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Biotechnology and exploitation of the green alga Scenedesmus obliquus in India
Biomass 4 (1984) 1-19
Biotechnology and Exploitation of the Green Alga Scenedesmus obliquus in India E. W. Becker Institut for Chemische Pflanzenphysiologie~ Universit/it Ttibingen, 7400 Ttibingen, Federal Republic of Germany (Received: 13 September, 1982)
ABSTRACT The Governments o f lndia and West Germany have initiated and supported a joint microalgae research project in lndia from 1973 to 1981. Data are presented about the green alga Scenedesmus obliquus, pertaining to its cultivation, processing, chemical composition, nutritional and toxicological properties. Optimal growth rates were obtained from algae supplied with C02 and molasses as carbon sources. Various analyses showed a nutritionally excellent composition o f the alga. Among the different processing methods tested, drum-drying gave the best results which could be confirmed by nutritional evaluations such as PER (Protein Efficiency Ratio), NPU (Net Protein Utilization), B V (Biological Value) and DC (Digestibility Coefficient). Preliminary toxicological studies with rats did not show any adverse symptoms or indications which would limit the utilization o f Scenedesmus as a supplement in food and feed. Key words: India, Scenedesmus, mass cultivation, feed, food.
INTRODUCTION The production and utilization of bacteria, yeasts and algae as sources o f unconventional protein are being tested in several projects worldwide. Since 1970 the Ministry for Economic Cooperation of West Germany has been supporting three microalgal research projects in 1
0144-4565/84/$03.00 - O Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
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E.W. Becker
Thailand, Peru and India. The Indo-German Algal Project was launched in 1973 at the Central F o o d Technological Research Institute (CFTRI) at Mysore, India with the aim to establish for the green algae: (a) the production capability (growth rate, contamination by other organisms, etc.); (b) digestibility and product safety as far as health is concerned; (c) acceptability as human food. The German participation in this project was terminated in 1981. It should be emphasized here that the primary task o f the Indian project was to develop an algal production system with minimum inputs o f energy and equipment, suitable to the potential and requirements of this country. The research activities were started with the cultivation o f the green alga Scenedesmus obliquus (strain 276-3a) by adopting a production process developed in Germany. ~ This paper summarizes the results obtained on the cultivation and utilization of this alga in India.
RESULTS AND DISCUSSION Cultivation A series o f different horizontal ponds were used for the o u t d o o r cultivation o f Scenedesmus. Initially the project was equipped with 5 ponds of 5 m 2 (500 litres), 8 ponds o f l l m 2 (2000 litres) and 2 ponds of 55 m 2 (10 000 litres). They consisted either of PVC-coated nylon fabric stretched over a metal frame (Fig. 1) or o f PVC sheets sealed on polyurethane blocks which formed the side walls o f the pond (Fig. 2). The ponds were built in the common raceway form with culture depths varying between 15 and 30 cm. Agitation o f the culture medium was maintained by motor-driven paddle wheels for 24 h day -1 giving a flowrate of 30 cm s-1 which is sufficient for adequate distribution of the algal cells to solar radiation and to prevent sedimentation o f the algae. Originally the algae were aerated with compressed air (10 litres min -1 m-2); however, the aeration was stopped after some time because it was found that this treatment did not improve algal growth as compared to cultures agitated only. These plastic ponds were employed for the immediate production o f sufficient amounts o f algal material to perform nutritional and toxi-
Biotechnology and exploitation of
Fig. 1.
Scenedesmus obliquus
3
11 m 2 cultivation pond made of PVC-coated nylon fabric stretched over iron frame. Agitation of algal suspension by paddle wheel.
cological studies. They were not considered as a permanent type of construction to be used in India; for this purpose various simpler ponds were built and tested. An elliptical pond with a total area of 94 m 2 (12 000 litres) was constructed with the intention to harvest the algal biomass by flocculation (Fig. 3). This pond was divided into four parts: a central sump pit, a storage section, an inner and an outer cultivation section. Each part was separated by a 30 cm high wall with several holes which could be closed with plugs; the floor o f the pond had a gradual slope to the centre. The storage section was provided for retaining the total culture
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Fig. 2.
E . W . Becker
55 m2 cultivation pond made of PVC sheets sealed on polyurethane blocks. Agitation of algal suspension by paddle wheel.
volume and also for collecting the supernatant o f the medium after harvesting the algae by flocculation or sedimentation. During cultivation the medium was pumped from the sump pit to the outer section of the pond from where it flowed back through the holes in the partition walls. This flow speed could be regulated by the number of open holes. To test another system, a 31 m 2 (6000 litre) cement tank o f oblong form was built, divided into two sections by a central wall (Fig. 4). Both parts o f this pond had a continuous gradient from the upper point of culture delivery to the lower part where the medium is collected and pumped back to the upper end. To further simplify the cultivation system, a series o f cement ponds was built which might be appropriate for algal cultivation on a rural level in India (Figs 5 and 6). The ponds were small ditches with a size of 0.9 X 11 m each (2000 litres) and a depth o f 40 cm, without partition
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Fig. 3. 94 m 2 cultivation pond made of bricks and cement. Agitation of suspension by pump and gravity flow. Aerobic digester for farm waste in foreground. (For details see text.)
walls or agitation devices. Mixing was done b y hand with a b r o o m from time to time during the day. Since the stirring of the culture was not continuous, the Scenedesrnus cells started to settle, this resulted in low growth rates. Hence this pond design is more suited for the cultivation o f algae such as Spirulina, where continuous agitation is not required. A still simpler and cheaper construction of algal ponds would be earthen pits lined with polyethylene sheets, as described b y Richmond and Preiss for Israel. 2
Nutrients To be independent o f costly pure chemicals, utilization of locally available mineral salts of commercial grade was tried as nutrients for the cultivation of Scenedesmus. A simple medium was developed with considerable success in reducing the amount of nutrients which are
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Fig. 4.
E . W . Becker
31
m 2
cultivation pond, constructed with bricks and cement. Agitation of suspension by pump and gravity flow.
normally supplied in standard media (Table 1). Crude sea salt is being used to provide undefined amounts of micronutrients. In search of cheap ingredients for algal media, some waste products such as whole blood and blood plasma were studied for their growth stimulating effects. Sheep's blood at 0.5% level resulted in optimal growth stimulation. Since blood carbonicanhydrase might be one of the effective ingredients, active and inactivated crude carbonicanhydrase were tested. Growth increase was less than with whole blood and addition of serum albumin instead of carbonicanhydrase produced similar results indicating that protein or its degradation products may be responsible for the effect. The supply of sufficient amounts of carbon to the algae is one of the major obstacles in algal mass cultivation. At Mysore, CO2 was obtained in gas cylinders and bubbled into the culture through perforated pipes. By constantly supplying CO2 for 8 h day-1, more gas was added into the medium at certain times of the day than could be utilized by the algae.
Biotechnology and exploitation of Scenedesmus obliquus
Fig. 5.
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10 m 2 cultivation pond constructed with bricks and cement. Agitation of suspension manually with a broom.
By using a pH-stat and monitoring the total amount o f CO2 consumed by the algae, it was found that the number o f times when CO2 had to be added to the culture to maintain a given pH value increased during the course of the day. It reached its maximum in the early afternoon hours. By totalling the daily CO2 additions, it was observed that the gas was supplied for only 4 h day -1 altogether. No difference in growth could be found between cultures gassed continuously with CO2 for 8 h day -1 and cultures supplied in intervals as regulated by the pH-stat. Based on this finding, the CO2 was added by manual control at 1 h
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Fig. 6.
E. IV. Becker
9 m2 cultivation pond constructed with mortar. Agitation of suspension manually with a broom.
intervals for 4 h day -1 and no marginal variations in growth could be observed between these two methods (Fig. 7). To exploit cheaper sources o f CO2 it was attempted to produce this gas by biodegradation o f cow dung by aerobic fermentation. The CO2-enriched air, produced in a simple fermenter, was sucked into the culture by the circulating medium. 100 mg CO2 h-1 kg-1 dung could be produced by this method from actively fermenting material. Fresh dung has to be supplied to the fermenter every two weeks. Based on the results that a minimum CO2 supply o f 3 litres m -2 h -1 is required to
Biotechnology and exploitation of Scenedesmus obliquus TABLE 1
Composition of Media for Cultivation of Scenedesmus obliquus
Nutrients
Urea (NI-h)2SO4 K2CO3 NaaPO4 Crude sea salt MgSO4.7H20 FeSO4.7H20 EDTA CaC12.2H20 K2HPO4 KH2PO4 As solution B6 solution
Standard medium (mgflitre )
Simplified medium (mgflitre )
300-0
60.0 20.0 25.0 25.0 25.0 10.0 2.0
120.0 10.0 10.0 7-0 144.0 92.0 1 ml/litre 1 ml/litre
As solution (g/litre): H3BO3, 2.86; MnC12.4H20, 1.80; ZnSO4.7H20, 0.22; MOO3, 0.01; CuSO4.5H20, 0.08. B6 solution (g/litre): NI-hVO3, 22.9; NiSO4.7H20, 47.8; Na2WO4, 17.9; Ti2(SO4)3, 40.0; Co(NOa)2.6H20, 4.40. sustain algal growth, approximately 60 kg o f dung are necessary to provide a comparable amount of CO2 in the form of 'CO2-enriched biogas'. The mode of carbon nutrition in Scenedesrnus can be shifted from autotrophic to heterotrophic, because this alga is able to utilize b o t h inorganic and organic carbon. 3 Different experiments were performed to test the feasibility of using sugar cane molasses as a cheap carbon source in algal mass cultivation. It was endeavoured to find a molasses concentration, which after addition to the medium in the evening, is absorbed b y the algae during the night so that a period o f heterotrophic growth (night) is followed b y a period of photoautotrophic growth (day). a At an average addition o f 1 0 0 - 1 5 0 m g l i t r e -1 suspension all sugar was absorbed b y the algae overnight and the bacterial count in the medium did not rise substantially. Maximum growth rates of Scenedesmus were obtained b y providing b o t h CO2 and molasses to the
E. W. Becker
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o Reference: Air(100ml/rnin) • CO2 (250ml/min) continuous & CO2 (500ml/min) continuous CO2 (500ml/min) in 1hr intervals i CO (ll/min) continuous
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C02(111min) inlhr
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Time [doys] Fig. 7.
Effect of different C02 concentrations on the growth of Scenedesmus obliquus.
100 80 t~
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--day
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/
Fig. 8.
o Air (100t/rain}, C02(0.511min},molosses (140rag/I)/''-~° • Air(1001/min) +C02(0.51/min) / ,~ Air(1001/min)÷ molasses(140mg/I) o......._..o/" / Air (1001/rain) ~-night Took volume 1~00i
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Growth of Scenedesmus obliquus under different autotrophic and heterotrophic culture conditions.
Biotechnology and exploitation of Scenedesmusobliquus
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culture (Fig. 8). Thus, an increase in yield of about 30% could be achieved by the addition of molasses to the normally supplied amount of COs.
Harvesting To harvest the algae, the suspension was Concentrated by continuous centrifugation into a slurry containing about 15% suspended solids which was the best concentration for the subsequent drying process. Since centrifugation requires high capital expenditures and energy costs, other harvesting methods were tried. Induced sedimentation by the addition of chemicals offers a promising alternative, however, residues of these chemicals in the harvested algal material may limit the further utilization of the biomass. Therefore, the non-toxic carbohydrate chitosan, obtained as deacetylated chitin of marine arthropods, was tested as flocculant for the separation of Scenedesmus. 5 A concentration of 50 ppm of this compound at pH 8.5 was optimal and resulted in better sedimentation rates of the algae than obtained with conventional flocculants. Drying If the concentrated wet algal slurry is not utilized immediately, it has to undergo a heat treatment. For Scenedesmus and other green algae this step is essential because it both dries and sterilizes the algal material and simultaneously ruptures the algal cells thus making the algal protein digestible. Different methods of drying Scenedesmus were tested, viz. sun-drying, sun-drying after previous cooking, freeze-drying, and drumdrying. While all methods gave a dry algal powder (6-10% moisture), only drum-drying resulted in a hygienically safe and fully digestible product. It seems that this method is the only one (aside from spraydrying) by which green algae can be processed into acceptable proteinaceous matter. Yield Strictly speaking, outdoor mass cultivation of algae represents a special form of agriculture, being subjected to similar variations in yield, quality and composition of the product. It is obvious that algal yields
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E. W. Becker
o f short-term cultivation periods o f 1-2 (summer) months are higher than o f long-term periods. For Chlorella and S c e n e d e s m u s growth rates in the range o f 5 - 5 0 g m -2 day -1 can be found in the literature. 6 The results of the Indian project demonstrated that under the given conditions an average yield of 15-20 g m -2 d a y -1 c a n be obtained for S c e n e d e s m u s provided all inputs are optimized. Cultures aerated with compressed air only (500 litres m -2 day -1) showed growth rates below 5 g m -2 day -/. Moreover, algae grown under this condition had lower protein (30%) and higher ash (up to 20%) contents than cultures grown with additional CO2 supply. According to experiences and theoretical considerations 7 it seems that the upper limit in yield o f 3 0 - 4 0 g m -2 day -a is a given characteristic of the photosynthetic process of algal growth which cannot be improved upon very much in large-scale cultivation. Algal production in India was performed in a semi-continuous way by periodic harvesting o f the algae and addition of recycled or fresh medium. The cultures were grown for 4-5 days up to a concentration of 0.8-1.0 g litre -I. The ponds were harvested partially until the remaining volume was sufficient to start a fresh culture with an initial concentration o f 0.2-0.3 g litre -1. The effluent from the centrifuge was made up with fresh nutrients and recycled. However, the same medium could not be reused more than 3 - 4 times, otherwise reduced algal growth and enhanced bacterial contamination occurred.
Chemical composition Data on chemical analyses of S c e n e d e s m u s are given in Table 2. It was observed that the pronounced dark and light phases at the latitude of Mysore resulted in a partial synchronization o f the o u t d o o r algal cultures, which influenced the chemical composition of the algae. Their protein content was always higher in the morning than in the evening, while the carbohydrate concentration was low in the morning and high in the evening; the difference in the protein content was between 10 and 15% o f the total biomass. The amino acid pattern of S c e n e d e s m u s is given in Table 3. The amount o f lysine and sulphur containing amino acids is slightly deficient compared with a recommended standard. Analyses of the algal lipids revealed an unusually high proportion of unsaturated fatty acids which amounted to 80% of the total lipid content. Linolenic acid (C 18 : 3) and
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TABLE 2 Chemical Composition of Drum-dried Scenedesmus obliquus
Overall composition
(g/1 O0 g algae)
Crude protein (N x 6.25) Total nitrogen Non-protein nitrogen Ether extractables Carbohydrates Starch Reducing sugars Non-reducing sugars Other polysaccharides Crude fibre RNA DNA
52.0 8.3 1.0 9.1 12.5 5.3 0.4 1.3 5.5 5.3 4.~ ~'~.,~" 1.6
Moisture Ash Phosphorus Iron Calcium Sodium Magnesium Potassium
7.1 8.0 3-9 0.7 0.5 0.3 0.3 1-0
Vitamins Thiamine Riboflavin Pyridoxyl-HC1 Cobalamine Biotin Folic acid Nicotinate d-Ca-Pantothenate Ascorbic acid /3-Carotene
Fatty acids C 14 : 0 C 16 : 0 C 16 : 1 C 16 : 4 C 18 : 0 C 18 : 1 C 18 : 2 C 18 : 3 C 20 : 0 C 20 : 2 C 22 : 0 C 24 : 0
(mg/kg algae) 8.2 36.6 2.5 0.44 0.2 0.7 120.0 16.5 17-7 230.0
(% of total lipids) 1.3 12.1 9.5 13.1 0.9 15.0 9-0 30.7 0.6 2.7 0-4 0.6
t h e p o l y - u n s a t u r a t e d f a t t y acid h e x a - t e t r a e n i c acid (C 16 : 4) c o n t r i b u t e 30 and 13% respectively. Nutritional studies Since the u l t i m a t e utilization o f algal biomass d e p e n d s p r e d o m i n a n t l y o n its n u t r i t i o n a l p r o p e r t i e s , a wide s p e c t r u m o f studies was p e r f o r m e d at C F T R I to establish t h e n u t r i t i o n a l value o f Scenedesmus. T h e following p a r a m e t e r s were e x a m i n e d b y feeding various c o n c e n t r a t i o n s o f Scenedesmus to rats: P r o t e i n E f f i c i e n c y R a t i o ( P E R ) , Biological Value
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E. W. Becker TABLE 3
Amino Acid Profile of Scenedesmus obliquus Protein Compared with FAO Recommended Pattern Amino acid
Isoleucine Leucine Valine Phenylalanine Tyrosine Lysine Methionine Cystine Tryptophan Threonine Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Proline Serine
FAO a {g/16 g N)
4.0 7-0 5.0 ~ 6.0 ~ 5.5 3.5 , ,..... 1.0 4.0
Scenedesmus (g/16 g N) 3.6 7-3 6.0 4.8 3.2 5.6 1.5 0-6 0-3 5.1 9.0 7-1 8.4 10-7 7.1 2.1 3.9 3.8
Energy and protein requirement report of a 'Joint FAO/ WHO ad hoc Expert Committee', No. 52 (1973).
a
(BV), Digestibility Coefficient (DC) and Net Protein Utilization (NPU). The results o f these experiments are summarized in Table 4. From the data on PER it is evident that S c e n e d e s m u s has a high nutritional quality (when compared with casein) and that drum-drying is superior to all other drying methods, a Diets supplemented with methionine showed PER values even closer to that o f casein. Cooking (20 min at 100°C) would be expected to improve the PER; however, no beneficial effect could be observed. Similar findings are reported for Chlorella by Cook. 9 A similar trend can be seen in the other parameters (NPU, BV, DC). Here too, drum-drying gave the best results, confirming that this method - although expensive - seems to be the most suitable process
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TABLE 4 Nutritional Studies with Differently Processed Scenedesmus obliquus. Results of Feeding Experiments on Rats Protein source
Protein
PERaorr
BV b
DC b
NPU b
10 10
2.50 1.99
87.8 80-8
95.1 81.4
83.4 65.8
10 15 20 10 10 20
2.20 2-00 1.68 1-14 1.20 1.52
72.1 71-9 -
72.5 77.1 -
52.0 55-5 -
(~) Casein Algae (drum-dried) Algae (drum-dried) a 0-03% methionine Algae (drum-dried) Algae (drum-dried) Algae (sun-dried) Algae (cooked, sun-dried) Algae (cooked, sun-dried)
a Values are the mean of 10 rats per group. b Values are the mean of 8 rats per group.
to produce a fully digestible algal product. Comparable studies with the same alga reported by other authors support the results from the Indian project. 1° Toxicological considerations The commercial distribution of unconventional proteins presupposes their toxicological safety. In default of unambiguous laws, recommendations o f international organizations such as the former Protein Advisory Group (PAG) or the International Union o f Pure and Applied Chemistry (IUPAC) have to be considered as guidelines for establishing the safety o f algae. These toxicological evaluations include analyses of chemical composition, tests for biogenic and non-biogenic toxins, shortterm and long-term (multigeneration) animal studies, sanitary analyses and clinical studies. To ensure the absence of biogenic toxins, S c e n e d e s m u s samples were tested for the following compounds: aflatoxin, ochratoxin A, sterigmatocystin, citrinin, patulin, penicillic acid, zearalenone, diacetoxyscirpenol and trichothecene. Additional dermatological and toxicological tests
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E. W. Becket
with trichothecene, as well as toxicity studies with mice on tremorgens (fumitremorgen and verrucolugen), were conducted. None of the above listed toxins could be detected and all biological tests gave negative results. Among the non-biogenic toxins, special attention has been given to the accumulation of anthropogenic pollutants, particularly heavy metals and pesticides. The concentration of these compounds in the algae depends on the origin of the sample and is not a characteristic of the algae itself, because all these toxins are accumulated from a polluted environment. Analyses for the pesticides a-HCH, /3-HCH, 7-HCH, HCB, dieldrin, DDE, DDD, DDT and PCB showed very low concentrations which were far below the permitted limits for foodstuffs. Dialkyl-nitrosamines of dimethyl- and dipentyl nitrosopiperidine, nitrosomorpholine and nitrosopyrrolidine could not be detected in the alga (detection limit 0.1 #g kg-1). After overcoming certain initial difficulties it was possible to keep the heavy metal content in the algae below the limits which are recommended for bacterial single cell protein (SCP) (Pb, 5-0; Cd, 1.0; Hg, 0.1 and As, 2.0 mg kg-1). Single cell protein has received special attention from toxicologists because it has a higher nucleic acid content than the normal ingredients of the human diet. Excessive uptake of nucleic acids leads to elevated uric acid concentrations in the blood which may cause gout or hyperuricemic nephropathy. Therefore the PAG recommended that the daily nucleic acid uptake from any unconventional source should not exceed 2 g (total intake from all sources, 4 g). Assuming a nucleic acid content of 6% in S c e n e d e s m u s the upper limit for the daily consumption of this alga would be 30 g for adults. By considering the number of persons with latent hyperuricemy, the daily uptake should be restricted to about 20 g or 0.3 g kg-1 body weight. As an initial step in establishing the toxicological safety of Scened e s m u s , 12 week trials were performed by feeding differently processed algae at various concentrations to rats and examining the animals afterwards. The results of these experiments did not reveal any harmful histological or haematological effects in the rats. The animals fed with 20% algal protein consumed more feed and gained more weight than the animals of the other groups. The consumption of sun-dried algae
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was much less and the animals of these groups showed a weight gain of only 35 g, while the rats of the drum-dried diet group (20%) gained 225 g.ll Besides this effect which is due to the physical method of processing, no adverse symptoms or indications have been observed which could be attributable to the algae and which would limit the utilization of Scenedesmus as supplement in feed or food.
Production cost
Due to the small number of projects connected with the commercial production of microalgae, information on cost-benefit analyses of algal mass production is only fragmentary. In India, the early idea of developing a semi-industrial system of algal production has given way to the more rural oriented technology with independent small-scale production units. However, to estimate the prospective costs of large-scale production and to point out the problems of such ventures, some cost computations have been made based on the data collected at the pilot plant at Mysore. For this calculation a plant size of 5 ha was assumed and a production technology which included agitation by paddle wheels, addition of self-produced CO2, harvesting by centrifugation, and processing by drum-drying. The yield was estimated at 280 t yr -1. Based on calculations and quotations, a price of 8 US $ m-2 can be obtained in India for the construction of the algal cultivation ponds (bricks and cement). This estimation is comparable with cost computations performed in Peru for sloped algal ponds made of concrete ( 1 0 - 5 U S $ m -2) or with adobe bricks (9.0US$m-2) 12. As long as no cheap CO2 sources in the form of waste gas, etc. are available, the COz production from fuel seems to be the most convenient method. The Indian results demonstrated that, for the production of 1 kg of algal dry matter 3 kg of CO2 are necessary. The CO2 production from fuel yields approximately 3 kg CO2 kg-~ oil so that 1 kg of fuel would be required to produce 1 kg of algae. The heat produced by this system can be utilized for drying of the algae. Harvesting of Scenedesmus contributes to 25%, production of CO2 to 20%, and drying of the algae to 10% of the total production costs, which are calculated at 2.30 US $ kg-1.
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CONCLUSIONS The most promising bases for an immediate utilization of microalgae seem to be those cases where the algae can be obtained either as byproduct or where they can be produced by a very simple technology. The development of such low level technology is the major objective of the Indian algal project. It was demonstrated that Scenedesmus obliquus can be grown successfully under Indian conditions at growth rates which are comparable to results reported from other algal research plants. It turned out that in the case of Scenedesmus the development of the outdoor cultivation technology has reached a stage which leaves only marginal space for further improvements. Design and construction of the ponds cannot be modified significantly anymore, agitation and aeration may be changed here and there but will not meliorate the system considerably. Some improvements can be seen in the development of new harvesting technologies. If other methods such as the application of flocculants or microstrainers can be standardized to replace the costly centrifugation, a reduction of the production cost can be expected. No alternate method has come up so far for the processing technique used at present. As long as Scenedesmus is designated for a utilization as food or feed, this alga has to be dried by hightemperature treatments. Although the nutritional tests (including supplementation studies) confirmed the high nutritional quality of Scenedesmus and the various toxicological evaluations did not show any toxic symptoms or abnormalities in the test animals, it was considered that the cultivation of this alga is not suitable in India. This conclusion is mainly due to the fact that the technological requirements for a proper production of Scenedesmus are too expensive and sophisticated for the purpose aimed at: the cultivation of microalgae on a rural level in small, independent units. The algal species of choice for this intention seems to be the Cyanobacterium Spirulina, which is being tested already at CFTRI. The immediate utilization of algae in India will be as feed, the application as human food remains a long-term goal.
ACKNOWLEDGEMENTS This work was supported by the German Agency for Technical Cooperation (GTZ) and the Department of Science and Technology, India.
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REFERENCES 1. Soeder, C. J. (1976). Primary production of biomass in freshwater with respect to microbial energy conversion. In: Microbial energy conversion, ed. H. G. Schlegel and J. Barnea. Oxford, Pergamon Press, pp. 59-68. 2. Richmond, A. & Preiss, K. (1980). The biotechnology of algal-culture, lnterdisciplin. Sci. Rev., 5, 60-70. 3. Mineeva, L. A. (1961). The use of various organic compounds by Chlorella vulgaris and Scenedesmus obliquus cultures. Mikrobiologiya, 30,586-92. 4. Shamala, T. R., Drawert, F. & Leupold, G. (1982). Studies on Scenedesmus growth. I. Effect of autotrophic and mixotrophic conditions on the growth of Scenedesmus acutus. Biotechnol. Bioeng., 24, 1287-99. 5. Nigam, B. P. & Venkataraman, L. V. (1980). Application of chitosan as a flocculant for the alga Scenedesmus acutus. Arch. Hydrobiol., 88,378-87. 6. Goldman, J. C. (1979). Outdoor algal mass cultures. I. Applications. Water Res., 13, 1-19. 7. Goldman, J. C. (1979). Outdoor algal mass cultures. II. Photosynthetic yield limitations. WaterRes., 13, 119-36. 8. Becker, E. W., Venkataraman, L. V. &Khanum, P. M. (1976). Digestibility coefficient and biological value of the proteins of the alga Scenedesmus acutus processed by different methods. Nutr. Rep. lnt., 14,457-66. 9. Cook, B. B. (t962). The nutritive value of waste-grown algae. Am. J. Pub. Health, 52,243-51. 10. Payer, H. D., Pabst, W. & Runkel, K. H. (1980). Review of the nutritional and toxicological properties of the green alga Scenedesmus obliquus as a single cell protein. In: Algae biomass, ed. C. J. Soeder and G. Shelef. Amsterdam, Elsevier, North-Holland Biomedical Press, pp. 787-97. 11. Venkataraman, L. V., Becker, E. W., Rajasekaran, T. & Mathew, K. R. (1980). Investigations on toxicology and safety of algal diets in albino rats. Food Cosmet. Toxicol., 18,271-5. 12. Schulz, P., Heussler, P., Moya, R. & Merino, F. (1980). Production cost of Scenedesmus and the economical impact of technological improvements and plant size. Third lnt. Conf. of Microalgae, Trujillo, Peru.
Biotechnology and Exploitation of the Green Alga Scenedesmus obliquus in India E. W. Becker Institut for Chemische Pflanzenphysiologie~ Universit/it Ttibingen, 7400 Ttibingen, Federal Republic of Germany (Received: 13 September, 1982)
ABSTRACT The Governments o f lndia and West Germany have initiated and supported a joint microalgae research project in lndia from 1973 to 1981. Data are presented about the green alga Scenedesmus obliquus, pertaining to its cultivation, processing, chemical composition, nutritional and toxicological properties. Optimal growth rates were obtained from algae supplied with C02 and molasses as carbon sources. Various analyses showed a nutritionally excellent composition o f the alga. Among the different processing methods tested, drum-drying gave the best results which could be confirmed by nutritional evaluations such as PER (Protein Efficiency Ratio), NPU (Net Protein Utilization), B V (Biological Value) and DC (Digestibility Coefficient). Preliminary toxicological studies with rats did not show any adverse symptoms or indications which would limit the utilization o f Scenedesmus as a supplement in food and feed. Key words: India, Scenedesmus, mass cultivation, feed, food.
INTRODUCTION The production and utilization of bacteria, yeasts and algae as sources o f unconventional protein are being tested in several projects worldwide. Since 1970 the Ministry for Economic Cooperation of West Germany has been supporting three microalgal research projects in 1
0144-4565/84/$03.00 - O Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain
Biomass
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E.W. Becker
Thailand, Peru and India. The Indo-German Algal Project was launched in 1973 at the Central F o o d Technological Research Institute (CFTRI) at Mysore, India with the aim to establish for the green algae: (a) the production capability (growth rate, contamination by other organisms, etc.); (b) digestibility and product safety as far as health is concerned; (c) acceptability as human food. The German participation in this project was terminated in 1981. It should be emphasized here that the primary task o f the Indian project was to develop an algal production system with minimum inputs o f energy and equipment, suitable to the potential and requirements of this country. The research activities were started with the cultivation o f the green alga Scenedesmus obliquus (strain 276-3a) by adopting a production process developed in Germany. ~ This paper summarizes the results obtained on the cultivation and utilization of this alga in India.
RESULTS AND DISCUSSION Cultivation A series o f different horizontal ponds were used for the o u t d o o r cultivation o f Scenedesmus. Initially the project was equipped with 5 ponds of 5 m 2 (500 litres), 8 ponds o f l l m 2 (2000 litres) and 2 ponds of 55 m 2 (10 000 litres). They consisted either of PVC-coated nylon fabric stretched over a metal frame (Fig. 1) or o f PVC sheets sealed on polyurethane blocks which formed the side walls o f the pond (Fig. 2). The ponds were built in the common raceway form with culture depths varying between 15 and 30 cm. Agitation o f the culture medium was maintained by motor-driven paddle wheels for 24 h day -1 giving a flowrate of 30 cm s-1 which is sufficient for adequate distribution of the algal cells to solar radiation and to prevent sedimentation o f the algae. Originally the algae were aerated with compressed air (10 litres min -1 m-2); however, the aeration was stopped after some time because it was found that this treatment did not improve algal growth as compared to cultures agitated only. These plastic ponds were employed for the immediate production o f sufficient amounts o f algal material to perform nutritional and toxi-
Biotechnology and exploitation of
Fig. 1.
Scenedesmus obliquus
3
11 m 2 cultivation pond made of PVC-coated nylon fabric stretched over iron frame. Agitation of algal suspension by paddle wheel.
cological studies. They were not considered as a permanent type of construction to be used in India; for this purpose various simpler ponds were built and tested. An elliptical pond with a total area of 94 m 2 (12 000 litres) was constructed with the intention to harvest the algal biomass by flocculation (Fig. 3). This pond was divided into four parts: a central sump pit, a storage section, an inner and an outer cultivation section. Each part was separated by a 30 cm high wall with several holes which could be closed with plugs; the floor o f the pond had a gradual slope to the centre. The storage section was provided for retaining the total culture
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Fig. 2.
E . W . Becker
55 m2 cultivation pond made of PVC sheets sealed on polyurethane blocks. Agitation of algal suspension by paddle wheel.
volume and also for collecting the supernatant o f the medium after harvesting the algae by flocculation or sedimentation. During cultivation the medium was pumped from the sump pit to the outer section of the pond from where it flowed back through the holes in the partition walls. This flow speed could be regulated by the number of open holes. To test another system, a 31 m 2 (6000 litre) cement tank o f oblong form was built, divided into two sections by a central wall (Fig. 4). Both parts o f this pond had a continuous gradient from the upper point of culture delivery to the lower part where the medium is collected and pumped back to the upper end. To further simplify the cultivation system, a series o f cement ponds was built which might be appropriate for algal cultivation on a rural level in India (Figs 5 and 6). The ponds were small ditches with a size of 0.9 X 11 m each (2000 litres) and a depth o f 40 cm, without partition
Biotechnology and exploitation of Scenedesmus obliquus
5
Fig. 3. 94 m 2 cultivation pond made of bricks and cement. Agitation of suspension by pump and gravity flow. Aerobic digester for farm waste in foreground. (For details see text.)
walls or agitation devices. Mixing was done b y hand with a b r o o m from time to time during the day. Since the stirring of the culture was not continuous, the Scenedesrnus cells started to settle, this resulted in low growth rates. Hence this pond design is more suited for the cultivation o f algae such as Spirulina, where continuous agitation is not required. A still simpler and cheaper construction of algal ponds would be earthen pits lined with polyethylene sheets, as described b y Richmond and Preiss for Israel. 2
Nutrients To be independent o f costly pure chemicals, utilization of locally available mineral salts of commercial grade was tried as nutrients for the cultivation of Scenedesmus. A simple medium was developed with considerable success in reducing the amount of nutrients which are
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Fig. 4.
E . W . Becker
31
m 2
cultivation pond, constructed with bricks and cement. Agitation of suspension by pump and gravity flow.
normally supplied in standard media (Table 1). Crude sea salt is being used to provide undefined amounts of micronutrients. In search of cheap ingredients for algal media, some waste products such as whole blood and blood plasma were studied for their growth stimulating effects. Sheep's blood at 0.5% level resulted in optimal growth stimulation. Since blood carbonicanhydrase might be one of the effective ingredients, active and inactivated crude carbonicanhydrase were tested. Growth increase was less than with whole blood and addition of serum albumin instead of carbonicanhydrase produced similar results indicating that protein or its degradation products may be responsible for the effect. The supply of sufficient amounts of carbon to the algae is one of the major obstacles in algal mass cultivation. At Mysore, CO2 was obtained in gas cylinders and bubbled into the culture through perforated pipes. By constantly supplying CO2 for 8 h day-1, more gas was added into the medium at certain times of the day than could be utilized by the algae.
Biotechnology and exploitation of Scenedesmus obliquus
Fig. 5.
7
10 m 2 cultivation pond constructed with bricks and cement. Agitation of suspension manually with a broom.
By using a pH-stat and monitoring the total amount o f CO2 consumed by the algae, it was found that the number o f times when CO2 had to be added to the culture to maintain a given pH value increased during the course of the day. It reached its maximum in the early afternoon hours. By totalling the daily CO2 additions, it was observed that the gas was supplied for only 4 h day -1 altogether. No difference in growth could be found between cultures gassed continuously with CO2 for 8 h day -1 and cultures supplied in intervals as regulated by the pH-stat. Based on this finding, the CO2 was added by manual control at 1 h
8
Fig. 6.
E. IV. Becker
9 m2 cultivation pond constructed with mortar. Agitation of suspension manually with a broom.
intervals for 4 h day -1 and no marginal variations in growth could be observed between these two methods (Fig. 7). To exploit cheaper sources o f CO2 it was attempted to produce this gas by biodegradation o f cow dung by aerobic fermentation. The CO2-enriched air, produced in a simple fermenter, was sucked into the culture by the circulating medium. 100 mg CO2 h-1 kg-1 dung could be produced by this method from actively fermenting material. Fresh dung has to be supplied to the fermenter every two weeks. Based on the results that a minimum CO2 supply o f 3 litres m -2 h -1 is required to
Biotechnology and exploitation of Scenedesmus obliquus TABLE 1
Composition of Media for Cultivation of Scenedesmus obliquus
Nutrients
Urea (NI-h)2SO4 K2CO3 NaaPO4 Crude sea salt MgSO4.7H20 FeSO4.7H20 EDTA CaC12.2H20 K2HPO4 KH2PO4 As solution B6 solution
Standard medium (mgflitre )
Simplified medium (mgflitre )
300-0
60.0 20.0 25.0 25.0 25.0 10.0 2.0
120.0 10.0 10.0 7-0 144.0 92.0 1 ml/litre 1 ml/litre
As solution (g/litre): H3BO3, 2.86; MnC12.4H20, 1.80; ZnSO4.7H20, 0.22; MOO3, 0.01; CuSO4.5H20, 0.08. B6 solution (g/litre): NI-hVO3, 22.9; NiSO4.7H20, 47.8; Na2WO4, 17.9; Ti2(SO4)3, 40.0; Co(NOa)2.6H20, 4.40. sustain algal growth, approximately 60 kg o f dung are necessary to provide a comparable amount of CO2 in the form of 'CO2-enriched biogas'. The mode of carbon nutrition in Scenedesrnus can be shifted from autotrophic to heterotrophic, because this alga is able to utilize b o t h inorganic and organic carbon. 3 Different experiments were performed to test the feasibility of using sugar cane molasses as a cheap carbon source in algal mass cultivation. It was endeavoured to find a molasses concentration, which after addition to the medium in the evening, is absorbed b y the algae during the night so that a period o f heterotrophic growth (night) is followed b y a period of photoautotrophic growth (day). a At an average addition o f 1 0 0 - 1 5 0 m g l i t r e -1 suspension all sugar was absorbed b y the algae overnight and the bacterial count in the medium did not rise substantially. Maximum growth rates of Scenedesmus were obtained b y providing b o t h CO2 and molasses to the
E. W. Becker
10
_soJ
o Reference: Air(100ml/rnin) • CO2 (250ml/min) continuous & CO2 (500ml/min) continuous CO2 (500ml/min) in 1hr intervals i CO (ll/min) continuous
.}
C02(111min) inlhr
,oo
~ /'~
• Q
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intefval~~ ~ ~IJ / . M. . . . . . _ _ ,,o / 7 . , ,
3O
//,~ /
°
/
///./o/O
{3n
I0
Time [doys] Fig. 7.
Effect of different C02 concentrations on the growth of Scenedesmus obliquus.
100 80 t~
E
--day
t-
60 ~0
/
Fig. 8.
o Air (100t/rain}, C02(0.511min},molosses (140rag/I)/''-~° • Air(1001/min) +C02(0.51/min) / ,~ Air(1001/min)÷ molasses(140mg/I) o......._..o/" / Air (1001/rain) ~-night Took volume 1~00i
I
1
/
/
/i/~) .~:~l~ o~O
"I
/
i
2 3 Ti me [days]
//A.______A
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i
4
i
5
Growth of Scenedesmus obliquus under different autotrophic and heterotrophic culture conditions.
Biotechnology and exploitation of Scenedesmusobliquus
11
culture (Fig. 8). Thus, an increase in yield of about 30% could be achieved by the addition of molasses to the normally supplied amount of COs.
Harvesting To harvest the algae, the suspension was Concentrated by continuous centrifugation into a slurry containing about 15% suspended solids which was the best concentration for the subsequent drying process. Since centrifugation requires high capital expenditures and energy costs, other harvesting methods were tried. Induced sedimentation by the addition of chemicals offers a promising alternative, however, residues of these chemicals in the harvested algal material may limit the further utilization of the biomass. Therefore, the non-toxic carbohydrate chitosan, obtained as deacetylated chitin of marine arthropods, was tested as flocculant for the separation of Scenedesmus. 5 A concentration of 50 ppm of this compound at pH 8.5 was optimal and resulted in better sedimentation rates of the algae than obtained with conventional flocculants. Drying If the concentrated wet algal slurry is not utilized immediately, it has to undergo a heat treatment. For Scenedesmus and other green algae this step is essential because it both dries and sterilizes the algal material and simultaneously ruptures the algal cells thus making the algal protein digestible. Different methods of drying Scenedesmus were tested, viz. sun-drying, sun-drying after previous cooking, freeze-drying, and drumdrying. While all methods gave a dry algal powder (6-10% moisture), only drum-drying resulted in a hygienically safe and fully digestible product. It seems that this method is the only one (aside from spraydrying) by which green algae can be processed into acceptable proteinaceous matter. Yield Strictly speaking, outdoor mass cultivation of algae represents a special form of agriculture, being subjected to similar variations in yield, quality and composition of the product. It is obvious that algal yields
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E. W. Becker
o f short-term cultivation periods o f 1-2 (summer) months are higher than o f long-term periods. For Chlorella and S c e n e d e s m u s growth rates in the range o f 5 - 5 0 g m -2 day -1 can be found in the literature. 6 The results of the Indian project demonstrated that under the given conditions an average yield of 15-20 g m -2 d a y -1 c a n be obtained for S c e n e d e s m u s provided all inputs are optimized. Cultures aerated with compressed air only (500 litres m -2 day -1) showed growth rates below 5 g m -2 day -/. Moreover, algae grown under this condition had lower protein (30%) and higher ash (up to 20%) contents than cultures grown with additional CO2 supply. According to experiences and theoretical considerations 7 it seems that the upper limit in yield o f 3 0 - 4 0 g m -2 day -a is a given characteristic of the photosynthetic process of algal growth which cannot be improved upon very much in large-scale cultivation. Algal production in India was performed in a semi-continuous way by periodic harvesting o f the algae and addition of recycled or fresh medium. The cultures were grown for 4-5 days up to a concentration of 0.8-1.0 g litre -I. The ponds were harvested partially until the remaining volume was sufficient to start a fresh culture with an initial concentration o f 0.2-0.3 g litre -1. The effluent from the centrifuge was made up with fresh nutrients and recycled. However, the same medium could not be reused more than 3 - 4 times, otherwise reduced algal growth and enhanced bacterial contamination occurred.
Chemical composition Data on chemical analyses of S c e n e d e s m u s are given in Table 2. It was observed that the pronounced dark and light phases at the latitude of Mysore resulted in a partial synchronization o f the o u t d o o r algal cultures, which influenced the chemical composition of the algae. Their protein content was always higher in the morning than in the evening, while the carbohydrate concentration was low in the morning and high in the evening; the difference in the protein content was between 10 and 15% o f the total biomass. The amino acid pattern of S c e n e d e s m u s is given in Table 3. The amount o f lysine and sulphur containing amino acids is slightly deficient compared with a recommended standard. Analyses of the algal lipids revealed an unusually high proportion of unsaturated fatty acids which amounted to 80% of the total lipid content. Linolenic acid (C 18 : 3) and
Biotechnology and exploitation of Scenedesmus obliquus
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TABLE 2 Chemical Composition of Drum-dried Scenedesmus obliquus
Overall composition
(g/1 O0 g algae)
Crude protein (N x 6.25) Total nitrogen Non-protein nitrogen Ether extractables Carbohydrates Starch Reducing sugars Non-reducing sugars Other polysaccharides Crude fibre RNA DNA
52.0 8.3 1.0 9.1 12.5 5.3 0.4 1.3 5.5 5.3 4.~ ~'~.,~" 1.6
Moisture Ash Phosphorus Iron Calcium Sodium Magnesium Potassium
7.1 8.0 3-9 0.7 0.5 0.3 0.3 1-0
Vitamins Thiamine Riboflavin Pyridoxyl-HC1 Cobalamine Biotin Folic acid Nicotinate d-Ca-Pantothenate Ascorbic acid /3-Carotene
Fatty acids C 14 : 0 C 16 : 0 C 16 : 1 C 16 : 4 C 18 : 0 C 18 : 1 C 18 : 2 C 18 : 3 C 20 : 0 C 20 : 2 C 22 : 0 C 24 : 0
(mg/kg algae) 8.2 36.6 2.5 0.44 0.2 0.7 120.0 16.5 17-7 230.0
(% of total lipids) 1.3 12.1 9.5 13.1 0.9 15.0 9-0 30.7 0.6 2.7 0-4 0.6
t h e p o l y - u n s a t u r a t e d f a t t y acid h e x a - t e t r a e n i c acid (C 16 : 4) c o n t r i b u t e 30 and 13% respectively. Nutritional studies Since the u l t i m a t e utilization o f algal biomass d e p e n d s p r e d o m i n a n t l y o n its n u t r i t i o n a l p r o p e r t i e s , a wide s p e c t r u m o f studies was p e r f o r m e d at C F T R I to establish t h e n u t r i t i o n a l value o f Scenedesmus. T h e following p a r a m e t e r s were e x a m i n e d b y feeding various c o n c e n t r a t i o n s o f Scenedesmus to rats: P r o t e i n E f f i c i e n c y R a t i o ( P E R ) , Biological Value
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E. W. Becker TABLE 3
Amino Acid Profile of Scenedesmus obliquus Protein Compared with FAO Recommended Pattern Amino acid
Isoleucine Leucine Valine Phenylalanine Tyrosine Lysine Methionine Cystine Tryptophan Threonine Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Proline Serine
FAO a {g/16 g N)
4.0 7-0 5.0 ~ 6.0 ~ 5.5 3.5 , ,..... 1.0 4.0
Scenedesmus (g/16 g N) 3.6 7-3 6.0 4.8 3.2 5.6 1.5 0-6 0-3 5.1 9.0 7-1 8.4 10-7 7.1 2.1 3.9 3.8
Energy and protein requirement report of a 'Joint FAO/ WHO ad hoc Expert Committee', No. 52 (1973).
a
(BV), Digestibility Coefficient (DC) and Net Protein Utilization (NPU). The results o f these experiments are summarized in Table 4. From the data on PER it is evident that S c e n e d e s m u s has a high nutritional quality (when compared with casein) and that drum-drying is superior to all other drying methods, a Diets supplemented with methionine showed PER values even closer to that o f casein. Cooking (20 min at 100°C) would be expected to improve the PER; however, no beneficial effect could be observed. Similar findings are reported for Chlorella by Cook. 9 A similar trend can be seen in the other parameters (NPU, BV, DC). Here too, drum-drying gave the best results, confirming that this method - although expensive - seems to be the most suitable process
Biotechnology and exploitation of Scenedesmus obliquus
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TABLE 4 Nutritional Studies with Differently Processed Scenedesmus obliquus. Results of Feeding Experiments on Rats Protein source
Protein
PERaorr
BV b
DC b
NPU b
10 10
2.50 1.99
87.8 80-8
95.1 81.4
83.4 65.8
10 15 20 10 10 20
2.20 2-00 1.68 1-14 1.20 1.52
72.1 71-9 -
72.5 77.1 -
52.0 55-5 -
(~) Casein Algae (drum-dried) Algae (drum-dried) a 0-03% methionine Algae (drum-dried) Algae (drum-dried) Algae (sun-dried) Algae (cooked, sun-dried) Algae (cooked, sun-dried)
a Values are the mean of 10 rats per group. b Values are the mean of 8 rats per group.
to produce a fully digestible algal product. Comparable studies with the same alga reported by other authors support the results from the Indian project. 1° Toxicological considerations The commercial distribution of unconventional proteins presupposes their toxicological safety. In default of unambiguous laws, recommendations o f international organizations such as the former Protein Advisory Group (PAG) or the International Union o f Pure and Applied Chemistry (IUPAC) have to be considered as guidelines for establishing the safety o f algae. These toxicological evaluations include analyses of chemical composition, tests for biogenic and non-biogenic toxins, shortterm and long-term (multigeneration) animal studies, sanitary analyses and clinical studies. To ensure the absence of biogenic toxins, S c e n e d e s m u s samples were tested for the following compounds: aflatoxin, ochratoxin A, sterigmatocystin, citrinin, patulin, penicillic acid, zearalenone, diacetoxyscirpenol and trichothecene. Additional dermatological and toxicological tests
16
E. W. Becket
with trichothecene, as well as toxicity studies with mice on tremorgens (fumitremorgen and verrucolugen), were conducted. None of the above listed toxins could be detected and all biological tests gave negative results. Among the non-biogenic toxins, special attention has been given to the accumulation of anthropogenic pollutants, particularly heavy metals and pesticides. The concentration of these compounds in the algae depends on the origin of the sample and is not a characteristic of the algae itself, because all these toxins are accumulated from a polluted environment. Analyses for the pesticides a-HCH, /3-HCH, 7-HCH, HCB, dieldrin, DDE, DDD, DDT and PCB showed very low concentrations which were far below the permitted limits for foodstuffs. Dialkyl-nitrosamines of dimethyl- and dipentyl nitrosopiperidine, nitrosomorpholine and nitrosopyrrolidine could not be detected in the alga (detection limit 0.1 #g kg-1). After overcoming certain initial difficulties it was possible to keep the heavy metal content in the algae below the limits which are recommended for bacterial single cell protein (SCP) (Pb, 5-0; Cd, 1.0; Hg, 0.1 and As, 2.0 mg kg-1). Single cell protein has received special attention from toxicologists because it has a higher nucleic acid content than the normal ingredients of the human diet. Excessive uptake of nucleic acids leads to elevated uric acid concentrations in the blood which may cause gout or hyperuricemic nephropathy. Therefore the PAG recommended that the daily nucleic acid uptake from any unconventional source should not exceed 2 g (total intake from all sources, 4 g). Assuming a nucleic acid content of 6% in S c e n e d e s m u s the upper limit for the daily consumption of this alga would be 30 g for adults. By considering the number of persons with latent hyperuricemy, the daily uptake should be restricted to about 20 g or 0.3 g kg-1 body weight. As an initial step in establishing the toxicological safety of Scened e s m u s , 12 week trials were performed by feeding differently processed algae at various concentrations to rats and examining the animals afterwards. The results of these experiments did not reveal any harmful histological or haematological effects in the rats. The animals fed with 20% algal protein consumed more feed and gained more weight than the animals of the other groups. The consumption of sun-dried algae
Biotechnology and exploitation of Scenedesmusobliquus
17
was much less and the animals of these groups showed a weight gain of only 35 g, while the rats of the drum-dried diet group (20%) gained 225 g.ll Besides this effect which is due to the physical method of processing, no adverse symptoms or indications have been observed which could be attributable to the algae and which would limit the utilization of Scenedesmus as supplement in feed or food.
Production cost
Due to the small number of projects connected with the commercial production of microalgae, information on cost-benefit analyses of algal mass production is only fragmentary. In India, the early idea of developing a semi-industrial system of algal production has given way to the more rural oriented technology with independent small-scale production units. However, to estimate the prospective costs of large-scale production and to point out the problems of such ventures, some cost computations have been made based on the data collected at the pilot plant at Mysore. For this calculation a plant size of 5 ha was assumed and a production technology which included agitation by paddle wheels, addition of self-produced CO2, harvesting by centrifugation, and processing by drum-drying. The yield was estimated at 280 t yr -1. Based on calculations and quotations, a price of 8 US $ m-2 can be obtained in India for the construction of the algal cultivation ponds (bricks and cement). This estimation is comparable with cost computations performed in Peru for sloped algal ponds made of concrete ( 1 0 - 5 U S $ m -2) or with adobe bricks (9.0US$m-2) 12. As long as no cheap CO2 sources in the form of waste gas, etc. are available, the COz production from fuel seems to be the most convenient method. The Indian results demonstrated that, for the production of 1 kg of algal dry matter 3 kg of CO2 are necessary. The CO2 production from fuel yields approximately 3 kg CO2 kg-~ oil so that 1 kg of fuel would be required to produce 1 kg of algae. The heat produced by this system can be utilized for drying of the algae. Harvesting of Scenedesmus contributes to 25%, production of CO2 to 20%, and drying of the algae to 10% of the total production costs, which are calculated at 2.30 US $ kg-1.
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E. W. Becker
CONCLUSIONS The most promising bases for an immediate utilization of microalgae seem to be those cases where the algae can be obtained either as byproduct or where they can be produced by a very simple technology. The development of such low level technology is the major objective of the Indian algal project. It was demonstrated that Scenedesmus obliquus can be grown successfully under Indian conditions at growth rates which are comparable to results reported from other algal research plants. It turned out that in the case of Scenedesmus the development of the outdoor cultivation technology has reached a stage which leaves only marginal space for further improvements. Design and construction of the ponds cannot be modified significantly anymore, agitation and aeration may be changed here and there but will not meliorate the system considerably. Some improvements can be seen in the development of new harvesting technologies. If other methods such as the application of flocculants or microstrainers can be standardized to replace the costly centrifugation, a reduction of the production cost can be expected. No alternate method has come up so far for the processing technique used at present. As long as Scenedesmus is designated for a utilization as food or feed, this alga has to be dried by hightemperature treatments. Although the nutritional tests (including supplementation studies) confirmed the high nutritional quality of Scenedesmus and the various toxicological evaluations did not show any toxic symptoms or abnormalities in the test animals, it was considered that the cultivation of this alga is not suitable in India. This conclusion is mainly due to the fact that the technological requirements for a proper production of Scenedesmus are too expensive and sophisticated for the purpose aimed at: the cultivation of microalgae on a rural level in small, independent units. The algal species of choice for this intention seems to be the Cyanobacterium Spirulina, which is being tested already at CFTRI. The immediate utilization of algae in India will be as feed, the application as human food remains a long-term goal.
ACKNOWLEDGEMENTS This work was supported by the German Agency for Technical Cooperation (GTZ) and the Department of Science and Technology, India.
Biotechnology and exploitation of Scenedesmus obliquus
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REFERENCES 1. Soeder, C. J. (1976). Primary production of biomass in freshwater with respect to microbial energy conversion. In: Microbial energy conversion, ed. H. G. Schlegel and J. Barnea. Oxford, Pergamon Press, pp. 59-68. 2. Richmond, A. & Preiss, K. (1980). The biotechnology of algal-culture, lnterdisciplin. Sci. Rev., 5, 60-70. 3. Mineeva, L. A. (1961). The use of various organic compounds by Chlorella vulgaris and Scenedesmus obliquus cultures. Mikrobiologiya, 30,586-92. 4. Shamala, T. R., Drawert, F. & Leupold, G. (1982). Studies on Scenedesmus growth. I. Effect of autotrophic and mixotrophic conditions on the growth of Scenedesmus acutus. Biotechnol. Bioeng., 24, 1287-99. 5. Nigam, B. P. & Venkataraman, L. V. (1980). Application of chitosan as a flocculant for the alga Scenedesmus acutus. Arch. Hydrobiol., 88,378-87. 6. Goldman, J. C. (1979). Outdoor algal mass cultures. I. Applications. Water Res., 13, 1-19. 7. Goldman, J. C. (1979). Outdoor algal mass cultures. II. Photosynthetic yield limitations. WaterRes., 13, 119-36. 8. Becker, E. W., Venkataraman, L. V. &Khanum, P. M. (1976). Digestibility coefficient and biological value of the proteins of the alga Scenedesmus acutus processed by different methods. Nutr. Rep. lnt., 14,457-66. 9. Cook, B. B. (t962). The nutritive value of waste-grown algae. Am. J. Pub. Health, 52,243-51. 10. Payer, H. D., Pabst, W. & Runkel, K. H. (1980). Review of the nutritional and toxicological properties of the green alga Scenedesmus obliquus as a single cell protein. In: Algae biomass, ed. C. J. Soeder and G. Shelef. Amsterdam, Elsevier, North-Holland Biomedical Press, pp. 787-97. 11. Venkataraman, L. V., Becker, E. W., Rajasekaran, T. & Mathew, K. R. (1980). Investigations on toxicology and safety of algal diets in albino rats. Food Cosmet. Toxicol., 18,271-5. 12. Schulz, P., Heussler, P., Moya, R. & Merino, F. (1980). Production cost of Scenedesmus and the economical impact of technological improvements and plant size. Third lnt. Conf. of Microalgae, Trujillo, Peru.