Abstract. This paper presents the experimental results of the effect of microorganism fertilizer (MF) on growth, yield and accumulation of Cu in lettuce grown on soil contaminated by Cu. The results show that applying MF on soil that has a Cu content of 17.03-106.4 ppm has a positive affect on the height, fresh weight, total chlorophyll content and catalase activity of lettuce grown on that soil. The amount of Cu found in leaves of lettuce grown on soil treated with MF was significantly less than that of lettuce grown on soil which was not treated with MF. Therefore, if lettuce was grown on soil contaminated with Cu, the leaves of the lettuce would be safer to eat if that contaminated soil was treated with MF.
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JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2015-00085
Chemical and Biological Sci. 2015, Vol. 60, No. 9, pp. 104-111
This paper is available online at
Received November 9, 2015. Accepted November 28, 2015.
Contact Tran Khanh Van, e-mail address: vantk@hnue.edu.vn
104
THE EFFECT OF MICROORGANISM FERTILIZER ON THE GROWTH
AND Cu ACCUMULATION OF LETTUCE (Lactuca sativa)
GROWN ON SOIL CONTAMINATED BY Cu
Tran Khanh Van a d Pham Thi Hau
Faculty of Biology, Hanoi National University of Education
Abstract. This paper presents the experimental results of the effect of microorganism
fertilizer (MF) on growth, yield and accumulation of Cu in lettuce grown on soil
contaminated by Cu. The results show that applying MF on soil that has a Cu content of
17.03-106.4 ppm has a positive affect on the height, fresh weight, total chlorophyll
content and catalase activity of lettuce grown on that soil. The amount of Cu found in
leaves of lettuce grown on soil treated with MF was significantly less than that of lettuce
grown on soil which was not treated with MF. Therefore, if lettuce was grown on soil
contaminated with Cu, the leaves of the lettuce would be safer to eat if that contaminated
soil was treated with MF.
Keywords: copper accumulation, lettuce, microorganism fertilizer, growth
1. Introduction
Environmental pollution and especially heavy metal pollution are growing problems.
Heavy metals that have polluted agricultural land used to grow vegetables are accumulating in
the bodies of livestock and people who consume these vegetables and a variety of health
problems have become apparent. Such soil pollution is the result of industrial operations
which treat their waste poorly or not at all. Offenders include the mining industry, businesses
operating in industrial zones and family businesses in craft villages. The environmental
pollution near industrial zones and craft villages has been bad and it is getting worse [1, 2].
Farmers in these areas have only two choices: continue to plant on their polluted soil or
abandon their farm and move to an urban area. In their quest to maintain farm productivity on
weaken soils, farmers have taken to using an increasing amount of pesticides, herbacides and
inorganic fertilizers, all of which is having a serious impact on the soil, the consumers of the
produce, and the environment in general. Thus there is a worsening problem of heavy metal
contamination of green vegetables.
Green vegetables are eaten daily and those which have absorbed heavy metals from the
soil will negatively affect human health [3]. Given this situation, the question of how
vegetables grown on polluted soil can be made safer is gaining a lot attention.
The effect of microorganism fertilizer on the growth and Cu accumulation of lettuce...
105
Initially, we tested soils being used to grow green vegetable in the suburbs of Hanoi (Bac
Tu Liem District, Minh Khai Ward, Phuc Ly village) for Cu content. We also tested samples
of lettuce, basella alba and mustard spinach being grown on these soils for Cu content. We
found that the Cu in our soil samples exceeded the limit allowed by Vietnamese government
regulations (QCVN) [4] and we found that the Cu content in our vegetable samples exceeded
the maximum level presented by the FAO/WHO [5]. Thus, we endeavored to find a way to
grow vegetables in soil which is highly contaminated with Cu which are safer for animal and
human consumption,
The application of measures to improve the quality of contaminated soils by biological
means is emerging as a new and promising solution. In consideration of the above, we have
carried out a study which we titled:Th effect of microorganism fertilizer on Cu
accumulation in lettuce (Lactuca sativa) grown on Cu contaminated soil.
2. Content
2.1. Materials and methods
2.1.1. Materials
- Plant: Lettuce (Lactuca sativa) providing by C.H Vietnam Co., Ltd.
- Soil:
+ Copper contaminated soils taken at a vegetable field in Phuc Ly village, Minh Khai
Ward, Bac Tu Liem District, Hanoi, coordinates N 21o03‟52.9”, E 105o44‟30.1”. The Cu
content in this soil was 106.45 ppm.
+ Control soil: alluvial soil taken from the alluvial ground of Red River, Dong Ngac
Ward, Bac Tu Liem District, Hanoi. The Cu content in this soil was 17.03 ppm.
- Copper: a CuSO4.5H2O solution was added to soil to increase Cu content,
- Microorganism fertilizer (MF)
- Substrate Source: Acne coconut (cocos)
- Place of Origin: Soils and Fertilizers Research Institute
- Ingredients: Bacteria BHCM7 _ VK2; Mycorrhizal DHCM20 _ AMF4
- Density of useful microorganism: > 5.00 x 108 CFU/g
- Subjects treated: Soil contaminated by Cu
2.1.2. Methods
- Plastic pot size: 20 x 20 x 16 cm.
- 5 kg alluvial soil and soil taken from Phuc Ly/1 bag.
Sowed 15 seeds/pot. Irrigation and after 15 days plants were removed to keep plant
density to 4 plants/pot. Plants were cared for until harvest 40 days after planting. They were
watered with “ditch water”.
A Vietnamese National Standard has been created which limits heavy etals in soils
(QCVN 03:2008/BTNMT), the limit for Cu being 50 ppm [4]. The Ministry of Agriculture
and Rural Development recommends that 50 kg of MF be applied per ha and we applied the
equivalent of 4 g/pot for this experiment [6].
The following experiments, so called treatment have been conducted:
- Treatment CT1: Control soil (alluvial soil from the Red River with the Cu metal
contents of 17.03 ppm);
- Treatment CT2: Control soil + Cu 50 ppm;
- Treatment CT3: Control soil + Cu 50 ppm + microorganism fertilizer (4g);
- Treatment CT4: Phuc Ly soil (Cu 106.45 ppm);
- Treatment CT5: Phuc Ly soil (Cu 106.45 ppm) + microorganism fertilizer (4 g).
Each treatment was repeated 3 times.
Tran Khanh Van a d Pham Thi Hau
106
The experiment was conducted at the Experimental Gardens of the Faculty of Biology,
Hanoi University of Education. Parameters were conducted at the Laboratory of the
Department of Plant Physiology and Application, Faculty of Biology, Hanoi University
of Education.
Parameters and monitoring methods
Height of plant: Plants were measured from base of stem to tip of longest leaf, plus or
minus 1 mm.
Plant weight: Plants were weighed on a balanced beam scale that had a 2.10-2g egree of
precision.
Total chlorophyll content in leaf: UV spectrophotometer Visible Model SPECORD 200
Plus with equation of Wettstein 1957 was used.
Total copper content in soil: TCVN 6496, ISO11047:1995.
Copper content in parts of the plant: AAS - Atomic Absorption Spectrophotometric
NovAA 350 Analytik Jena. Destruction of plant sample involved use of wet ash method at
550 oC.
Catalase activity in leaf: Use of Bach and Oparin method
Data processing
The data were processed by statistical one–way ANOVA analysis (Turkey‟s – b) with a
significance level of α = 0.05.
2.2. Results and discussions
2.2.1. Effects of microorganism fertilizer (MF) on the height of lettuce grown in soils
with different Cu concentrations
The capability of a plant to grow in contaminated soils is important to the uptake and
accumulation of heavy metals in its biomass. The effect of microorganism fertilizer on the
height of lettuce grown in soils with different Cu concentrations is shown in Table 1.
Table 1. The effects of microorganism fertilizer on the height of lettuce grown in soils
with different Cu concentrations
Treatments Height (cm) % compared to
control
CT 1 (control soil) 7.70a ± 0.43 100
CT2 (control soil + Cu 50 ppm) 7.17a ± 0.32 93.12
CT3 (control soil + Cu 50 ppm + MF) 6.98a ± 0.24 90.06
CT4 (Phuc Ly soil) 6.91a ± 0.27 89.74
CT5 (Phuc Ly soil + MF) 8.81b ± 0.29 111.44
(Note: Values in the same column followed by the same letter
are not significantly different at 0.05)
The results (Table 1) show that in soil without microorganism fertilizer (MF), the h ight
of lettuce decreased as the Cu content in the soils increased. Plant height was 6.9% and 10.3%
less in treatment CT2 and CT4, respectively, compared with that in the control (treatment
CT1). Comparing with MF and without MF application to soils, we found that average lettuce
The effect of microorganism fertilizer on the growth and Cu accumulation of lettuce...
107
height in treatment CT2 was greater than in treatment CT3. However, when the Cu content
was the same as in the soil taken from Phuc Ly, plant height in treatment CT4 was
significantly less than in treatment CT5.
When soil is contaminated with heavy metal Cu, lettuce growth is reduced. With an
increase of Cu in soils in CT1 (Cu 17.03 ppm) to CT2 (Cu at CT1 + 50 ppm) to CT4 (Cu
106.4 ppm), there was a significant decrease in plant height. However, we saw no difference
in the morphology of lettuce grown in soils with different levels of Cu content. The lettuce in
treatment CT3 was not as tall as that in treatment CT2, and perhaps that‟s because Cu is an
essential microelement that was lacking in CT3. Nevertheless, too much Cu in the soil might
have a bad influence on plant growth. Because plant height was greatest in treatment CT5, it
appears that applying MF to Cu contaminated Cu soil may reduce the toxicity of Cu resulting
in improved plant growth.
2.2.2. Effects of microorganism fertilizer (MF) on fresh weight of lettuce grown in soils
with different Cu concentrations
Fresh weight is one parameter to estimate plant yield and photosynthetic efficiency as a
whole. Therefore, fresh weight is importance parameter to evaluate the growth and
development of plant.
Figure 1. Effects of microorganism fertilizer on fresh weight of lettuce
grown in soils with different Cu concentrations
The results indicate that when Cu concentration in the soil increases and MF application
remains the same, the fresh weight of lettuce increases. And, when Cu concentration in the
soil increases but there has been no MF application, the fresh weight of lettuce decreases.
That mean the possitive effect of MF to the accumulate biomass in lettuce, and clear y seen t
treatment CT5.
With the MF application in treatment CT3 and CT5, the fresh weight of lettuce was
9.05% and 27.3% than that in the treatment control (CT1). According to the One-way
ANOVA analysis, the fresh weight of lettuce in these treatments was sta i tically significant
to α = 0.05, compared to treatments CT1, CT2 and CT4. In treatments CT2 and CT4, the
plants had lower fresh weights, 85.20% and 68.24% less than that of the control (CT1).
Perhaps the microorganisms in the fertilizer enhanced the uptake of nutrition to increase the
biomass of the plants [7]. With an increase in fresh weight, farmers will increase their income.
Tran Khanh Van a d Pham Thi Hau
108
2.2.3. Effects of microorganism fertilizer (MF) on total chlorophyll content of lettuce
grown in soils with different Cu concentrations
Plants have a photosynthetic pigment absorbing function and can convert light into
chemical energy in organic compounds. These photosynthetic pigments have an important
role in the formation of the products of photosynthesis. Photosynthetic pigme s include
chlorophyll, carotenoids, and phycobilins.
Of the photosynthetic pigments, chlorophyll is the most important.
Total chlorophyll content is related to photosynthetic capacity and therefore plays an
important role in the growth and quality of harvested products. Total chlorophyll content
depends on many factors, including seed quality, water quality and quantity, fertilizer and the
weather.
The results of the influence that MF had with different Cu soil concentrations to total
chlorophyll content in lettuce leaves are presented in Table 2.
Table 2. Effects of microorganism fertilizer on total chlorophyll content of lettuce
grown in soils with different Cu concentrations
Treatments Total chlorophyll content
(mg/g fresh weight)
% compared
to control
CT 1 (control) 0.130a ± 4.50.10-4 100
CT2 (Cu 50 ppm) 0.132a ± 5.11.10-4 101.54
CT3 (Cu 50 ppm + MF) 0.168d ± 5.52.10-4 129.23
CT4 (soil taken at Phuc Ly) 0.139b ± 6.49.10-4 106.92
CT5 (soil taken at Phuc Ly + MF) 0.153c ± 7.97.10-4 117.69
(Note. Values in the same column followed by the same letter
are not significantly different at 0.05)
Total chlorophyll content varied from 0.130 - 0.168 mg/g fresh weight, the highest being
treatment CT3 (0.168mg/g fresh weight) and the lowest being tr a ment CT1 (control).
Comparing CT1, CT2 and CT4, the total chlorophyll content in leaves increased as the Cu
content of the soils increased. When Cu content in soils remained the same, we found that
total chlorophyll content in leaves increased in treatments that received an MF application
(CT3 vs CT2, CT5 vs CT4. However, the effect of MF was less when there was a higher Cu
concentration in the soil (CT5 vs CT4). This indicates that when Cu concentration in soils is
0 - 106.45 ppm, the Cu promotes the ynthesis of chlorophyll with a consequent increase total
chlorophyll content in leaf lettuce.
The microelement Cu increases chlorophyll biosynthesis and chlorophyll production.
However, when the concentration of Cu in soil exceeded the Cu tolerance level of the plant),
MF was less effective. It appears that when present in high concentration, Cu is toxic to lettuce.
Microorganism fertilizer can help fix Cu in rhizomes but the amount fixed is limited and
Cu concentrations in soils can be expected to remain quit high. When a large amount of Cu
is taken up by a plant, it is toxic to plant. Thus the lower chlorophyll content in treatment CT4
compared to treatment CT5.
The effect of microorganism fertilizer on the growth and Cu accumulation of lettuce...
109
2.2.4. Effects of microorganism fertilizer (MF) on catalase activity in leaves of lettuce
grown in soils with different Cu concentrations
Catalase is a common enzyme found in nearly all living aerobic organisms (bacteria,
plants and animals). It catalyzes the decomposition of hydrogen peroxide to water and oxygen. It
protects the cell from oxidative damage by reactive oxygen species (ROS).
The reaction of catalase in the decomposition of hydrogen peroxide in plant tissue is as
follows:
2 H2O2 → 2 H2O + O2
The effect of microorganism fertilizer on catalase activity in the leaves of l ttuce grown
in soils with different Cu concentrations is presented in Figure 2.
Figure 2. The effects of microorganism fertilizer (MF) on catalase
activity in the leaves of lettuce grown in soils with different Cu concentrations
We can see from Figure 2 that catalase activity in lettuce leaf ranged from 0.13 to 0.33 mg
H2O2/g/min. Catalase enzyme activity in all treatments is lower than that of the control,
except for treatment CT3. In addition, with treatments that had an MF application, catalase
activities were higher than those in treatments without MF application. This indicates that
high Cu concentration in soil is toxic to lettuce plants but more catalase activity will
detoxify the plant. However, if the Cu soil concentration is sufficiently high, the Cu will
inhibit synthesis of catalase with a reduction of catalase initiated activities. In treatments
that had as MF application, microorganisms in the fertilizer enhanced catalase activity.
Nevertheless, catalase activity in treatment CT5 was lower than that of treatment CT3
implying that the advantages of the MF were negated by the toxicity of the Cu.
2.2.5. Effects of microorganism fertilizer on the accumulation of Cu in lettuce leaf grown
in soils with different Cu concentrations
This study was carried out in hopes that an MF applied to the soil might counteract CU
toxicity and vegetables grown on CU polluted soil could be made safe to eat. To measure the
Cu content of lettuce leaves, fresh leaves were analyzed when the lettuce plants were 40 days old.
From Table 3 and Figure 3 it can be seen that the concentration of Cu in lettuce leaf is in
direct proportion to the concentration of Cu in the soil. Cu accumulation in the leaves in
treatments CT2, CT3, CT4 and CT5 increased 13.1 %, 6.14 %, 47.31 % and 31.30 %,
respectively, compared to the control. The lowest amount of Cu found in lettuce leaves was
29.17 mg/kg fresh weight, and that was for CT1, the control. Samples grown on Cu
contaminated soils (including CT4 with 106.4 ppm Cu in the soil) accumulation of Cu in the
leaf was high, the worst being 42.97 mg/kg fresh weight. And such lettuce is being sold daily
to families in Hanoi who have young children. In Vi tnam, it is legal to sell good looking
food that is highly contaminated.
Tran Khanh Van a d Pham Thi Hau
110
Table 3. Cu in fresh lettuce leaf from lettuce grown in Cu contaminated soils
Treatments Concentration of Cu in leaf
(mg/kg fresh weight)
% compared
to control
CT 1 (control) 29.17a ± 0,48 100.0
CT2 (Cu 50 ppm) 32.99c ± 0,47 113.10
CT3 (Cu 50 ppm + MF) 30.96b ± 0,22 106.14
CT4 (soil taken at Phuc Ly) 42.97e ± 0,26 147.31
CT5 (soil taken at Phuc Ly + MF) 38.30d ± 0,47 131.30
(Note: Values in the same column followed by the same letter
are not significantly different at 0.05)
A study which measured Cu, Pb and Zn in lettuce was done by Chung et al. and they
found that the presence of Cu, Pb and Zn in lettuce increased in proportion to the
concentration of the heavy metal contaminant found in the soil [8].
According to WHO/FAO, if more than 40 mg/kg of Cu is present in leafy vegetables,
those vegetables are unsafe to eat [5]. In his study, the average Cu content of the lettuce
leaves in treatments CT1, CT3, CT2 is lower than the WHO‟s maximum amount (29.17,
30.96 and 32.99) while in that of treatment CT4 (soil taken from Phuc Ly), on average the Cu
content of the leaves exceeded the WTO‟s threshold by 13%.
In treatment CT5, microorganism fertilizer reduced the Cu uptake in lettuce l aves but
the level is very close to that set by the WHO/FAO.
Figure 3. Cu in fresh lettuce leaf from lettuce grown in Cu contaminated soils
Comparing the accumulation of Cu in the leaves in treatments CT2 and CT3, CT4 and CT5,
we find that Cu accumulation in the leaves was significantly reduced due to the
microorganism fertilizer that contained BHCM7-VK2 and DHCM20-AMF4 bacteria. These
useful bacteria helped improve the soil and reduce Cu toxicity. It is thought that Cu in the soil
is taken up by the bacteria and stored in its mycelium and spores. This, at least temporarily,
reduces the amount of Cu in the soil land resulting in less Cu being absorbed by plants
growing in that soil. It is also been proposed that a plant would also absorb Cu in its tems
and roots and there would then be less Cu in the soil to be absorbed into the leaves, but this is
a silly idea. Those who are growing vegetables in soil that is highly contaminated by Cu (the
Cu accumulation in leaf
(mg/kg fresh weight)
The maximum limit of Cu
concentration in vegetation
issued by WHO/FAO
(mg/kg fresh weight)
Cu concentration
(mg/kg fresh weight)
Treatments
The effect of microorganism fertilizer on the growth and Cu accumulation of lettuce...
111
Vietnam Ministry of Agriculture and Rural Development has set the maximum „safe‟ level at
50 ppm) [7], should make use of microorganism fertilizers to reduce heavy metal
accumulation in the foods that they are selling. Unfortunately, near 0% of all produce grown
is ever tested, and a good washing will do nothing to lower the heavy metal content of the
foods we eat.
3. Conclusion
With a Cu concentration in the soil ranging from 17.03 to 106.4 ppm, the height, fresh
weight, total chlorophyll content and catalase activity of lettuce was adversely affec