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Nature and Science 2010;8(10


Carotenoids Accumulation in the Green Alga Scenedesmus sp. Incubated with Industrial
Citrate Waste and Different Induction Stresses
El-Sayed, A.B

Fertilization Technology Department, National Research Centre, Dokki, Cairo, Egypt
Abstract: A laboratory experiment was conducted in the algae-station of the Fertilization Technology Department,
National Research Centre, Dokki, Cairo, Egypt with the green alga Scenedesmus sp. to study the effect of citrate
waste in the nutrient solution on vegetative growth and pigments accumulation. The alga was incubated in 2000 ml
culture with 10 ml citric acid waste characterized by high CNPK contents. After maximum growth rate was achieved
induction was performed (by the day ten) and separate addition of NaCl, FeSO4 and chelated FeSO4 was performed
and citrate volume was raised to be 50ml.l-1. Growth measurements were dry weight, total chlorophyll and
carotenoids. Results showed that maximum vegetative growth was reached after approximately 10 days of
incubation. The maximum growth rate during this period was 0.29 and 0.26 d-1, while average growth rate was 0.14
and 0.16 d-1for dry weight and total chlorophyll, respectively. Except of cultures received chelated FeSO4, all other
treated cultures (control, metal FeSO4 and NaCl) resulted in complete degradation of chlorophyll and massive
accumulation of carotenoids. The cultures were completely greenish yellow and yellow colored. Maximum
chlorophyll ratio was found in control cultures which received citrate only (50ml.l-1), however maximum
carotenoids ratio was detected in the algae supplied with metal FeSO4, followed by 2.0% NaCl. [Nature and Science
2010;8(10):34-40]. (ISSN: 1545-0740).

Key words: Citrate wastes; Green algae; Vegetative growth; Carotenoids

1. Introduction
Recycling of particulate organic wastes into A pilot plant called 'Biocoil' has been set up in
marketable production matrix is highly the UK and in Australia (Robinson, 1987). Their
recommended to meet the safe re-use and utilization biocoil system is claimed to be used in conjunction
of wastes which daily increased due to the high with bacterial or algal growth to break down toxic
industrial development (Stirling and Okumug, 1995). wastes and to extract metals from liquids.
A promising technique in this field is the leaching Different microorganisms, fungi, algae and
with microorganisms. With this technique both higher plants are able to synthesize carotenoids.
energy requirements and environmental damage are Production of secondary carotenoids from
kept low (Schinner and Burgstaller, 1989). microalgae surpasses all other sources including
The use of autotrophic microorganisms is yeast and fungi. Production of carotenoids from
advantageous in this respect, because no organic micro-algae for human consumption is of more
carbon source is needed for their growth. On the interest during the last years, however, more studies
other hand, heterotrophic microorganisms can be should be done to make it economically competitive
used with higher pHs (alkaline, acid-consuming (Bhosale, 2004, Pulz and Gross, 2004 and Spolaore
materials). Another advantage of leaching with et al., 2006).
heterotrophic microorganisms is the precipitations Factors affecting the bio-accumulation of such
avoidance when high concentrations of metals and pigments were fully understood. Carotenoids
alkaline pHs are present because of the formation of accumulation by microalgae depend on both
complexes between metals and metabolites. At the nutritional status (Cero'n et al., 2005 and Tittel,
same time, complication often reduces the toxicity 2005) and environmental conditions, such as high
of metal ions (Avakyan and Rabotnova 1971 & light intensity (Bhosale, 2004), type of light (Janhke,
Babich and Stotzky 1980). Because of their capacity 1999). In all cases, the induced algal cells must be
for oxidizing the substrate only partially and used by the log phase growth to avoid the dry
secreting it again in partly oxidized forms, fungi are weight failure. Also, shifting of photosynthetic
of particular interest for leaching. This incomplete metabolism to carotenoids accumulation by lipid
oxidation is strongly influenced by the composition biosynthesis should be considered (El-Shafey et al.,
of the media and causes the accumulation of organic 1999).
acids, which are able to extract metals from solid In the present work, the green alga Scenedesmus
substrates. The most important primary metabolites sp. (El-Sayed, 2004) was used to study the effect of
for leaching is citric acid (Bosecker, 1987). citrate wastes in the medium on both vegetative 34

73:1 heated in water bath to 70oC for 5 min. Chlorophyll absorbance in Table 1. Chemical prosperities of citrate waste Carotenoids were determined based on the O.13 395 21 9 13 residual was dissolved again by 5-10ml of 95% DMSO. filters were upper hold throughout 3mm polyethylene tube. Absorbance was measured at 468nm and By the 10th day. kept over anhydrous calcium chloride till 2.l-1) 0.1:0. The Polyethylene cylinders containing 2000 ml of difference between weights monitored the net dry nutrient growth media were used for incubating weight of the grown alga within define sampling Scenedesmus sp.l -1 50m.001 g. Growth measurements Daily measurement of dry weight was routinely suitability of such media to Scenedesmus sp due to its chemical composition or to the adaptation http://www.95 0.Materials and Methods minutes.. homogenized and 4.8(10) growth and carotenoids accumulation in the carried out by filtering a defined volume of the algal presence or absence of some induction factors. 1972.C C:N pH containing algal biomass were soaked into 5% (w/v) dS. where the high ratio during wastes + + wastes vegetative growth explain the high growth rate 0. specific growth rate (µ) showed the long exponential phase as dry weight accumulation. Supernatant P K Fe Zn Mn Cu was discarded and 5 drops of concentrated acetic % ppm acid were added to recover carotenoids and the 0. accumulation.2. filtered and Chemical characteristics of the used waste are listed re-extracted if necessary. Result and Discussion of citrate wastes with different induction factors 3. Nature and Science 2010.47 0.56: 0. soaked within 5-10ml of 95% DMSO.l-1 + which in turn monitored the high nutrient FeSO4 chelated Fe 2%NaCl consumption rate with high dry weight Wuxal. induction growth was 1976. Filters E..l-1 0. Growth media was composed of time . Here.1. full media were used (per each) aiming at enhancing of chlorophyll to obtain the biggest bulk which resist algal decay formation during the vegetative growth period and during induction period.. Induction factors used and their delivered by such time as a result of chlorophyll concentrations development curve. 1973. a micronutrients suspension (3% Fe. phosphoric acid and Total chlorophyll was determined based on the potassium sulphate.65 0.0% NaCl) or iron (Fe) as well as recorded their log phase by the day eleven. Table 1. the recommended retention time could be Table 2. Vegetative growth including NaCl (2%) metal FeSO4 and chelated Citrate wastes were previously used to enhance FeSO4 (Table 2).l-1 rate of nutrient consumption or biomass citrate citrate wastes citrate wastes citrate accumulation. Filters were dried at 105oC for 30 2 . (g.013 . Here. Thus.sciencepub. slurry (5-10 ml) over pre-weighted dried membrane filter (0. was performed using the growth media and kept the sediment algal bulk to methods adopted by Pirt. Sodium acetate was added in both vegetative growth of the green alga Scenedesmus sp. the induction growth media composed of tap enriched by 50 ml-1 3. 1992. Illumination was provided by day 95% extract. was measured at 666nm and concentration was calculated according to Seely et al.01 from urea.N modification done by Boussiba et al. the relationship Control FeSO4 *Fe(chelate) NaCl between chlorophyll and carotenoids defining the 50m.87 4.1. the next induction growth. carotenoids bioaccumulation during induction.3% Zn. 3% Mn and 14%N) As for growth analysis. Chlorophyll content represented the same by the 10th day. At 70oC.l -1 50m. respectively plus 10 ml of methods described by Burnison 1980 on DMSO citrate wastes. mainly specific. growth periods at a final concentration of 45mM in NPK depleted media. Otherwise.001 g.80 35 naturesciencej@gmail. explains the high 2. maximum performed by discarding the upper clear layer of and average growth rate. Alga and growth conditions room temperature and then re-weighted. Growth analysis.45 µm).M O. Daily yield analysis decomposition of chlorophyll in the presence of showed that dry weight accumulation was obviously salinity (2.m-1 % ratio KOH in 30% (v/v) methanol. A modification was done by light lamps (12x40w) reflexes from one side.C T. which in turn. precipitating the algal biomass over membrane filter Aeration was performed by compressed air from the after dry weight determination.l-1 50m. as cultures reached their concentration was calculated according to Davies maximum vegetative growth.

Here. b When control cultures received only 50 ml-1 of citrate waste represented a slight dry weight decreases as compared with the time of citrate T. maximum growth rate by growth as dry weight was enhanced at first by the the third day was 0. The effect of citrate and other potential of the growth medium. where chlorophyll was drastically decreased and carotenoids were Fig. all cultures were supplied by extra amount of citrate wastes (40 ml. Chlorophyll decomposition mainly nitrogen are required to overcome the dry was also engaged with extra carotenoids http://www.l-1 of citrate wastes represented the same afore-obtained result concerning dry weight and total chlorophyll. Chlorella and Haematococcus inhibitory effect on dry weight as compared with showed that addition of at least 10% of nutrients citrate induced cultures. The main effect different macronutrients including carbon. The ratios here are not conditioned by the same rate of dry weight acceleration (Fig. might be goes back to the high initial biomass. The main reason could be ascribed to observed by the days 5 and 7.37mg : 7. growth rate of Scenedesmus sp. accumulation rate was 0.Ch (mg. salt potential.W (g. grown complete chlorophyll decomposition was observed with 10ml. Nature and Science 2010. on growth enhancement could be attributed to the phosphorous.63mg) at the first cultivation time that explain the high initial biomass c as dry weight or total chlorophyll. Cultures received 50 ml-1 of citrate waste plus Previous studies on carotenoids accumulation by sodium chloride (2.l-1) addition (following to the 10th day).l-1).29 d-1 and the calculated average addition of citrate wastes even at the higher was 0. the inhibitory effect on chlorophyll 3. Here. in particular. Of this result. Full BG-II a medium which received the recommended NPK ratio when enriched by 10 ml. Comparing both data showed the slight decrease on chlorophyll content against the increase on µ (d-1) carotenoids content.1 and Tab. 3). By the D. As for total chlorophyll. potassium and some micronutrients.l-1) 10th day.8(10) potential of this alga to the given nutritional weight accumulation failure (El-Shafey 1999). The decrease on dry weight accumulation due to salting effect was accompanied by chlorophyll decomposition and carotenoids accumulation. conditions. A slight decline in the growth rate was . Induction growth decomposition by NaCl could be ascribed to the Different hypothesis on carotenoids presence of citrate wastes with its initial properties. accumulation by green microalgae were mentioned. At the end of vegetative growth. 2010).27d-1 with calculated Addition of extra citrate amount increased the salt average of 0.sciencepub. maximum carbohydrate metabolism and green pigments.31) by the same aforementioned respect. b) total chlorophyll and c) massively increased.. The ratio showed the drastic effect of NaCl on Time (day) growth metabolites. Under conditions of 2%NaCl. but the growth.0g : 19. a) Dry weight. Different treatments including salinity (NaCl) and different iron forms represented variable results due to the kind of stress factor used. even though they like-wastes on algal growth was considerably are serve as utilizable nutrients as a source of studied (Ruiz-Marin et al..2. which explains the the presence of organic carbon that allows the fast difference between both µ values (maximum and carotenoids accumulation on the expence of average). initial content of some macro and micro-nutrients Growth showed a slight delayed due to such high especially carbon and nitrogen.13 d-1.06:13. nitrogen. 1992 with Haematococcus pluvialis.l-1 of citrate wastes by Boussiba et al.16 d-1.0%) represented an extra Scenedesmus. such ratio was found as (1:17.1. Dry weight: chlorophyll: carotenoids ratio was found as ( 36 naturesciencej@gmail.

2). Kobayashi et al.31 at small quantities. It is well known that active hydroxyl group (active oxygen species). appearing of visual carotenoids suggesting the rapid Chlorophyll decomposition was slightly inhibited as chlorophyll decomposition. 2007). while decreases in (1.1991) or happened. algal growth parameters including dry weight and 1983) and astaxanthin in the yeast Paffia rhodozyma pigments...37 7.37 .8(10) accumulation rate.06 13.57 44.45 inducing cyst formation and astaxanthin accumulation triggered by a high carbon/nitrogen V = ratio at the end of vegetative growth I = ratio at the end of induction growth (C/N) ratio (Kakizono et al. Na. slightly inhibited.63 Droop (1954) and Borowitzka et al.0% other organic acids which stimulate both vegetative of NaCl to growth medium (Boussiba et al. acetate- I 1 17. in particular is known to easy to penetrate the life cells and also used to give rise to free radical formation (especially remove toxicity of heavy metals even in human hydroxyl radicals (HO•) via Fenton chemistry.W T.37 7. These Results protein content and biomass yield. required for enhanced astaxanthin biosynthesis in Treatments Ratio D.0 : 14. the stability of dry probably due to less adaptability of the organism to weight could be attributed to the effect of extra high salinity (Vazquez-Duhalt and Arredondo-Vega citrate addition. The afore-mentioned ratio was Radical initiation of cellular processes via signal found as (1.1985).63 carotenogensis.64) at the end of induction cascade systems is thought to be an integral part of period.sciencepub. The main reason for such results The massive effect could be oriented as Fe.14 37. however dry weight was well as the inhibition of carotenoids accumulation. is fully established by many investigations even in although nitrogen exists in the culture medium. but markedly increasing I 1 37 naturesciencej@gmail. As mentioned before. During carotenoids showed the highly drastic effect of haloadaptation of such alga their hydrocarbon metal Fe on cell metabolism.63 excess may generate a relative shortage of nitrogen I 1 12. decrease was observed due to its utilization by the Comparing the ratio of dry weight: total chlorophyll: alga (Ranga Rao et al. where Fenton reaction is with citrate wastes. could be ascribed to the effect of ferrous ion on Fe-chelate and control.64 concentration dependent. As mentioned Furthermore. enhancing both growth and NaCl (2%) V 1 19. in the absence of essential nutrients was increased with the rise of sodium chloride including nitrogen and phosphorous. growth and carotenoids accumulation. The algal cells seem to reduce their nitrogen uptake and begin to The effect of salinity against growth metabolism use the cellular nitrogen as in typical N-deficiency.. Acetate addition in Fe-Chelate V 1 19. 1991 and Ben-Amotz et al. the effect of acetate was I 1 14. 1992).. Car Haematococcus pluvialis.52 20. Dry weight. body.63 inhibiting growth. while phosphate of the growth media. (1993) reported that increased astaxanthin formation Table 3. As observed early by Control V 1 19. increasing of concentration and maximum biomass was achieved acetate concentrations also rise the salinity potential in 17mM and 34mM salinity. however.l-1) Haematococcus pluvialis.52 : 20. 1995).. halotolerant algae species such as Dunaliella.9). (Schroeder & Johnson. Carbohydrates mean that complete chlorophyll decomposition and and lipids remain unchanged may be due to the extra accumulation of carotenoids are possibility decrease in photosynthetic rate (Hart et al. (1991). (Tab. suggesting the HO• formed under different carotenogensis conditions by an iron-catalyzed.Ch T.37 7. total chlorophyll and caused by Fe+2 was inhibited by potassium iodide carotenoids ratios of Scenedesmus sp grown which scavenges HO•.The chelating prevent the free charge of ions and make it ferrous iron form (Fe+2). 1992). In Citrate wastes at high concentration (50 ml. The fast cultures except those of control cultures. Cultures received 50 ml-1 citrate waste with Ferrous sulfate induced cultures were drastically chelated Fe represented a moderate response and the changed and the fast yellow color was observed by rate of dry weight accumulation surpasses all other the fourth day of induction period.Amotz & Avron. Higher concentrations FeSO4 V 1 19. the biomass of Botrycoccus braunii before. 3 and Fig. Sodium induced cultures which became many biological systems and it was suggested that nearly orange in color suggesting the extra free radicals may play a role in the accumulation of salting-out potential that affect all the determined β-carotene in Dunaliella (Ben. appeared to be an important carbon source.0: 3. carotenoids accumulation support growth media by high organic carbon and (astaxanthin) was triggered by the addition of 2. where it was found as profile was highly increased.9 astaxanthin content per cell.57: 44. http://www. Nature and Science 2010.

l-1 of citrate wastes with different the induction effect suggested to mainly factoring induction factors. it is possible to quickly check the N status of the cell as an indicator to astaxanthin accumulation. This suggests that the synthesis T.14) in which. Nitrogen starvation is an effective way to enhance astaxanthin accumulation in Haematococcus.Ch (mg. It can Research Centre.l-1 (Orosa c et al.2. Our data could be supported by that Corresponding Author obtained by El-Sayed 1999. kind and chelating dependent rather than the salt concentration used. Technical University of Munich (Prof. Dr. FeCl3 triggered El-Sayed. Dokki-Cairo. grown on enhancement of the induction growth. it is accompanied with the same content of Zn. b) total chlorophyll and c) enhancement of algae vegetative growth is very growth rate (dry weight and total advisable to reduce production cost and chlorophyll) of Scenedesmus sp. concentrations. Conclusion Time (day) As it supports growth media by organic carbon and several nutrients. Scenedesmus sp. The ratio chlorophyll-a / total carotenoids could be a good indicator for the physiological status of the culture. 1991). indicating nutrient replete conditions. 4. National agents neutralize the ionic charges of the used ions which in turn prevent the photo-reaction producing 5. In This work was carried out as a part of the activities this context. Z.Car (mg. .0 : 12. and most likely reflects the need for continuous synthesis of protein in order to support the massive accumulation of pigments. 2005). D.W (g. Cairo (Coordinator: stated that EDTA-chelated FeC13. and Chlorella sp. however. no Fenton reaction could be ascribed other Plant Nutrition Problems in Egypt” conducted as mentioned by Borowitzka et al. Dr. In addition. The afore-obtained ratio found as (1. . El-Fouly) and the Institute of Plant produce any significant differences in astaxanthin Nutrition. it explains the inhibitory microalgae in batch and continuous systems. where. The optimum nitrate concentration to avoid this problem seems to be 0. 1991.sciencepub.M. (1988).net/nature 38 naturesciencej@gmail. Amberger).B the carotenoids accumulation by Haematococcus Fertilization Technology Department. Refrences 1. Thus.6H20 did not Prof. when the chelated iron form was used of the Egypto-German Project” Micronutrients and in this action. 50ml. a value below 3 implied limitation of growth by reduced nutrient supply or another kind of stress. a) Dry weight.14 : 37. Concerning the chelated Fe form used.. in: http://www. it may be concluded that chelating prevent the high solubility of ferrous in which became slow Acknowledgment released ion and delay the free radical generation.8(10) a effect of EDTA against drastic chlorophyll decomposition. formation when added at three different A.l-1) Vonshak. this value was about 4. Nitrate concentration played a very important role in the cell division rate and in the accumulation of secondary carotenoids of H.l-1) of astaxanthin requires nitrogen.utilizing of citrate wastes for Fig. Nature and Science 2010. A. Biosorption of heavy metals by free radicals.l-1) which minimizes the toxicity of extra Fe addition and also contains 14% of N delivered from urea that enhance vegetative growth and support growth medium by alternative carbon source (El-Sayed et al. Aksu.. 2001). Boussiba and T. Thus. 1991 added that in favorable growth conditions. pluvialis (Boussiba b and Vonshak. Egypt be also mentioned that EDTA and other chelating bokhair@msn.15 g.. where he by National Research Centre.

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