Bio Remediation of Industrial Effluents Using Constructed Wetland Technology

By Sukumaran Dipu¹, and Salom Gnana Thanga²
February 2009

  1. Research Scholar, Department of Environmental Sciences, University of Kerala, Kariavattom Campus, Thiruvananthapuram, Kerala, India
  2. Senior Lecturer, Department of Environmental Sciences, University of Kerala, Kariavattom Campus, Thiruvananthapuram, Kerala, India
Heavy metal pollution in soil and water is becoming a serious problem for agriculture and health. Some heavy metals which are natural micronutrient (Fe, Zn, Mn etc) in the vegetable kingdom are rapidly absorbed by the roots and translated into the whole plant. The toxic effect is checked when the heavy metals concentration in soil is higher than the normal trace amounts. However, many plant species have developed some physiological mechanisms that allow their survival in such environments. Such potential can be harnessed for remediation of waste water containing heavy metals. The advantage is minimal fossil fuel is required and no chemicals are necessary. Being low cost, constructed wetlands are potential alternative or supplementary system for waste water treatment in developing countries. In India there is abundance of wetland macrophytes which can be used for phytoremediation, but unfortunately the studies are at its primary level. Constructed Wetlands for waste water treatment involve the use of engineered system that are designed and constructed to utilized natural process. These systems are designed to mimic natural wetland system, utilizing wetland macrophytes, soil, and associated microorganisms to remove contaminants from waste water effluents. In this study, the main focus is on the percentage of absorption of heavy metals and the biochemical changes in macrophytes are done. The wetland macrophytes used for this study are hyper accumulators of heavy metals and the most important is that they can be utilized after phytoremediation for different useful purposes.

Key words: Heavy metal, constructed wetlands, wetland macrophytes, waste water, micro organisms, hyper accumulators.

1. Introduction

The accelerating industrialization in developing countries with an enormous and increasing demand for heavy metals causes a high anthropogenic emission of pollutants into the biosphere (Cheng et al., 2002). Constructed Wetland System (CWS) for waste water treatment involve the use of engineered system that are designed and constructed mimic natural wetland system, utilizing wetland plants, soil, and associated microorganisms to remove contaminants from waste water effluents (EPA, 1993). Most constructed wetlands emulate marshes because soft stemmed plants in the marshes require the shortest time compared to plants in bogs and swamps for full development and operational performance (Suresh and Ravishankar. 2004).Constructed wetland systems works on the principle of phytoremediation taking advantages of the unique and selective up take capabilities of plant root system together with the translocation, bio accumulation and contaminant storage or degradation abilities of the entire plant body (Lasat, 2000).

Constructed wetlands are inexpensive systems for waste water treatment and have They are used not only to degrade organic substances and nutrients from municipal sewage, storm water, and agricultural runoff (Lakatos et al.,1997), but also to remove metals from mining effluents and special industrial waste water (Greenway, 1999., Obarska, 1999).

These systems have been recommended for use in tropics and subtropics. The plants after waste water treatment are used for biogas production also be composted or can be used as animal feed. Tanaka et al., (1992) noted copper absorption by Lemna species, Nasu et al., (1984) observed differential mode of uptake response from a bimetallic medium of Cadmium and Copper by Lemna. Though these plants were small in size, they appear to have a remarkable inbuilt resistance capacity against metals. Phragmites australis and Typha latifolia are commonly used in this system. Typha species usually grow in marshy areas with long erect strap shaped leaves. Tayler and Crowder (1984) used typha for the removal of copper and nickel. These planted soil filters have become well established in water treatment, with various technological designs of constructed wetlands (Wiebner et al., 2002).

Though this technology is already in use in many countries for waste water treatments containing metallic effluents, in India it is still at its infancy. This study was done to find out heavy metal uptake of locally available wetland plants, their adaptation in heavy metal rich environment, biochemical changes and finally their potential utilization after metal sequestration.

In most developing countries, there is very few waste water treatment plants due to high costs of treatment process, lack of effective environmental pollution control laws or law enforcement (Newman and Reynolds, 2004). Being low cost and low technology system constructed wetlands are potential alternative or supplementary system for waste water treatment plants in developing countries.

2. Materials and Methods

Five artificial wetland plots were constructed in the Department of Environmental Sciences, University of Kerala using plastic crates. The sizes of the crates were 18 x 18 x 24 cm. These crates were filled with soil from the wetland and water up to a level of 9 cm. The filling was with marshy soil and kept for one month for stabilization.

The plants used for the experiment are: one emergent plant ie. Typha sp., floating plant like Pistia. These plants were collected from local wetlands. The plants were planted in control another crates and after one month, the second generation plants were used for the experiment.

Plant, soil and water samples were initially analyzed for nutrients and heavy metal contents by Standard Methods by APHA (1995). The plants were then planted in the stabilized constructed wetlands. After one week, 1ppm of each heavy metal were added to the constructed wetlands. Two replicates were used for each experiment. Duplicates were maintained for each experiment. The plants were analyzed for heavy metals in the fifth, tenth and fifteenth day after the heavy metal treatment. The heavy metal content in plant species were analyzed using di acid method and by AAS. The hydro peroxide activity was analysed using idometric assay. The enzyme activities of plants were analysed using methods of biochemical analysis by Patterson and Lazarow (1995).

3. Results and Discussion

3.1. Heavy metal uptake by plants in CWS

Heavy metal pollution in soil and water is becoming a serious problem for agriculture and health. Some heavy metals are natural micronutrients in the vegetable kingdom. They are rapidly absorbed by the roots and translated into whole plant. The toxic effect is checked when the heavy metals concentration in soil is higher than normal trace amounts. However, many species have developed some physiological mechanisms that allow the survival also in that states. Physiological studies on plants able to live in presence of high concentration of heavy metals have explained that metals attachment and binding are main mechanisms for detoxification. In the present study, heavy metals like Arsenic, zinc and copper were added to both emergent and free-floating CW systems at a rate of 1.0 ppm per treatment. All the heavy metal studied was taken up by the plants in the system, whereas from soil and water, a decrease in heavy metal content was observed. Among the plant systems used, the emergent system was more efficient in heavy metal uptake than free floating system. Among the different heavy metal studied, copper was found to have the maximum uptake percentage by the plants followed by arsenic. The results are shown in the table’s I to III.

Soils may become polluted with high concentration of toxic metals and their remediation may often involve excavation and removal of soil to secured landfills, an expensive technology that requires site restoration (Glick, 2003). Moreover, besides being an expensive and labor intensive effort, cleaning up contaminated sites has been accompanied by secondary environmental and legal problems. The phytoremediation of heavy metal contaminated soil basically involves the extraction or inactivation of these metals in soils. However, some metals such as Pb are largely immobile in soil and their extraction rate is limited by solubility and diffusion to the root surface (Lombi et al., 2001). In phytoextraction and phytomining, accumulated toxic metals in plant tissues are harvested for metal recovery and reuse. Normally, the plants termed hyper accumulators are preferably used, since they have the ability to withstand and build up high concentrations of metals, when compared to other plants. These plants can be processed to recover the metals accumulated during the phytoremediation process. Although it is cheaper than the conventional methods, phytoremediation is not an easy technology that consists of simply planting and growing some hyper accumulating plants in the metal polluted area (Alkorta et al., 2004). It is in fact a highly technical strategy, requiring expert project designers with field experience that choose the proper species and cultivars for particular metals and regions (Alkorta et al., 2004).

3.2. Changes in plant biochemistry due to heavy metal uptake

3.2.1. Ascorbic acid analysis

Ascorbic acid is one of the most studied and powerful antioxidants (Smirnoff, 2000). It has been detected in the majority of plant cell types, organelles and in the apoplast. Under physiological conditions ascorbic acid exists mostly in the reduced form (90 % of the ascorbate pool) in leaves and chloroplasts and its intracellular concentration can build up to mill molar range. In chloroplasts, ascorbic acid acts as a cofactor of violaxantin de-epoxidase thus sustaining dissipation of excess excitation energy. Ascorbic acid carries out a number of non-antioxidant functions in the cell. It has been implicated in the regulation of the cell division, cell cycle progression from G1 to S phase and cell elongation (De Tullio et al., 1999). Ascorbate is a major metabolite in plants. It is an antioxidant and, in association with other components of the antioxidant system, protects plants against oxidative damage resulting from aerobic metabolism, photosynthesis and a range of pollutants. Recent approaches, using mutants and transgenic plants, are providing evidence for a key role for the ascorbate±glutathione cycle in protecting plants against oxidative stress. The biosynthetic pathway of ascorbate in plants has not been identified and evidence for the proposed pathways is reviewed. Ascorbate occurs in the cell wall where it is a first line of defense against ozone. Cell wall ascorbate and cell wall-localized ascorbate oxidize have been implicated in control of growth. High ascorbate oxidize activity is associated with rapidly expanding cells and a model which links wall ascorbate and ascorbate oxidize to cell wall extensibility is presented (Tanaka et al. 1992). There is a need to increase our understanding of this enigmatic molecule since it could be involved in a wide range of important functions from antioxidant defense and photosynthesis to growth regulation (Smirnoff, 2000). Given the pivotal role of ascorbate in photosynthesis and its possible role in cell division and expansion, it is surprising that so little is known about its metabolism. Further investigation of ascorbate biosynthesis and its role in cell growth are needed. There is now scope for molecular genetics to complement biochemical and physiological approaches. We may then be in a position to increase the vitamin C content of food plants and possibly enhance their tolerance to photo-oxidative stress.

The ascorbic acid content of Typha, Pistia, were found increased after treatment with heavy metals like Arsenic and Cadmium. In Typha it was 0.62 mg/g FW of sample in control. When it was treated with Arsenic the value was increased to 2.56 mg/g FW of sample and with cadmium it was 2.79mg/ g FW of sample. The results are shown in the table IV.

3.2.2. Enzyme analysis

Another important role that plant play in degradation of heavy metals involves the release of enzymes. These enzymes are capable of transforming contaminants by catalyzing chemical reactions in the soil Schnoor et al., (1995) identified plant enzymes as the causative agents in transformation of contaminants mixed with sediments and soil. This suggest that plant enzymes have significant spatial effects extending beyond the plant itself and temporal effects continuing after the plant has died (Cunningham et al.,1996).The production of stress enzymes are high in plants when treated with heavy metals.

A tripeptide glutathione is an abundant compound in plant tissues. It has been found virtually in all cell compartments: cytosol, endoplasmic reticulum, vacuole and mitochondria (Jimenez et al., 1998). Together with its oxidized form glutathione maintains a redox balance in the cellularcompartments. The latter property is of great biological importance,since it allows fine-tuning of the cellular redox environment under normal conditions and upon the onset of stress, and provides the basis for Growth Stimulating Hormone (GSH) stress signaling. The production in glutathione in both free floating and emergent plant were found increased when treated with cadmium and arsenic. This may be the stress produced by the heavy metals.

The intracellular level of Hydrogen peroxide (H2O2) is regulated by a wide range of enzymes, the most important being catalase and peroxidase (Willekenes et al., 1995). In addition to defense against active oxygen compounds, plants peroxidases have other important cellular roles. However, in different cases endogenous auxin levels are regulated by the enzymes auxin oxidase and peroxidase (Reinecke, and Banddurski, 1988). The activities of some antioxidant enzymes increase during stress treatment, and the types of enzymatic activities that increase are dependent on the form stress imposed. The enzymes whose activities increase during stress treatment may play an important role in defense against that particular stress. Under anoxia a differential response of the peroxidase system has been observed in coleoptiles and roots of rice seedlings.There was a decrease in activity of cell wall-bound guaiacol and syringaldazine peroxidase activities, while soluble peroxidase activity was not affected in coleoptiles. In this study different plant enzymes Viz: Glutathione, lipid peroxidation, diene conjugates, hydroperoxidase etc.were analysed.

In the case of lipid peroxidation enzyme the values of the control plants were much lower than the treated plants .This may due to the stress produced by the heavy metals. The variation in enzymes of treated plants and control plants are shown in the tables V to VIII.

3.4. Rhizosphere effect

Heavy metal pollution of soil is a significant environmental problem and has its negative impact on human health and agriculture. Rhizosphere, as an important interface of soil and plant, plays a significant role in phytoremediation of contaminated soil by heavy metals, in which, microbial populations are known to affect heavy metal mobility and availability to the plant through release of chelating agents, acidification, phosphate solubilization and redox changes, and therefore, have potential to enhance phytoremediation processes. Phytoremediation strategies with appropriate heavy metal-adapted rhizobacteria have received more and more attention. Large microbial populations in the rhizosphere are sustained by exudation of carbohydrates and amino acids from the root and decertification of the root (Wiebner et al., 2002). Once established, microbial population may be passively nourished by the root exudation and decaying plant matter. Continual change at the root – soil interphase, both physical and chemical produces constant alteration in the soil structure and microbial environment. Fibrous root structures of wetland plants provide a larger surface area for colonization than tap root systems (Altas and Bartha, 1993).

This rhizosphere effect is often quantified as the ratio of microorganisms in rhizosphere area to the number of microbes in non microbial area, the R/S ratio (Katznelson, 1946). The increase in numbers of microorganisms is dependent on plant species, plant age, and soil. The interaction between plants and microbial communities in the rhizosphere is a complex relationship. Plants sustain large microbial population in the rhizosphere by rhizodeposition, root cap cells, which protect the root from abrasion, may be lost tom the soil at a rate of 10000 cells per plant (Campbell, 1985).In addition, root cells excrete mucigel, a gelatinous substance that is a lubricant for root penetration through the soil during growth. In return for receiving exudates, microbes in the root zone can help to solublise insoluble nutrients and recycle organically bound nutritive elements.

In the present study the microbial colonies are quantified by colony forming units (CFU). It was found that the emergent species used for the study (Typha) showed maximum R/S ratio than the free floating macrophytes. The R/S ratio in different constructed wetland systems are shown in the table IX.

4. Utilization of Phytoremediation Byproducts

Combustion and gasification are the most important sub routes for organized generation of electrical and thermal energy. Recovery of this energy from biomass by burning or gasification could help make phytoextraction more cost-effective. Thermochemical energy conversion best suits the phytoextraction biomass residue because it cannot be utilized in any other way as fodder and fertilizers. Combustion is a crude method of burning the biomass, but it should be under controlled conditions, whereby volume is reduced to 2–5 % and the ash can be disposed properly. This method of plant matter disposal is often mentioned by many authors (Bridgewater et al., 1999, Raskin, et al., 1997). Gasification is the process through which biomass material can be subjected to series of chemical changes to yield clean and combustive gas at high thermal efficiencies. This mixture of gases called as producer gas and/or pyro-gas that can be combusted for generating thermal and electrical energy.

Cow dung is used as the base material for gasification. One Kilograms of fresh cow dung is used to make the slurry and 35 grams of plant material is also used. Plants are macerated to make them into slurry form. Four different concentrations of slurry are used for gasification, namely; 100% cow dung, 75% cow dung and 50% cow dung and 0% cow dung. The rest percentage is plant material. They are taken in triplicates. The amount of gas produced after fourth day upto ninth day is taken. It was found that the amount of gas produced is high in the initial days in the cow dung slurry but after sixth day the amount is decreased .In case of plant based slurry the amount of gas produced is low in the initial stages and increased as the day’s progress. Among the plants based slurry, Typha based slurry showed the highest amount of gas produced followed by pistia based slurry. In plant based slurry, the initial decrease in production of gas may due to the time taken to decay the plant materials. The results are shown in tables X and XI.

4. Concluding Remarks and Outlook

Several processes can limit the performance of plants in phytoremediation, such as the availability of the toxic metal ions in the soil for uptake by plant roots, the rate of uptake of the contaminants by plant roots, their translocation from roots to shoots and the extent of tolerance, or the rate of chemical transformation into less toxic, possibly volatile compounds. Full utilization of plant resources after they have been used for phytoremediation is an unsolved problem. Therefore the testing of the plants used in phytoremediation is necessary and may support their continuous use in contaminated soils. While phytoremediation processes hold a great promise as a way to remediate contaminated soils, there are disadvantages and limitations that must be carefully considered.

It is evident that phytoremediation has benefits to restore balance to a stressed environment, but it is important to proceed with caution. Phytoremediation technology is still in its early development stages and full scale applications are still limited. The results already obtained have indicated that the plants are effective and could be used in toxic metal remediation. Although it appears to be common sense among scientists, engineers, and regulators about the more widespread future use of this technique, it is important that public awareness about this technology is considered and clear and precise information is made available to the general public to enhance its acceptability as a global sustainable technology to be widely used.

5. List of Tables

Table I · Treatment of Arsenic in Constructed Wetland Systems
CWS Control (mg/l) After treatment (mg/l)
Day 1 Day 5 Day 10 Day 15
Typha based CWS
Typha 0.621 0.867 1.24 1.617 (+61.5)
Soil 0.173 0.315 0.11 0.011 (-93.64)
Water 1.000 0.612 0.444 0.166 (-83.4)
Pistia based CWS
Pistia 0.412 0.488 0.666 0.717 (+42.5)
Water 1.000 0.924 0.746 0.697 (-30.3)
Values in parentheses indicate change in heavy metal uptake
Table II · Treatment of Zinc in Constructed Wetland Systems
CWS Control (mg/l) After treatment (mg/l)
Day 1 Day 5 Day 10 Day 15
Typha based CWS
Typha 0.320 0.424 0.498 0.643 (+50.2)
Soil 0.111 0.141 0.285 0.312 (-64.4)
Water 1.000 0.866 0.648 0.478 (52.2)
Pistia based CWS
Pistia 2.410 2.468 2.588 2.843 (+15.2)
Water 1.000 0.942 0.822 0.427 (-57.3)
Values in parentheses indicate change in heavy metal uptake
Table III · Treatment of Copper in Constructed Wetland Systems
CWS Control (mg/l) After treatment (mg/l)
Day 1 Day 5 Day 10 Day 15
Typha based CWS
Typha 0.013 0.158 0.354 0.467 (+97.22)
Soil 0.007 0.066 0.102 0.277 (-97.47)
Water 1.000 0.796 0.564 0.276 (-72.4)
Pistia based CWS
Pistia 0.103 0.265 0.398 0.612 (+83.17)
Water 1.000 0.838 0.705 0.490 (-51.0)
Values in parentheses indicate change in heavy metal uptake
Table IV · Variation in Ascorbic Acid due to Application of Heavy Metals to Wetland Plants
Plants Control mg/g FW Treated plants mg/g FW
As Cu Zn
Typha 0.62 2.56 2.42 2.79
Pistia 0.50 2.12 2.08 0.98
Table V · Variation in Glutathione
Plants Control mg/100g FW Treated plants mg/100g FW
As Cu Zn
Typha 46.69 69.63 77.38 57.75
Pistia 39.63 85.63 67.56 76.64
Table VI · Variation in Lipid Peroxidation
Plants Control μmol MDA/100 g FW Treated plants μmol MDA/100 g FW
As Cu Zn
Typha 3.45 5.24 5.68 4.28
Pistia 0.92 0.80 2.14 1.54
Table VII · Variation in Diene Conjugates
Plants Control nmol/g FW Treated plants nmol/g FW
As Cu Zn
Typha 141.27 260.32 260.32 245.24
Pistia 136.51 226.98 257.76 224.43
Table VIII · Variation in Hydroperoxidase
Plants Control nmol/g FW Treated plants nmol/g FW
As Cu Zn
Typha 169.94 798.84 558.38 453.36
Pistia 58.96 717.92 267.43 342.46
Table IX · R/S Ratio in different Constructed Wetlands
Plants Before Treatment (BT) After Treatment (AT) R/S Ratio (BT) R/S Ratio (AT)
rhizosphere area (CFU*10) non rhizosphere area (CFU*10) rhizosphere area (CFU*10) non rhizosphere area (CFU*10)
Typha 7.64 1.68 9.88 1.86 4.55 5.31
Pistia 2.86 8.6 3.86 1.12 3.33 3.45
Table X · Gas Production in Typha based Slurry
Slurry Amount of water displaced (gas production)/day
% of cow dung % of plant material 1 4 5 6 7 8 9
100 0 0 60 45 40 30 15 5
75 25 0 50 40 30 45 20 5
50 50 0 30 30 20 50 30 20
25 75 0 20 25 25 45 45 25
0 100 0 25 20 25 60 50 30
Table XI · Gas Production in Pistia based Slurry
Slurry Amount of water displaced (gas production)/day
% of cow dung % of plant material 1 4 5 6 7 8 9
100 0 0 60 45 40 30 15 5
75 25 0 40 25 30 25 20 10
50 50 0 35 20 25 35 25 20
25 75 0 30 20 25 45 30 25
0 100 0 30 25 30 50 40 30

5. List of Figures

Fig 1 · Standard deviation of plant uptake from control when treated with Arsenic
Fig 1

Fig 2 · Standard deviation of plant uptake from control when treated with Zinc
Fig 2

Fig 3 · Standard deviation of plant uptake from control when treated with Copper
Fig 3


The authors acknowledge with gratitude the help and support from Prof. & Head Department of Environmental Sciences, University of Kerala. Thanks are also due to University Grants Commission, India for the financial support to complete the project.



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