Chlorella (PROSEA)

Plant Resources of South-East Asia
Introduction

List of species

Chlorella vulgaris - 1, vegetative cell; 2, sporangium with autospores; 3, discharge of autospores.

Chlorella Beij.

Protologue: Bot. Ztg. 45: 758 (1890).

Family: Selenastraceae

Chromosome number: x= unknown

Major species and synonyms

Chlorella ellipsoidea Gerneck, Bot. Centr. 212: 250 (1907). Now, synonym of Chloroidium ellipsoideum (Gerneck) Darienko et al. (2010).

Chlorella pyrenoidosa Chick, Proc. Roy. Soc. London, Ser. B 71: 458 (1903). Now, synonym of Auxenochlorella pyrenoidosa (H.Chick) Molinari & Calvo-Pérez in Calvo-Pérez & Molinari (2015).

Chlorella vulgaris Beij., Bot. Ztg. 45: 758 (1890), synonym: Pleurococcus beyerinckii Artari (1892).

Vernacular names

The designation "Chlorella" is in common use.

Origin and geographic distribution

Chlorella is ubiquitous and found in both aquatic (freshwater, brackish and marine) and terrestrial habitats, including soil and concrete walls. It is a common contaminant of containers of water left undisturbed for long periods.

Uses

In the 1950s Chlorella was used as a source of unconventional proteins (i.e. single cell proteins). Presently, the alga is consumed as a health food, and is used in food additives to milk products, fermented soya beans, liquors and other drinks, noodles and cakes. Some marine Chlorella spp. are cultured in hatcheries and several are used as aquaculture feed or feed additive, especially as larval diets for shellfish and as diets for living animal feed. Chlorella microalgae are often combined with other microalgae because a readily amenable species is not always nutritionally excellent. Chlorella extracts have been shown to stimulate biosynthesis of chlorophylls in terrestrial plants. When sprayed onto the fields, these extracts stimulate growth and rooting of fruit trees, vegetables, rice and turf grass in golf links. These extracts were approved as a growth-regulating agent by the Japanese Plant Growth Regulator Research Association in 1984.

Certain strains of Chlorella develop many red or orange pigments in a nitrogen-limited and/or hypersaline culture medium. This is mainly due to the accumulation of the red carotenoid pigment astaxanthin. These strains can be used as an effective feed supplement for pigmentation of cultured fish and shellfish. Chlorella spp. can be cultivated in combination with treatment of agro-industrial waste water.

Many health-promoting effects of Chlorella have been suggested. These include therapeutic efficacy for gastric ulcers, wounds, constipation, leucopenia, anaemia, hypertension, diabetes, infant malnutrition and neurosis. All these effects have been validated by clinical tests and may be attributable to composite effects not only of nutritive components such as vitamins, minerals, dietary fibres and proteins, but also to a preventive action against artheriosclerosis and hypocholesterolemia by glycolipids and glycoproteins, peptides, nucleotides and related compounds. Many of the claims, however, are still not fully backed up by detailed scientific and medical research, making the claims rather controversial.

Production and international trade

A first pilot plant of Chlorella mass culture was constructed in the United States, in 1951. The first commercial plant was established in Japan around 1960 and Taiwan in 1964. In 1977, there were 48 largescale Chlorella factories in Asia, with a combined production of more than 1000 kg of dried alga per month. After that period, however, many plants closed down due to a glut of Chlorella production. In the same period a photosensitization disease affected those who ingested Chlorella tablets, prepared with ethanol and water from Chlorella biomass that had not been subjected to health treatment at the time of harvest. Most probably, chlorophyllases that remained intact induced the formation of photosensitizing pheophorbides. Heat treatment at harvest and a dry tablet-making method (without using ethanol) have solved this problem. However, a successful algal production factory for Chlorella powder in Peninsular Malaysia closed down in that period because of a collapsing Chlorella market. Today, Chlorella based health food is being produced and marketed mainly by more than 70 companies in Japan under various trade names (e.g. "Sun Chlorella" and "Albeille d'Or").

The total quantity of Chlorella powder traded in Japan in 1996 was about 2000 t dry weight, which included 30 t from Indonesia and 900 t from Taiwan. In Taiwan the total production of Chlorella powder is about 1600 t. In eastern Java, Indonesia, a Japanese health food company has built a plant which will have a future annual production of 300 t. Chlorella spp. are already extensively grown in large quantities using fermentation techniques in Japan and Korea.

Properties

Per 100 g edible portion (dry weight basis) C. vulgaris contains: protein 51-58 g, fat 14-22 g, carbohydrates 12-17 g, nucleic acids 4-5 g and ash 6-15 g. The major amino acids (% of total protein nitrogen) are glutamate (7.8%), arginine (15.8%), lysine (10.2%), alanine (7.7%), leucine (6.1%) and valine (5.5%). The dominant fatty acids (% of total fatty acids) in C. vulgaris are 18:1 (25%), 18:2 (29%) and 18:3 (14%). However, the fatty acid biosynthesis changes under different concentrations of KNO₃ or NH₄Cl. Chlorella contains chondrillasterol as well as other sterols. The chlorophyll-a content of Chlorella ranges from 0.3-2.0% (dry weight basis), and the chlorophyll a / b ratio ranges from 2-3. Total carotenoid content averages 0.2% (dry weight basis), with lutein making up 50% of the total content. Other carotenoids produced include violaxanthin, neoxanthin, αcarotene and βcarotene. Vitamin B₁₂ can be isolated from Chlorella spp. Chemical components of the cell wall in Chlorella include sporopollenin (a carotenoid polymer) and sugars such as glucose, mannose and glucosamine.

Environmental conditions greatly affect the chemical composition of Chlorella. Fat or carbohydrate content may increase 2-3fold at the expense of protein in some species when the cells are nitrogenstarved.

Description

Cells usually small, 2-12 μm long, either spherical (with ratio of the two axes equalling 1) or ellipsoidal (with ratio of the long axis to the shortest axis of 1.45-1.60).
Chloroplasts parietal, adhering to the cell wall and covering most of the periphery, saucer, band, cup or mantelshaped.
Pyrenoids common in some species.
Only asexual reproduction is known.

C. ellipsoidea. Cells ellipsoidal, sometimes asymmetrical; chloroplast forming a folded plate covering part of the cell wall; vegetative cells 9-9.5 μm long, 7-8 μm in diameter.

C. pyrenoidosa. Similar to C. vulgaris but cells smaller, 3-5 μm in diameter.

C. vulgaris. Cells always spherical, 4-10 μm in diameter, with cupshaped chloroplasts; single pyrenoid occasionally present.

Growth and development

Reproduction in Chlorella is asexual, producing 4, 8 or sometimes 16 autospores which are released by rupture or dissolution of the parental walls. Spores are not motile. Both light and temperature affect cell division; high light intensity and high temperatures are favourable.

Other botanical information

The taxonomy of Chlorella is subject to much controversy. It is often difficult to classify Chlorella spp. based solely on morphology. Recently, molecular techniques have been used to sort out the taxonomic confusion, resulting in the transfer of former Chlorella spp. to classes other than the green algal Trebouxiophyceae. This classification, however, is still far from being generally implemented.

Ecology

There are both mesophilic and thermophilic strains of C. pyrenoidosa, with temperature optima for growth ranging from 25-26 °C and 38-39 °C, respectively. Certain freshwater Chlorella spp. can tolerate a salinity of 30‰.

Propagation and planting

Chlorella grows well in most inorganic media (e.g. Bold's, Knop) using NO₃, NH₄⁺ or urea as a nitrogen source. The cultures are bubbled with air enriched by 5% CO₂to enhance growth. Growth and biomass production can be further enhanced by adding organic carbon sources such as glucose and acetate. Some species are capable of growing in the dark with organic carbon supplementation under heterotrophic conditions. Stock cultures can be maintained on 2% agar slants. Illumination by fluorescent lamps can be continuously or intermittently (normally 12:12 hours lightdark cycle) supplied at an intensity of 34-50 μmol photon m^-2s^-1. Micronutrients required for the growth of these green microalgae are Mn, Fe, Zn, Cu, and occasionally also Mo. These micronutrients are particularly essential for autotrophic growth, less so for heterotrophic growth.

Phycoculture

Mass cultures of Chlorella are carried out in round (diameter 36-50 m), shallow, concrete ponds agitated by rotating arms in the centre. The Japanese Chlorella factory in East Java has 16 open-air ponds of 36 m in diameter. To increase the biomass yield, the cultures are supplemented with CO₂, acetic acid or glucose. These algae can also be grown in high-rate algae ponds, which consist of an open and very shallow raceway mixed by paddle wheels. These are capable of producing very high yields.

In recent years, Chlorella has also been grown in fermentors or in combinations of fermentors and tubular photobioreactors for continuous sequential heterotrophic/autotrophic production of biomass, and the yield attained can be 10 times higher than in open cultures. The closed systems can solve contamination problems but due to cost constraints, they are only economically feasible for the production of highvalue chemicals. There is an optimum cell density in each culture for obtaining best growth yield in sunlight; above this concentration, the yield decreases due to increased respiration and limitation by light saturation.

Growth in photobioreactors was studied in several cases with different Chlorella spp. The effect of the inclination of flat plate reactors on productivity of outdoor cultures has been studied in Singapore for C. pyrenoidosa. The productivity was not much influenced, but the findings of the researchers cannot yet be generalized. Other forms of bioreactors in which Chlorella spp. can be grown are tubular bioreactors, which may be able to be placed in vertical "biocoil" facilities. The cost of dry product obtained in these closed systems is usually higher than that of open air systems, although contamination problems and thus maintenance costs of the cultures are less. Costs of heterotrophically grown algal biomass, however, are usually much lower than that of autotrophic cultivation.

Diseases and pests

Contamination of open air cultures of Chlorella by other microalgae is a general problem.

Harvesting

The cultures of Chlorella are pumped out of the pond and undergo repeated centrifugation and washing. This is generally a costintensive stage of microalgae cultivation. The algae are concentrated 50-200fold, giving an algal slurry of 5-15% dry weight. Combined algal production and fish culture, especially together with waste-treatment systems, are successful especially because the low capital and maintenance costs are in these cases enhanced by the elimination of harvesting problems as the algae are utilized in situ by fish.

Yield

The concentration of Chlorella biomass in open-pond cultures ranges from 1-5 g/l, while daily productivity varies between 15-40 g/m², due to seasonal changes in light, temperature and salinity.

Handling after harvest

The Chlorella slurry is dehydrated after which the algal cell walls are broken by physical disintegration and kept at low temperature. A new insect-detection immunoassay is available to assess insect contamination of the yield. The concentrates are spraydried to produce Chlorella powder, which is moulded into tablets by a direct press machine.

Genetic resources

Maintenance of Chlorella strains is very important for the future of commercial cultivation of microalgae. It is not only sufficient that the organisms be preserved, but also important that the special and unique characters of the strains be maintained, in order to ensure that the strain is genetically stable. Cryopreservation of Chlorella cultures allows long-term preservation of frozen algae with no significant reduction of viability for up to 22 years of storage. Recently developed recombination and transformation techniques for Chlorella have potential use in direct commercial application.

Prospects

Research on the culture of Chlorella using agroindustrial wastes has been carried out in various parts of Asia. Apart from treating the wastes, the biomass generated can be used as a highquality animal feed. Research in Malaysia showed that high biomass concentration and efficient removal of pollutants such as ammonia and phosphate as well as lowering of Chemical Oxygen Demand (COD) can be achieved by growing C. vulgaris in highrate algal ponds treating rubber effluent or palm-oil mill effluent. The microalgae can also concentrate heavy metals in industrial waste water. Thus, the use of Chlorella to treat wastes appears to be a prospective area that deserves more intensive research in the coming years.

Literature

Huss, V.A.R., Frank, C., Hartmann, E.C., Hirmer, M., Kloboucek, A., Seidel, B.M., Wenzeler, P. & Kessler, E., 1999. Biochemical taxonomy and molecular phylogeny of the genus Chlorella sensu lato (Chlorophyta). Journal of Phycology 35: 587-598.
Kamiya, A. & Miyachi, S., 1982. General characteristics of green microalgae: Chlorella. In: Zaborsky, O.R. (Editor): CRC Handbook of biosolar resources. Vol. 1(2). CRC Press, Boca Raton, United States. pp. 25-32.
Ogbonna, J.C., Masui, H. & Tanaka, H., 1997. Sequential heterotrophic/autotrophic cultivation - An efficient method of producing Chlorella biomass for health food and animal feed. Journal of Applied Phycology 9: 359-366
Oh-Hama, T. & Miyachi, S., 1988. Chlorella. In: Borowitzka, M.A. & Borowitzka, L.J. (Editors): Micro-algal biotechnology. University Press, Cambridge, United Kingdom. pp. 1-26.
Shihira, I. & Krauss, R.W., 1965, Chlorella: physiology and taxonomy of forty-one isolates. Port City Press, Baltimore, Maryland, United States. 92 pp.
Takeda, H., 1991. Sugar composition of the cell wall and the taxonomy of Chlorella (Chlorophyceae). Journal of Phycology 27: 224-232.

Sources of illustration

Hori, T. (Editor), 1994. An illustrated atlas of the life history of algae. Vol. 1. Green algae. Uchida Rokakuho Publishing Company, Tokyo, Japan. Fig. 164, p. 336. Redrawn and adapted by P. Verheij-Hayes.

Authors

S.-M. Phang