conditions on the content changes of saturated and ... · Introduction of fatty acid Fatty acid is a kind of carboxylic acid that has a long aliphatic tail. Fatty acids are divided

J. Appl. Environ. Biol. Sci., 2(10)537-548, 2012

© 2012, TextRoad Publication

ISSN 2090-4274 Journal of Applied Environmental

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*Corresponding Author: Soleymanian S., Deparetmnt of Biology, Gorgan Branch, Islamic Azad University, Gorgan, Iran

The Effect of Aluminum presence in heterotrophic and autotrophic conditions on the content changes of saturated and unsaturated fatty

acids in micro algae Chlorella vulgaris Beijernick

*Soleymanian, S., Sateei, A., Shokravi, Sh.

Deparetmnt of Biology, Gorgan Branch, Islamic Azad University, Gorgan, Iran

ABSTRACT

In this research, after we collect and purify Chlorella vulgaris Beijernick, algae they were grown in BG-11 medium containing aluminum ( AICI3 ) and under the condition of heterotrophic and autotrophic. In research survival and growth we studied in the logarithmic growth phase dry weight, internal aluminum content of the sample, the amount of fatty acids accumulated in the algae and sprinkled into the culture medium. According to the results, compare to the other treatments, aluminum cause significant growth (p< 0.05) and dry weight in autotrophic treatment containing 300 micro molar aluminum. Also the presence of aluminum reduces the fatty acids accumulated in the algae and also increase sprinkled fatty acids into the culture medium in the heterotrophy treatment in comparison with autotrophy treatment. The survival of sample was preserved in the heterotrophic condition and in this condition showed more willingness to glucose. The samples in the heterotrophic glucose condition, without latency, grow which showed rapid habituation of organisms with heterotrophic condition. It seems that the mentioned characteristics, made single-celled green algae Chlorella vulgaris Beijernick. more efficient in the different aspects. Key words: Autotrophic, Fatty acids, Green algae Chlorella vulgaris Beijernick. , Heterotrophic, Internal

aluminum content

INTRODUCTION

Micro – algae were used as a source of protein and fat since hundreds of years ago (kay, 1991). A large number of researches have been done about food and medical value of Chlorella vulgaris Beijernick. The cells of algae use sunlight for photosynthesis. In the Chlorella vulgaris Beijernick photosynthesis rate is 10 to 100 times faster than in take rate. The dry weight of these algae approximately is 95%. The protein content depends on the algae species, cultivation method, different harvesting and about 15% to 88% dry weight and in average 50% (merchant, et. al., 2000). 7 percent aluminum makes up of the composition of earth’s crust. In general, the effect of aluminum is associated with low ptt (Amonette et al., 2003).

Aluminum at the PH 5 and lower than 5 is solution and sediments at PH higher than 5 response to aluminum stress is with the production of reactive oxygen’s such as superoxide, hydroxyl radicals, oxygen radicals, oxygen syngleh and toxic molecules of peroxide and hydrogen (Panda et al., 2003).

Heavy metals directly increase harmful effects of reactive oxygen’s through increasing cell concentration and also increase cellular antioxidant capacity (Teresa, 2001). Reactive oxygen’s which are produces in cells are detoxified by enzymatic and non-enzymatic antioxidative systems (Hirayma et al., 2002).

The response of sandsemus green algae to aluminum and low pH showed that the protein content decrease with the increase of aluminum concentration and pH, in 5/6 pH the amount of protein is more than 5/5 pH and in 5/5 pH is more than 5/4 pH. Also the intensity of the antioxidant response increased with the increase of aluminum (Jusu et al., 2004) Other indigestible of Chlorella binds to heavy metals and takes them from their environment. When these metals cause toxicity, Chlorella is the diet use to clean the blood, gut and liver for 3 month or more depending on the toxicity (Hasegawa et al., 1990). Introduction of fatty acid

Fatty acid is a kind of carboxylic acid that has a long aliphatic tail. Fatty acids are divided into two groups: saturated and unsaturated. Unsaturated fatty acids, especially linoleic acid (c18:3) and a linolenic acid (C18:3) which are the combination of Omega 6 and Omega 3 and have a key role in metabolic

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Soleymanian et al., 2012

processes of many human body cells because there is no synthesis mechanism for them in the body (Sayanova and Napier, 2004; Funk, 2001).

Therefore external source is needed to provide fatty acids for human body. The majority of researches which have been conducted on the processes of fatty acids were about photoautotrophic growth (Sanches et al., 2002). Algae’s which are in the lowest food level in aquatic systems are the first determining of food quality. Green algae Chlorella is important for food industry applications such as unsaturated fatty acids which is produced by C.sorokiniana and lutein which is produced by C.protothecoides. C.zofingiensis is important as a valuable producer of carotenoid (Orosa et al., 2000). Due to the use of fatty acids in health and nutrition, it is very important to analyze them (Kanchanamayoon and Kanenil, 2007).

The most common process in fatty acid analysis is conversion of them into methyl ester in order to improve their volatility. Several articles focus on have been focus on the analysis of fatty acids (Miller, 1962; Milner, 1948; Nichols, 1965; Nielsen, 1953; Osterlinid, 1948; Otauka and Morimura, 1966). Comparison autotrophy and heterotrophic cultivation in the production of fatty acids The subject of many investigations is the direct relationships between culture conditions and the percentage of carotenoids, proteins and fatty acids (Piorreck, et al., 1984). The amount of fatty acids in autotrophy culture is low because of in sufficient light for algae (means that algae’s prevent sufficient light reach each other) (Barclay et al., 1994). For increasing the production of fatty acids by algae’s, it is more appropriate to increase heterotrophic growth (wen, 2001).

The result of El-Sheekh and Fathy research (2009) showed that chlorella vulgaris Beijernick has simpler fatty acids composition compared with other species of Chlorophyceae and natural food production and natural pharmaceutical products using Chlorella vulgaris Beijernick autotrophy cells is more considerable than heterotrophic cells. Chlorella as a biofuel: Recently, due to limited fossil resources and environmental pollution concerns, the concern increase for the production of bio fuels such as biodiesel from vegetable ails animal fat and algae. It has been found that lipid production is algae are higher than other land-based products. Biodiesel production reaction

Biodiesel production takes place during chemical reactions with alcohol in the presence of catalysts. A final product is methyl ester (which is actually biodiesel) and glycerol (a byproduct) and this process is called transesterification. Catalysts are alkali. Reactions are took place at high pH. Used alcohols are ethanol and to a lesser degree methanol. This chemical reaction is sensitive to water. The presence of water leads to saponify reaction which effective on biodiesel production and its quality.(Figure1) Laboratory studies indicate that micro-algae are capable to produce 70% lipid, however tests in industrial scale showed 30% maximum efficiency.( Figure2)

The presence study investigates the possibility Chlorella vulgaris Beijernick green algae survival and growth in heterotrophic conditions. Aluminum content and fatty acids content accumulated in algae and secreted into the culture medium was investigated in heterotrophic conditions compared with autotrophy conditions.

MATERIALS AND METHODS

1- Algae cultivation Chlorella vulagaris Beijernick algae got from micro-algae collection of applied sciences research

institute of shahid Beheshti University and were cultivated in BG-11 medium. Types of treatments which are considered in this study include:

a) Heterotrophic treatment Glu,Al 300 µM control : heterotrophic Al0 µM, Glu b) Heterotrophic treatment Suc,Al 300 µM control : heterotrophic Al0 µM,Suc c) Autotrophy treatment control : autotrophy Al0 µM

Inoculated was conducted in 250 ml Erlenmeyer flask (with three replicates). Autotrophy treatments were grown in brightness conditions 1500 lux and temperature between 27-25 ̊c and light frequency 20 hours lightness and 4 hours darkness and heterotrophy treatments in the incubator with a temperature of 28 degree for 21 days.

2- Measurement of growth Survival and growth was measured based on opacity survey by spectrophotometers (spectronic LABOMED, INC, USA) (Mirbehbahani et al., 2006).

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3- Measurement of dry weight Algae cells were dried in test tube in oven with temperature of the 108 ̊c. Dry weight of algae (algae suspension ml/mg 10) were obtained through follow relationship: Tube weight – (weight of tube + algae) = dry weight.

4- Measurement of aluminum by using acid extraction with atomic absorption Spectrophotometers model VARIAN AA 240 model: first of all, algae were mixed with nitric acid and hydrochloric acid and after centrifugation; the supernatant solution absorption was read by atomic absorption.

5- Lipid extraction (Blight and Dyer, 1959) We added on the dried sample chloroform solvent to a ratio of two to one and it got ( سونیکھ).

After that, put catharsizes solvent into (Separateory Funnels) and chloroform and distilled water was added. Removed the lower phase and put it in spinning evaporate apparatus at the temperature of 50 degrees until its chloroform got evaporated.

Removed the balloon containing a few drops of oil from machine and added alcoholic potash and connected it to soxhlet and heated it gently. Then added distilled water to the balloon contents and put it in a (Separateory Funnels) and added petroleum ether to it and mixed them well in (Separateory Funnels). Put it way for a few minutes so that ether segment containing soap substances to be separated acidified. The soap solution in ( Separateory Funnels) through using chloridric acid then added petroleum rather into it and put acidified soup solution into (Separateory Funnels), waited for a while until two phase got composed. Poured the ether phase containing the fatty acid into balloon and was connected into evaporation apparatus with the temperature of 50 degree Celsius. The methylation of fatty acids for injection into gas chromatography

We poured 100 ml methanol without water into graduated cylinder, slowly added 5ml of concentrated sulfuric acid and regularly mixed it. Then we added 50 ml Toluene as a lipid solver material and take 5 ml to dried fatty acid bottle to shake. Transferred the solution into the cap test tube and put for one hour in the boiling water bath. After tubes got cooled we added 5ml distilled water and 5ml diethyl ether to each of them and completely mixed them. The upper phase (organic phase) was separated by using samper and added about 1gr sodium sulfate and have kept at temperature below zero degrees Celsius. After concentration the extracts were injected into the GLC column.

6- Statistical considerations : The analysis of data was conducted through a completely randomized design and comparison

between treatment and controls was done according to Duncan and T2 tamhane test at 5% level. Results were presented based on SDX .

RESULTS

The effect of autotrophic and heterotrophic treatments on the growth in the presence of aluminum according to figure 3, a significant difference was observed in autotrophic and heterotrophic sucrose treatment compared to the control group (p < 0.05).

While a significant difference in growth were not observed in heterotrophic treatment (glucose). Identical letters indicate no difference between treatments and their controls. The effect of autotrophic and heterotrophic treatment on dry weight in the presence of aluminum. According to figure 4: In all the treatments there is a significant difference in terms of dry weight compared with to their controls (p < 0.05). The effect of autotrophic and heterotrophic treatment on the content of inner aluminum. According to figure 5, the amount of internal aluminum in autotrophic treatment compared with other treatments has a significant reduction in the 05/0 ml. The effect of autotrophic and heterotrophic treatments on fatty acids content in the presence of aluminum. According to the figure 7, about all fatty acids, the amount of them increases in 300 micro molar aluminum treatment. In the absence of aluminum, the amount of acid and linolenic acid increase a bit in algae.

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The effect of aluminum increase all of fatty acids secreted into culture medium compared with control group. The presence of aluminum decrease all fatty acids accumulated in algae compared with its control. The presence of aluminum reduced all fatty acids secreted into the culture medium compared with the control. The presence of aluminum and light increase fatty acids accumulated in algae in autotrophic treatment compared with heterotrophic treatment. The presence of aluminum and light decreased fatty acids secreted into culture medium in autotrophic treatment compared with heterotrophic treatment.

DISCUSSION

According to the results, aluminum made accumulated saturated and unsaturated fatty acids increase in algae in heterotrophic treatment compared to the control.

In this treatment, the amount of oleic acid and linoleic acid has considerably increased compared to the control. Also aluminum increase saturated and unsaturated fatty acids secreted into the culture medium in heterotrophic treatment compare to the control. The amount of oleic acid, linoleic acid and linolenic acid also increased compared to the control.

In autotrophic treatment the presence of aluminum decreased saturated and unsaturated fatty acids accumulated in algae compared to the control. Also, the presence of aluminum decreased saturated and unsaturated fatty acids secreted into the culture medium in autotrophic treatment compared to the control. The amount of oleic acid, linolenic acid and linoleic acid decrease in this treatment compared to the control.

The comparison of heterotrophic treatments with autotrophic treatments showed that the presence of light has increased saturated and unsaturated fatty acids accumulated in algae in autotrophic treatment compared to heterotrophic treatment. Also the amounts of oleic acid, linoleic acid and linolenic acid have increased significantly in autotrophic treatment compared to heterotrophic treatment. The presence of light decreased saturated and unsaturated fatty acids secreted into the culture medium in autotrophic treatment compared to heterotrophic treatment. Also, the amount of oleic acid, linoleic acid and linolenic acid in this treatment has been reduced substantially compared to heterotrophic treatment.

El-sheekh and Fathy (2009) mentioned that the biological importance of the mechanism for glucose consumption by Chlorella is not clear, but in normal condition the growth of Chlorella autotrophic changes into heterotrophic growth. This condition may use exogenous carbon source possible.

In the presence of exogenous carbon source glucose, growth and production of fatty acids has increased.

Still the important question is that whether there is any explanation for qualitative changes in the fatty acid species (phenotypes) of Chlorella. The results of El-Sheekh and Fathy (2009) revealed that Chlorella vulgaris qualitatively has a quite simple fatty acids composition compared to other species of Chlorophyceae. Qualities composition of fatty acids in not only different in species but also is different in various phenotypes of one species (Nichols, 1965; petkov & Garcia, 2007).

In 2007, petkov emphasized that no difference is exist in the qualities composition of fatty acids in the Chlorella, but he believes that stability of fatty acids as a specific characteristic of Chlorella is important. Although, he revealed the application of the qualitative composition of fatty acids as a taxonomical adjective in the Chlorella species, so in this way proved that changes exist in the qualitative composition of fatty acids.

For example, he showed that the presence of fatty acids 15:0, 77:0, 11:1 occurs in the culture along with bacteria (Wacker et. al., 2002).

In addition, he explained that the presence of fatty acids 20:0, 20:1, 20:2 in the presence of impurities with a relation time ( the exits of material from GC column which is important in GC), with the result similar to GLC and others, including Homova et al. (1986) and Antonyan et al. (1986).

Dickson et al., (1969) conducted qualities and quantities analysis of Chlorella fusca important fatty acids in autotrophic and heterotrophy cultures. Increase in environment CO2 concentration (from 1 to 30 percent) in heterotrophic cultures lead to 40% increase in lipids and approximately 50% increase in fatty acids.

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Spoehr and Milner (1949), reported that carbon assimilation as CO2 or organic carbon will probably continue after some environmental factors (such as limited nitrogen) led to cell division stop. CO2 has a direct inhibitory effect on Chlorella cell division which has proved by Sorokin 1965. The production of new protoplast and its growth may be very sensitive to environmental stresses, whereas the carbon can be assimilated and accumulated as a storage product like triglyceride. Conclusion

Autotrophic growth condition significantly increased growth and dry weight compared to heterotrophic growth condition. The presence of aluminum and light in autotrophic treatment reduced saturated and unsaturated fatty acids that was accumulated in algae and was secreted into the culture medium. The pretense of aluminum in heterotrophy treatment (GLU) increase saturated and unsaturated fatty acids that was accumulated in algae and was secreted into the culture medium. The result is that the presence of aluminum reduce the accumulation fatty acids in algae and also increase the fatty acid secreted into the culture medium in heterotrophic treatment compared to autotrophic treatment.

The survival of sample was preserved in heterotrophic condition and in this condition showed more willingness to glucose. The sample in heterotrophic glucose condition without latency phase grew and this represented the rapid habituation of organisms with heterotrophic condition. The models for Chlorella valguris Beijernick behavior for fatty acids synthesis in the presence of aluminum in the heterotrophic and autotrophic culture medium

Model 1. The behavior of Chlorella vulgaris Beijernick algae on fatty acids synthesis in the presence of

aluminum in heterotrophic culture medium.

Modl 2. Chlorella vulgaris Beijernick algae behavior for fatty acids’ synthesis in the presence of aluminum

in the heterotrophic culture medium.

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Acknowledgment The authors thank all of those who have cooperated in this study, especially Mr.Maziar Ahmadi

Golsefidi, Mrs.Kiaee (head of research laboratory) and Mr.Aeeneh (the expert of analysis laboratory)

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Figure1: Biodiesel production and glycerol

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Figure 2: Biodiesel manufacturing process

Figure3: The histogram for comparison between treatments (Al 300) and their controls (Alo) in green algae Chlorella vulgaris Beijernick.

Figure 4: The histogram for comparison of dry weight between treatments (Al 300) and their controls (AIO) in green algae Chlorella vulgaris Beijernick

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Figure 5: The histogram for comparing inner aluminum content in different treatments of Chlorella vulgaris Beijernick under the condition of aluminum treatment 300 micro molar.

Figure 6: GC chromatogram of fatty acids extracted from algae Chlorella vulgaris Beijernick, the observed

growth was under heterotrophic condition containing 05/0 gr glucose and 300 micro molar aluminum.

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Figure 7: The histogram for fatty acids content accumulated in Chlorella vulgaris Beijernick heterotrophic

glucose (Al 300) and its control.

Figure 8: The histogram for fatty acids content secreted into the culture medium in heterotrophic glucose

treatment (Al 300) and its control.

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Figure 9: The histogram for fatty acids content accumulated in algae Chlorella vulgaris Beijernick in autotrophic treatment (Al 300) and its control.

Figure10: The histogram for fatty acids content secreted into the culture medium in the autotrophic treatment (Al 300) and its control.

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Figure 11: The histogram for fatty acids content accumulated in algae Chlorella vulgaris Beijernick. in autotrophic (Al300) and heterotrophic glucose treatments.

Figure 12: The histogram for fatty acids content secreted into the culture medium in autotrophic (Al 300) and heterotrophic (glucose) Al 300 treatments.

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Documents

conditions on the content changes of saturated and ... · Introduction of fatty acid Fatty acid is a kind of carboxylic acid that has a long aliphatic tail. Fatty acids are divided