Abstract

       Water pollution is recognized as a major threat to aquatic ecosystems globally. Phycoremediation is cost effective and a continuous treatment technique that uses microalgae to clean up the polluted water from pharma industry. In the present study, two predominant fresh water microalgae Chlorella sp. and Scenedesmus sp. were isolated from the pharmaceutical industrial effluents of Kanchipuram district that are potential phycoremediators both as individuals and as consortium for these contaminated waters.. Preliminary results revealed that these microalgal species to be highly efficient in reducing BOD, COD, TSS, TDS, and metals like sulphate, zinc and copper from pharmaceutical industrial effluents. Therefore, both the species can be used as eco-friendly adsorbent in the treatment of polluted waste water.

Introduction

         Heavy metals are metallic elements and metalloids that have relatively high density compared to water. In recent years, there has been an increasing ecological and global public health concern associated with environmental contamination by heavy metals and toxic effluents. Assuming heaviness and toxicity are interrelated an exponential increase of their use in several industrial, agricultural, domestic and technological applications has increased risk of exposure to these elements. Environmental pollution is very prominent in point source areas such as mining, foundries, smelters and other metal-based industrial operations. Toxic metals sometimes imitate the action of an essential element in organisms, interfering with their metabolic process. Waste waters from the above industries are recognized as a major threat to aquatic ecosystems globally (Oswald and Gotaas, 1957). Some of the pollutants like lead (Pb), arsenic (As), mercury (Hg), chromium (Cr) especially hexavalent chromium, nickel (Ni), barium (Ba), cadmium (Cd), cobalt (Co), selenium (Se), vanadium (V), oils and grease, pesticides, are very harmful and toxic even in ppb (parts per billion) range to ppm (Kaplan et al., 1987).

         Most pharmaceutical effluents are known to contain varying concentrations of organic compounds and total solids including heavy metals such as lead, mercury, cadmium, nickel, chromium and other toxic organic chemicals or phenolic compounds that are mutagenic and carcinogenic to humans (Idris et al., 2013). Bioremediation using biological systems of the contaminated sites to catalyze the degradation or transformation of various chemicals to non toxic forms is an ideal approach to deal with water pollution. Algal bioremediation is most effective in waste water treatment as an inexpensive biomaterial for removal of contaminants. Hence, development of biological based treatment system is considered as economically cheaper and environment friendly (Prabha et al., 2016).

         Microalgae are best known for biological treatment and have capability to grow in nutrient rich waste waters thus contributing to reducing pollutants and help in the maintenance of environmental sustainability and carbon neutrality (Mohan et al., 2011). Waste water treatment using microalgae is gaining prominence, due to its potential in efficiently uptaking the nutrients and organics by heterotrophic nutritional requirement coupled with the production of potentially valuable biomass, which can be further used in several processes to generate value added products (Park et al., 2011; Mohan et al., 2015 & 2016). Based on the above background, an attempt was made for bioremediation of waste water through identification of potential algal species from the polluted water itself.

Materials and Methods

         Kandigai in Kanchipuram district of Tamil Nadu is a pharmaceutical industrial region with polluted water bodies by these industrial effluents. Water samples were collected and cultured to identify algal diversity present in it. Temperature and pH were recorded from collection sites using YSI Multi parameter water quality instrument.

         The samples were subjected to microscopic studies by simple wet mount preparation and cultured for microalgae isolation in Bold Basal Medium (BBM). Samples 1ml each were serially diluted from 10⁻¹ to 10⁻¹⁰ and from each dilution 0.01 ml of samples were transferred to BBM containing 2% agar in Petri plates. The plates were incubated at room temperature for 10 days and observed for growth. Later discrete green colonies were selected and transferred to liquid medium (BBM) and maintained for 21days at room temperature.

         The growth rate experiments were done for isolated algal species at temperature of 28˚C with cycles of light: dark (12:12h) illumination, measuring cell density at constant intervals using Spectrophotometer (Hitachi U 2900) at wavelength of 680nm and their dry weights were estimated. Effluents were treated with selected algal cultures in BBM with different ratios such as 100:400, 200:300, 250:250, 300:200 and 400:100 v/v respectively. The same experiment was repeated with combination of algal mixture in equal volume to determine the effectiveness of consortium in treatment of pharma effluents.

         The physico-chemical parameters were analyzed for both raw and microalgae treated effluents following APHA standard methods (APHA, AWWA, and WEF. 1998). Microalgae were estimated for pigments such as Chlorophyll a, b and β-carotene following methods of Jeffrey and Humphrey (1975) and MacKinney (1941) using different wavelengths of UV visible spectrophotometer (Hitachi U-2900). Bio-Chemical analysis such as carbohydrate (Dubois et al., 1956), protein (Bradford, 1976) and lipid (Folch et al., 1957) was also determined for selected microalgae. The absorption of metals by microalgae was studied using Scanning Electron Microscopy.

Results & Discussion

         The physico-chemical parameters of water samples such as temperature ranged from28°C to 32°C, pH between 5.8 to 6.7, dissolved oxygen (DO) in range of 4.1 to 9.52 mg/L, chemical oxygen demand (COD) and biological oxygen demand (BOD) were in average about 1240 mg/L and 374 mg/L respectively. The effluent samples predominantly contained two micro algae Chlorella sp. and Scenedesmus sp. However, other algal species though present were in lesser numbers. Hence, further studies were carried out using Chlorella sp. and Scenedesmus sp that had quick adaptation and high growth rates.

         Chlorella sp., Scenedesmus sp. and consortium showed biomass productivity of 622.88 ± 14.03 mg dry weight L^-1, 142.50 ± 4.52 mg dry weight L^-1 and 680.88 ± 98.03 mg dry weight L^-1 respectively after 21 days of incubation. The percentage of protein content in these microalgae ranged from 18 – 45 % of dry matter. The highest percent of the protein was measured in the Scenedesmus sp. (45.10 ± 0.70%) followed by consortium (39.00 ± 0.62%) and least in Chlorella sp. (18.65 ± 0.70 %). Carbohydrate content was quantitatively evaluated and was found to be high in Consortium of micro algal samples 17.12 μg ml^-1 followed by Chlorella sp. and Scenedesmus sp. as 15.07 μg ml^-1 and 15.67 μg ml^-1 respectively. The total lipid contents for these microalgae ranged from 18.89 ± 1.2 % to 40.49 ± 2.58 % of the dry weight. Maximum lipid content was observed in the Consortium (40.49 ± 2.58%) followed by Chlorella sp. (38.10 ± 2.58 %) and Scenedesmus sp. (18.89 ± 1.2 %) suggesting that the members of the class Trebouxiophyceae accumulated higher lipid content than the Chlorophyceae members (Figure 1).

Figure 1: Graph showing the biochemical composition of selected microalgal species isolated from effulent water samples of pharmaceutical industry.

         Photosynthetic pigment chlorophyll a was quantitatively high in consortium (18.12 μg ml^-1) followed by Chlorella sp. (15.343 μg ml^-1) and Scenedesmus sp. (11.16 μg ml^-1). Chlorophyll b content was found high in Chlorella sp. (25.134 μg ml^-1) compared with Consortium (23.31 μg ml^-1) and Scenedesmus sp. (16.13 μg ml^-1). Carotene content was accumulated high in consortium (192.15 mg ml^-1) compared with Chlorella sp. (185.23 mg ml^-1) and Scenedesmus sp. (145.8 mg ml^-1) (Figure 2).

Figure 2: The graph showing the quantitative analysis of pigment composition of selected microalgal strains.

         The morphology of two different micro algae were examined through scanning electron microscopy (SEM) from treated effluents. It was found that there was a change in the morphology of structure indicating that algae has taken up metals from the treated samples, a process known as bioaccumulation. The pharmaceutical industry has shown great interest in the use of algae as a source of biochemically active substances. At present, outcome and toxicity of pharmaceutical residues in the aquatic environment pose difficulties. The pharmaceutical waste water effluent sample collected from Kandigai was found to contain numerous species out of which Chlorella sp. and Scenedesmus sp. were predominant with bioremediation capabilities.

Figure 3: The scanning electron microscopic image of selected micro algae A. chlorella sp.and B. scenedesmus sp. The marked places show changes in morphology due to bioaccumulation of metals from samples.

Conclusion

         Pharmaceutical manufacturers must operate under strict regulations by FDA in different countries and ought to maintain acceptable water quality standards for use, discharge or reuse. Growing microalgae on wastewater offers new insights for the microalgae industry as well as the wastewater treatment industry. The selected microalgae like Chlorella sp., Scenedesmus sp. and consortium is applied in treating the pharmaceutical wastewater and it will help in reducing the toxicity and facilitate recycling and reutilization of polluted water. The use of wastewaters for cultivating microalgae is necessary in order to reduce the cost of microalgae production. Furtherrmore, India being a tropical country with plenty of sunshine it is well suited for implementation of Phycoremediation as a technology and also can be carried out at an industrial scale for effective utilization of biomass.

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