JOURNAL OF AGRICULTURE & SOCIAL SCIENCES

ISSN Print: 1813–2235; ISSN Online: 1814–960X

08–012/AWB/2008/04–4–165–169
http://www.fspublishers.org

Full Length Article

Effect of Chlorella vulgaris as Bio-fertilizer on Growth

Parameters and Metabolic Aspects of Lettuce Plant
FAYZA A. FAHEED1 AND ZEINAB ABD-EL FATTAH
Botany Department, Faculty of Science, Sohag University, 82524 Sohag, Egypt
1
Corresponding author’s e-mail: [email protected]

ABSTRACT

Seeds of Lactuca sativa were germinated in culture medium containing microalga (Chlorella vulgaris) and grown for 3, 6, 9,
12 and 15 days in order to study its effect on growth parameters and some physiological response of seed germination and
growth. In general, microalgal treatment significantly increased the growth with a significant decrease in soluble carbohydrate,
soluble protein and total free amino acids compared with those of the control (sterilized culture medium) of seed germination.
Addition of microalga to the culture medium or soil significantly increased fresh and dry weight of seedlings as well as
pigments content. The best treatments were 2 and 3 g dry alga kg-1 soil.

Key Words: Lactuca sativa; Chlorella vulgaris; Carbohydrates; Amino acids; Proteins

INTRODUCTION fresh and dry green microalgae (Chlorella vulgaris) as soil

additive on growth and some physiological responses of
In Egypt, soil fertility is diminishing gradually due to lettuce seedlings and to determine the importance of bio-
soil erosions, loss of nutrient, accumulation of salts and fertilizer application in order to improve the yield quality
other toxic elements, water logging and un-balanced and productivity and avoid environmental pollution.
nutrient compensation. Organic wastes and bio-fertilizers
are the alternate sources to meet the nutrient requirement of MATERIALS AND METHODS
crops and to bridge the future gaps. Farming regions that
emphasizing heavy chemical application led to adverse Plant material. The experimental plant used in this study
environmental, agricultural and health consequences. Many was (lactuca sativa cv.) lettuce. The seeds were obtained
efforts are being exercised to combat the adverse from the Agricultural Research Center (ARC), Cairo, Egypt.
consequences of chemical farming. Algal culture. Microalga (Chlorella vulgaris Beijerinck)
The bio-fertilizer, organic manuring and bio-control of was isolated from River Nile Sohag, Egypt and enriched
agricultural have emerged as a promising component of using algae nutritive medium specific for chlorophyta
integrating nutrient supply system in agriculture. (Stein, 1966). The final pH of this medium was 6.8, after
Microbiological fertilizers are important to environment being autoclaved. The nutritive media were inoculated, with
friendly stainable agricultural practices (Bloemberg et al., algal suspension from agar slants. The algal cells were
2000). Bio-fertilizers include mainly the nitrogen fixing, grown at a temperature of 25±10C and surface light of about
phosphate solubilizing and plant growth- promoting 2500 lux (measured by Gossen Luxmeter). Filtered dry air
microorganisms (Goel et al., 1999). Among bio-fertilizers was let to bubble in the culture vessels to provide carbon
benefiting the crop production are Azotobacter, dioxide and to prevent settling of cells.
Azospirillium, blue green algae, Azolla, P-solubilizing Effect of culture media after growth of green microalga
micro organisms, mycorrhizae and Sinorhizobium (Hegde on seed germination of lettuce. In this experiments, culture
et al., 1999). Green manures were also found to stimulate media of green microalga after growth of alga for 3, 6, 9, 12
root growth and produce good yields (Boussiba, 1987; and 15 days were harvested for assay and analytical
Mandimba et al., 1998). Dry green algae contain high procedures.
percentage of macronutrients, considerable amount of Determination of cell number and pigments content of
micronutrients and amino acids (El Fouly et al., 1992; alga. Cell number was determined by taking 0.1 mm deep
Mahmoud, 2001). They can be conveniently produced on having Improved Naubauer Hemocytometer ruling (A.O.
sewage and brackish water and partially substituted the Spencer "Bright Line"). Data were given as cell per mL.
chemical fertilizers to avoid environmental pollution. The pigments were determined spectrocoloimetertcally
Objectives of this work were to study the effect of using the method recommended by Metzner et al. (1965).

To cite this paper: Faheed, F.A. and Z. Abd-El Fattah, 2008. Effect of Chlorella vulgaris as bio-fertilizer on growth parameters and metabolic aspects of
lettuce plant. J. Agri. Soc. Sci., 4: 165–69
 FAHEED AND ABD EL-FATTAH / J. Agri. Soc. Sci., Vol. 4, No. 4, 2008

The data were given as µg/mL algal suspension. RESULTS AND DISCUSSION
Treatment of lettuce seeds. Seeds were surface sterilized
with 5% sodium hypochlorite for 8 min, then rinsed with Growth parameters (cell number & pigment contents) of
distilled water several times before germination. The seeds alga. The growth (cell number & pigments) of alga after 15
were classified into two groups. The first group of 50 seeds days, was followed. A gradual increase in cell number was
were floated in each petridish containing 10 mL of sterilized recorded after 3, 6, 9, 12 and 15 days (Table Ia). The
culture medium of Chlorella vulgaris. The second group of pigment (chlorophyll a, chlorophyll b & carotenoids) and
50 seeds were floated in each petridish containing 10 mL of consequently the total pigments were generally increased
supernatant after centrifuged culture medium and after after 3, 6, 9, 12 and 15 days (Table Ib). This increase was
grown the alga for 3, 6, 9, 12 and 15 days. Petridishes were highly significant (p<0.0001). Also, the pigment fraction
maintained in green house under a 16 h photoperiod at 20- (chl. a/chl. b) was significantly increased (p<0.05).
250C for one week. At the end of the experiment total fresh Seed germination and seedling growth of lettuce plants.
weight, length of shoots and roots per plant were The results of growth parameters obtained for the
determined. The seedlings were washed twice with distilled germination of lettuce seeds subjected to culture media after
water, oven-dried at 750C for 72 h to obtain the dry weight growth of microalga for 3, 6, 9, 12 and 15 days treatments
and then ground to a fine powder. Three replicates were
Fig. 1. Effect of culture medium containing Chlorella
used.
vulgaris grown for 3, 6, 9, 12 and 15 days on growth
Chlorella vulgaris as bio-fertilizers for lettuce seedlings
parameters (a: fresh and dry weight, b: shoot and root
growth. The algal culture were grown in two big container
length) of seed germination of lettuce plants
(5 L) for 7 days. After this period the algal suspension of the
first container was centrifuged and the clear growth medium (a)
was decanted. The algal cells were precipitated. 1/2, 1, 2 and
3 g of fresh algal cells were added to the pots containing 1
1.2
kg soil before sowing. The algal suspension in the second
container was centrifuged and the algal cells were dried over
night in an oven at 1050C. 1/2, 1, 2 and 3 g dry algal cells 1.0
Fresh and dry weight (g/ plant)

were added to another groups of pots containing 1 kg soil

before sowing and then 20 seeds were planted in each pot 0.8

and placed in greenhouse under a 16 h photoperiod at 20-

250C. After 30 days of sowing, samples of each treatment 0.6

were taken for studying the metabolic analysis.

Metabolic samples analysis. The photosynthetic pigments 0.4
(chlorophyll a, b & carotenoids) were determined using
method of Metzner et al. (1965). The dry matter yield was 0.2
used (in replicate) for chemical analysis for the
determination of water-soluble sugars, a known weight of 0.0
the powdered tissue was hydrolyzed in distilled water for 2 medium 3 day 6 day 9 day 12 day 15 day

h in a boiling water bath. After cooling, the hydrolysis was Treatment

filtered and filtrate was made up to a known volume, after Fresh weight Dry weight

which the water-soluble sugars were determined by the

anthrone sulphuric acid method (Fales, 1951). Free amino (b)
acids were extracted from the plant tissues and determined 5
according to the method of Moore and Stein (1948).
To estimate the soluble proteins, powdered tissue
4
samples were boiled in distilled water for 2 h. After cooling
Length of shoot and length of root (cm)

the water extract was centrifuged and supernatant was

decanted and made up to known volume with distilled 3

water. The water insoluble protein residue was treated with

NaOH. The plant protein content was determined according 2

to Lowry et al. (1951), using bovine serum albumin as

standard.
1
Statistic analysis. The measurements of growth parameters
and metabolic aspects were subjected to one-way analysis of
variance (ANOVA) to test difference among means 0
medium 3 day 6 day 9 day 12 day 15 day
corresponding to alga levels via STATISTICA computer Treatment

software distributed by Stat Soft. Inc. Length of shoot Length of root

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 Chlorella vulgaris AS BIO-FERTILIZER / J. Agri. Soc. Sci., Vol. 4, No. 4, 2008

Table I. Growth parameters (a) cell number 104 mm3/mL algal suspension and (b) pigments content µg/mL) of
Chlorella vulgaris for 3, 6, 9, 12 and 15 days under control laboratory conditions (Mean ± SD, n=3)
(a)
Treatment (day) Cell number
0 time 4
3 6
6 15
9 20
12 43
15 66

(b)
Treatment (day) Chl.a Chl. b Carot Total Chl.a/Chl.b
0 Time 2.472±0.084 1.159± 0.137 0.995±0.001 4.627±0.227 2.149±0.192
3 6.023±0.068 2.667±0.438 2.244±0.109 10.934 ±0.417 2.301±0.532
6 8.664±0.116 3.685±0.265 3.633±0.173 15.982 ±0.341 2.363±0.207
9 12.239±0.032 5.711±0.208 5.072±0.105 23.023±0.226 2.146±0.193
12 14.088±0.171 6.815±0.114 6.290±0.214 27.193±0.437 2.067±0.019
15 22.633±0.167 10.326±0.529 10.544±0.113 43.503± 0.431 3.661±1.291

Table II. Effect of culture medium containing Chlorella vulgaris grown for 3, 6, 9, 12 and 15 days on pigments
content (mg/g f. wt.) of seed germination of lettuce plants (Mean ± SD, n=3)
Treatment Day Chl a Chl b Carot Total Chl. a/Chl. b
Sterilized culture medium 0 time 0.202±0.002 0.089±0.001 0.073±0.007 0.372±0.017 2.389±0.172
Culture medium after growth alga 3 0.202±0.002 0.089±0.001 0.071± 0.001 0.362±0.002 2.280±0.007
6 0.230±0.004 0.094±0.004 0.088±0.008 0.412±0.012 2.440±0.040
9 0.268±0.008 0.121±0.001 0.092±0.004 0.481±0.001 2.209±0.009
12 0.371±0.001 0.089±0.009 0.109±0.002 0.568±0.009 4.216±0.008
15 0.270±0.005 0.125±0.000 0.094±0.003 0.489±0.003 2.168±0.002

Table III. Mean values of pigment contents (mg/g fresh weight) in seedling of lettuce plants as affected by different
levels of fresh and dry alga as soil additives (Mean ± SD, n=3)
Treatment Chl.a Chl.b Carot Total Chl.a/Chl.b
Control 0.673±0.091 0.236±0.068 0.197±0.112 1.105± 0.047 2.992±0.472
Fresh alga 1/2 g/kg soil 0.597±0.121 0.258±0.056 0.211±0.020 1.066±0.197 2.320±0.039
1 0.823±0.036 0.345±0.024 0.271±0.011 1.438±0.061 2.390±0.058
2 0.662±0.035 0.264±0.006 0.130±0.013 1.056±0.043 2.516±0.182
3 0.551±0.056 0.303±0.107 0.098±0.011 0.951±0.062 1.818±0.084
Dry alga 1/2 g/kg soil 0.602± 0.064 0.306±0.067 0.096±0.002 1.003±0.129 2.017±0.229
1 0.570±0.054 0.234±0.051 0.114±0.004 0.918±0.008 2.610±0.794
2 0.574±0.029 0.262±0.051 0.117±0.001 0.952± 0.021 2.336±0.581
3 0.577±0.003 0.289±0.050 0.120±0.001 0.986±0.049 2.062±0.368

are given in Table II, Fig. 1a and b. Fresh weight and of alga as bio-fertilizer on some growth parameters of
chlorophyll contents were more increased (p<0.0001) after lettuce are in accordance with the results obtained by Rani
3, 6, 9, 12 and 15 days, compared with those of control, and Sathiamoorthy (1997). Mahmoud and Amara (2000)
while dry weight, shoot and root length indicated no who found that all treatments significantly increased plant
pronounced increase except at 3 day. growth parameters compared with un-treated plant. In
In case of seedling growth of lettuce plants, addition, similar trend was observed by Mekki et al. (1999)
fertilization treatment by fresh and dry green microalga and Galal et al. (2000). Such increases might reduce
increased the values of growth parameters and pigments chemical fertilizers and consequently reduce pollution and
content (Table III & Fig. 3) as compared with those of un- health hazard as reported for pearl millet Verma (1996).
fertilized plants. Fresh and dry weights (Fig. 3) were found Furthermore, enhancement in the growth parameter
to be high significantly increased (p<0.0001) with the attributes leads to improved crop productivity (Ghosh &
addition of increased fresh and dry alga levels. Most Mohiuddin, 2000). These results are in agreement with
effective treatments were 2, 3 g dry alga kg-1 soil than fresh those obtained by Mehta et al. (1995), Snehal et al. (1998),
alga. Increase in fresh and dry weight resulted from the Mahmoud and Amara (2000) and Das et al. (2001).
improved nutrient status of plant due to the presence of alga Carbohydrates content in seed germination and seedling
in the soil. Similar trends were found by Al-Gosaibi (1994) growth of lettuce plants. The seed germination of lettuce
and Shaaban and Mobarak (2000). The stimulatory effects plants germinate under culture media after growth of green

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Fig. 2. Effect of culture medium containing Chlorella Fig. 4. Mean values of soluble carbohydrate (mg/g dry
vulgaris grown for 3, 6, 9, 12 and 15 days on soluble weight) in seedling of lettuce plants as affected by
carbohydrate, soluble protein and total free amino different levels of fresh and dry alga as soil additives
acids (mg/g dry weight) of seed germination of lettuce 36
plants
34
38
32
36

Soluble carbohydrate (mg/g dry weight)

34
30
32
30 28
total free amino acids (mg/g dry weight)

28
Soluble carbohydrate, soluble protein,

26 26
24
22 24
20
18 22
16
14 20
12
10
18
8
16
6
4
14
2
0
control 1/2 g 1g 2g 3g
medium 3 day 6 day 9 day 12 day 15 day
Treatment
Treatment
Fresh algae+soluble carbohydrate Dry algae+soluble carbohydrate
Soluble carbohydrate Soluble protein Total free amino acids

Fig. 3. Mean values of fresh and dry weight (g/plant) in Fig. 5. Mean values of total free amino acids (mg/g dry
seedling of lettuce plants as affected by different levels weight) in seedling of lettuce plants as affected by
of fresh and dry alga as soil additives different levels of fresh and dry alga as soil additives
12 4.5

11

10
4.0
Total free amino acids (mg/g dry weight)

9
Fresh and dry weight (g/ plant)

8
3.5
7

5 3.0

3
2.5
2

0 2.0
control 1/2 g 1g 2g 3g control 1/2 g 1g 2g 3g
Treatment Treatment
Fresh algae+fresh weight Dry algae+fresh weight
Fresh algae+dry weight Dry algae+dry weight Fresh algae+total free amino acids Dry algae+total free amino acids

microalga for 3, 6, 9, 12 and 15 days, significantly (p<0.05) (2001) also concluded that, the increase yield productivity
decreased the soluble carbohydrate compared with those of with bio-fertilizer application is due to microelement and
control (sterilized culture medium) seeds germination (Fig. plant growth regulator contained in the fertilizer.
2). The results of soluble carbohydrate in lettuce seedling Amino acids and proteins content in seed germination
grown under different fertilizers (1/2, 1, 2 & 3 g kg-1 soil) and seedling growth of lettuce. Little investigations were
levels are given in Fig. 4. The soluble carbohydrates were carried out to study in case of seeds germination. The
significantly (p<0.01) lower in fertilized compared with addition of microalga after growth of on the total free amino
unfertilized plants. This reduction was more pronounced in acids, protein contents revealed that total free amino acids
dry alga (p<0.001) than in fresh alga (p<0.01) especially at and soluble proteins (Fig. 2) were significantly (p<0.001)
levels 2 and 3 g kg-1 soil. These results are in accordance increased on germinated seeds in control compared with
with those obtained by Goel et al. (1999) who reported that those of seeds treated with Chlorella vulgaris for 3, 6, 9, 12
the inoculation with certain plant growth-promoting and 15 days.
rhizobacteria may enhance crop production either by Total free amino acids of fertilized lettuce plants with
making the other nutrients available or protecting plant from dry alga were significantly (p<0.0001) lower than those of
pathogenic micro organism (allelopathic effects). Zodape the un-fertilized control plants (Fig. 5). A significant

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