Nitrogen and phosphate removal by the use of Chlorella

vulgaris and Scenedesmus quadricauda from waste water:

potential of microalgae

Maham saleem(0309-mphil-m-20)
Graphical abstract:
 Problem statement:
• Water quality of aquatic ecosystems has been analyzed by the physical, chemical and biological interactions. The net balance
of the aquatic ecosystems has been reduced by human interventions, which increase the pollution of water and ultimately
kill the aquatic species, bitter taste, odour, color and introduction of invasive weeds. Quantity of waste is affected by level of
hardness, decrease and increase in Ph and free CO2 in different levels of water. Heavy metals such as cadmium, copper, lead,
chromium, nickel and zinc are considered as pollutants that cause poisoning conditions. Good quality water is a right of every
citizen and it helps to prevent the disease and improve life quality (Oluduro and Adewoye, 2007). Mostly fresh water bodies
like rivers and lakes are being contaminated because several industrries like petro-chemical, fertilizer industry, oil, pulp,
paper, and textile and tannery industries dumped their effluents directly into water bodies. Most of the farmers used
effluents of industries for irrigation purposes, and they contain harmful chemials (Wang 2011).
• In bioremediation, use of algae as waste water treatment is commonly used such as blue green algae like cyanobacteria
considered more ancient prokaryotes which are supposed to appear before pre Cambrian period (Ash and Jenkins, 2006).
Use of different types of microalgae helps to remove nitrogen, phosphorus and chemical oxygen demand from different
waste effluents.
• In developing countries sewage waste water in urban and semi urban areas become a major problem (Moore, 1998). In
waste water major effluents comes from industrial and residential discharge, which finally dumped into rivers and cause
major water problems (Kumari et al, 2006). Industrial discharge of wastewater cause water pollution and ultimately increases
biological oxygen demand and nitrification, which destabilized the aquatic ecosystem (Morrison et al, 2001; DWAF and WRC,
1995). So the use of microalgae as a bioremediation technique to treat waste water is an urgent and powerful solution.
Objectives:
• To examine the efficiency of microalgae strains in removal of nitrogen and phosphate to
prevent further deterioration of water quality of domestic wastewater.
• To determine the kinetics of N and P elimination as well as simultaneous growth of
microalgae strains in waste water treatment.
• To evaluate wastewater treated with culture of C. vulgaris and S. quadricauda and wastewater
treated without culture of C. vulgaris and S. quadricauda (Control)
Literature review:
• For treatment of decontamination in waste water bioremediation is a newer approach. This technique used the
living organisms to remove most polluted components from waste water. Heavy metal removal from industrial
waste water is most important as it cause environmental and human health problem. Metal accumulation by
bioaccumulation process is very important as it use the cell active metabolism (Volesky, 1990; Wase and Foster,
1997).
• Use of microalgae in bioremediation is not only a unique process; also it provides numerous benefits with other
biological methods. In specific bio removal application, micro algae strains are used as cultivated approach to
remove worldwide metal problems (Edward and Benemann, 1993). For the decontamination of metal effluents
bio sorption is used as a cost effective and alternative technique (Kratochvil and Volesky, 1998). Bio sorption
technique has been used as metal recovery and removal in aqueous solution (Kaewsarn, 2002). In agricultural
and industrial secondary effluents, Chlorella vulgaris is used for the ammonia and phosphorus removal as a bio
treatment. These microalgae were extracted from stabilization ponds. Microalgae have ability to remove
ammonia from waste waters to some extent, but they are not used for the treatment of bivalent ions like
cadmium, copper and lead, and dead dries algae removed these bivalent ions by 50% volume/volume ratio in
solution (Al-Qunaibit, 2009).
• Due to good performance, low cost and mostly available algae have been increasing attention day by day for
heavy metal removal and recovery (Wang and Chen 2008). For the nutrients removal in tertiary waste water
treatment microalgae has been used. The pH increases due to mediated algae helps in phosphorus and
ammonia precipitates in air and act as a disinfectant in waste water (Karin Larsdotter, 2006).
• Micro algae strains have been used for toxic heavy metals removals support the bioremediation technique in
waste water treatment (Monteiro et al 2009). To investigate the algae role in waste water algal samples were
collected from different waste waters. This helps us to identify the pollution tolerant algae species from
waste water such as C. vulgaris and S. quadricauda. Valderramna et al. (2002) suggest in an experiment that
61% COD is removed by the C. vulgaris in the citric acid and ethanol production industry, and it also reduced
BOD and COD values of the industriual effluents and its possible when algae has high growth rate and intense
photosynthetic activity (Colak and Kaya, 1988).
• Zhang et al. (2008) in a study predicts that Scendesmus sp. has potential to remove inorganic nutrients from
municipal waste water. (Azeez, 2010) suggests that C. vulgaris has capacity to remove BOD and COD 88 and
89% respectively. C. vulgaris and S. quadricauda has ability to remove 78 and 70% nitrate removal from waste
water (Tam and Wong, 1990). Removal of phosphate by C. vulgaris in bioremediation is due to the utilization
of phosphorus for growth (Rao et al., 2011).
Chlorella vulgaris Scenedesmus quadricauda
Methodology:
1. Collection of different waste water samples from industrial area
A 2 liter bottle washed with 10% HNO3 for 48 hours and few drops of HNO3 will be added to prevent metal loss.
2. Study waste waste-water samples in order to;
2a) Physical and chemical parameters:
Physical and chemical parameters of waste water must be checked before and after the treatment by using Punjab
Environmental Quality Standards for Drinking Water.
i. Color and pH:
 Color:
Color of waste water samples must be tested from naked eye.
 pH:
It is measured by pH meter. Calibration of pH should be accomplished by electrodes dipped in pH 7 buffer. pH should
be measured by placing electrode in 6-7cm deep into the sample and then recording meter after stabilization.
BOD and COD:
Biological oxygen demand (BOD):
Reagents:
o Phosphate buffer
o Magnesium sulphate solution
o CaCl2 solution
o FeCl3 solution
o Sodium sulphite solution
Procedure:
Water dilution will be prepared by adding all the reagents in 2ml distilled water. 2ml sample water will be
aerated. Undiluted sample should be analyzed for the DO and denoted as Doo. A bottle filled with mixture and
designed as DO1. Other one is considered as blank as designed DO2. Both of 2 bottles will be incubated at
temperature of about 20C for 5 days after that BOD will be determined by the following formula.
BOD (mg/l) = [{(DO2-DO1) x 100} (DO2-DO0)}]
Lab test of microalgae:
Chemical oxygen demand:
Reagent which we used:
o 0.1M potassium dichromate solution
o Sodium thiosulphate
o H2SO4 (2M)
o 10% potasium iodide solution
o Starch solution 1%
Procedure:
100ml of water samples will be taken in 100ml flask and make 6 samples and also blank samples
will be prepared. Add 5ml K2CrO7 Solution in each 6 flasks.these flasks kept at 100C in hot water
bath for 1 hour. Samples were cool for 10 minutes and then add 5ml KI in samples and then add
10ml H2SO4. Each flask will be titrated against 0.1 M sodium thiosulphate solution until color
turned into pale yellow. 1ml starch solution added and color changed from yellow to blue and
titrate utill blue color disappears.
COD of the sample (mg/l) = 8xCx (B-A) S
Selection of microorganisms:
algal species which we select to isolate from the nearby river are the
Chlorella vulgaris and Scenedesmus quadricauda used as test organisims.
Procedure
A 2ml of suspension of C. vulgaris and S. quadricauda as initial inoculums a 9
days old culture containing in each flask with 200ml of waste water sample.
Experiment conducted under controlled conditions Ph, BOD, COD And nitrate
were checked after 5 days interval like 0th ,5th , 10th , 15th and 20th
respectively for a total duration of 20 days, and checked the physiochemical
parameters of waste water using standard methods.
Research Utilization

 Bioremediation with the help of algae

has significant importance, and
considered as an efficient and
environment friendly technology to
remove effluents from waste water.
Algae bioremediation in waste water
has low cost and environment
friendly results for the removal of
decontamination.
 Year Year 2
Activity
1 Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug
Course Work
Literature
Review
Sample
Collection
Sample
Preservation
& Storage
Sample
Preparation
Chemical
Analysis
Statistical
Analysis &
Interpretation
of Result
Thesis
Write-up