The effect of microorganism fertilizer on the growth and Cu accumulation of lettuce (Lactuca sativa) grown on soil contaminated by Cu

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