Bioremediation of different waste waters is a relatively new technology that has undergone more intense investigation in recent decades. As rapid industrialization and urbanization releases numerous toxic compounds into natural water bodies, polluting both fresh water resources as well as marine water. This process is focused on destroying or immobilizing toxic waste materials present in these water sources. Several techniques have been proposed for efficient wastewater treatment, most of them presenting some limitations, such as poor capacity, the generation of waste products, incomplete mineralization and a high operating cost. The bioremediation of waste waters can be divided into two broad categories: In-situ and Ex-situ treatment. Both methods have significant advantages and disadvantages. Many microbial detoxification processes involve the efflux or exclusion of metal ions from the cell, which in some cases can result in high local concentrations of metals at the cell surface, where they can react with biogenic ligands and precipitate. Although microorganisms cannot destroy metals, they can alter their chemical properties via a surprising array of mechanisms. The problem is with the heavy metals associated with environmental contamination, for instance, lead (Pb), cadmium (Cd), and chromium (Cr), which are potentially hazardous to ecosystems.
Introduction
The definition of bioremediation is the use of living organisms, primarily microorganisms, to degrade the environmental contaminants into less toxic forms. It uses naturally occurring bacteria and fungi or plants to degrade or detoxify substances hazardous to human health and/or the environment. The microorganisms may be indigenous to a contaminated area or they may be isolated from elsewhere and brought to the contaminated site.
Not all contaminants are easily treated by bioremediation using microorganisms. For example, heavy metals such as cadmium and lead are not readily absorbed or captured by organisms. The assimilation of metals such as mercury into the food chain may worsen matters. The phytol - remediation is useful in these circumstances, because natural plants or transgenic plants are able to bio-accumulate these toxins, which are then harvested for removal. The heavy metal in the harvested biomass may further be concentrated by incineration or even recycled for industrial use.
For effective bioremediation, the microorganisms must enzymatically attack the pollutants and convert them to harmless products. As bioremediation can be effective only where environmental conditions permit microbial growth and activity, its application often involves the manipulation of environmental parameters to allow microbial growth and degradation to proceed at a faster rate. Environmental contamination by heavy metals from anthropogenic and industrial activities have caused considerable irreparable damage to aquatic ecosystems. Sources include the mining and smelting of ores, effluent from storage batteries and automobile exhaust, and the manufacturing and inadequate use of fertilizers, pesticides, and many others. The metals and metalloids that contaminate waters and are most commonly found in the environment include lead, chromium, mercury, uranium, zinc, arsenic. These metals are the subject of concern due to their high toxicity. It can also consist of biological factors such as various pathogens like bacteria (Salmonella, Vibrio etc.), viruses (enterovirus), protozoa (Giardia, Entamoeba), parasites such as Ascaris and hookworm and several other non-pathogenic microorganisms. Apart from being hazardous to human health, they also have an adverse effect on the fauna and flora, and they are not biodegradable in nature. Thus, there is a need to seek new approaches in developing treatments to minimize or even eliminate metals present in the environment. The way in which microorganisms interact with heavy metal ions is partially dependent on whether they are eukaryotes or prokaryotes, wherein eukaryotes are more sensitive to metal toxicity than prokaryotes. The possible modes of interaction are (a) active extrusion of metal, (b) intracellular chelation (in eukaryotes) by various metal-binding peptides, and (c)transformation into other chemical species with reduced toxicity. For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products. Bacteria and higher organisms have developed mechanisms associated with resistance to toxic metals and rendering them innocuous. Most bioremediation systems are run under aerobic conditions, but anaerobic conditions make it possible for microbial organisms to degrade otherwise recalcitrant molecules. In aerobic process, bacteria that are oxygen dependent is converting the contaminants in the water. Aerobic bacteria can only convert compounds when plenty of oxygen is present, because they need it to perform any kind of chemical conversion. Usually the products they convert the contaminants to are carbon dioxide and water.
Methodology
Azolla microphylla fronts were cultured in Espinase and Watanabe (E and W) medium in 20*25*5cm plastic trays in polyhouse to produce healthy biomass. The biomass produced was harvested, washed with distilled water and gently blot dried to remove excess moisture and used as inoculant for further experiments. Partially treated municipal effluent (primary and secondary) were obtained from Nilothi sewage treatment plant, New Delhi. Fresh Azolla fronds (10g) were grown in trays for seven days. Tap water, E and W medium, primary and secondary treated municipal water (in triplicates) served as growth medium. Experiments were conducted with inoculated primary and secondary treated water which served as controls, to check the natural attenuation (due to the presence of natural microflora in water). After seven days Azolla was harvested and blot dried and final weight, doubling time and RGR were calculated. The biomass harvested was dried in excess at 60 degrees Celsius till constant weights were obtained. Known amount of dry biomass from each replication was digested with triacid (HNO3+HClO4+H2SO4 in ratio 9:2:1) mixture on a hot plate till a clear solution was obtained and suitably diluted with distilled water. Percentage of phosphorus in dry Azolla was calculated. The growth characteristics of Azolla was studied by inoculating in primary and secondary treated sewage effluents. These were compared with the growth obtained in medium and tap water. Initial and final levels of nutrients like total and available phosphorus (P), Total Organic Carbon (TOC), nitrite and ammonia were analyzed following American Public Health Association standard (APHA, 1999) methods for water and wastewater. Accordingly, total and available P was analyzed by ascorbic acid method, TOC by Wakley and Black method, ammonia by nesslerization method and nitrite by colorimetric method. Physical parameters including color and pH were noted.
Objective 1
Growth Characteristics of Azolla microphylla on different media The temperature dependence of the Azolla species on the growth was analyzed by growing the Azolla samples in three different temperatures to find out the optimum temperature for the growth of Azolla sps. The comparison was made between three temperature ranges.
When the temperature was 10-20 degree Celsius, the maximum relative growth rate and minimum doubling time was shown by the medium (0.15 & 4.57 respectively) followed by tap water (0.14 & 4.93 resp.). The secondary treated wastewater had shown a lower doubling time compared to primary treated wastewater. This may be due to the presence of reduced levels of ammonia in secondary treated wastewater. The percentage P was found to be highest in Azolla grown in medium followed by tap water and secondary treated water.
Maximum relative growth rate and minimum doubling time among the samples were observed when temp was around 20-35 degree Celsius. A least doubling time of 3.66 days was shown by Azolla grown in secondary treated effluent. This was comparable to Relative Growth Rate (RGR) of medium and tap water (3.67days and 3.68 days). The highest RGR was shown by Azolla grown in primary treated wastewater. Thus the secondary treated wastewater supported better growth than standard media and tap water. The highest percentage P was found in tap water followed by secondary treated wastewater (0.28 and 0.27 resp.). When the temperature ranged from 35-45 degree Celsius, the temperature was unfavorable for the growth of Azolla. The doubling time was found to be too high for all the four effluents. However, the growth rate of Azolla was found to be comparatively more in secondary treated wastewater compared to primary treated effluents. Due to the reduced growth of Azolla there was also growth of green algae in 1degree and 2 degree treated effluents. This has further inhibited the growth of Azolla. The Azolla samples were also dried in the oven and percentage P was calculated from the dry weight. The percentage P was higher in primary treated water followed by secondary (0.48 and 0.44 resp.). This higher value shown was due to the presence of green algae along with Azolla.
Objective 2: Physico-Chemical Parameter Analysis
The water samples collected were analyzed for physical parameters like color and pH, and chemical parameters like Total Organic Carbon, Organic Matter, Total P, Available P, Nitrite and Ammonia. The pH of the samples was in range of 7.5 to 8.9 and growth of Azolla has caused a slight increase in the ph. The color of the primary treated wastewater was brown which had turned colorless after growth of Azolla. However, the secondary treated wastewater was colorless initially and finally. Growth of Azolla was able to bring down the TOC, nitrite, total and available phosphorus and ammonia levels (Table 2). Growth of Azolla had brought down the total organic carbon level to 50 per cent for secondary treated water. Organic matter was 33 per cent. The Biological Oxygen Demand of both primary and secondary treated wastewater was very much within limits. Total P value has shown a reduction of 80 per cent in secondary treated effluent. The reduction in the phosphorus values can be attributed to the fact that phosphorus is the limiting nutrient for growth of Azolla. The ammonia levels of secondary treated wastewater had shown a reduction of 54.8 per cent. This shows that ammonium ions did not always inhibit growth and nitrogen fixation of Azolla (Kitoh et al,1993). Nitrite content of the wastewater was also analyzed. Nitrite was found to be higher in secondary treated wastewater. This may be supported by the nitrification process of conversion of ammonia to nitrite, as ammonia content of the secondary treated wastewater was low. There was 71.4 percent removal in nitrite content after Azolla growth. This may be attributed to the microflora present in the wastewater and in association with Azolla Microphylla.
DISCUSSION
The ability of Azolla to carry out bioremediation of wastewater was analyzed and the study has shown good results of removal of total organic carbon, phosphorus and nitrogen and proves to be efficient in removal of ammonia. The presence of nitrogen and phosphorus leads to the growth of algae (eutrophication) which consumes oxygen of water bodies and destroys natural flora and fauna leading to their death. Azolla growths which were found to be at peaked 20-35degree Celsius did not allow the growth of algae while the uninoculated controls maintained had algal growth in them. At 35-45degree Celsius when growth and multiplication of Azolla was negligible due to unfavorable temperature, there was growth of algae in both primary and secondary treated sewage water. Azolla uses a low cost Espinase and Watanabe Medium without nitrogen added to it. This is due to the presence of Anabaena Azollae which is in association with Azolla. It fixes atmospheric nitrogen for Azolla. The biomass analysis shows that the greater nitrogen, phosphorus, potassium and organic content would favor the use of Azolla as a bio fertilizer, especially when it grows in domestic wastewaters, and also when it is harvested from natural environments. Since Azolla can accumulate phosphorus, the concentration of this element in the plant was as high as the growth medium was rich in phosphate. The used Azolla biomass can be applied as green manure after testing for heavy metal presence. If the presence of heavy metal is detected then it should be dried and extracted for heavy metal extraction or incinerated to prevent further recycling in the environment (Soodet al., 2012). Further the potential of Azolla can be made use of in developing operational constructed wetlands. Constructed wetlands have been set up with rooted plants like typha, phragmites, canna but the option of using Azolla in combination with these rooted plants will enhance the removal of nutrients and heavy metals with less time.
Conclusion
Improvement in water quality, soil and removal of pollution with the added advantage of dual cropping proves Azolla to be a promising plant for maintaining a sustainable environment. Policy makers and the scientific community are equally challenged to seek solutions to mitigate the environmental issues of ecosystem services loss including water, biomass and global climate regulation. Azolla can prove to be a promising organism for these problems. Azolla is thus an ideal plant for polishing wastewater and can be efficiently used in constructed wetland.
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