasses, two by-products of sugar refineries, were separately

diluted to 1% concentration by addition of distilled water and their pH
adjusted accordingly. For the control, Tris-buffered inorganic medium (pH
9) was used. Aliquots (290 ml) of the various media were dispensed in sterile
500-ml culture bottles and the latter plugged with cotton. Media requiring
sterilization were autoclaved at 20 psi for 15 minutes Chemical analyses of
uninoculated media followed involving the following nutrients: total nitro-
gen by acidimetric method; available phosphorus (PO4=), stannous chloride
method; calcium and magnesium, EDTA titrimetric method. All analyses
had two replicates except for nitrogen and phosphorus with three.
Chlorella vulgaris M-3 was obtained from the BIOTECH culture

collection at UPLB. Ten-day old cultures were rinsed and suspended in
sterile distilled water to make an inoculum of density 12.7 x 106 cells/ml. The
final volume of the medium after inoculation was 320 ml.
Cultures were maintained inside a greenhouse where temperature and

light conditions fluctuated naturally. They were gently swirled and reori-
ented twice daily. Data on cell density were obtained at four-day intervals
for two weeks using a completely randomized design. All randomly selected
replicates of a treatment were discarded after cell density determination. On
the 14th day, which followed the last day of cell density determination,
cultures were filtered and their dry weight obtained. Then, the total
nitrogen and available phosphorus contents of the harvested biomass were
determined using the Kjeldahl and molybdovanadophosphoric acid meth-
ods, respectively.
RESULTS
Cell density and dry weight
The trends in growth response of Chlorella vulgaris M-3 measured in

terms of cell density are shown in Fig. 1. Growth was minimal in all types
of sterilized media during the first four days. On the other hand, all
unsterilized media exhibited better algal growth than did the control.
Apparently, Chlorella underwent a period of adaptation in sterilized media
70
11
LEGEND:
A - Sterile (S) TBIM (control)
B - Sterile (S) Rice hull
10.5 C - Sterile (S) Rice straw
D - Sterile (S) Mollasses (1%)
1 - Unsterile (U) Rice hull
2 - Unsterile (U) Rice straw
10 3 - Unsterile (U) Slop (1%)
I C - Initial cell count
Culture and Use of Algae
9.5
9.0
LOG CELL NUMBER

7.5
7.0
0
IC A BCD 1 2 3 A BC D 1 2 3 A B CD 1 2 3 A BCD 1 2 3
1 " 4 9 10 13
AGE OF CULTURE ( DAYS )
Fig. 1. Trends in growth response of Chlorella vulgaris M-3 in different substrates.
during the first four days which was characterized by a pronounced slowing
down of growth rate. This was less expressed in unsterilized media.
Indications of substrate suitability became appreciable on day 7. Sta-

tistical analyses (Table 1) showed that around the said day, unsterilized rice
straw filtrate (30 x 109), TBIM, the control (16 x 109), unsterilized slop (15 x
1()9), and unsterilized rice hull (13 x 109), in that order, were suitable
substrates for Chlorell production. Results of Duncan's multiple range test
also showed that in general, day 7 is the ideal time for harvest.
The cell doubling time (Stockner & Costella, 1976) of the alga in various
media was shortest (7 h) in the control and longest (24 h) in unsterilized slop.
An 8-h cell doubling time is normal for the alga (Kingsbury, 1968). Consid-
ering the computed averages on day 7, a desired amount of harvest may be
achieved at regular intervals if a careful manipulation of the amount of
inoculum is made. The possibility of this, however, depends on whether or
not the adaptation period of four days as seen in some media could be
shortened by preconditioning stock cultures in appropriate media.
The impressive growth behavior of the alga in the slop medium is of

particular interest. Compared with molasses, slop has a lighter brown color
which does not disappear in two weeks. If this brown color interfered with
the degree of illumination of algal cells to an extent that was not determined,
then the alga may have compensated for light limitation through heterotro-
phic growth which is normal in cultures supplied with organic nutrients.
Another possible explanation for growth in slop is that it may have supplied
certain nutritive substances, possibly organic, that promoted algal multipli-
cation.
Statistical analysis of the dry matter yield (Table 2) showed no differ-

ence among the various substrates. This result, which runs counter to
Table 1. Growth of Chlorell vulg ris M-3 after seven and thirteen days; cell density
at day 0, 0.013 × 109
Cell density (x 109)

Media 7 days 13 days
Sterilized TBIM (control) 16b 7d

Sterilized molasses 3' 3d
Sterilized rice hull 2d 4d
Sterilized rice straw 9c 24d
Unsterilized slop 15d 51d
Unsterilized rice hull 13bc 6d
Unsterilized rice straw 30a 38b
Means with the same letter(s) are not significantly different from each other at
0.05% level, DMRT.
72
expectations, is attributed to the presence of bacterial contaminants and

suspended particles inherent in the media. These interfering substances
notwithstanding, it is well to take a cursory look at the dry matter yield data
for the purpose of comparing them with those in literature.
Thin-layer (1 cm) cultures of Chlorella, considered by Milner et al.

(1978) as impractical, yield greater than 50 g dry weight/l/da. The present
yields range from 0.008 g/l/da in sterilized rice straw to 0.016 g/l/da in slop
and molasses. Assuming the bacterial contaminants to be negligible, the
computed biomass closely approximates local harvests from production
ponds (0.02 g/l/da) (Rigor et al. 1980; Martinez 1980).
To further evaluate the suitability of the substrates for algal SCP

production, the nitrogen content of the resulting biomass was also analyzed
(Table 2). Algae grown in sterile or non-sterile rice hull filtrate yielded the
highest nitrogen content of from 7.3 to 7.5%, this range exceeding the control
by at least 62%. All other yields including that of the control had lower
nitrogen content ranging from 3.8 to 5.9%. Multiplying the total nitrogen
values by 6.25, the gross protein content was obtained (Table 2). Chlorella
in rice hull filtrates had roughly 50% protein while the others had from 24
to 37%. The protein content of the former compares favorably with the
normal quantities cited by Endo & Shirota (1972) for the alga.
Table 2. Dry matter yield, nitrogen and gross protein contents of Chlorella vulgaria
M-3 grown in different substrates
Dry matter Gross

N content
Media yield protein
(%)
(mg) content (%)
Sterilized TBIM (control) 27.87a 4.5ab 25.6

Sterilized molasses 34.83a 3.8ab 23.8
Sterilized rice hull 17.90a 7.3ab 45.6
Sterilized rice straw 24.80a 5.9ab 36.9
Unsterilized slop 35.17a 4.5ab 25.6
Unsterilized rice hull 32.66a 7.5ab 46.9
Unsterilized rice straw 24.83a 4.1ab 25.6
Means with the same letter(s) are not significantly different from each other at
0.05% level (DMRT).
Media Analysis
To explain the above results on cell density and protein content, mineral
analysis of the uninoculated media was undertaken (Table 3). The highest
level of nitrogen was in unsterilized rice straw filtrate which had an average
of 2.91 ppm total nitrogen, roughly eight times that of the control (0.37 ppm).
Unsterilized slop and sterilized molasses had moderate levels, 1.87 and 1.59
ppm, respectively, whereas filtrates of unsterilized rice hull, sterilized rice

straw and sterilized rice hull together with the control had low levels of 0.99,
0.47, 0.37 ppm, respectively.
With respect to phosphorus, sterilized rice hull filtrate contained the

highest amount at 969 ppm followed by unsterilized rice hull and unsteril-
ized rice straw with 813 and 532 ppm, respectively. The P content of slop also
exceeded that of the control by over 50%, but molasses is relatively "impov-
erished" in P with an average of 13.3 ppm. It is quite easy thus to appreciate
the fact that rice plant remains are promising materials, nutrient-wise, for
use in preparation of media for algal SCP production.
The calcium and magnesium levels of the media were also analyzed.
Amounts of these nutrients are similarly abundant (Table 3).
The nutrient content of all media is apparently sizeable, even excessive,

for some nutrients. More algal biomass could have been harvested had the
density of inoculum been increased. This could have been realized with
greater certainty if the algal inoculum had been preconditioned to the
appropriate culture medium.
Correlation
Attempts to correlate cell density with the independent variables

revealed two interesting relationships: nitrogen and pH positively corre-
lated with cell density. The relationship with respect to N suggests that this
nutrient may have limited algal response to various media. The proportion
of P to N is tremendously lopsided (Table 3).
The positive correlation between pH and cell density on day 7 invites

attention. Among the media, rice straw with or without sterilization
underwent the least departure from its initial pH of 9. Unsterilized slop
Table 3. Initial levels of nutrients and final pH of different substrates
Media Nutrient concentration (ppm)

N P Ca Mg pH
Sterilized TBIM (control) 0.37c 177.7c 182c 179.8ab 10.0a

Sterilized molasses 1.59b 13.3f 683a 179.8ab 4.5f
Sterilized rice hull 0.37c 968.7a 78c 212.2a 7.6d
Sterilized rice straw 0.47c 532.0c 117c 185.0ab 9.3b
Unsterilized slop 1.87b 230.7c 455b 95.6ab 8.4c
Unsterilized rice hull 0.99bc 812.7b 455b 41.7b 7.3e
Unsterilized rice straw 2.91a 417.7d 390b 38.6b 9.5°
Means with the same letters) are not significantly different from each other at
0.05% level (DMRT).
74
followed with pH 8.4. Rice hull dropped to around neutral, pH 7.5, while
molasses became acidic with pH 4.5. The control became more alkaline, pH
10 (Table 3). These findings seem to indicate that in addition to the combined
growth effects of undetermined factors in the medium, the pH factor exerts
an appreciable effect on the ability of algal cells to multiply. Thus among the
media, rice straw filtrate elicited better growth probably because it retains
a pH level closest to that preferred by Chlorella, around pH 9. The same
reasoning probably holds for the slop medium. There is also the possibility
that unchecked pH levels had pronounced effects upon 2-week old cultures.
These results emphasize the importance of a sustained pH optimum to
ensure maximum sustainable yield.
DISCUSSION
The current trends in human population growth reiterate the under-

scored needs of the past decade for the formulation of policies that would
solve the food, energy, and population problems. Single-cell proteins have
time and again been turned to, reportedly as unconventional protein sources.
Setting aside the issue of individual taste preferences and cultural taboos on
food, it is incumbent upon the SCP researcher to recommend foodstuffs like
algae which are grown under hygienic conditions. Not only must the culture
be hygienically acceptable by the best of human standards; it also must
generate the minimum of affront to human senses. Domestic and animal
wastes clearly hold a lot of promise in terms of their ability to generate
biomass fit enough for farm animal consumption. But knowing the vast
areas required to meet the necessary production rate, it is not difficult to
imagine the degree of pollution of the air over the production site involving
animal wastes.
Rice straw and rice hull as sources of culture media have a good
potential for use in algal SCP production. They are hygienic substrates in
that they do not lead to fouling of the air. They have the following
advantages, namely, 1) cheapness, 2) availability, 3) simplicity of prepara-
tion, 4) nutrient sufficiency, 5) pH adequacy, 6) potentially high protein yield
of the algal product, and 7) recycleable residues. Slop is another medium
with a high nutrient content. A cheap hygienic substrate, it has the
consistency and taste of molasses, not to mention its appetizing aroma. This
substrate also enables heterotrophic growth of the algal SCP, supplying as
it does the energy and carbon dioxide requirements of the culture organism.
Preliminary as these findings are, some important thrusts of immedi-

ate efforts in algal SCP production become identifiable. First is the need to
establish the optimum concentrations of the plant filtrates and slop used.
Second is the need to undertake a complete nutrient analysis of the media
in order to determine what nutrient enrichment procedures are necessary.
Residual nutrients do have to be determined to establish the optimum

amount of the inoculum. Third is the need to determine what size of
inoculum is optimum for the highest sustainable yield to be realized. Fourth
is the need to establish what culture maintenance procedure to undertake
for maximum sustainable yield. Lastly, the need is felt for a systematic
enumeration of microorganisms that contaminate the algal cultures. It is
only after these immediate problems shall have been solved that one may
embark on the formulation of algal food preparations fit for human consump-
tion. The next few years will see renewed efforts to accomplish these goals.
Financial support by the National Institutes of Biotechnology and Applied

Microbiology (BIOTECH) and the National Science Development Board (NSDB) is
gratefully acknowledged.
LITERATURE CITED
Endo, H. and M. Shirota. 1972. In: Fermentation Technology Today. Proceedings

of the Fourth International Fermentation Symposium, Kyoto, Japan. p. 533.
Kingsbury, J.M. 1968. The biology of the algae. (Lecture Notes). Mimeographed
copy.
Lee, S.K., H.M. Fox, C. Kies, and R. Dam. 1967. The supplementary value of algae
protein in human diets. Journ. Ser., Nebraska Agric. Exper. Sta. p. 281-285.
Martinez, M.R. 1976. Studies on Chlorella production and utilization. Terminal

Report, PCARR 130. 199 pp.
Martinez, M.R. 1980. Algal technology for RP. PCARR Monitor 8(5):2-3.
Mateles, R.I. and S.R. Tannenbaun. 1967. Single-cell protein. MIT Press, Massa-
chusetts.
Milner, M., N.S. Scrimsha, and D.I.C. Wang. 1978. Protein resources and technol-
ogy: Status and research needs. AVI Publishing Co., Inc. Westport, Connecti-
cut.
Pantastico, J.B. and R. Sulabo. 1974. Utilization of hog manure for production of
Chlorella. Paper read in the 3rd Annual Meeting Phil. Soc. Microbiol. April,
1974.
Rigor, E.M., J.A. Eusebio, and B.R. Garcia. 1980. Chlorella as protein feed
supplement for swine. NSDB Technol. Jour. 1980:9-16.
Stockner, J.G. and A.C. Costella. 1976. Marine phytoplankton growth in high
concentrations of pumpmill effluents. J. Fish. Res. Board Can. 33(12):2758-
2765.
Tamiya, H. 1968. Green Micro-algae. In: Food Protein Sources. N.W. Pine, Ed.
Cambridge University Press, New York. p. 35-39.
UTILIZATION OF SEAWEED RESOURCES
Gloria J.B. Cajipe

Marine Sciences Center
University of the Philippines
Diliman, Quezon City, Philippines
SEAWEEDS AS SOURCES OF INDUSTRIAL GUMS
The commercial importance of seaweeds derives principally from their

use as sources of industrial gums such as agar, carrageenan, and alginic acid.
These gums are made up of the structural polysaccharide material found in
red seaweeds in the case of agar and carrageenan, and in brown seaweeds in
the case of alginic acid. Such gums have many industry applications.
Seaweed gums are unique in that they impart to various processed

products special physical properties relating to viscosity, texture, and
gelling ability. These properties are dictated by the chemical structure of the
polysaccharide. Seaweed gums are made up of unique sugar molecules.
Agar is made up of repeating units of B-D-galactose and 3,6-anhydro-B-L-
galactose; carrageenan of B-D-galactose and 3,6-anhydro-B-D-galactose
sulfated at various positions depending on the type of carrageenan; and
alginic acid of mannuronic and guluronic acid residues. These different
polysaccharidal structures can be distinguished from one another by means
of their infrared (IR) spectra. Each spectrum is essentially a "fingerprint" of
the polysaccharide. Thus, the spectrum of agar is distinct from that of
carrageenan or alginic acid. A spectrum arises as a result of vibrations
unique to each kind of molecule. There are peaks that are unique to each
polysaccharide and which are indicative of certain structural features
present in the molecule, e.g., the 3,6-anhydro galactose moiety gives rise to
the peak at 930 cm 1 ; the various ester sulfate groups to peaks between 800
and 850 cm 1 .
Seaweed polysaccharides are extracted by various processes. Common

to all is the extraction of the polysaccharides into water. This indicates that
seaweed gums are fairly soluble in water, a property that is again dictated
by molecular forces arising from the preponderance of such groups as -OH
(hydroxy), -OSO3- (sulfate ester) and -COO (carboxylate) in the macromole-
cule. In the Philippines, Eucheuma is presently the most important source
of carrageenan. Eucheuma alvarezii yields kappa carrageenan while E.
denticulatum yields the iota form. There are, however, other carrageenan-
containing seaweeds that can be found in tropical waters. The carrageenan
isolated from Acanthophora appears to be of the lambda form, while that
78
from Hypnea appears to be a variant of kappa carrageenan. The cultivation

of these seaweeds is a matter that should be further investigated as these are
sources of carrageenans that are of great commercial importance. The
carrageenan from Hypnea can form very strong gels; that from Acanthophora
possibly can be used as a stabilizing agent in non-viscous liquid products.
The extraction process for agar involves a freezing-thawing process.

Gracilaria verrucosa appears to be the most promising species. However,
other species also have potential economic value, at least in the production
of food-grade if not bacteriological-quality agar.
The extraction process for alginate essentially involves the conversion of

naturally-occurring alginate into its soluble form, i.e., sodium alginate.
Most, if not all, of the alginates that can be bought in the world market today
come from kelp. However, there are tropical seaweeds that are potential
sources. Sargassum appears to be most promising.
CARRAGEENAN AS SUBSTITUTE
FOR MICROBIOLOGICAL AGAR
Recent investigations on the use of carrageenan in microbiological media

show that this application is indeed feasible. Carrageenan processed from
Eucheuma has been especially formulated and tested as a medium for the
growth of a wide spectrum of microorganisms - bacteria, yeast, and other
fungi. The carrageenan medium has been tested in a number of research and
teaching laboratories and satisfactory results have been obtained. The use
of carrageenan as a substitute for bacteriological agar may be a boon to
developing countries such as the Philippines where the rising cost of agar has
been most strongly felt.
SEAWEEDS AS BINDERS OF HEAVY METAL POLLUTANTS
The use of seaweeds as a pollution-control agent has also been investi-

gated at some length. Basic chemical studies conducted to date show that
such an application is feasible. The approach used in the study consists of
the following methodologies: 1) dialysis of solution of pure seaweed polysac-
charides against solutions of heavy metal salts; and 2) elution of heavy metal
salt solutions through a column of ground, dried seaweed. The metals that
have been investigated are lead, cadmium, copper, zinc, iron, and mercury.
The affinities of both carrageenan and alginate for these metals have been
examined. Both polysaccharides exhibit a preferential affinity for lead,
although the affinity for the other metals are not insignificant. The affinity
for copper, in fact, almost matches the affinity for lead. Sargassum is
Seaweed Resources Utilization 79
presently being developed for this particular application because of its ease
of handling. Preliminary experiments with actual industrial effluents
contaminated with lead and cadmium indicate that an industrial system for
wastewater treatment that uses Sargassum as metal binder can be devel-
oped.
OTHER POTENTIAL SEAWEED APPLICATIONS
Although the use of seaweeds as fertilizer in agriculture and horticulture

has been introduced on a commercial scale in some European countries, this
particular application still has to be developed in Asian countries. There are
reports that some coastal communities in the Philippines do use Sargassum
occasionally as a fertilizer. However, scientific studies that will lead to its
more widespread use have been limited. Pre-development studies involved
chemical studies on auxin-like substances from Sargassum polycystum.
Auxins are plant growth hormones and their presence in Sargassum may
partly account for the fertilizing property of this seaweed. Substances which
exhibit auxin-like activity have indeed been isolated and partially character-
ized. Chemical formulas have been obtained although their exact identities
have not yet been established. None of the compounds isolated were found
to be indolic in nature (most known auxins possess the indole group).
The use of seaweeds as food is not as widespread in the Philippines as it

is in Asian countries, particularly Japan. A study that looks into the
nutritive value of some edible Philippine seaweeds is presently being
undertaken. Nutritive value is being analyzed in terms of crude protein
content, amino acid composition, and mineral and vitamin content. Results
to date indicate that seaweeds are not the best sources of protein. However,
they are excellent sources of minerals and do contain some vitamins.
CULTURE AND UTILIZATION OF FRESHWATER
ALGAE AS PROTEIN SOURCE
Bienvenida R. Rodulfo
Division of Microbiology
National Institute of Science & Technology
Manila, Philippines
INTRODUCTION
Mankind is faced with great challenges in the years ahead. The future
prospects would include an even higher incidence of hunger, starvation, and
malnutrition. The production of food from unconventional sources may
alleviate some of these problems. It is predicted that the earth's population
will increase by at least 50% to a total of 6 billion by the end of the century.
It is expected to double to 8 billion in the 21st century. Large cities such as
Mexico City, Sao Paulo, and Calcutta will have population of 30,26, and 16
million, respectively, by the year 2000 (Blume 1979).
With the world's increasing population, large amounts of protein will be

required for human and animal feed (Meadows et al. 1972). The protein
deficit is markedly seen in many Third World countries and has led to
massive investments in research on single-cell (microbial) protein (SCP).
However, it is desirable that SCP be produced in a very large scale and with
the cheapest available substrates (Meyer 1980).
UTILIZATION OF ALGAE FOR INDUSTRIAL

PHOTOSYNTHESIS
The scientific and economic evaluation for the commercial production of

algae in mass culture on inorganic media has been thoroughly studied by
different groups. A comprehensive survey of the relevant work published
was compiled by the Carnegie Institution of Washington (Burlew 1953).
Tamiya (1957) gave a detailed review of the technical aspects of mass culture
of algae and found reason to be moderately optimistic about the commercial
future of the process.
With the advances in algal culture, three lines of development are

distinguished: (1) culture of algae for the production of useful organic
materials as food, feed, and some special organic materials; (2) culture of
nitrogen-fixing algae for increasing soil fertility; and (3) culture of sewage
algae in symbiosis with bacteria with a two-fold purpose - to accelerate
stabilization of organic sewage material, and to utilize the harvested algae
as in (1).
The culture techniques and species of algae are more or less different. In
(1), fast-growing green algae such as Chlorella and Scenedesmus; in (2), blue-
green algae which are strong nitrogen fixers like Nostoc, Anabaena, Toly-
pothrix, etc.; and (3), mixed cultures of various algal strains in harmony with
sewage bacteria.
Although different in aims, these projects have many common problems

related to the physiology and biochemistry of algae under carefully con-
trolled laboratory conditions. The main physiological characteristics of the
algae involves the variety and flexibility of their nutrient requirements and
the chemical composition of their cells.
Pioneering investigations on large-scale culture of algae under con-

trolled conditions in inorganic media were conducted in the US, Germany,
and Japan in the 1950s. In the 60s, cultivation of algae was investigated as
a means of bioregeneration of wastes in chemocycle systems for extended
space exploration missions or in treatment of wastes in seawage oxidation
ponds (Oswald & Golueke 1968). More recently, algae were again considered
for producing protein as food or feed in developing countries (Durand-
Chastel & Clement 1975).
Considered algal pilot plant operations were those conducted at the

University of California; Czechoslovakia; the Indian/West German System
in Mysore, India; the Sosa Texcoco Process in Mexico City; that in Technion,
Haifa, Israel; and in Kyoto University in Japan.
SOCIOECONOMIC IMPLICATION OF ALGAL MASS CULTURE
Out of about 17,000 algal species that have been described since the turn
of the last century, only a few have been investigated and described as
excellent for possible sources of protein. Among the important ones are
species of Chlorella (C. pyrenoidosa, C. vulgaris, C. ellipsoidea), Scenedesmus,
and the nitrogen-fixing blue-green algae (Anabaena variabilis, A. cylin-
drica, Nostoc commune, N. muscorum, N. punctiforme, Phormidium molle,
Tolypothrix, Stigonema, Nodularia).
The fertility of rice fields in Southeast Asia and the Malay Archipelago
is said to depend in some measure on the nitrogen-fixing species of Toly-
pothrix. In India, mixed cultures of Aulosira and Cylindrospermum are
used.
Freshwater Algae as Protein Source 83
Chlorella has been studied in great detail due to its high rate of
photosynthesis, carbon dioxide consumption and release of equimolar quan-
tity of oxygen, and high content of protein (40-60%, dry weight). That
Chlorella could be used in a bioregenerative life support system of a space
craft implies that it could serve also as an important source of food.
Detailed data on the chemical composition in terms of assimilation and

biological value of Chlorella and Scenedesmus have been reported and
discussed (Table 1) (Cook 1962). Assimiliation of Chlorella and Scenedesmus
mixture (1:10) was insignificantly bettered by boiling and autoclaving.
In recent years, people in certain countries (e.g., Taiwan) have incorpo-

rated relatively small amounts of extracted algal cells in their diet. Unex-
tracted algae produced a very disagreeable flavor when used as food ingre-
dient. However, Hayami et al. (1969) found that Japanese women would
accept 30 grams metnanol-extracted algae per day in their diet A drawback
here is that the green color of algae is difficult to mask in most food mixtures.
The cell walls of algae are fibrous and can irritate the gastrointestinal
tract. Algae used in Taiwan are steam-treated to rupture the cell walls and
release the cell content. As much as 150 g of methanol-extracted algae per
day could be consumed without complaints. The estimate of the nutritive
value of Chlorella and Scenedesmus abated the original proposal on their
utilization for human consumption and directed further studies along two
lines: (1) examination of the practicability of using Chlorella and Scenedesmus
as animal feed, and (2) preparation from the algae biomass of protein
concentrates for human consumption.
However, difficulties involved in protein extraction, poor food properties

and costly technological processes suggest that the preparation of protein
products from Chlorella and Scenedesmus is not yet a primary and beneficial
solution to the protein problem.
A study of the protoplast of various algae may give the key to a greater
use of algal protein. The single-celled Dunaliella and Cosmarium whose
protoplasts may become a potential protein source have been described.
Interesting reports on the preparation of Chlorella protoplasts by means of
Table 1. Biological value of algae (After Cook 1962)
Protein Source Biological Value
Dry algae 54.2 ±3.2

Autoclaved algae 55.5 ±2.6
Algae boiled for 30 min. 56.0 ±2.7
Algae boiled for 180 min. 48.7 ±2.6
hydrolytic enzymes had been published (Gibbs & Dorffres 1976; Berlines &
Wenc 1976; Bruan & Aach 1975).
Of particular interest is the unicellular blue-green alga Spirulina. This

alga is relatively large (about 100 times longer than Chlorella). There are
good grounds for believing that it may be will suited for human consumption
because the Africans on the shores of Lake Chad consume it as part of their
diet (Table 2). The alga was also eaten by ancient Aztecs in Mexico.
Table 2. Chemical analysis of algal cakes on sale in the Republic of Chad (Azoulay
& Senez 1960; Champynot 1965)
Protein 45-46%
Fats 5-6%
Carbohydrates 16-20%
Experiments to assess Spirulina assimilation have been conducted in

France and Mexico (Table 3) (Clement et al. 1968). The results were so
convincing that the Mexican government permitted the sale of Spirulina in
1973 (Clement 1975).
Table 3. Chemical analysis of Spirulina (Clement et al. 1968)
Protein 62-65%
Pats 2-3%
Carbohydrates 18-20%
The value of traditional recycling methods used in rural areas of Asia is

also being increasingly widely recognized. An example is the Indonesian
village pond in which wastes are converted to algae which are then eaten by
fish. Lately, with the energy crisis in the 70s, the Asian Pacific countries got
involved in a technique which started out as a means of waste utilization and
opened the way to bioenergy through aquaculture on any scale desired. In
principle, one and the same stock of mineral nutrients can maintain a
constant supply of algae and methane. At present, capacities of 80 tons dry
weight per hectare per year have been obtained but a further increase is still
expected. These agricultural practices of recycling minerals by waste
conversion to useful production opened up a new outlook toward aquafarming
by rendering land more productive and making these minute plants acces-
sible for human use.
FOOD SITUATION IN THE PHILIPPINES
The Filipinos are suffering from a deficiency of protein since our average
daily protein intake of 42.5 g is far below our daily requirement of 68 g. Our
total production of high protein foods seems barely sufficient to supply 50%
of our protein needs. Even with all our importation of high protein foods such
as eggs, milk, fish and meat products and our eating of low-protein foods, our
deficiency in protein is still enormous. If we add to this picture the fact that
our population is increasing very fast and that to cope with this situation we
have to constantly extend land area under cultivation, the thought naturally
occur that a time will come when our fertile land will be limited by crowded
population and could no longer supply our need for food. As a means of
alleviating the threat of famine, particularly protein famine in this country,
we should also explore the culturing of high-protein food algae not only for
human but also for fish and animal nutrition.
The Philippines, being a tropical country, is naturally rich in algal flora.

In raising high-protein algal food it is advantageous to use our local species
as they are already accustomed to living under the existing local conditions,
and therefore, their culture may not necessitate drastic modifications and
adjustments of these conditions in order to favor their development. Strains
which can tolerate high temperature or which may not be affected by
diversified environment can be used. With such algae, the use of a cooling
device similar to that used in culturing algae in temperature countries may
be obviated.
NIST'S RESEARCHES ON ALGAL CULTURE
Early investigation was conducted in 1954 at the Biological Research

Center, Institute of Science and Technology to explore the algal flora of
Manila and nearby provinces for high-protein strains of unicellular green
algae. The exploration yielded several Chlorella isolates in pure state. A
detailed study of a select isolate, C. pyremoidosa Chick was made (Palo et al.,
1965). This alga was found to contain more than 60% protein (dry weight
basis), with all the essential amino acids and vitamins present; very prolific
in growth (rate of more than 33 times the initial cell number in 4 days); and
tolerate high temperatures (up to 46°C), fully exposed to sunlight during
summer.
Later, further work was continued on four local Chlorella strains. The
effects of media with low and high available nitrogen on the protein and lipid
contents of the algae were determined (Rodulfo et al. 1972). The results were
in agreement with those of Milner (1948, 1951) who showed that the
chemical composition of Chlorella "can change in response to change in
environment." Harvests with as high as 36.4% lipid and 22.7% fat were
obtained by this method: The methanol extracts of Chlorella were active
against Micrococcus aureaus, Bacillus cereus and B. subtilis. This confirmed
the presence of some antibiotic substances in their cells. While the culture
of algae did not reach pilot plant scale level at NIST, stock cultures of
Chlorella were made available to other research institutions and to institu-
tions of higher learning, fish culturists, etc. who would like to venture in
algal culture.
Some other researches done at NIST were on Scenedesmus obliguus,

another fast growing green alga which is equally rich in protein (50%, dry
weight). A comparative study on bench-scale production of Chlorella and
Scenedesmus was made for a year. Environmental conditions such as those
of temperature and light were not controlled. Results showed growth of
these algae to be favorable at summer time although proliferation of rotifers
and other protozoa could not be avoided.
In the 70s, the NIST pursued further research on cultivation of the

nitrogen-fixing blue-green algae with emphasis on edible forms commonly
utilized by natives in Northern Luzon. One filamentous edible species,
Nostoc linckia, locally called tabtaba was studied (Rodulfo 1980). This alga
grew best in a nitrogen-free inorganic medium, had a protein content of 40-
45% (dry weight), and grew well in an alkaline pH (7.5-8.0). Another very
interesting species, Nostoc commune, was isolated from Albay in the Bicol
region. The richness of the soil in the region could be due to the presence of
this alga which was very prolific in the area. Growth was better in a nitrogen-
free solution at pH 6.8-7.2 Maximum increase in colony size was 2.5 cm after
two weeks (from initial pin size). Blue-green algae promise to be a very good
source of protein fitted for human consumption.
LITERATURE CITED
Azoulay, E. and J. Senez. 1960. Ann. Inst. Pasteur Paris 98:868-71.
Berlines, M.D. and K.A. Wenc. 1976. Appl. Env. Microb. 32:436-37.
Blume, M. 1979. The population crisis: The bomb is still tickling. International
Herald Tribune (19 Nov.): 1, 5.
Braun, E. and H.C. Aach. 1975. Planta 126:181-185.
Burlew, J.S. (ed.) 1953. Algal culture from laboratory to pilot plant. Carnegie Inst.
Wash. Publ. No. 600.
Champynot. A. 1965. Sci. Amer. 213:13.
Clement, G. 1975. Single-cell protein II. MIT Press. Cambridge Mass.
Clement, G., H. Durand-Chastel, and M. Henny. 1968. Rep. Inst. Francois Pet.
Cook, B. 1962. Am. Jour. Pub. Health. 52:243-249.

Durand-Chastel, H. and G. Clement. 1975. Spirulina algae. Food for tomorrow.

Proc. 9th Int. Cong. Nut. 3:85-90.
Gibbs, N. and C.M. Dorffres. 1976. Appl. Env. Microb. 31:602-4.
Hayami, H., Y. Matsuno, and K. Shino. 1969. Studies on the utilization of Chlorella
as a source of food. Annu. Rep. Natl. Inst. Nutr. Japan. Part 8, p. 58.
Meadows, D.H., D.L. Meadows, J. Randens, and W.W. Behrens. 1972. The limits to
growth. Universe Books, N.Y.
Meyer, O. Using carbon monoxide to produce single-cell protein. Bioscience. June

1980.
Milner, H.W. 1948. The fatty acids of Chlorella. J. Biol. Chem. 176:813-17.
Milner, H.W. 1951. Possibilities in photosynthesis methods for the production of oils
and proteins. J. Amer. Oil Chem. Soc. 28:363-65.
Oswald, W.J. and C.G. Golueke. 1968. Large scale production of algae. In R.I.
Mateles and S.R. Tannenbaum, ed. Single-cell protein. p. 271-305. MIT Press,
Cambridge.
Palo, M.A.,B.R. Rodulfo, and C.B. Balita. 1965. A study on a local high protein strain
of Chlorella. Phil. Jour. Sci. 94(2).
Rodulfo, B.R. 1980. Studies on some local species of nitrogen-fixing blue-green algae
with special reference to an esculent freshwater strain of Nostoc known in Ilocos
region as "tabtaba" (Thesis).
Rodulfo, B.R., P.S. Santos, and C.P. Navarro. 1972. Studies on protein and fat
contents of four local Chlorella strains. Proc. NSTW, July 10-16, 1972. NSDB
Doc. Div.
Tamiya, H. 1957. Mass culture of algae. Annu. Rev. Plant Physiol. 8:309-334.
PHILIPPINE ALGAL TAXONOMY:
PAST, PRESENT, AND FUTURE
Paciente A. Cordero, Jr.

Phycology Section, Botany Division
National Museum, Manila, Philippines
ABSTRACT
This paper presents a historical account of the development of

algal taxonomy in the Philippines, from its early beginnings in
1800 to the present, with emphasis on marine forms.
Marine algal taxonomists in the country are urged to shift em-

phasis from the classical morphologic approach to the chemotaxon-
omic and cytologic method in attempts at resolving the classifica-
tion and phylogeny of important marine groups such as the poly-
morphic and economically important Caulerpa, Ulva, Codium,
Sargassum, and Gracilaria. Chemotaxonomy has close affinity
with the morphological approach, hence is given priority over
cytology with the use of the scanning electron microscope.
INTRODUCTION
This paper attempts to present an overview of the status and future

direction of algal taxonomy in the Philippines, with emphasis on marine
algae. This is in recognition of the significant role the taxonomists play in
providing baseline data that link them with other scientists.
A developing country such as the Philippines expects to benefit from this

symposium on the culture and utilization of algae. The information to be
gained will constitute a significant addendum to the rather anemic algal
literature of the country.
HISTORICAL ACCOUNT OF ALGAL TAXONOMY
Available literature and actual explorations show that the Philippines

is rich in algal materials for biological laboratory studies as well as for
commercial utilization. Most of the early studies were taxonomic works by

foreign or visiting scientists.
1800-1900
During the early nineteenth century, various foreign expeditions as well

as individuals undertook botanical collecting in the Philippines.
A. von Chamisso, botanist of the Romanzoff Expedition (1817-1818),

collected the first Corallopsis specimen in Manila. The Philippines was not
in the itinerary of this Russian exploration, but the ship Rurik was forced to
take shelter in Manila following a heavy storm in the Pacific.
C.A. Agardh (1820) described and illustrated the type species under the
name Sphaerococcus salicornia which was later assigned to Corallopsis and
more recently to Gracilaria. R. Greville (1830) monographed Corallopsis
based on the morphology of Chamisso's Manila material.
The Prussian East Asia Expedition headed by F.J.E. Meyen visited the
Philippines in 1831. From among the materials collected in Rizal and
Laguna, Georg von Martens found two new Cladophora species, the freshwater
Cladophora diluta and C. luzoniensis.
The year 1837 might be considered the birth of algology as a science in

the Philippines. It was then that the earliest mention of the algae of the
country was made by Blanco (1837) in his book Flora de Filipinos. Two more
editions of the book followed, one in 1845 by Blanco himself, and a posthu-
mous edition in 1877-1883, completed by Fr. Ignacio Mercado and Fr.
Antonio Llanos. Blanco revised a number of algal identifications he himself
had made. The scarcity of botanical literature at the time and the limited
academic contact with foreign algal taxonomists caused some misidentifica-
tions and duplications of scientific names.
A Manila-based Londoner, Hugh Cumings, made some valuable collec-

tions of algae that were believed worked on by Montagne (1844-1846). A
great number of the Cumings collections are deposited in the Kew Herbar-
ium in England.
An American expedition in 1854 collected mostly flowering plants and a

few algae; there was no algologist in the group. The algal specimens were
turned over to Bailey and Harvey who reported new records like Dictyota
dichotoma and a host of new species, among them Amphitetras favosa,
Campylodiscus kutzingii, Lagena williamsonii and Triceratium orientale.
The collecting site, as reported by Charles Wilkes, head of the exploration,
was confined to Marongas Island, northeast of Jolo in southern Philippines.
Georg von Martens (1866), in his compilation of algae described or

Taxonomy of Philippine Algae 91
reported from tropical Asia and the Pacific, made some revisions of the
nomenclature in Blanco's Flora de Filipinos, e.g., Fucus gulaman Blanco,
renamed Fucus edulis, to Sphaerococcus gelatinus Agardh.
After the British Challenger Expedition (1874-1875), Dickie (1876,

1877) cited in his enumeration Polyphysa spicata Kutz. from Mactan Island
in the Visayas.
In 1919-1921, Shaw published his exceptional study of new volvocine

genera: Campbellosphaeria, Janetosphaeria, Merrillosphaeria, and Copelan-
dosphaeria. Shaw (1923) also reported M. africana from Manila.
The last recorded exploration that reached the Philippines in 1800-1900

was that of the Italian Vettor Pisani. The collection yielded new species as
described by Piccone (1886).
1900-1941
The year 1900 saw the coming of the Dutch Siboga Expedition which
undertook intensive dredging in the Sulu Sea. The specimens were studied
by different investigators. Van Bosse & Foslie worked on the corallinaceous
group; Barton on Halimeda; A. & E.S. Gepp (1911) on Codiaceae. Weber van
Bosse (1913-1928) published in two parts her annotated listing of blue-
green, green, brown, and red algae. She made special mention of the
abundance of Bornetella sphaerica (Zanard) Solms-Lauback. The bulk of the
collection was preserved supposedly in Holland and elsewhere in Europe.
Unlike previous expeditions, the Italian group failed to turn over duplicate
materials to the Philippine Government.
Between 1907 and 1910, the United States Fish Commission boat
Albatross visited the Philippines and some collecting was done. The
Chlorophyceae was partly worked out by Gilbert(1941, 1942a,b, 1946, 1947).
Velasquez (1963) cited most of the blue-greens years later.
In 1913, Merill and Shaw sent to the United States some marine algae
now deposited in the New York Botanical Garden. Most of the green algae
were loaned to and studied by Gilbert who described a new species, Acetabu-
laria philippinensis.
Collection trips that followed were rather small-scale joint ventures with
the Philippine Government. Outstanding were those by Barlett on two
occasions. In 1935, Bartlett, who at that time was with the University of the
Philippines as exchange professor, undertook extensive algal collecting from
Batanes down to Sulu. His collections were sent to the University of
Michigan.
Manza (1937a-c), a Filipino marine botanist, worked on articulated

corallinaceous algae and described such new genera as Bossea and Joculator
as well as some new species.
Bartlett made his second visit to the Philippines in 1940-41 as a full-time

agronomist. Nevertheless, he included seaweeds in his collections, with the
assistance of J.S. Domantay, then with the Bureau of Fisheries, and a
Filipino Muslim diver. The second batch of marine algal specimens was also
sent to the University of Michigan and identified and distributed under the
supervision of W.R. Taylor. The chlorophyceans appeared in the papers of
Gilbert (1941, 1942a,b, 1946, 1947); phaeophyceans in Taylor's (1961, 1962,
1963, 1966); myxophyceans in Velasquez's (1940, 1941a,b, 1962).
1942-1945
The outbreak of World War II set back algological study in the Philip-
pines. The country, scene of some of the world's fiercely fought battles, saw
its herbarium, then under the Bureau of Science, reduced to ashes during the
liberation of Manila. Nothing could be salvaged from the debris; algal and
flowering plant materials and valuable references disappeared in the flames.
A reported study during the period was that of Dawson (1954) who
collected Corrallopsis salicornia along the sea wall of the Manila Harbor.
His material later became the topotype of the present Gracilaria salicornia
(C. Agardh) Dawson and, therefore, confirmed the real type locality of
Chamisso's erstwhile Corallopsis material which was doubted by Ruprecht
in 1851.
1956 to the present
Rebuilding Philippine phycology was a very challenging task. The initial

step was taken by Velasquez who, under a grant-in-aid program from the
American Philosophical Society, continued his research on the myxophyceae.
A number of his masteral students assisted in the rebirth of research and
accumulation of references on algae.
The next step was the organization of the Phycological Society of the
Philippines under the initiative of Dr. Velasquez. The aim was to build a
"bank" of well-duplicated numbered specimens, only roughly identified as to
family or genus, from which specialists might receive materials for use in
monographic or regional studies.
In November 1964, one of the biggest post-war expeditions arrived in the

Philippines. This was the joint exploration-collection of the Kagoshima
University of Japan and the National Museum of the Philippines. The places
of collection were the islands of Batan, Batanes, and Camiguin, and the
provinces of Cagayan and La Union (San Fernando), all in the northern part
of the country. The expedition lasted for one month and yielded a very
substantia] number of algal materials including freshwater forms. Tanaka

(1967) described some new species out of the collected materials, like
Avrainvillea capituliformis and Dictyopteris camiguensis from San Pioquinto,
Camiguin Island, Cagayan Province, and Claudea batanensis from Basco,
Batan Island, Batanes Province.
No notable expedition has been undertaken after the 1964 joint explora-
tion. However, the University of the Philippines, through Dr. Velasquez and
his students, has organized collection trips aboard the university training
ship Pampano. The Philippine National Herbarium has had its share of
algal collection trips through the initiative of the author and his co-workers
in the Division of Botany.
The Philippine National Herbarium shortly after the war was trans-
ferred to the National Museum. The algal section was left with no one to
restore the precious specimens that formed part of one of the richest herbaria
in the world. Except for few duplicates recovered from foreign herbaria, very
few materials were added. Numerous marine forms from various rich and
previously unrepresented collecting grounds have been accumulated since
1963. The Philippine National Herbarium today boasts of algal specimens
from different parts of the country as well as of foreign duplicates kept as
exchange materials.
The 60s witnessed the turning point in algal taxonomy in the Philip-
pines. It marked the initial active participation of Filipino phycologists in
taxonomic studies especially those dealing with marine algae.
CURRENT STATUS
The algae of the Philippines are probably the best known taxonomically
in the tropical Pacific. Earlier works have already been mentioned above.
Cordero (1977) studied the red algae, while the freshwater plankton became
the subject of research by Pantastico (1977) and Martinez & Eakle (1977).
The brown algae group is being studied by R. Modelo, Jr. at Kyoto University.
Contribution by Dr. G. Trono, Jr. and his students at the University of the
Philippines and by Dr. E. Menez of the Smithsonian Institution (including
his students at Siliman University and University of San Carlos) also
advanced significantly marine biology research in the country.
Velasquez et al. (1975) listed 229 genera and 824 species for the
Philippines based on 88 publications, the earliest by Rumphius (1750) and
the latest by Cordero (1977).
No less than 20 genera contain several species considered as potentially

of economic importance. Some of these are the green Enteromorpha, Ulva,
Caulerpa, Codium, Monostroma, and Chaetomorpha; the brown Hydro-

clathru8 and Sargassum; and the red Asparagopsis, Acanthophora, Eu-
cheuma, Gelidiella, Gracilaria, Hypnea, Laurencia, and Porphyra.
Caulerpa, Sargassum, Eucheuma, Gracilaria, and Porphyra promise to

yield economic benefit once maricultured intensively. Except for the Philip-
pine species of Sargassum, the taxonomy of species in these genera has been
worked out substantially by both foreign and Filipino phycologists.
To date, only Caulerpa racemosa, Eucheuma striatum, E. spinosum, and

Gracilaria verrucosa have been farmed vegetatively using cuttings. There
were previous attempts in 1980 to mariculture Porphyra by the Bureau of
Fisheries and Aquatic Resources (BFAR), but with little success due to
unsustained funding. Porphyra in the country is expensive, its supply being
dependent only on natural growth in the coastal waters of northern Luzon.
It promises to be a dollar-earning marine commodity. Three species occur in
the country (Cordero 1974, 1976), namely, P. crispata Kjellman, P. subor-
biculata Kjellman, and P. marcosii Cordero. The latter two are recom-
mended for sea farming.
FUTURE DIRECTION
The paucity of algal taxonomists in the Philippines is very pronounced.

This becomes even more apparent when we consider that the phycologists in
the country are based in Luzon. The Philippine Archipelago, with its 7,100
islands and a coastal line more extensive than that of continental U.S.A., is
a virtual paradise for phycologists. It is, however, a difficult place to work
in. Distance between islands has to be reckoned, logistic support is often
entirely self-borne, and the security in some areas is unstable.
It was only in the late 70s when phycology graduates started to prolif-
erate. Before, taxonomy and most biological sciences were taboo to Filipino
students. As one writer puts it, taxonomy is not a "fashionable" science; even
in terms of funding it plays second fiddle to its sister sciences like physiology
and ecology. There was also the misinformed notion that taxonomy is highly
specialized, and the dim chance of getting a good-paying job was most feared
by students. There is some truth to this because taxonomy contends with the
difficult problem of classifying biological organisms. Still, the problem
involving shortage of manpower adept in algal taxonomy and absence of a
well-defined national algal research program must somehow be alleviated
soon.
For the present, the Philippines needs a redirection of its approach to

algal taxonomy from the present classical method. This is not to say that we
do away with the latter. Rather, we recognize the classical method as
integral part of the taxonomic scheme, but we must gradually shift to the
cytological approach using the scanning electron microscope (SEM). How-
ever, considering the prohibitive cost of the SEM it is advisable for Filipino
algal taxonomists to give more emphasis on chemotaxonomy as a tool toward
improving taxonomic output. Chemotaxonomy has a close affinity with the
classical morphological method. It requires team effort - a histochemist,
someone to do immuno-electrophoresis, and another to do spectroscopic
analysis of polysaccharides, proteins, and other pigments in the cytoplasm
and cell wall of algae.
We are now doing the chemotaxonomy of brown algae in one area in

Luzon. Later, we hope to resolve with the same approach some fundamental
problems in the taxonomy of such economically important genera like Ulva,
Caulerpa, Gracilaria and Sargassum. For these genera, life-history studies
are also needed. Sargassum needs utmost attention. Velasquez etal.(1975)
list 27 species for the country, but the veracity of this record is difficult to
ascertain in the absence of a more intensive taxonomic study of the genus,
itself highly polymorphic. Sargassum is the country's answer to the algin-
rich kelp (Macrocystis) of temperate waters.
The issue, therefore, is how fast the Filipino algal taxonomists can accept
and shift to chemotaxonomy and cytology as fundamental tools toward
improvement of our research output.
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LENGER" - chiefly Polynesian. Journ. Linn. Soc. Bot. 15:235-246.
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H.M.S. "CHALLENGER" from various localities. Journ. Linn. Sco. Bot. 15:486-
489.
Gepp, A. and E.S. Gepp. 1911. The Codiaceae of the Siboga Expedition. Siboga
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Gilbert, W.J. 1941. Notes on Caulerpa from Java and the Philippines. Pap. Mich.
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Gilbert, W.J. 1942a. Studies on the marine Chlorophyceae of the Philippines.

Unpublished doctorate thesis. 217 pp., 17 pls., 45 figs., 1 map.
Gilbert, W.J. 1942b. Studies on the Philippine Chlorophyceae, I. The Dasyc-

ladaceae. Pap. Mich. Acad. Sci. Arts & Lett. 28:15-35.
Gilbert, W.J. 1946. Studies on Philippine Chlorophyceae, II. Survey of literature and
list of recorded species prior to 1940. Bull. Tor. Bot. Club 73:73-79.
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Torrey Bot. Club 74:121-132.
Greville, R. 1830. Algae Britannicae. 218 pp., 99 pls. Edinburg.
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23:44-48.
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Africa. Proc. Nat. Acad. Sci. 23:561-567.
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Nat. Acad. Sci. 23:568-572.
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p. 1-152, 4 phos., 3 pls.
Martinez, M.R. and T.W. Eakle. 1977. Fluctuations in algal population densities in
fishponds and fishpens, Laguna de Bay, Philippines. National Research Council
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dosphaeria, 21(2):207.
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218.
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Proc. 9th Pac. Sci Congress, Bangkok (Bot.)4:234-238.
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ALGAL PRODUCTION AND UTILIZATION
RELEVANT TO AQUACULTURE
IN THE PHILIPPINES
J.B. Pantastico
J.P. Baldia
C.C. Espegadera
and
D.M. Reyes, Jr.
Aquaculture Department, SEAFDEC
Binangonan Research Station
Binangonan, Rizal
INTRODUCTION
Phycological researches in support of aquaculture are a recent develop-

ment in the Philippines. Progress in this area gained momentum with
increased efforts to expand fish farming in the Philippines as a means of
producing more animal protein. The Aquaculture Department (AQD) of
SEAFDEC, having taken the lead in larval rearing of penaeids and economi-
cally important fish species, intensified its search for promising algal species
as natural feed. Imported and indigenous algal species were screened and
tested for use in hatchery and nursery operations. The vital role of
microalgae to sustain growth of larvae during critical stages of development
was demonstrated.
This paper presents the researches on algal culture and utilization

conducted at AQD, SEAFDEC from 1974 to date. Both brackishwater
and freshwater species are covered, with emphasis on freshwater algae.
SELECTED ALGAL SPECIES AS NATURAL FEED
Brackishwater Algae
Diatoms species were considered highly acceptable to Penaeus monodon

at the early zoea stages. For this reason, early attempts at growing natural
feed mentioned unidentified diatoms at 20,000 to 50,000 cells/ml for hatch-
ery operations (AQD Annu. Rept. 1974). Nitzschia, Navicula and Thalas-
siosira in washings from the seaweed Sargassum were also supplied as
natural feed in combination with bread yeast.
Mixed diatoms, predominantly Chaetoceros sp., were given at 1-5 x 103

cells/ml (AQD Annu. Rept. 1975). A new technique of growing natural feed
was applied wherein Chaetoceros sp. and Skeletonema sp. were grown
separate from the larval rearing tank (AQD Annu Rept. 1976). Skeletonema
costatum imported from Japan had a low temperature requirement which
limited its use in the Philippines.
Of the naturally occurring diatom species collected from Buyuan Bay,

Iloilo, Chaetoceros calcitrans was most promising because of its small size (4-
5 μm diam.) and stability in culture under different environmental condi-
tions. This indigenous species has proved very effective as natural food.
More recently, algal species other than those belonging to the Bacillario-
phyceae have been used as natural feed. Tetraselmis chuii and two strains
of Isochrysis galbana were imported from other laboratories. Local strains
of Tetraselmis sp. and Dunaliella sp. were also tested (AQD Annu. Rept.
1980).
Freshwater Algae
Expansion of the research program to freshwater led to the establish-

ment of the Binangonan Research Station. Here, emphasis on hatchery and
nursery operations for tilapia and milkfish necessitated algal production.
Algae representing different major groups were selected, namely: Chlorella
ellipsoidea, Chlorophyta; Chroococcus dispersus, Cyanophyta; Navicula
notha, Chrysophyta; and Euglena elongata, Euglenophyta.
The algal composition of "green" water usually varies. The predominant

species are Chlorella spp. and Scenedesmus spp. Ankistrodesmus sp. and
Nannochloris are also present in lesser numbers.
CULTURAL METHODS
Brackishwater
Batch cultures of algae, being the simplest, were used during the early
attempts at larval rearing of P. monodon (AQD Annu. Rept. 1976). Seawater
was enriched with commercial fertilizers (NPK or urea), and naturally
occurring algal species were made to "bloom" in rearing tanks. However,
problems were encountered when excessive diatom blooms resulted in left-
over algae which decayed and polluted the water. Furthermore, fertilizers
seemed to be toxic to the larvae. Thus, the procedure was modified so that
the algal culture was sand-filtered and the diatom concentrated was pumped
into the larval rearing tank (Platon 1978).
More improvements were made later in the algal production system.

Stock cultures of different species isolated from Buyuan Bay were main-
Utilization of Algae in Aquaculture 101
tained in the laboratory. Chaetoceros calcitrans was among the first algae
to be studied extensively and utilized effectively as live, natural food for P.
monodon larvae. It was established in a culture medium containing macro-
and micro-nutrients (Table 1). There were two sources of silicon in the
medium, the inorganic salt and "Agrimin" which is a commercially available
mixture of micro-nutrients. Growth of C. calcitrans in this medium was
monitored. When nutrients were replenished daily, growth per day for the
first three days was significantly higher (148.7%) than in the control (35.6%)
without replenishment (Fig. 1).
Improvements of the basal Chaetoceros medium were made later with

good results (Platon, 1978). Simplication of culture media for large-scale
tank cultures utilized commercial fertilizer (e.g., urea). Silicon was always
provided to enhance growth of diatoms.
Table 1. Chaetoceros medium
NaNO3 - 0.1 g/l

K2HPO4 - 1.0 g/l
FeCl4 - 0.2 mg/1
Na2SiO3 - 0.1 mg/l
Vitamins (B 12 & B1) - 1.0 mg/1
Agrimin* - 1.0 mg/1
Seawater (boiled/filtered) - 500 ml
Freshwater - 500 ml
*Agrimin, a brand name: Manganese, 15%, Boron, 5%; Iron, 8%; Calcium, 3%;
Zinc, 10%; Molybdenum, 5-10%; Copper, 5-10%; Potassium, 3%; Silicon, 36%.
In Freshwater
Laboratory cultures. Recent experiments were conducted to compare the

growth of selected algal species in three types of media: a) organic, b)
inorganic, and c) semi-synthetic (Table 2). Inexpensive organic sources of
nutrients such as ipil-ipil {Leucaena leucocephala) leaf meal extract and
duck manure extract were used. Chemical analyses of the three types of
media show some differences in the amounts of major and minor elements
required for algal growth (Table 3).
Growth rates of Chroococcus dispersus in the different types of media

were comparable (Fig. 2). However, the lag phase was longest in the organic
medium. It took about eleven days for the logarithmic phase to be reached
as compared to only seven days in the inorganic and semi-synthetic media.
This may be explained in terms of the slow release of nutrients in the organic
medium.
Duncan's Multiple Range Test did not show significant differences

among the different media tested for C. dispersus (Table 4).
Chlorella ellipsoidea showed the best growth rate in semi-synthetic

(K= 1.16) and inorganic (K=1.12) media (Table 4). The organic medium was
relatively poor for Chlorella (Fig. 3).
Navicula notha* preferred the organic and semi-synthetic media over

the inorganic one. With the inorganic medium, the log phase was reached
only after 12 days of culture (Fig. 4).
Euglena elongata showed significantly different growth rates based on

the type of medium: best in semi-synthetic, moderate in organic, and poor
in inorganic (Fig. 5, Table 4).
Fig. 1. Growth of Chaetoceros calcitrans in Chaetoceros medium (see Table 1

for composition).
*Verification of identification courtesy of Dr. Milagrosa Martinez, University of the

Philippines at Los Banos.
Table 2. Media for growing selected species of freshwater algae
Inorganic Medium
g/l
CaNO2 .1258
MgCl2 .0664
MgSO4 .0450
KC1 .0191
NaCl .0812
NaHPO4 .0229
NaNO3 .2573
Na2SiO3 .1861
FeCl3 .0003
Micronutrients* l ml/l
Organic Medium
Prepare following stocks separately: ml stock /l
- Ipil-ipil leaf meal extract
Grind 500 g ipil-ipil leaves; squeeze through cheese-

cloth in 500 ml distilled water; autoclave at 20 psi for
15 min. 10
- Duck manure extract
Pulverize 500 g duck manure; squeeze through cheese-

cloth in 500 ml distilled H2O; autoclave at 20 psi
for 15 min. 10
- Agrimin** - 10 g/100 ml 1
- Water 159
Semi-synthetic medium
ml stock /l
Inorganic medium (without micronutrients) 800

Soil water extract 200
Agrimin** 1
*Composition/100 ml: H3BO3, 200 mg; MnCl2.H2O, 150 mg; ZnSO4.7H2O, 20 mg;
CuCl2.5H2O, 10 mg; NaMoO4, 1 mg; Hormex, 1 ml.
**For composition, see footnote for Table 1.
Table 3. Chemical analyses of different media (ppm)
Parameters Organic Semi- Inorganic

synthetic
Total inorganic N .096 .051 .283

Orthophosphate .00017 .0032 .0153
Silica 20 82.5 91.6
Total Hardness (CaCO3) 158 158 -
Calcium 5.58 4.64 30.73
Magnesium 35 35.5 25.5
Sodium 25 180 118.2
Potassium 7.0 28.0 10.02
Manganese .87 .14 416.4
Iron .231 .046 .103
Table 4. Mean generation rates (K) of four algae in different media (Figures are
means of three replicates)
A. Algal Species
B. Media C. dispersus C. ellipsoidea N. notha E. elongata
(A1) (A2) (A3) (A4)
Organic media (B1) 0.74a 0.85b 0.89a 0.81a

Inorganic media (B2) 0.61a 1.12a 0.58b 0.61b
Semi-synthetic (B3) 0.65a 1.16a 0.75a 0.87a
Means of the same superscript in a column are not significantly different from
one another.
ANOVA for species (A), media (B) and AxB are highly significant. The
organic medium exerted the same effect on all the species, i.e., comparable
growth rates were shown by the four algal species (Table 5). In the inorganic
and semi-synthetic media, Chlorella showed significantly faster growth
rate. In general, the organic and semi-synthetic media proved best for all
the algal species representing different major groups (Table 6).
Based on the foregoing, the possibility of growing selected algae singly

or in combination in inexpensive media seems to be a promising alternative
in the production of natural food for use in aquaculture.
Outdoor tank cultures. A simplified, continues culture technique was

followed in producing phytoplankton for fry-to-fingerling production. Ma-
rine plywood tanks (1,000 liters capacity) were field with water to a depth of
40 cm only. NPK(14-14-14) was added at 0.1 g/l every three days to sustain
algal bloom. Furthermore, one-third of the old culture medium, including
algal cells that settled at the bottom, was siphoned out every three days
prior to fertilizer application. The same amount of tap water was added as
replenishment.
Fig. 2. Growth of Chroococcus dispersus in different media. Fig. 3. Growth of Chlorella ellipsoidea in different media.
Fig. 4. Growth of Navicula notha in different media. Fig. 5. Growth of Euglena elongata in different media.
Table 5. Duncan's Multiple Range tests of growth rates of different algae in different
media (Means underlined are not significantly different from one another)
Organic medium
C. dispersus E. elongata C. ellipsoidea N. notha
.74 .81 .85 .89
Inorganic medium
N. notha C. dispersus E. elongata C. ellipsoidea
.58 .61 .61 1.12
Semi-synthetic medium
C. dispersus N. notha E. elongata C. ellipsoidea
.65 .75 .87 1.16
Table 6. Duncan's Multiple Range test for media based on total growth rate means
of four species (Means underlined are not significantly different)
Organic Semi-synthetic Inorganic

(B1) (B3) (B2)
3.29 3.43 2.92
With the method described above, "green" water with a cell density of
150-175 x 103 cells/ml was produced. This optimum concentration of algal
cells can be maintained up to 60 days with proper management.
UTILIZATION OF MICROALGAE IN AQUACULTURE
Larval Rearing of P. monodon
One of the major problems in the operation of P. monodon hatcheries is

the need for a continuous and adequate supply of the right kind of live food.
Reports of high fry mortality triggered the all-out effort to conduct extensive
feeding studies and screen promising algal species.
Earlier laboratory studies (AQD Annu. Rept. 1976) used live and frozen
Chaetoceros calcitrans as feed for up to zoea 3 giving as high as 93% and 98%
survival, respectively. Thus, there is the possibility of harvesting and
storing diatoms for future use to augment the supply of natural feed during
times of scarcity. In the same experiment, diatom consumption was
determined by monitoring the cell density of the medium with or without
larvae. Results showed average diatom consumption per larva at the zoea
stages as follows: zoea 1 = 6,000 cells; zoea 2 = 13,100 cells; and zoea 3 =
14,000 cells (Fig. 6).
Fig. 6. Estimated algal consumption (Chaetoceros calcitrans) of different zoea

stages of P. monodon.
Later, feeding experiments explored the use of a variety of algal species

(AQD Annu. Rept. 1980). Five algal species which include local strains of
Chaetoceros calcitrans (10 x 104 cells/ml), Tetraselmis sp. (5 x 104 cells/ml),
Dunaliella sp. (5 x 104 cells/ml), two imported strains of Isochrysis galbana
(7 x 104 cells/ml), and Skeletonema costatum (10 x 104 cells/ml) were used as
natural feed for P. monodon larvae. Highest survival was obtained with C.
calcitrans.
Sunaz (1980) compared growth and survival of P. monodon zoeas given

different diatom feeds. Highest mean survival rates were obtained using
Chaetoceros gracilis (62.90%) and mixed diatoms (60.43%).
Fry to Fingerling Production
Tilapia nilotica. "Green" water consisting of Nannochloris sp., Chlorella

spp. and Scenedes spp. was given to T. nilotica fry at various concentrations:
a) high density - 150-175 x 10 3 cells/ml; b) moderate density - 90-120 x 10 3

cells/ml; and c) low density - 50-60 x 10 3 cells/ml.
There was a proportionate increase in growth and survival of tilapia fry

with increased density of phytoplankton. "High", "moderate" and "low" algal
densities gave growth rate of 13.3, 8.9 and 4.7 mg/day, respectively (Table 7).
Growth of tilapia fry given limited amounts of phytoplankton was poor and
comparable to that given rice bran as feed.
Table 7. Mean weight, survival rate and growth rate of tilapia fry fed phytoplankton
at various density levels
Algal Weight (g) Survival Growth

rate rate
density 0 2nd wk 4th wk 6th wk
(%) (g/day)
High 0.008 0.164a 0.481a 0.546a 93a 0.0133a

Moderate 0.008 0.098b 0.330b 0.363b 93a 0.0089"
Low 0.008 0.033c 0.099c 0.192c 62b 0.0047c
Rice bran
(control) 0.009 0.024c 0.045c 0.186c 36c 0.0045c
F values
(ANOVA) . 23.35* 29.04** 14.40** - -
Means with the same superscript in a column are not significantly different from
one another (DMRT).
*Significant. **Highly significant.
Gut analysis of T. nilotica fry grown in various algal concentrations gave

an estimate of the relative intake of algal food at various levels of feeding
(Fig. 7). Results showed decreasing algal food in the gut of T. nilotica with
decreasing amount of phytoplankton in the rearing medium.
Milkfish (Chanos chanos). Stage 1 milkfish fry reared in aquaria with

different algal species for five days showed high survival in the Chlorella-
Chroococcus-Euglena combination and Oscillatoria alone (Table 8). These
algal species were shown to be suitable natural feeds up to ten days of culture
for the Chlorella-Chroococcus-Euglena combination and up to 15 days for the
treatment Oscillatoria alone. Older milkfish fry, 20 days in culture (stage
IV), showed poor survival in all treatments.
More experiments are being conducted to pursue the preliminary results

described above.
CONCLUSION
Accelerated pace in aquaculture to produce fish protein for the people of

Southeast Asia calls for support from all disciplines. Phycology, despite its
Fig. 7. Remaining algal food after 24 h in gut of T. nilotica fingerlings in "green

water" with different phytoplankton densities.
Table 8. Mean survival (%) of different stages of milkfish fry given different algal
feeds (Figures are averages of three replications)
Stage
Algal feed I II III IV
(Sept. 3-8) (Sept. 9-14) (Sept. 16-21) (Sept. 21-27)
Chlorella 41.7 75.0 50.0 8.0

Euglena 75.0 41.7 0 8.0
Oscillatoria 100.0 100.0 100.0 50.0
Navicula 91.7 100.0 75.0 16.7
Chroococcus 41.7 91.7 41.7 8.0
Euglena-Oscillatoria-
Navicula 58.0 100.0 66.7 8.0
Chlorella • Chroococcus -
Euglena 100.0 100.0 58.0 50.0
Rice Bran 91.7 91.7 66.7 33.0
being a very basic discipline, is most relevant to fish farming. It is in the area
of natural food production where the micro-algae have become very impor-
tant to sustain high fry and fingerling survival.
There is need to integrate efforts in the culture and utilization of algae

for greater impact to fisheries development. Manpower and physical
resources should be pooled effectively. Only then can we go beyond the
laboratory scale and find application in the field.
LITERATURE CITED
SEAFDEC, AQD, Annual Reports 1974-1980.
Sunaz, F.P. 1980. Growth and survival of Penaeus monodon Fab. zoeae on different
diatom feeds. Quart. Res. Rept., SEAFDEC, AQD. 4(3).
Platon, R.R. 1978. Design, operation and economics of a small-scale hatchery for the
larval rearing of sugpo, Penaeus monodon Fab. Aquaculture Ext. Manual No. 1.
SEAFDEC, AQD.
CULTURE AND USE OF ALGAE IN SOUTHEAST ASIA
in Southeast Asia
AQUACULTURE DEPARTMENT
SOUTHEAST ASIAN FISHERIES DEVELOPMENT CENTER

Culture and Use of Algae in Southeast Asia Compressed

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CULTURE AND USE OF ALGAE

Proceedings of the Symposium on

Published and Printed by

Seaweed resources in the developing countries of Asia:

Utilization and farming of seaweeds in Indonesia 9

Present status of seaweed culture in Korea 21

Utilization of seaweeds in Thailand 27

Life history of Acrothrix pacifica and Sphaerotrichia divaricata

Antithamnion sparsum, its life history and hybridization

Water quality assessment of the Langat River, Selangor, Malaysia

Growth and development of Trentepohlia odorata in culture 55

Agroindustrial waste products as sources of cheap substrates for

Utilization of seaweed resources 77

Philippine algal taxonomy: Past, present, and future 89

Algal production and utilization relevant to aquaculture

Efforts to organize the Symposium started in early 1981 under the

Attendance in the Symposium included fifteen full participants from

Thirteen papers were presented covering a wide variety of topics from

This late the Aquaculture Department of SEAFDEC decided to publish

Gavino C. Trono, Jr.

The bulk of world seaweed production today comes from devel-

In the Philippines, development of the farming technology on

The socioeconomic implications of the development of the sea-

Although no accurate data are available, world production of seaweeds

In contrast, developing countries contributed very little to the total sea-

Present estimates (Naylor 1976) show that seaweed production in devel-

STATUS OF SEAWEED PRODUCTION IN ASIA

Except for Japan, China, and Korea which have a well-established

The harvesting of natural seaweed stocks is very unreliable. Production

The potentials of seaweed production in the developing countries of Asia

Table 1. Principal seaweed genera of economic potential in Asia

Country Genera Uses Status of

Philippines Caulerpa food pond culture &

Indonesia Gracilaria agar, food wild crops

Singapore Eucheuma carrageenan wild crops

Brunei Gracilaria agar wild crops

East Malaysia Porphyra food wild crops

West Malaysia Gracilaria agar wild crops

Thailand Gracilaria agar wild crops

Vietnam Gracilaria agar wild crops

Hong Kong Sargassum alginate wild crops

Sri-Lanka Gracilaria agar wild crops

India Gracilaria agar wild crops;

Burma Gracilaria agar wild crops

Pakistan Gracilaria agar wild crops

SEAWEED PRODUCTION IN THE PHILIPPINES

The Philippine seaweed export profile is reflected in Table 2. No official

Pond culture of Caulerpa is presently done in Mactan, Cebu. Although

Table 2. Philippine seaweed export, 1967-1980

Year Metric Ton Value (Pesos)

1967 674.5 351 989

All Sargassum productions in Central Visayas are being exported to

The data on Philippine seaweed exports indicate an almost twenty-fold

be hastened through the influence of international agencies such as the

The socioeconomic impact of the development of the seaweed resources

A Philippine experience, as exemplified by the development of the

The production in northern Bohol in 1979 was, however, small compared

As an entrepreneur, the seaweed farmer is subject to both favorable and

Since seaweed farming is labor-intensive, the industry can employ the

A great variety of seaweeds grow abundantly along the 81 000-

At present, seaweeds collected in Indonesia are mainly used

The paper reviews the state and problems of seaweed utiliza-

The Indonesian archipelago is situated between the Asian and the

Early marine research in Indonesia started when Amboina was a