Biotecnologia de Microalgas (1988 R) - (de La Noue and de Pauw) - The Potential of Microalgal Biotechnology a Review of Production and Uses of Microalgae

8/19/2019 Biotecnologia de Microalgas (1988 R) - (de La Noue and de Pauw) - The Potential of Microalgal Biotechnology a R…

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Biotech. Adv. Vol. 6, pp. 725-770, 1988 0734-9750/88 0.00 + .50

Printed in Great Britain. All Rights Reserved Copyright (~) 1988 Pergamon Press plc

THE POTENTIAL OF MICROALGAL

BIOTECHNOLOGY: A REVIEW OF PRODUCTION AND

USES OF MICROALGAE

JOEL DE LA NOUE and NIELS DE PAUW

Groupe de Re c h e rc he e n Re c y c lage B io log ique Un iv e r s i t~ L av a l Quebec,

C a n a d a G 1 K 7 P 4

L abora tory

fo r

M a r i c u l tu r e S t a t e U n i v e r s i t y o f G h e n t G h e n t B e l g i u m

ABSTRACT

An overview of the various aspects, promises and limitations of microalgal

biotechnology is presented. The factors of importance n microalgal cultivation

as well as the culture systems are briefly described. Microalgal biomasses can

ful f i l the nutritional requirements of aquatic larvae and organisms. The

biochemical composition of algae can be improved by the manipulation of culture

conditions. The nutritive value of the microalgal biomasses for human and animal

consumption is also commented upon as well as some socio-economical aspects.

Among the sources of required nutrients N, P), wastewaters and manures can

upgraded as culture media for microalgae he safety of which has to be evaluated.

Harvesting of the biomass is one of the bottlenecks. The various techniques,

physical, physico-chemical and biological are outlined and their feasibility and

economic interest examined. Microalgal biomasses can be submitted to various

technological transformations. Various processes are reviewed in the light of

their effects on safety and nutrit ional value. The possible extraction of fine

chemicals and the preparation of protein concentrates is also reported on. The

various uses of microalgae lead to a possible competition, to be evaluated,

between systems for the production of food, energy and chemicals. The review

finally covers the application of genetic manipulation to microalgae.

725


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7 6

J D E L A N O U E a n d N . D E P A U W

KEYWORDS

Microalgae - Solar biotechnology - Culture conditions Microbiological

safety Harvesting Nutr it ional value Technical transformations - Fine

chemicals - Feeds/Foods - Genetic manipulations -

IMPORTANCE AND POTENTIAL OF MICROALGAE

Microalgae, as well as bacteria and yeasts, belong to those promising

microorganisms which deserve attention within the vast array of tradi tional as

well as new biotechnologies. Along with basic research, applied algology has

been developing rapidly over the last 40 years, star ting in Germany and extended

in the United States, Japan, Israel , I tal y, with the aim of producing single cel l

protein (SCP} and fat (see Burlew, 1953). As a result, already in the 1960s, the

commercial production of Chlorella in Japan and Taiwan as a novel health food

item was a success (Kawaguchi, 1980; Soong, 1980). In the 1950s the idea of

using microalgae fo r wastewater treatment was launched and in the 1960s interest

grew in developing ex tra ter rest ri al l i fe support systems. In the 1970s attention

went to the production of microalgal biomasses for fuel and fer t i l i zers. A new

trend in the 1980s is to use microalgae as a source of common and fine chemicals.

Furthermore, microalgae have since the 1940s been playing a role of increasing

importance in aquaculture. For histor ica l reviews on applied algology, the

reader is referred to Goldman (1979a) and Soeder (1980, 1986).

An important feature of microalgal systems is thei r v er sat i l i ty, making i t

possible to l ink di ff erent applications within the same process, for example

wastewater treatment and production of food, feed and chemicals. Another

at tracti ve characteristic of microalgae, in comparison with other microorganisms,

is the ir photosynthetic capabi li ty to convert solar energy into valuable biomass

with an interesting biochemical composition. As such, microalgae could play an

important role in solar biotechnology. Hereby, annual yields of 25 T and more,

even up to 200 T dry weight algae per hectare have been forwarded (Dubinsky e_tt

a ..., 1978; Goldman, 1979a; Shelef and Soeder, 1980; Soeder, 1980; Sant il lan,

1982; Richmond, 1986c). Moreover,more than 60 of the dry weight can be made up

by protein. Under favourable conditions, microalgal cultures can produce up to

20 to 35 times more protein than soybean and more than 50 times more than rice,

wheat or maize for the same area (Switzer, 1982; Ci ferr i , 1983). Of part icular

importance is also that microalgae can be grown yearound and harvested on a

continuous basis and be cultured on marginal lands in arid regions of the world,

ut i l iz ing waters unsuitable for conventional agriculture (Thomas, 1983; Cife rr i

and Tiboni, 1985; Gauthier et al_~.., 1985; Hal l, 1986; Richmond, 1986c). Algal


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MICROALGAL BIOTECHNOLOGY 7 7

cultures also have a lower water consumption than that required by tradit ional

cultivars (Heussler e_t_t al___:., 1978c). I f one considers that the water used for

algal cultures can be used afterwards for i r r igat ion, algal cultures are even

more advantageous.

Although there is yet no real breakthrough in this f ie ld , due to a number

of fundamental drawbacks (Benemann and Weissman, 1984; Benemann e .t al_._~., 1986;

Soeder, 1986), interes t in applied algology has never decreased i f we look at the

recent appearance of several important books on this matter: Algae Biomass

(Shelef and Soeder, 1980), Biotechnology and Exploi ta tion of Microalgae (Becker

and Venkataraman, 1982), CRC Handbook of Microalgal Mass Culture (Richmond,

1986a), Algal Biomass Technologies (Barclay and Mclntosh, 1986), Algal

Biotechnology (Stadler e_t_t al___~., 1988) and Microalgal Biotechnology' (Borowitzka

and Borowitzka, 1988). In connection one can mention the establishment in 1980

of an International Association fo r Applied Algology (IAAA) on ,the occasion of

the International Conference on Microalgae Production in Tru j i l l o , Peru (G.

Shelef, person, commun.) and the French Association fo r Applied Algology ,

active since 1982 (R. Fox, person, commun.). Also a newsletter cal led Applied

Phycology Forum , edited by W.R. Barclay in the US, is dist ri bu ted worldwide

since 1984 (Barclay, 1984-1985).

PRODUCTION OF MICROALGAE

Figure l schematically shows the major pathways followed in microalgal

production and ut i l iza t ion of the biomasses for di ff erent purposes. Production

of microalgae f i r s t involves the cu lt iv at io n, followed in most cases by harvest-

ing and processing o f the algae (Soeder, 1980; Becker and Venkataraman, 1982).

From a systematic point of view, the microalgal group includes several

thousand species belonging to two major groups: the prokaryotes inc luding

blue-green algae (cyanobacteria) and the eukaryotes, including a.o. green algae

(Chlorophyta), red algae (Rhodophyta), and diatoms (Bacil lariophyta). Of these,

only 30-40 species have been considered for mass cu lt iv at ion and only few are

presently of real commercial importance (Borowitzka, 1988a; Richmond, 1986c). To

these belong representatives of the genera Chlorella, Scenedesmus, (green algae),

Spirul ina (a blue green alga) and a number of phytof lagellate and diatom species

which are used as live food in larval mariculture (De Pauw and Persoone, 1988).

Other algae of commercial in terest in the future could be Dunaliella (a green

flagel late), Porph~ridium (a red alga) and Botr¥ococcus (a green alga) (Richmond,

1986c). Expressed in quanti ties, the world production of microalgae (mainly

Spirul ina and Chlore lla) only amounts to about one thousand tons per year


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7 2 8

[ DE LA NOUE and N DE PAU W

~I ~ CULTIVATION POND

m c cc ~ ~ 0m;~nolm.ra~ ~

~ ccMP~ I

l i

F ~F ~

Figure I. Flow diagram of algal mass cultivation systems (from Becker and

Venkataraman, 1982).

(Kawaguchi, 1980; Soong, 1980; Ci ferr i and Tiboni, 1985) but is expected to

increase markedly in the near future (Venkataramanand Becker, 1988).

Determinants of Algal Growth

Major factors of importance in the cult ivat ion process are l ight,

temperature, nutrients, pH and agitation (Becker and Venkataraman, 1982).

Although maximum growth rates are achieved in conditions of l ight saturation,

maximum yields wi l l be obtained only when l ight is the l imi ting nutrient since

production is proportional to solar energy conversion eff iciency. Light

limi ta tion is established as a function of irradiance by adapting the areal

density of the culture and thus the algal concentration. In continuous cultures

this is achieved by changing the detention time or d ilu tion rate of the culture

(Goldman, lgTgb). The maximal theoretical conversion efficiency for total l ight

energy has been established at 6.6 (Shelef and Soeder, 1980). However,

sustained efficiencies of conversion of total incident radiation are more l ikely

to be within the range of l to 2 , or expressed in PHAR (Photosynthetic Active

Radiation) about 2 to 4.5 (Benemann eta)..., 1977; Goldman, 1979b; Shelef and

Soeder, 1980; Soeder, 1980; Pouliot and de la NoUe, 1985). Translated into dai ly

yields, these conversion ef fic iencies correspond to less than lO g up to 30 g and

in exceptional cases even 50 g dry weight per m (Goldman, 1980). Althoughunder


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MICROALGA L BIOTEC HNOLOG Y 7 9

l ight limiting conditions, the effect of temperature is lessened with regard to

the growth rate, too low temperatures may also become limiting especially during

the daytime or in winter Toerien and Grobbelaar, 1980; Vonshak e.t_t al., 1982;

Bedell, 1985). For this reason heating up of the cultures may be beneficial

mainly in areas with a l ot of incident radiation. Optimal temperatures for most

species range between 15 and 30°C.

Several kinetic as well as empirical models predicting algal productivities

as a function of irradiance and/or temperature have been developed Goldman,

1979b; M~rkl, 1980; Toerien and Grobbelaar, 1980; Hil l and Lincoln, 1981; Grobbe-

laar e al_~., 1984). Other models also take nutrients Shelef, 1981, in Grobbe-

laar et a l. , 1984) and mixing into account Erickson and Lee, 1986). In open air

cultures, yields are pract ically linearly correlated with the incident radiation

Paelinck, 1978, in De Pauw and Van Vaerenbergh, 1983; Castillo e_t_tal_~., 1980).

For optimal growth, the culture must be provided with nutrients in adequate

amounts Borowitzka, 1988b). These include several macronutrients such as

carbon, nitrogen and phosphorus, sulfur , potassium, for diatoms also si licon)

and a number of trace elements like minerals e.g. Co, Mo, Mn) and several

vitamins e.g. Bl2, thiamin). Besides quantit ies, the right proportions among

nutrients e.g. N:P; N:Si) are also important. Basic information on algal

nutr ition is given by Kaplan et al. 1986). With regard to the source of carbon,

apart from some algae like Spirulina which are capable of using bicarbonate at

alkaline pH values Ci fe rri, 1983), most photo-autotrophically growing algae

prefer to ut i l ize free CO2 Heussler e al., 1978a; Richmond e al., 1982; Kaplan

e_t_t al.__~., 1986). Yields may, however, be increased two- or threefold by using

organic sources of carbon like glucose or acetate mixotrophic growth) which of

course adds substantially to the production costs Soong, 1980).

Present attention is also directed towards growing microalgae

heterotrophically in the complete absence of l ight of which certain species e.g.

Chlorogonium elongatum and Chlorella p~renoidosa) are capable Kawaguchi, 1980;

Becker and Venkataraman, 1982; Kreuzberg et al. , 1985; Kaplan et al ., 1986).

This process could also be exploited under conditions of low illumination where

glucose can replace l ight as the energy source Folmann et al., 1978). The type

of C-source is related to the pH Richmond, 1986b). This factor substantially

controls the bio-avai labil ity of nutrients. A too high pH for example wi l l ,

through the photosynthetic activity, make the free carbon dioxide unavailable to

most algae. Phosphorus, too, be may precipitated and ammonia nitrogen is also

stripped to the air De Pauw and Van Vaerenbergh, 1983). For this reason,

addition of free carbon dioxide is beneficial to algal growth Becker and


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730 J DE LA NOUE an d N DE PA UW

Venkataraman, 1982). Moreover, norganic carbon levels play an important role in

affecting individual species growth rates (Novak and Brune, 1985).

Agitation is also very important to microalgal cult ivat ion, not at least to

avoid sedimentation, photoinhibit ion, nutrient limita tion and thermal

st ra tifi cation but also to increase the l ight conversion efficiency (M~rkl, 1980;

Persoone e_t_t al. , 1980; Richmond, 1986d). Since mixing requires energy, however,

the most economical regime wi l l have to be sought for each cult ivat ion system,

for which the algal output is maximal (e.g. De Pauw et al . , 1983; de la NoUe e.t_t

al~, 1984). Even i f the above conditions are optimally fu l f i l led, a number of

biological problems can and wi l l arise in the mass cult ivat ion of microalgae.

These include contamination, grazing, diseases, premature collapse and lack of

species control (Shelef and Soeder, 1980; Becker and Venkataraman, 1982; De Pauw

e_t_t al~, 1984; Richmond, 1986d). Control measures for avoiding contamination by

bacteria and other algal species are ster il izat ion and ul tr af i lt ra t io n of the

culture medium (Ukeles, 1976). Grazing by protozoans and diseases like fungi can

eventually be treated chemically (Heussler et al . , 1978b; Becker and

Venkataraman, 1982; De Pauw, pers. commun.; Becker, 1986). Larger zooplankters

and insects can be removed mechanically by screening (De Pauw et al . , 1983;

Becker, 1986), eradicated chemically (Loosanoff et al . , 1957) or by changing the

culture conditions (Lincoln et al . , 1983; SchlUtter and Groenweg, 1981).

Premature collapse of pure algae cultures during upscaling can be avoided by

establishing balanced growth conditions. A protocol for defining such conditions

has been presented by Pruder (1981). Control over the species composition of

large scale cultures on the other hand can be obtained to a certain extent by

proper operational management (Azov et al ., 1980; Richmond et al . , 1982; Vonshak

et al. , 1982; De Pauw et al . , 1983).

Cultivation Systems

Depending on the purpose of the mass production, the technology employed

may vary from a state close to agriculture to elaborate biotechnology (Soeder,

1980).

With regard to the origin of nutrients involved in the cultivation, a

dist inct ion can be made between clean water and wastewater-based production

processes (Soeder, 1980). In the former case str ict ly defined media are usually

employed in which bacteria are of no significant metabolic importance; in the

latter case (Fig. 2) less defined media such as sewage and manure are used in

which mixed cultures of bacteria and microalgae are act ive. While for small

scale culturing one usually relies on media which are complex and expensive, in


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MICROALGAL BIOTECHNOLO GY 7

good effect. I t has also been shown that greenhouse technology could make

yearound algal production feasible in northern climates with low temperatures but

suffi cient solar irradiance Pouliot and de la NoUe, 1985). Recently proposals

have also been made to ut i l ize geothermal water Bedell, 1985; Goldstein, 1986).

With regard to the sophistication of the cul tivation technology, the process may

be semi-natural, without inoculation of algae, and a natural bottom, e.g. the

cult ivat ion of Spirulina at Sosa Texcoco) or a r t i f ic ia l , with inoculation of

precultured algae and lining of the bottom e.g. Spirulina cultivation in Taiwan)

Ciferri and Tiboni, 1985).

Finally, i t must be mentioned that the cultivation process can be

batchwise, semi-continuous or continuous Vonshak, 1986).

Harvestin9 of microalgae

One of the major bottle-necks, limi ting the further expansion of most

microalgal biomass applications, is the cost-effective harvesting Table l ) .

Except for a few larger algae like Spirulina, which can be easily recovered by

simple gravity f i l t rat ion and inexpensive microstraining Becker and

Venkataraman, 1982), this is not at all the case for most species which are

indeed small in size less than 20 ~m). For this reason, many efforts have been

devoted to the development of suitable technologies for harvesting these small

particles Mohn, 1980; Richmond and Becker, 1986). Though technically solved,

the handicap s t i l l remains the incompatibil ity between the eff iciency of the

proposed methods and their cost-effectiveness Benemann e_t_tal~, 1980).

Table I.

Advantages and disadvantages of different harvesting methods

modified from Benemann e _tal.~., 1977, 1979 in De Pauw and Van

Vaerenbergh, 1983).

Method Re liabil ity Cost energy Quality for

requirement bioconversion

Centrifugation good high good

Chemoflocculation good high poor

Sandfiltration fai r low poor

Ultrafi l tration good high good

Microstraining poor low poor

Bioflocculation poor low good

The most successful techniques are centrifugation, f i l t rat ion and

flocculation Mehn, 1980). In practice, a combination of techniques is often

used to preconcentrate and/or concentrate.the algae. For preconcentrating the


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734 J DE LA NOUE an d N DE PAU W

algae, chemical flocculation eventually followed by flotation is common practice

in many operations Becker and Venkataraman, 1982; De Pauw and Van Vaerenberg,

1983; Lavoie et al., 1984; Richmond and Becker, 1986). However, a number of

problems with the use of flocculants, including toxicity and carcinogenicity,

have been recognized Dodd, 1979). This aspect is certainly important when the

objective is to use the algal biomasses as food or feed. A promising non-toxic

flocculant in this regard could be chitosan which is a natural product derived

from chitin, avail able worldwide Nigam et al., 1980; Becker and Venkataraman,

1982). Presently n its disfavour, is its high price. Process optimization and

recycling of the flocculant, however, could increase the cost-effectiveness of

chitosan Lavoie and de la NoUe, 1983; Lavoie et al., 1984; Morales et al.,

1985). To reduce the high inorganic flocculants demand, the use of polymers or

ozone treatment prior to the flocculation process has also been recently proposed

Shelef et al., 1986).

Another important step forward would be to get control of the process of

bioflocculation autoflocculation) of microalgae without addition of chemicals

Benemann e___al~., 1980). Bioflocculation is the formation of cellular aggregates

by means of exocellular polymers. Research is in progress to unravel the

mechanism of this phenomenon Sukenik and Shelef, 1984; Lavoie, 1985; Sukenik e_.tt

al., 1985). Bioflocculation can also be enhanced by continuous mixing of the

cultures, promoting the succession of readily settl ing self-flocculating species

or inhibiting photosynthesis in standing populations Lincoln and Koopman, 1986).

Recently the great potential of tangential flow fi l tration for concentrating

marine microalgae has also been demonstrated Welsh e_t_t al., 1985). The equipment

is, however, st i l l expensive. Finally, for harvesting Dunaliella, several

procedures have been proposed which exploit the high salinity-dependent

physiological and behavioural characteristics of this species Borowitzka and

Borowitzka, 1988). Another method is exploiting the salinity-dependent

hydrophobicity of the Dunaliella cell membrane Curtain and Snook, 1983).

UTILIZATION OF MCROALGAE

MicroalBae for Aquaculture

Microalgae are one of the live foods which are essential in aquaculture for

hatchery rearing of bivalve molluscs and peneid shrimp as well as the culturing

of several zooplankters rotifers, cladocerans, brine shrimp, copepods) which are

themselves live food organisms for larvae of marine fish and curstaceans De Pauw

and Pruder, 1986). Numerous, more or less sophisticated systems, have been

developed for culturing some 40 algal species to feed these larvae and


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MICROALGAL BIOTECHNOLOGY 7 5

zooplankton organisms (Ukeles, 1976; Watson, 1979; De Pauw, 1981; De Pauw and

Pruder, 1986; De Pauw and Persoone, 1988). In particular cases where pure

nutrient-rich well water or deep sea water is available, i t has been shown that

large scale production of monospecific algal cultures to feed oysters and clams

up to market size would be feasible (e.g. Roels et al., 1976; Scura e_tt al_~.,

1979). Also, i t has been demonstrated hat closed-cycle rearing of the American

oyster, from the larva up to the market size, is possible (Pruder and Greenhaugh,

1978)o However, in the latter case, the economic feasibility can be put in

question. For this reason, when large quantities of microalgae are needed, the

alternative for pure algae cultures may consist of bloom induction of natural

phytoplankton (De Pauw, 1981; De Pauw and De Leenheer, 1985).

De Pauw et al. (1983) demonstrated hat with proper operational management,

i t is possible to steer the composition of the natural assemblages towards

species suited to the consumers. Based on that principle, an industrial model

for nursery culturing of bivalve molluscs has been worked out (Claus et al.,

1984). The cultivation of microalgae none the less requiring specific skills,

the harvest and eventually the storage of algae grown at latitudes with ample

sunshine would represent a major breakthrough in aquaculture hatchery and nursery

operations (De Pauw et al., 1984). Apart from the constraints of economical

harvesting, however, are the processing and storage of the algal harvest and the

(re)treatment of the stored algae to make these acceptable again to the

consumers. More specifically, microalgae entangled n the matrix of a flocculant

are of too large particle size to be ingested by most filter-feeders, and

techniques for declustering such algal masses need to be developed (COST,

1983).

Apart from technical and economic problems involved with the mass

production of microalgae, the major problems in aquaculture are nutrition-

related (De Pauw e_t_tal_~., 1984). On the one hand, there is the lack of knowledge

of the nutritional requirements of the microalga consumers (molluscs,

crustaceans, fish) which are diff icult to assess, and on the other hand, there is

the biochemical composition of the microalgae which is determined by the culture

conditions (Webb and Chu, 1982). Of particular importance here is the presence

of essential fatty acids and the degree of fatty acid unsaturation. These

quantities can be modified by changing the culture conditions (Samson, 1980;

Enright e_t al.__~., 1986b). Of importance, for example, are l ight, temperature,

N-source, N:P ratio, etc (Sansregret, 1986). The same is true of the content of

amino acids and carbohydrates which are also of importance (Enright e_tt a__~.,

1986a; Terry e_t_tal_~., 1983; Terry, 1986).


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Microal~ae for Wastewater Treatment

The increasing deterioration of our environment and the need for energy and

food is forcing us to explore the feasibi l i ty of wastewater recycling and

resource recovery. Within this context, bio-treatment with microalgae is

part icular ly at tract ive because of thei r photosynthetic capabi li ties, converting

solar energy into useful biomasses and incorporating nutrients such as nitrogen

and phosphorus causing eutrophication (Fig. 2). This fascinating idea launched

some thirty years ago in the U.S. by Oswald and Gotaas (1957) has since been

intensively tested in many countries (see examples n Goldman, 1979a; Shelef and

Soeder, 1980; De Pauw and Van Vaerenbergh, 1983). Thereby, emphasis may be put

on wastewater treatment and/or algal biomass production. Depending on the

options taken, deep oxidation ponds or shallow high rate oxidation or algal ponds

(HIROP, HRAP) are used for this purpose. For a review on the variables playing a

role in the design and operation of microalgal wastewater treatment systems we

refer to Azov and Shelef (1982), De Pauw and Van Vaerenbergh (1983) and

Abeliovich (1986). Processes involved in the removal of nutrients are

precipitat ion, stripping and (luxury) uptake by algal biomass. The efficiency of

removal of a part icular nutrient (for example N or P) wil l also depend on whether

or not these nutrients are limi ting in the wastewater to be treated. In this

regard i t was shown by de la No~e et al. (1980) that a two-phase culture system

with preconditioning of the algae could increase the nitrogen uptake of the

starved algae. In practice, with adequate stirring more than 90 nitrogen and/or

phosphorus can be removed (e.g. De Pauw e__t_tal__~.., 1978; Lincoln and Hi l l , 1980;

Shelef e_t_tal__~., 1980; Martin et al . , 1985a,b).

Provided suff ic ient solar irradiance and space are available, (non-toxic)

wastewaters of various or igin and nature (municipal, industr ia l, agricultural ,

aquacultural) may be treated with microalgal systems. Part icular ly promising

seem to be the combined treatment and upgrading of animal manures from

bio-industries with algal biomass production possibly in combination with biogas

production (Chung e_t_ta___~., 1978; Taganaidese__t_ta___~., 197g; Lincoln and Hi l l , 1980;

Groeneweg e_t.tal___~., 1980; De Pauw et al . , 1980a; Pieterse and Le Roux, 1980; Soong,

1980; Duerr, 1985; Martin e_t_tal~, 1985b). Light and temperature being the major

factors determining yi eld, wastewater treatment with algae wi l l be part icular ly

suited for application in tropical and subtropical countries (Soeder, 1984).

Light often being the limiting factor in Northern climates during winter time,

the use of ar t i f i c ia l il lumination to increase the performance of wastewater

treatment in Quebec, Canada, has also been considered (Pouliot and de la NoUe,

1985). However, the cost of such a process is presently prohibi tive, i f not

forever. I t is also important to stress that in the treatment process, a large


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MICROALGAL BIOTECHNOLOG Y 7 7

array of microalgal species developing in different aquatic environments e.g.

fresh-, brackish-, sea-, alkaline-, brine-water) adapted to specific high

nutrient loads can be utilized De Pauw and Van Vaerenbergh, 1983).

To be effective in wastewater treatment, the algal biomasses must be efficiently

removed. Different pathways may be followed to this end. Usually, harvesting

includes a solid-liquid separation followed by dewatering and drying Moraine e_t_t

a_._~., 1980; Richmond and Becker, 1986). Although harvesting is not presently

cost-effective, i t has been shown that under tropical and subtropical conditions

the cost-benefit of microalgal wastewater treatment processes may compare

favourably with classical wastewater treatment systems such as activated sludge

systems. Particularly in favour of microalgal wastewater treatment systems is

the fact that the harvested algal biomasses can be upgraded n numerous ways, for

example to animal feed, and that no sludges have to be handled Shelef e_t_t al.,

1978).

Wastewater grown microalgae may be used not only as a supplement n animal

feed Chung e_t_t a.__~., 1978; Lincoln and Hil l , 1980; Sandbank and Hepher, 1980;

Soong, 1980; Saxena e_t_t al_~., 1983) but also in aquaculture for feeding fish,

molluscs and crustaceans see several examples in Grobbelaar et al., 1981; De

Pauw and Van Vaerenbergh, 1983 and de la NoUe et al., 1986), and as a source of

energy, fuel, fertilizers and chemicals Benemann e_t_t a___~., 1977). Indirect

harvesting of the microalgae by fil terfeeders through art i f icial aquatic food

chains including zooplankton, bivalve molluscs or fish, has also been a promising

and realistic alternative e.g. Dinges, 1974; Goldman and Ryther, 1976; De Pauw

et al., 1980a,b; Pieterse and Le Roux, 1980; Edwards, 1980; Groeneweg and

Schl~tter, 1981; Tarifeno-Silva e_t_tal...~_., Ig82a; Proulx and de la NoOe, 1985a,b).

Depending on the destination of the algal biomass, different criteria will

have to be taken into account with regard to possible contaminants such as heavy

metals, pesticide residues, pathogenic bacteria and viruses. However, the few

results already available indicate that fear concerning potential risk might be

excessive Yannai e_t_tal., 1980; Tarifeno-Silva et al., 1982b; de la NoOe e___al.,

1986; Gauthier et al., 1985; Becker, 1986). Pathogens and bacteria eventually

remaining will be eliminated from the algal biomass at the processing stage. In

this context, microalgal production could also be used as a trap for toxic ions

and molecules Aaronson e_t_ta_._~., 1980).

Since the land-space requirements of microalgal wastewater treatment

systems are substantial De Pauw and Van Vaerenbergh, 1983) efforts are being

made to develop wastewater treatment systems based on the use of


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7 38 J D E L A N O U E a n d N D E P A U W

hyperconcentrated algal cultures. This process, called activated algae

(McKinney et al . , 1971), is highly efficient in removing N and P within very

short periods of time, e.g. less than one hour (Lavoie and de la No~e, 1985).

Concentrated algal cell suspensions experiencing severe light l imitat ion appear

to be more promising for tertiary treatment than for actual biomass production

purposes. The design of workable systems, not only with free microalgae, but

also with flocculated or algae immobilized for example n carrageenan beads, has

also been shown possible (de la NoUe et al . , 1983; Chevalier and de la NoUe,

1985a,b; 1986). Also, recycling part of the algae produced, to operate at the

shortest possible retention time, could increase system performance (de la No~e

and Ni Eidhin, 1988). The challenge will be to make these systems not only

reliable but also economically competitive with conventional wastewater treatment

systems. I t could well be that the fi rs t applications to effluent treatment wi l l

occur in the field of toxic metal removal from industr ial eff luents. Some

promising systems are currently under investigation (see, for example, Kosaric

and Ngcakani, 1988).

Microalgae as Human Food

Many ef forts have been made to promote microalgae as food directly for man.

This idea has been even more supported by the discovery that several native human

populations, at Lake Chad in Africa and Lake Tezcoco in Mexico among others, have

subsisted part ia ll y on the nutr it ional qualit ies of the naturally occuring

blue-green alga Spirulina (Durand-Chastell, 1980; Ci ferri, 1983; Bourges, 1986).

Representatives of this prokaryotic alga are indeed very rich in protein (Table

2) and except for some deficiency of sulphur-containing amino acids (methionine,

cysteine), have a f ai r ly balanced composition (Table 3) comparing favourably with

egg and milk protein. The same is true of essential fatty acid content

(Sant illan, 1982; Ci fe rr i, 1983; Bourges, 1986). The nut ri tional qual ity of

Spirul ina protein is even superior to that of soybean (Jaya et al . , 1980).

Table 2. Basic chemical composition of the microalgae, Scenedesmus obliquus

and Spirulina maxima, as compared to soya bean (whole seed) and

wheat (whole gra~-~. Modified af ter Soeder (1980).

Components Scenedesmus Spirulina Soya seed Wheat

Crude protein 50-60 56-62 34-40 13.4-13 .5

Water 4-8 I0 7-10 12 .8-13.5

Li pi ds 12-I 4 2-3 16-20 2. 1-2.4

Carbohydrates l O-I 7 16-I 8 19-35 78.6-80.5

Crude fibre 3-I0 0.I-0.9 3-5 2.1-2.4

Ash components 6-I0 6.4-9.0 4-5 1.6-2.8


15/46

Table 3.

M CROALGAL BIOTECHNOLOGY 739

Essential amino acid composition of Scenedesmus species compared o the

FAO pattern (g/16 gN).

Amino acid

S. acutus S__~.obliquus2 FAO3

VAL 4.7 5.7 5.0

LEU 7.0 8.3 7.0

ILEU 3.1 4.1 4.0

PHE+TYR 6.0 lO.l 6.0

LYS 4.6 5.9 5.5

MET+CYS 3.2 2.9 3.5

TRY 1.7 n.d. l.O

THR 4.9 8.6 4.0

l Art i f ic ial medium (Becker et al . , 1976).

2 Urban wastewater (Proulx a~dTla NoUe, 1985b).

3 FAO/WHO (1973).

Furthermore, representatives of eukaryotic microalgae such as Scenedesmus,

Chlorella and Coelastrum have more or less the same nutri tional characteristics

(EI-Fouly e _t al__~., 1985). Microalgae are also rich in vitamins and other growth

factors. For these reasons, several research groups have been endeavoring to

establish production units for these algae in tropical or subtropical

(developing) countries l ike Peru, Thailand, India, and Egypt but also in the USA

and Israel (Soeder, 1980; Castillo e_t_tal__~., 1980; Becker and Venkataraman, 1982;

El-Fouly e_t_t al . , 1985; Richmond, 1986c). In most of these projects, serious

attention has been paid to the nut ri ti onal qual ity and the possible toxicological

effects of the algae (Payer e al . , Ig80; de la NoUe e al.__~., 1986). The tests

involved humans as well as animals (Pabst, 1978; Becker, 1980; Ci ferri, 1983).

Extensive testing has indicated that microalgae are a valuable and safe source of

protein. Testing with malnourished children also showed promise (Gross et al . ,

1978). The high nucleic acid content, however, limi ts the admissible daily

consumption to about 5 of the human requirements (Becker, 1986). Based on a

recommended maximum daily supply of 2 g nucleic acids from SCP per adult

(FAO/WHO, 1973, in Becker, 1986), 46 g of dry Spirulina or 15 g Scenedemus would

pose no problems (Becker, 1978a, 1986; Soeder, 1980; Bourges, 1986). Some

sensi tivi ty reactions have been reported with Chlorella in humans but no major

adverse effects have been reported (Shubert and Larsen, 1985; see also Scrimshaw,

1986).

I t is also of importance that the di gestibi l i ty and the nut ri ti ve value of

microalgae are influenced by the processing technology used (Becker, Ig80~

Venkataraman et al . , 1980; Becker and Venkataraman, 1982). I t has been shown

that d igestibi l i ty especially of chlorococcalean algae could be markedly


16/46


increased by cracking the cell wall with appropriate treatments such as drum

drying, spray drying, etc (Mitsuda etal . , 1977a,b; Becker, 1980). However, he

high cost of production including the harvesting and processing, and the

potential consumer nonacceptance (determined psyche- culturally, poor sensorial

properties) are the major obstacles to a defini tive breakthrough. Direct

consumption is therefore presently limited to the use of expensive health food

sold for US $ 60 per kg and more on the international market (Bourges, 1986;

Richmond, 1986). Total production amounts to about one thousand tons per year

mainly involving Spirulina from Mexico and the U.S.A. (Ciferri and Tiboni, 1985)

and Chlorella from Japan and Taiwan (Kawaguchi, 1980; Soong, 1980). In contrast

with the health food cultus (Hills, 1980; Switzer, 1982), a different philosophy

is followed by several other scientists who endeavor to exploit inexpensive

algoculture systems to combat malnutrition in developing countries (Fox, R.,

1980, 1983, 1985; Olguin and Vigueras, 1981; Becker and Venkataraman, 1982). The

integrated vi llage health and energy systems involve cultivation of Spirulina

along with the production of biogas and compost.

Micro~lgae as Animal Feed

As in the case of human food, microalgae have also been successfully used

as an animal feed ingredient. Feeding experiments with rats, mice, poultry,

pigs, sheep and carp, demonstrated unequivocally that microalgal meals produced

from various strains or species of Chlorella, Scenedesmus and Spirulina are

valuable protein sources lacking any acute toxicity (Becker, 1980, 1986). As

already mentioned s the utilization of wastewater grown microalgal biomasses for

which hygienical cr iteria are not so stringent as for human food part icularly

promising (see examples given in section on Microalgae for wastewater

treatment ). Primary and secondary toxicological testing of sewage grown algal

biomasses with regard to heavy metals demonstrated the likelihood that routine

use wi ll turn out to be toxicologically safe (Becker, 1980, 1986). Moreover

these biomasses are a by-product of wastewater treatment and thus cost-

competitive in with conventional feeds (Shelef et al., 1978). In contrast,

though the potential is there, the cost of pure microalgal biomasses like

Chlorella and Spirulina presently prohibits their extensive application in

aquaculture (De Pauw etal____~., 1984).

Microalgae as a Source of Energy

At pilot scale i t has been demonstrated that microalgal biomasses can be

converted by fermentation into energy-rich products such as methane gas, alcohol,

or liquid fuel as vegetable oils or hydrocarbon (Benemann et al., 1977, 1986;


17/46

MICROALG AL BIOTEC HNOLOGY 74

Mitsui, 1980; Cohen, 1986). A promising alga in th is regard seems to be

Botrxococcus braunii for the production of long-chain hydrocarbons Gudin e al . ,

1983; Benemann and Weismann, 1984; Richmond, 1986c). Though technically

feasible, a major drawback to practical appl ication is that enormous algal plants

are needed o ensure even a small percentage of our energy requirements. Concepts

have been developed for the exploi tat ion of algal energy farms up to lO0 square

miles in size in which unreclaimable water and wastewater nutrients could be used

Oswald et al . , 1977). The microalgal fuel economics and engineering of such

systems are described by Goldman and Ryther 1977) and Benemann et al. 1986).

Aside from the fact that no one is presently running algal farms larger than a

few thousand square meters, major constraints to further development are the

economical harvesting of the algal biomasses and the lack of control over

unwanted algal consumers like zooplankters. Moreover, n the present situation,

methane from microalgal biomasses is not at all competitive with conventional

energy sources Dubinsk.v e__t_tal__=, 1978). Other problems are related to the actual

methane fermentation of the algal biomass Benemann et al . , 1977).

Many algal species can also be induced to produce hydrogen through

biophotolysis. Cultures of ni trogen-f ix ing heterocystous e.g. Anabaena) as well

as non-heterocystous blue-green algae e.g. Spirul ina, Osci llator ia) can be used

for this purpose Benemann and Weissman, 1976; Hallenbeck e_t_t al.__~., 1978; Mitsui,

1980; Kumazawa and Mitsui, 1982, Karube et al~, 1986). A team from the

university of Miami Mitsui and co-workers) demonstrated hat a part icular strain

of marine blue-green non-heterocystous alga Osci llator ia) in a chamber 20 feet

square and three feet deep, could produce enough hydrogen to yield lO00

kilowatt-hours of elect r icity per month. Technical as well as economic

constraints, however, presently rest rict the practical application of these

systems. Immobilization of microalgae or cyanobacteria by entrapment in various

matrices polyurethane and polyvinyl foams, for example) might prove to be an

interesting solut ion to the problem of production of fuels, energy and chemicals

Ha l l , 1 9 8 8 ) .

Microal~ae as Fertilizers

A promising idea gaining more and more interest is the production of easi ly

harvestable nitrogen-fixing blue-green algae in conjunction with wastewater

treatment which could be converted into organic nitrogen f er t i l i zer Benemann,

1979; L i, 1981; Padhy, 1985; Venkataraman, 1986). I t has also been shown that

unconsumed microalgae in a wastewater-fish production system could be directly

upgraded agriculturally for the production of maize Edwards e al~, 1981). This

could be part icular ly rewarding in tropical countries where microalgal wastewater


18/46


treatment systems could be easily applied. Presently, several companies nvolved

in algal biotechnology, are exploring the potential of blue-green algae as

agricultural fertilizers and soil conditioners (Applied Phycology Forum, 1985;

Metting, 1985). According to Curtin (1985) one company in the US has already

started producing and selling fertilizers composed of blue-green algae, which are

competitive with conventional agricultural fertilizers. One kg of algae could

replace 60 kg of conventional nitrogen ferti l izer.

Microal~ae as a source of common and fine chemicals

Our modern industrial world depends heavily upon petroleum and its

derivatives to obtain a vast array of useful chemicals. Production of microalgae

in arid and sunny parts of the world may be a solution to the economic and

material stress raised by the needs of the industry. It has been suggested that

those who wi ll benefit f irst from commercial marine biotechnology wi ll be the

producers of sugars and polysaccharides, pharmaceuticals, dyes, bioflocculants,

pigments, vitamins, lipids, oi l , etc. One of the most convincing examples is

that of phycobiliproteins which are fluorescent dyes used in certain immunoassays

and cell separation and worth 75/mg (Curtin, 1985). Two categories of products

can be obtained f~om microalgae (Gudin e_t_t al_~., 1983): endocellular substances

that act as osmoregulators in the cell (glycerol, sorbitol, mannitol, etc) or do

not (starch, amylase, amylopectin, glycogen) and exocellular products, mainly

polysaccharides (glucan, mannan, chitan), hydrocarbon or polyacrylates.

Obviously, the latter can be recovered more easily, and usually without

destruction of the cells.

The many products of interest find applications in the chemical, food,

and pharmaceutical industries and medecine (Tables 4 and 5). For reviews on

chemicals and products from microalgae, the reader is referred to Aaronson et

al. (1980), Benemann and Weissman (1984), Borowitzka (lg88a), Cohen (1986). The

diversity of exo-polysaccharides produced by microalgae is impressive and

undoubtedly represents considerable potential for the food industry as gums,

thickeners, gelling agents and stabilizers and many other diverse uses (Weiner e__

al_~., 1985). A carrageenan-like polysaccharide has been extracted from the marine

microalga Porph~ridium cultivated in outdoor saltwater ponds (Curtin, 1985) by

Israeli workers (Arad et al., 1986). It has been shown by Weissman and Benemann

(1980) that polysaccharide productivity can be markedly enhanced by nitrogen

starvation. This alga is currently one of the most promising, and efficient

culture systems have been designed (Gudin e_t_t al., 1983).


19/46

M I C R O L G L B I O T E C H N O L O G Y

Table 4. Application of microalgae as a source of chemicals.

7 4

Chemicals Example Application

Proteins Protein concentrates Food and feed industries

Lipids Glycerides (glycerol) Fuels, food additives

Pigments B-carotene, Phytol Precursor vitamins

Phycobiliproteins Dyes, cosmetics

Vitamins Biotin Vitamin-rich meals

Carbohydrates Mannitol, Sorbitol Art i f icial sweeteners

Polysaccharides Viscosifiers

Pharmaceuticals Sterols Steroid hormones

Chlorella extract Antibiotics

Toxins Anti-parasitic

Table 5. Valuable products from microalgae.

Source Product Reference

Anabaena flos-aquae Protein Molton et al., 1980

Chlorella sp.

Phaeodactxlum tricornutum

Dunaliella tertiolecta


Asteromonas 9racilis

Microphytes

Chlam~domonas agloeformis

Porphxridium cruentum

Bacillariophyceae d~atoms)

Botrxococcus braunii

Phaeocxstis p ~ i

Scenedesmus acutus

Spirulina

Dunaliella, Spirulina

Cyanobacteria

Spirulina

Lipids Molton et al. , 1980

Sansregr-6t~T986

Starch (insoluble)Williams et al., 1978

Glycerol Dubinskye-t-aT., 1978

Ben-Amotz~ta-a-T., 1982

Agar-Agar Bonin, 1983

Carrageenan

Polysaccharides Moltonet al., 1980

Gudin et--a.l~,Ig83

Chitan Gudin ~a l . , 1983

Hydrocarbons Gudin et-aT., 1983

Polyacrylates Gudin~aT., 1983

Pigments Partali e_t_ta___~., 1985

Phycocyanin Tel-or et al., 1980

g-carotene Tel-or e t aT., 1980

Chlorophyll a

Phycobiliprotein Curtin, 1985

Enzymes Tel-or et al. , 1980

Ferredoxin

Ferredoxin-NADP reductase

Cytochromes

Ribulose bi-phosphate

Carboxylase

Although the ini t ial investments appear high, even prohibit ive, the cost of

producing glycerol from Dunaliella could be competitive with that of

petrochemical methods (Ben-Amotz and Avron, 1980). The method is subject to

patent applications and pi lot-scale production has been undertaken in Israel

where long-term productivities of 4.5 g of glycerol/m2.day in 3.5 M NaCl have


20/46


been measured (Ben-Amotz et al . , 1982). An intracellular glycerol content of up

to 4.4 molar in cell has been reported for D. v ir idis (Williams e al._.~., 1978) and

7 molar with D. salina (Borowitzka, 1981). This is about 50% of the dry weight.

Cell concentrations of above l g/L can, however, be di f f i cul t to obtain since

optimal growth conditions do not necessarily maximize glycerol production

(Ben-Amotz e_~_ta___~., 1982). Dunaliella exhibi ts the added advantage of having no

rigid cell wal l, thereby fac i l i tat ing extraction. By bacterial fermentation, the

algal-glycerol mixtures can be converted into neutral solvents (Nakas e a___~.,

1986).

I t is, however, the B-carotene-producing capacity of Dunaliella bardawil

(Ben-Amotz and Avron, 1980, 1983) which has stimulated a large commercial

investment, i.e. 2.5 mi ll ion by Koor Foods of Israel, over the last 6 years

(Weiner, 1985). About 5 to 9% of the dry biomass may be B-carotene. The same

incentive has been triggering large companies like Roche Products and

Wesfarmers to sponsor a sc ientific team in Austral ia to develop an industr ial

production unit for B-carotene from Dunaliella (Borowitzka e_tt al~, 1984,

1986a,b). In the USSR, attention has also been paid to the mass production of

Dunaliella (Massyuk, 1966). Within the group of the carotenoid pigments,

xanthophylls are also particularly useful in pigmenting chicken skin, tissue

and egg yolks (Benemann and Weissman, 19@4). Other pigments derived from

microalgae and receiving attention from industrial researchers are the

phycocyanins (blue pigments) making up the photosynthetic apparatus of

Spirulina and valuable as dyes in food or cosmetics (Ciferri, 1983; Sasson,

1983) or even in the jeans industry (Bionov, 1986, personnal commun.). Even

i f the production cost is high ( 15/mg) production under ar t i f i c ia l li gh t is

retained (Curtin, 1985). Other potential products from microalgae are phytol

and a wide range of sterols. Phytol is a suitable precursor for the synthesis

of several vitamins (A, E, K, K ) and B-carotene while sterols can be used as

substrates for the synthesis of steroid hormones (Borowitzka, 1988a).

Algae as a source of edible oi ls have begun to receive attention, in

spite of fierce competition among tradit ional vegetal oils and a slowly

expanding market . Other non-food markets may be suitable for algal oi ls

because of the ir chemical diversity and thei r high l ipid content, which can be

comprised between l and 85% on a dry weight basis (Shifrin, 1984) depending

upon the species and the culture conditions. For example, Phaeodact~lum

tricornutum is a marine diatom that contains some highly unsaturated long-

chain acids, some of which could be used as substitutes of drying oils in paints

and lacquers (Molton e_~_tal__~., 1980). These authors made some economic projections

on a process incorporating manure hydrolysis and algal culture for


21/46

MICROALG AL BIOTEC HNOLOG Y 7 5

oil and feed protein production and calculatea a conservative simple return on

investment of 21%.

If microalgae are used for oil production, the production cost based on

very conservative figures for capital and operating costs would be within the

range of 0.25-2.00/gai ( 0.07-0.75/L), which is quite encouraging (Shifrin,

1984). Certainly one of the factors which has aroused interest is the enormous

productivity possible - up to 25 and more tons/ha.yr requiring however a huge

capital investment. The possibi lity of coupling production to sewage

treatment may satisfy economic requirements (Dubinsky e_t_t al_~., 1978; Shelef e_t_t

al__~., 1978). The oi l content of many microalgae is in the range of 40-70%

(Ratledge and Boulton, 1985) and includes a large proportion of neutral lipids

with fatty acids in the C12-22 range (Shifrin, 1984). Species of Chlorella

appear to offer promise as a source of edible oils (Ratledge and Boulton, 1985)

while Botr~ococcus braunii excretes oils (unsaponifiable lipids) and carotenoids

which may have various industrial uses (Gudin et al., 1983; Wolf, 1986). Much

research remains to be done both on the production of the oi ls by the algae in

question, and on their recovery and chemical characterization (Benemann and

Weissman, 1984).

Another area of potential development for microalgae is the extraction

of pharmaceuticals (Table 6). Despite some di fficulties, such as relative

inaccessibility and lack of characterization, lower product-yield than usual

sources, complexity of purification, the search for pharmaceuticals from algae

and marine organisms is under way and some very interesting biologically

active products have been found (Wright, 1984; Borowitzka, 1988a; Cohen,

1986). For example, a lipid antioxidant function may be responsible for the

purported action of B-carotene as an anti-cancer agent (Burton and Ingold,

1984). Extracts from Chlorella and Scenedesmus have been shown to have in vitro

antibacterial activity (Reichelt and Borowitzka, 1984) and stimulate the growth

and yield of yeasts and other microorganisms (Fingerhut e_t_t al~, 1984). This

property finds application in the fermentation industry to improve the growth of

lactic acid bacteria (Soong, 1980). Extracts of Scenedesmus and Spirulina could

also serve as a replacement for serum (Kumamoto, 1984). Certain compounds

extracted from Porpt~vridium could play a role in commercial abalone culture,

inducing the settlement and metamorphosis of the larvae (Morse and Morse, 1984).

Other valuable compounds of interest for the food industry which may be

produced from microalgae include other osmoregulatory substances such as

sorbitol, mannitol, and cyclohexanetetrol, bio-emulsifiers and various low-

molecular weight metabolites, e.g. amino acids. The latter may be released


22/46

74G J DE LA NOUE an d N DE PAU W

Table 6. Pharmaceuticals rom microalgae.

Source Product Reference

Amphiridiumcarterae 8-2 blocker

(dinoflagellate}

Nitzchia ovalis (diatom)


Cyanobacteria

(blue-green algae)

Rivularia firma

Scxtonema hofmani

Lyngbya majuscula

Lyngbya lutea

Gomphosphaeria aponina

Spirulina

Dinoflagellates

Ptxchodiscus brevis

Microphytes

Antibacterial

Antioedema

Bronchodilator

Polysynaptic

Blocker

Anticonvulsant

Hypotensive

Antiinflammatory

Analgesic

Anaesthetics

Antiallergic

Cyanobacterin (antibiotic)

Malyngolide (antibiot ic)

Antineoplastic

Antiamphetamine

Aponin (lysis of dinofl.)

Serum replacer

Toxins (saxitoxin)

Brevetoxin

Cosmetics

Baker et al., 1985

Maksimova et al., 1984

Baker e_~tal__.~., 1985

Norton and Wells, 1982

Norton and Wells, 1982

Wood et al. , 1982

Cardillo et al., 1981

Wright, 1984

Baker e_t_tal~, 1985

Lem and Glick, 1985

Lem and Glick, 19~5

Curtin, 1985

Wright, 1984

Bonin, 1983

from immobilized cyanobacteria (Synechocystis) by osmotic shock (Reed et al.,

1986). I t has long been known that some microalgae, such as Spirulina, are

among the richest known sources for vitamins, especially Bl2 (Lem and Glick,

1985). The proportion of an alga s vitamin production excreted to the medium

may be quite high and could be enhanced by manipulating the growth conditions

(Borowitzka, 1988a). Although the studies done with immobilized microalgae

or cyanobacteria are recent (see Rao and Hall, 1984) progress has been recently

made with carrageenan for Scenedesmus (Chevalier and de la NoUe, 1985a),

calcium alginate (Martinez e_~_t al., 1987) or chitosan (Proulx and de la NoUe,

1988) for Phormidium., or polyurethane for Porph~ridium (Gudin e_.t_t al___:., 1983).

One interesting possibi lity, at least on theoretical grounds, is that of the


23/46

M I C R O A L G A L B I O T E C H N O L O G Y 7 7

co-immobilization of algae with bacteria, the former producing oxygen for the

latter which produce the product of interest for the food industry (Chevalier and

de la No~e, 1986).

PROCESSING OF MICROALGAL BIOMASSES

As previously shown, microalgal biomasses can be used as such in animal

feeding, aquaculture or for human consumption, despite low digestibi l i ty in

some cases or deficiency in some amino acids, such as sulphur-containing ones.

In some cases, however, processing of the biomasses might be desirable or

necessary either to improve the nut ri tional qualit y or to allow the

conservation of the biomass for later use. I f one wishes to extract specific

substances or added value products, additional processing steps are obviously

required.

Processin9 Methods and their Effect on the Safet~ and Nutr it ional Value of

Algal Biomasses

Sometimes, i t is possible to obtain strains of microalgae which possess

characteristics that simplify thei r ut il izat ion. For example, a strain of

Chlorella vulgaris (CCAP no 211-la) lacks the resistant cell wall component

sporopollenin (Strain et al~_., 1984). This strain has a relat ively high

digest ibi l i ty (81.7 ) without any treatment and , therefore, no expensive

processing to increase it s digestibi l i ty is required. In most cases, however,

i t is necessary to break cell walls either by applying physical, physico-

chemical or enzymatic treatments. Breaking cell walls is not an easy matter.

For example, the breaking pressure (by gas decompression technique) is 95

atmospheres for Chlam,vdomonas as compared to only 30 atmospheres for cultured

cells of carrot in suspension (Carpita, 1985).

Drying the biomasses is one of the most common treatments applied but

degradation may account for some 30 of the to tal production cost (Lin, 1985).

The choice of technique used for dehydrating algae affects the appearance, the

texture, the nutr it ional value and the digest ibi l i ty of the fina l product.

Spray-drying appears to be more useful than freeze-drying since toxic

substances are more effectively destroyed (Lin, 1985). The resulting powder

is harder, an important characteristic for the production of hard tablets.

Although these processes are expensive, thei r overal l evaluation shows that

they are superior to drum-drying (Lin, 1985). For Spirulina, thermal

treatment has been reported to be effect ive for detoxi fication (Voronkova e

a___~., 1983).


24/46


With respect to true protein content only, drum-drying, deamination,

irradiation and autoclaving appear o be equivalent in the case of Ooc~stis (Lee

e a.__~., 1982). Rats fed with autoclaved algae showed a lower Net Protein Ratio

(NPR) and a lower protein digestibility than rats fed algae treated by other

methods (Becker, 1980; Lee e_~_ al., 1982). Improved digestibi lity after

gamma-irradiation may be expected since this treatment brings about cellulose

depolymerization (Campbell e_~_ a___~., 1986). Studies done in vivo with rats fed

Chlorella have shown that treatment processes give different results. For

dried cells, digestibility is 60 , and rises to 73 after heating to lO0°C for

30 minutes and to 80 for broken cells (Fujiwara-Arasaki, 1984). Considering

the high cost of most processing techniques, i t appears that the most

economical may be to evaporate water from algae by sun-drying on sand beds

(Lincoln and Koopman, 1986; Becker and Venkataraman, 1982; Richmond and

Becker, 1986). It is clear that the choice of the technique is a matter of

balance between cost, intended use and changes in the characteristics of the

processed biomass. In the case of algae intended for human consumption,

concern has been expressed that their color might hinder consumer acceptance.

It has been shown that decolorization may be achieved by photolysis

(15 000 lux, fluorescent lamp, lO h). Moreover, this treatment resulted in

the removal of the unpleasant odour of the blue-green alga used, Anabaena

flos-aquae (Choi and Markakis, 1981). Enzymatic treatments are st i l l not

widely used for microalgae. By analogy with what has been done with marine

macrophytes (Fujiwara-Arasaki, 1984), one can expect that treatment with

pepsin, pancreatin and pronase might lead to increased digest ibi lity of

alkali-soluble proteins.

Another aspect of biomass treatment is that of supplementation.

Microalgae are known to be deficient in sulphur-containing amino acids

(Becker, 1978b; Ben-Amotz and Avron, 1980; Saxena et al., 1983). Supplemen-

tation of sun dried Spirulina platensis biomasses with 0.2 methionine has

been shown to significantly improve the biological value and the net protein

utilization with the same digestibility (Narasimha e_t_t al~, 1982). It would

also be possible to improve the nutritional value of S__~ platensis by

compensating for the low content in methionine through the use of mutants that

have higher intracellular pools of this amino acid (Ciferri, 1983). It is

suggested that with appropriate genet ic enhancement, microalgae producing

desirable amino acids in sufficiently high concentrations could be produced

(Borowitzka, 1988a).


25/46

MICRO LG L BIOTECHNOLOGY

Preparation of ProteinConcentrates

7 9

Algal biomasses are generally high in protein content, values up to 60

or more being reported for Spirulina for example (Santillan, 1982; Ciferri and

Tiboni, 1985; Bourges, 1986). This has prompted some investigators to explore

the possibility of preparing protein concentrates that exhibit good functional

properties such as water and fat absorption, emulsification and foaming

capacities, and foam stabi lity. In general, these functional properties for

Spirulina protein concentrate are better than or similar to those of soybean

meal, except for foam stabi lity (Anasuya Devi and Venkataraman, 1984).

Preparation of protein concentrates from blue-green algae such as Anabaena

flos-aquae has been thought to be a means of overcoming its low digestibility

due to cell walls, its unattractive color and its strange flavor (Choi and

Markakis, 1981). Treating the biomass with 3N HCI at 95°C for lO minutes and

neutralizing afterwards has been found to be an effective way of obtaining a

solution containing 80 of the cell nitrogen (Choi and Markakis, 1981). It is

likely that enz~nnic treatments could be applied to such concentrates to

prepare partial ly hydrolyzed fractions. Unless such fractions yield highly

priced products, i t is unlikely that algal biomasses may constitute a

competitive protein source. However, as the price of traditional sources such

as fish or soybean meal continues to rise, microalgal SCP used as a protein

supplement may become increasingly attractive especially when produced as a

by-product of wastewater treatments (Aaronson and Dubinsky, 1982; Pouliot and

de la NoUe, 1985). Protein isolated from algae shows a much higher

digest ibi lity than entire cells (Choi and Markakis, 1981), a result that is

not surprising. In vi tro digestion of Chlorella proteins with trypsin, for

example, gave digest ibi lities of 45 for dried cells, 69 for frozen cel ls,

71 for broken cells and 86 for extracted proteins (Ishii e al., 1974;

Mitsuda et al., 1977a, b).

COMPETITION BETWEENDIFFERENT INDUSTRIES FORTHE USE OF ALGAL BIOMASSES

If commercial scale production of microalgal biomasses is to be

ini t ial ly limited to existing installations, e.g. sewage treatment fac il i ties

or to their extension as tertiary treatment systems, i t is conceivable that

supply may fall short of demand, given all the possible applications already

described. The price of the biomass produced will have to increase before the

number of production sites can increase substantially. This could arise

through other uses for microalgae to which biotechnology might contribute.

Possibili ties of low-to-medium value-added products include biosorption of

metals, with a wide range of applications (Darnall et al., 1986) ammonia


26/46


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M I C R O A L G A L B I O T E C H N O L O G Y 75

growth rate, capacity to grow at suboptimal temperatures, higher yields or higher

content of nutrit ionally important components. However, basic genetic research

has been slow to reach algae and studies on them are relatively few Lemieux et

a___~., 1985; Necas, 1985; Turmel e_t_t al__~., 1986; Chauvat e_t_t al_._~., 1987; Lee and Tan,

1987), since they do not appear to offer obvious advantages over the favourites,

i .e .E, coli , Bacillus sp. or Saccharomxces ei ther as a vehicle of expression for

genes of higher organisms or as a model for the genetics of higher eukaryotes.

However, the possibility of inserting new gene sequences in smal l linear DNA

demonstrated in Chlamxdomonas Turmel et al . , 1986) might open interesting

perspectives, even more promising than the use of plasmids G. Bellemare person.

comm. .

For the study of microalgal genetics the prokaryotic cyanobacteria offer

several advantages. Most can be grown on defined medium, with generation

times as short as f ive hours and can be handled and grown on,agar medium as

colonies. Transformation does occur, and to-date several shuttle vectors for

cyanobacteria can replicate stably in E. coli Baker e_t_t al__:., 1985; Lem and

Glick, 1985).

Algal pond culture may already be considered for a wider range of

products. Extension of the latest genetic techniques to the eukaryotic

microalga Dunaliel la is considered a high priority Baker e_t_t al___~., 1985).

Approaches to the genetics of this alga have been presented by Simon and

Latorella 1986). Use of genetic alterations in cyanobacteria for maximizing

H production have been recently demonstrated Spiller and Shanmugam, 1986).

Spirul ina, a tropical species with optimum growth temperature around 30°C, has

been manipulated genetical ly and a strain has been developed by Japanese

workers which grows at 4°C Anon., 1984). This might open vast possibi li ties

for culture systems under temperate or even cold cl imatic conditions.

Strain-specific differences in the chemical composition with respect to

l ight intensity have been demonstrated for Phaeodactxlum ricornutum Terry e

al._~., 1983). Genetic studies are also required in order to transfer the

capacity for the production of algal polysaccharides to faster growing

bacteria or to transfer t raits such as pesticide or herbicide resistance into

nitrogen- fi xing blue-green algae in rice paddies Erickson et al . , 1984)o

Genes coding for the production of polysaccharides can be amplified by

inclusion of smal l extrachromosomal elements and genetic manipulations,

including transfer to bacteria, according to one report Weiner e al_._~., 1985).

Other properties of algae to be exploited, pending genetic characterization


28/46

7 5 2 J DE LA NOUE and N DE PAUW

Gallagher, 1986), include the abi l i ty of certain species such as Chlorella to

grow under extreme conditions of sa lini ty and acioity and the super- sensi ti vi ty

of some strains of C_.~. saccharophila to the toxic heavy metal cadmium Kessler,

1985). These are only a few examples of possible interesting applications of

genetic manipulations with microalgae. A much more complete treatment of the

subject wi ll be found in the recent review contribution of Craig et al . 1988).

ACKNOWLEDGMENTS

The authors wish to thank deeply D. Proulx and S. Davids for thei r help in

preparing this paper, D. Ni Eidhin for revising i t and G. Gagnon for the typing.

Financial help was provided by FCAR-Equipe grant, NSERC and the minist~re des

Relations Internationales du Quebec.

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