Development of Sustainable Phycoremediation With the Help of Chlorella Pyrenoidosa for Textile Wastewater Analysis Using Response Surface Methodology

The uncontrolled utilization for the textile products is increasing year by year resulting with the elevating wastewater generated from the textile industries, which makes it among the prevalent sources of critical environmental deteoration issue globally. Products obtained from the dyes used are the primary toxic product for aquatic life, they cause aesthetic pollution, eutrophication, perturbation and increase in BOD and COD in aquatic life. Three types of textile wastewaters (Acid Yellow dye, Acid orange dye and Basic pink dye) has been used for wastewater treatment and microalgal (Chlorella pyrenoidosa) biomass production. Nitrogen content in textile wastewaters is very less, hence urea is used as nitrogen source in wastewater. Optimal growth condition (Urea-0.4g/L, wastewater- 40%(v/v)) is developed through Response surface methodology (RSM). The biomass productivity for chlorella sp. is 1.2-1.5 g/L/day in textile wastewaters. The reduction eciency of COD, Nitrate-N Ammonia-N, Phosphate-P, and Dye(color) removal for Chlorella is 90-95%, 75-85%, 90-98%, 65-74% and 40-65%.After harvesting the Biomass by occulation method it can be used for biofuel production by in-situ transesterication. eciency%


Introduction
Water pollutants may originate from point sources or from dispersed sources. Point-source pollutants are easier to control than dispersed-source pollutants, since they ow to a single location where treatment processes can remove them from the water. Such control is not usually possible over pollutants from dispersed sources, which cause a large part of the overall water pollution problem. Dispersed-source water pollution is best reduced by enforcing proper landuse plans and development standards. Excessive reactive oxygen species, would be produced by the algal cells which would cause cellular peroxidation, in this manner chloroplasts being destroyed, inhibiting chlorophyll synthesis and hence dropping photosynthesis. With increasing the time of exposure, irreversible algal damage may lead to death [1]. Signi cance of treatment of wastewater should be marked as a water use as it is interconnected with the other uses of water. Mass cultivation is the most critical step in terms of water and nutrient requirement. Wastewater from industries such as textile wastewater (TWW) is source for water which is easily available,cheap and additionally encompasses obligatory nutrients (nitrates, phosphate, micronutrients etc.) and organic dyes (potential carbon source) for cultivation of algae [2].
There are a number of textile industries which are dumping their wastewater untreated into the environment which are not only toxic but also changing the BOD COD levels and hence the entire physiochemical parametric texture. Many types of chemical dye used in textile industries are very harmful to the aquatic life and sh species. Most of the dyes have carcinogenic action and also cause other skin problems like allergies, dermatitis, skin irritation, etc [3]. The textile wastewater holds a typical characteristic of strong order high chemical oxygen demand (COD) high salinity variable PH and elevated temperature [4]. Coloured dyes in the wastewater adversely affect aesthetics, gas solubility, transparency of water and can equally prove to be hazardous to both aquatic ora and fauna resulting with critical environmental issues. Also, to be included that most synthetic azodyes and their metabolites are mutagenic and carcinogenic potent enough to be toxic to human health to be toxic to human health. The process of treatment of the textile e uents includes mainly physical and chemical ways, but they cost expensive. It becomes di cult to treat the dye wastewater by physical and chemical due to their complicated molecular structures. Not only this has the disposal of thickened sludge generated a bigger problem using biological methods for textile wastewater remediation is gaining interest now. Majority of the studies are focusing on the application of bacteria, algae and fungi to check coloured wastewater from the textile [5]. The research areas using microalgae in phytoremediation of coloured textile wastewater is gaining attention because of their signi cant role and xation of carbon dioxide, in addition it can be sustainably and economically used for production of biofuel [6]. In this research an attempt is to use a microalgae named Chlorella pyrenoidosa for treatment of wastewater using a complete in-situ eco-friendly operation on a low cost self -designed tubular photobioreactor. Although, many studies have been done earlier by different researchers earlier, an innovative approach of cost effectiveness to pilot technology is referred, yet an attempt is made for people, so that it becomes accessible and afforadable not only for the large scale industrialists on a large scale and the small scale textile manufacturers, to check before discharging the untreated wastewater which is extremely toxic not only for the environment but also for the Ganga inhabitants and people using the gangawater by any means.
The purpose of the study is to design and evaluate a sustainable way of bioremediation using Chlorella pyrenoidosa and analysis of pollutant removal from textile wastewater, making the technology accessible on a larger scale.

Culture Preparation
The microalgae strain Chlorella pyrenoidosa (NCIM Accession number 2738), was collected from National Chemical Laboratory, Council of Scienti c Research, Pune, India. 10% (v/v) of inoculums of exponentially growing algae culture is used for the biomass growth and optimization. Along with the parametric studies which are designed for the characterization and de ning optimal operating requirements for Chlorella pyrenoidosa as mass culture [7], also monometric studies were implemented to study if the pollutants could signi cantly reduce the 02-CO2 e ciency for gasexchange of the Chlorella.
A reformed Trypticase-glucose-yeast extract (TGYE) medium comprising 5.0 g of Tryptone-phytone,, 1 g of yeast extract, 2.5 g of glucose and 15 g of agar per liter of distilled water were used as the plating medium.Followed by which the samples were withdrawn from the photosynthetic gas-exchange unit when the steady state of growth were observed by the algal culture. All plates were counted after 24 hr of incubation at 39 C.

Sample Collection (textile wastewater) and characterization
The e uents as wastewater were taken from textile industries and their outlets into the river Ganga in Varanasi using the Grab Sampling method. Textile wastewater contains many dyes which affects the aquatic system. Three types textile dyes are collected named as Acid yellow 17 dye, Acid orange 7 dye and Basic pink dye containing textile wastewater. The Characterization of physical and chemical parameters and biological parameters like Titerimetric method for COD, Wrinkler method for BOD, Nesslers method for Nitrogen estimation of each batch sample collected was determined using standard protocols (APHA).

Experimental Studies
Three textile azo dyes were used in the research investigation, named as Acid yellow, Acid orange, Basic pink. To determine the wavelength giving a maximum absorption, the absorbance of these three dyes were examined from 300 to 700 nanometers using a spectrophotometer (Shimadzu UV/Vis 160). Acid yellow (AY) is an acidic dye with absorption maximum at 650 nm while acid orange (AO) is another acidic dye with maximum absorption at 670 nm whereas basic pink (BP) is a basic dye with absorption maximum at 580 nm.

Physiochemical Parameters
Titrimetric method is used for COD studies. Prior to completing the COD test, a series of known standards are prepared using KHP (potassium hydrogen phthalate). Most wastewater samples will fall in the high range, so standards of 100, 250, 500 and 1000 mg/L are typically prepared. COD standards can also be purchased. A COD reactor/heating (150 °C) block and a colorimeter are turned on so that both instruments are allowed to stabilize.
Pre-prepared low-range (3-50 ppm) or high-range (20-1500 ppm) vials are selected for the COD test based on expected results. Both ranges can be used if expected results are unknown.
One vial is marked as a "blank," and three or four vials are marked with known standard levels. Two vials are then marked for the wastewater sample to make a duplicate run. Note: If multiple wastewater samples are being run, at least 10% of samples are duplicated.
2 mL of liquid are added to each vial. In the case of the "blank," 2 mL of DI water are added. 2 mL of each standard are added to the corresponding vials. If the wastewater sample is tested at full strength, then 2 mL is added to the corresponding vial. If dilution is required, then serial dilutions are performed and 2 mL of the diluted sample are added to the corresponding vial.
Each vial is mixed well and placed into the reactor block for two hours. After two hours, the vials are removed from the block to a cooling rack for about 15 minutes.
The colorimeter is set and calibrated per the speci c instructions for that unit (i.e., proper wavelength, blank and standards) and each vial is placed in the unit and the COD concentration read. If the sample was diluted, the corresponding multiplication is made.
Winkler method for BOD and DO studies, the method involves lling a sample bottle completely with water (no air is left to bias the test). The dissolved oxygen is then " xed" using a series of reagents that form an acid compound that is titrated. Titration involves the drop-by-drop addition of a reagent that neutralizes the acid compound and causes a change in the color of the solution. The point at which the color changes is the "endpoint" and is equivalent to the amount of oxygen dissolved in the sample.
Nessler method for Nitrogen estimation studies, In the ammonia test, Nessler Reagent (K2HgI4) reacts with the ammonia present in the sample (under strongly alkaline conditions) to produce a yellow-colored species. The intensity of the color is in direct proportion to the ammonia concentration.

Microalgae strain and growth optimization
Shake ask study has been performed in inoculum in wastewater sample, samples were taken daily in 2 ml volumes from each shaker ask and analyzedfor soluble N for 10 to 20 days and was used to determine the `algal growth rate (k) . Experimental Setup -A Photobioreactor ( made of perspex ) of volume of 10.5 L, which was in continuous illumination by tubular uorescent lamps (40W PHILIPS tube lights). The light intensity kept 5400lux at the surface of culturing vessels so that the growth of microalgae can be maximum. The culture medium of textile e uent is diluted and divided in to runs according to the factorial design to calculate the optimum growth condition. The microalgal cells were inoculated with a concentration of 20% (V of inoculation/ V of solvent) in a10.8 L reactor incubated at room temperature with a 16/8-hour dark/light period for 8 days and optical density was recorded per day.

Cell Mass Extraction
After the treatment of wastewater in the PBR biomass is harvested with the occulation technique. The characterization of chemical, physical and biological parameter of every batch sample collected was analyzed performing the standard protocols (APHA). The major nitrogen and carbon elimination processing in the algal photobioreactor were assimilation into biomass (algal) and stripping ahead.
As soon as growth in algae was noticed and it started multiplying, both phosphorus and nitrogen content in wastewater decreased signi cantly. There was a rapid removal rate reported during the rst week of growth and more than of wastewater N and P was reduced. However, post the initial period, the removal rate slowed down. At the end of this study, more than 80% of total inorganic Nitrogen and Phosphorus removal was observed. At large, a higher inoculum size of algal cells provided more phosphorus and nitrogen removal than that in the lower inoculum. The ndings suggest that of Chlorella cultivation seems to be one of the viable approaches to decrease the amount of phosphorus and nitrogen entering the nearby shoreline water, thus checking the eutrophication problem. Also to be noticed, that algal ponds with higher inoculum size might be more suitable to be installed as a secondary process comparative to a tertiary treatment.

Textile wastewater characterization
The Physico-chemical characterization of textile wastewaters was done by the standard protocol given in APHA manual.
Sample was collected with method of discrete sampling at different location from Textile industry in or near Varanasi, India. The result of textile wastewater characterization conferred in Table 1 indicates that values of pH, colour, temperature, total dissolved solid (TDS), total suspended solid (TSS), alkalinity (Carbonate), total nitrogen(ammonia), total nitrogen (Nitrate), total Phosphorus, Biological oxygen demand (BOD), Dissolved oxygen(DO) and Chemical oxygen demand (COD). BOD content is high (67.2 mg/L) in the case of acid orange textile wastewater and in all the textile dyes the nitrogen content (ammonia + nitrate) is low. Table 3 shows that the values of pH, TSS, TDS, Nitrogen (ammonia) and

Microalgae growth optimization and Kinetics Study
The microalgae culture was inoculated in the shake ask (250 ml) under light control for 10 days and allowed for the growth in Batch Mode operation, after that the growth and Biomass content is calculated. The graph of 2(a) shows the growth kinetics curve for the Chlorella pyrenoidosa, the results obtained by cultivating the Chlorella pyrenoidosa in the Bolds basal media (BBM). Lag phase occurred for 3 days and log phase occurred for 7 days for the Chlorella pyrenoidosa. The growth constant (µ) is 0.21 g/L/day and maximum growth rate constant (µmax) is 0.29 g/L/day.
Growth curve ( Fig. 2(b)) of Chlorella pyrenoidosa in Acid yellow 17 dye, Acid orange 7 dye and Basic pink dye textile wastewater in composition of 10% of wastewater and BBM 90%, the biomass productivity is 0.98 ± 0.3 g/L/day, 0.96 ± 0.2 g/L/day, 1.2 ± 0.3 g/L/day respectively. As the nitrogen content is textile wastewaters is very low and the for the microalgae growth nitrogen content is very important, hence different types of nitrogen source is used like sodium Nitrate (NaNO 3 ), ammonium nitrate (NH4NO3), ammonium chloride (NH 4 Cl) and urea (NH 2 CONH 2 ) in the concentration 0.2 g/L, 0.4 g/L,0.4 g/L and 0.2 g/L respectively. The biomass productivity is 0.75 ± 0.2 g/l/day in all the sources showed in Fig. 2(c). Hence nitrogen content is enriched by urea. Urea is low cost source having the high biomass productivity. The physicochemical characteristics of textile wastewater is highly uctuating but it is comparable and within range as reported [8]. However, the components of textile wastewaters vary as per the raw materials and ingredients used in textile manufacturing. Nitrogen (Ammonia) and Nitrogen (Nitrate) in textile wastewaters are generally low, hence external nitrogen source is required. Low Nitrogen content has an added advantage as it enhances the lipid productivity in the Microalga biomass but low nitrogen content also affects the growth of microalgae. The urea is best nitrogen source for the textile wastewater as it is cheap and easily available. The maximum biomass density 0.79 g/l/day found in the case of Ammonium nitrate (NH 4 NO 3 ). Similarly, the carbon source is also optimized using different carbon source like Sodium carbonate (Na 2 CO 3), Sodium bi-carbonate (NaHO 3 ), and Potassium Bi-carbonate (KHO 3 ), the biomass productivity is 0.4 ± 0.1, 0.2 ± 0.05, 0.2 ± 0.05 g/L/day respectively. The maximum biomass productivity found in the case of sodium carbonate (Fig. 2(d)).
The biomass concentration of algae reaches to approximately 6 g/l by around 8th day after having a lag phase of 2 days from the day of inoculation in BBM while in the textile wastewaters (Acid yellow, Acid orange and Basic pink) when inoculated by the acclimatized algal species operated for 10 days monitoring the algal growth, in 20% dyes mixed with 10% BBM, the graph elevated to maximum of around 1 g/l biomass concentration on 8th day for pink dye treatment and around 10 g/l biomass concentration on around 8th day of yellow and orange dye which shows a signi cant increase in growth compared to BBM only. Biomass concentrations were optimized by various nitrogen sources where a number of nitrogen sources were exposed with algae for comparative analysis. The biomass concentrations were found increasing with NH 4 NO 3 in comparison to NaNO 3 , NH 4 Cl and Urea but it was interesting to nd out that Urea which did not show rise in graph initially (lag phase is long) but showed signi cant growth during log phase, not only this Urea is found to be the most cheapest Nitrogen source to be used (around 10 Rs/ kg) and very easily available. Amongst the various Carbon sources used for optimization, Sodium bicarbonate was found showing maximum biomass concentration 3.5 g/l on 5th day which was considerably higher with respect to Sodium Carbonate and Potassium bicarbonate with biomass concentration of 2 g/l on 5th and 6th day respectively. respectively.

Statistical Optimization
Surface plots obtained from the RSM optimization for Nitrogen removal e ciency % with correlation effect of Carbon source and pH, Nitrogen Source and pH, wastewater% and pH, Carbon Source and Nitrogen Source, wastewater% and Carbon source and Wastewater% and Nitrogen source are represented in the gure (6). The optimized value of pH, carbon source, Nitrogen source and wastewater % are 8.431 ± 0.6, 0.29 ± 0.1 g/L, 0.2 ± 0.05 g/L and 38.90 ± 5% Surface plots obtained from the RSM optimization for Phosphorus removal e ciency % with correlation effect of Carbon source and pH, Nitrogen Source and pH, wastewater% and pH, Carbon Source and Nitrogen Source, wastewater% and Carbon source and Wastewater% and Nitrogen source are represented in the gure (7).

Analysis of pollutant removal
Potential of microalgae in degrading speci c pollutants are being studied. Microalgae has been reported for degradation of large number of micropollutants, p-chlorophenol collected from a site where water contaminated with a number of aromatic pollutants can be degraded at a rate of 10 mg/L/day by Chlorella vulgaris and Coenochloris pyrenoidosa [9].
The analysis of parameters including COD, ammonia, colour and phosphorus were examined with respect to the methodology described in APHA (2000)  as their carbon sources and nally transform them into metabolites moreover these algae can equally be employed as bio sorbent as the dyes hold the potential to absorb onto their surface.
As can be seen in the graph shown in Fig. 8 (a) COD concentration was ranging from 250-300 mg/L for all the three dyes i.e. acid yellow, acid orange and basic pink on day 0 but as the growth continues a steady fall in the graph can be seen, as soon as it reaches the 8th day the COD level drops down to 50 mg/L which shows that Chlorella pyrenoidosa is e cient enough to remove COD. On the contrary when the same study was performed with phosphorus, the level of phosphorus in acid yellow and acid orange was around 2.5 mg/L while in basic pink it was around 4.0 mg/L way higher than the other two but as the growth reaches between 4 to 6 days the graph falls down to 1.0 mg/L which con rms the e ciency of the alga in removing phosphorus as can be seen in Fig. 8 (b). Figure 8 (c) shows removal of ammonia where the level for the three dyes having ammonia were as high as 14-16 mg/L which experiences a steep fall in the graph and falls down to 2 mg/L at around 8th day of inoculation. The removal of colour was studied for the three dyes which were ranging from 0.09 to 0.10 on the day 0 but the intensity of colour was signi cantly decreased to the level of 0.05 on the fth day and fell down to 0.04 on the 9th day of inoculation con rming the ability of Chlorella pyrenoidosa in removing colour of the dyes successfully.
The e ciency of nitrogen (ammonia) removal, Nitrogen (nitrate) removal, COD removal, colour removal, and phosphorus removal in three types textile wastewaters (acid yellow dye containing textile wastewater, acid orange dye containing textile wastewater, basic pink dye containing textile wastewater) is explained in the form of bar chart given in gure (9).
The e ciency of nitrogen (ammonia) removal, Nitrogen (nitrate) removal, COD removal, colour removal, and phosphorus removal in three types textile wastewaters (acid yellow dye containing textile wastewater, acid orange dye containing textile wastewater, basic pink dye containing textile wastewater) is explained in the form of bar chart given in Table (3).

Conclusions
Three types of textile wastewater were used for the study containing the Acid yellow, Acid orange and Basic Pink dye.
The growth of microalgae species Chlorella pyrenoidosa was low initially 0.3-0.4 g/L/day but after acclimatization biomass productivity increased up to 1.2-1.5 g/L/Day in textile dyes. Nitrogen content in all type textile wastewaters was very less, hence urea is used as nitrogen source in wastewater. Then optimized value for various parameter like pH, carbon source, Nitrogen source and wastewater % used for the biomass productivity, colour removal, Nitrogen removal, and Phosphorus removal are 8.431 ± 0.6, 0.29 ± 0.1 g/L, 0.2 ± 0.05 g/L and 38.90 ± 5% respectively. Obtained optimized results for biomass productivity, colour removal, Nitrogen removal, Phosphorus removal are 1.13 ± 0.2 g/L/Day, 65 ± 2%, 91 ± 3%, 65 ± 1% respectively. The COD removal e ciency obtained 92 ± 1% in nearly all three types of textile wastewater.
Harvesting ( occulation method) is optimized using RSM in which chitosan was used as occulant. The optimum condition achieved for occulation is 0.5 g/l at pH 10.0 having the mixing time and settling time 30 min and 120 min respectively. Low amount of Nitrogen enhances the lipid productivity in the algal biomass. Therefore, Chlorella pyrenoidosa can be an alternative for textile wastewater bioremediation and high biomass can be achieved using urea. The algal biomass produced may be advantageous as fertilizers, feedstock, and production of biofuel. This technology can be used for techno economic wastewater treatment and enhanced biomass production process.  Figure 1 Methodology of Bioremediation of textile wastewater using designed Bubble column tubular Photo-bioreactor.

Figure 9
Comparative Analysis of E ciency of Pollutant removal in Acid yellow, Acid Orange and Acid Pink dye wastewaters for nitrogen (ammonia) removal, Nitrogen (nitrate) removal, COD removal, colour removal, and phosphorus removal. A very little variation in nitrogen (ammonia) removal e ciency in acid yellow dye containing textile wastewater (91±3%), acid orange dye containing textile wastewater (90±3%), and basic pink dye containing textile wastewater (91±1%). A small variation found in nitrogen (Nitrate) removal e ciency in acid yellow dye containing textile wastewater (79±2%), acid orange dye containing textile wastewater (68±3%), and basic pink dye containing textile wastewater (70±4%).