Dissertation ppt biostimulation- a potential practice for wastewater treaatment by using selected species of chlorophyceae family

BIO-STIMULATION: A POTENTIAL PRACTICE FOR
WASTEWATER TREATMENT USING SELECTED SPECIES
OF CHLOROPHYCEAE
Dissertation submitted to Sardar Patel University in partial
fulfilment of the requirement for the degree of
MASTER OF SCIENCE (M.Sc.)
IN
ENVIRONMENTAL SCIENCE & TECHNOLOGY
Submitted By:
SUMER PANKAJ
Guided By:
Dr. MAYUR JOSHI
Mr. SUMEET MOHANTY
Mr. ANKIT PATEL
DEPARTMENT OF ENVIRONMENTAL SCIENCE & TECHNOLOGY
INSTITUTE OF SCIENCE AND TECHNOLOGY FOR ADVANCED STUDEIS AND RESEARCH
SARDAR PATEL UNIVERSITY
VALLABH VIDYANAGAR
GUJARAT
(2017-18)

Preface
Industrial
Consultancy
(B to B
projects)
Product
Development
Innovation
and
application
Restoration
of Rivers &
lakes (B to
G projects)
 Neutralization pH
 Reduction of C.O.D.,
Ammonical nitrogen,
colour, hardness, TDS
etc.
 Products like
Organic Green Live
(OGL).
 OGL boost.
 Mixture of nutrients
to increase the
setalability of MLSS
 To develop an algal
bacterial consortium
 Entrapment of Bacterial
MLSS to activated
carbon in Bio-towers.
 Restoration of ploouted rivers
like Mausam river at Mlegaon
Maharastra.
 Lake restoration at Alighar. (U.P.)
PhycolincTechnologiesPvt.Ltd.
• Phycolic Technologies Pvt. Ltd.
is a matured start up firm based at
Ahmedabad (Gujarat),
established in the year 2015.
They are committed to the use of
innovative, sustainable, green and
chemical-free technologies to
provide effluent treatment and
bio-remediation solutions to
complex waste water
management problems, which
conventional technologies cannot
address. The company is working
on Phycoremediation technology
which is based on applications of
Micro Algae in Industrial Effluent
Treatment, Polluted Lakes and
rivers remediation. The company
has provided solutions in the
industrial sectors like chemical,
Pharma intermediate,
agrochemical, textile,
electroplating, leather and oil and
drilling industries for treatment as
well as Zero Liquid Discharge for
High TDS effluent and RO reject.

Content
1. Introduction
2. Objectives
1.1 Distribution of water resources across the
Earth.
1.2 per capita availability of water in India, 2001-
2015.
1.3 Phycoremediation – a cleaner and greener
technology to treat industrial
wastewaters
3. Review of literature
4. Materials and method
4.1 Product Development and activation.
4.2 Possible applications of the product on industrial waste
water treatment.
4.3 Application of Algal biomass as a bio-fertilizer and aqua
feeds.
5. Result and discussion
5.1 Activation of OGL.
5.2 To check the effect of macro/micro-nutrients & growth
promoters on the cell growth of OGL.
5.3 To check the effect of growth promoters on Algal-
Bacterial (ALBA) consortia development.
5.4 Application of the Algal-bacterial (ALBA) consortium
to treat industrial Effluents
5.5 To study the application of algal biomass as a fertilizer
and aqua feed
6. Summary & conclusion
2.1 To design and develop a product
having the potential to
remediate the contaminants
from waste water streams.
2.2 To study the possible applications of
the developed product.
2.3 To study the application of Micro-
algae as bio-fertilizers and
aqua-feeds

INTRODUCTION
Figure 2 - per capita availability of water in India, 2001-
2015
The NCIWRD commission, based on a small sample of
industries and their water use, projected that industrial
water demand would increase from 30 BCM in 2000, to
about 101 and 151 BCM by 2025 and 2050, respectively
Figure 1 - Distribution of water resources across the Earth.
(Gleick, P. H., 1996). (Source - National Commission on Integrated Water
Resources Development, India)

Phycoremediation – a cleaner and greener technology to treat
industrial wastewaters
Figure 3 - commonly used wastewater treatment
processes.
Figure 4 - Dual application of microalgae for phycoremediation
and biomass production for sustainable biofuels production
and fertilizers.
(Source - Rawat, I., Kumar, R. R., Mutanda, T., & Bux, F.
(2011).
According to the United Nations Environment Program
(UNEP) definition of “Cleaner Production” is the continuous
application of an integrated, preventative environmental
strategy to processes, products and services to increase
efficiency and reduce risks to humans and the environment”
“Phycoremediation is the use of algae to treat wastes or
wastewaters”. The algae are distributed widely throughout the
earth and have adapted to a diversity of habitats. This
advantage led to the wide use of the algae in bioremediation of
wastes, resulting in treated waters as well as the production of
a useful biomass which can serve as feedstock.

Characteristics Chemical
Treatment
Biological
treatment
using
Bactria
Biological
Treatment
using
Algae
Biological
Treatmen
t using
Alba
(algae +
Bactria)
Sludge 30-50%
sludge
generation
10%
sludge
0-10%
sludge
generation
0-10%
sludge
generatio
n
Efficiency 50-70%
reduction
40-450%
reduction
50-90%
reduction
80%
reduction
Cost reduction 8rs per
liter
40% 50 % 60-80%
Nature of
Sludge
inorganic organic organic organic
Retention time 6-12 hours 24-48
hours
24-48
hours
24-48
hours
• Effective neutralization of Highly Acidic (Upto 1.5 pH) &
highly basic (Upto 13 pH) effluents without any
chemical addition and sludge generation.
• Reduction in COD & BOD up to 97% in effluents which
have extremely high TDS (1,00,000 to 1,50,000 ppm).
• Excellent reduction (Upto 99%) in Ammonical Nitrogen
(NH3-N) & Cyanide without any chemical
addition & hazardous waste generation.
• Bio-accumulation of Heavy metals (Cr, Ni, Ar, Zn, Fe, Cu,
Co etc.) & hardness (Ca, Mg) from polluted
water/effluent.
• Effective removal of Microbial Toxicity, Color, odor etc.
from polluted water/Effluent.
• Removal of Pathogenic bacteria like Total coliform and
Fecal coliform from polluted water bodies.
• Chemical Free process, Low operating process and low
Advantages of Phycoremediation on commonly available chemical
treatment –

Objectives of the study
To design and develop a product
having the potential to remediate
the contaminants from waste water
streams –
• To isolate algae from the waste
waters.
• Cultivation and harvesting of
desired algae.
• To develop a product by using the
microalgae isolated from the waste
water streams.
• To trace the desire activation
period required for OGL.
• To check the effect of micro
nutrients and growth promoters
on activation period.
• To monitor the cell count and
chlorophyll content continuously.
To study the possible
applications of the developed
product –
• To develop the algal-bacterial
consortium (ALBA) by
entrapment method.
• To collect different
wastewaters from industries.
• To analyze the physico-
chemical parameters of the
wastewaters collected.
• To check the efficiency of the
Algal-bacterial (ALBA)
consortium in different waste
waters.
To study the application of
Micro-algae as bio-fertilizers
and aqua-feeds –
• To check the fish toxicity of
generated algal biomass.
• To calculate its LD50 and LC50
value.
• To carry out a study to check
the applicability of the algal
biomass as a bio-fertilizer.
• To check the morphological
characters and biochemical
parameters of the grown
plant.

REVIEW OF LITERATURE
• It has been stated in by N. Renuka et.al., (2015) that Phycoremediation is the green technology that supports the direct use
of living green microalgae for in situ, or in place, removal, degradation, of contaminants in soils, sludge, sediments, surface
water and ground waters.
• It has been studied by J.B.K. Park, R.J. Craggs & A.N. Shilton (2010) that mass cultivation of microalgae can be
achieved by the construction of High rated algal ponds (HRAP) also know as race-way ponds by providing an co2 sump at
the bottom.
• I. Rawat et.al (2011), emphasized on the dual application of microalgae for Phycoremediation of domestic waste waters
and biomass production for sustainable biofuels production by using HRAP.
• Sathish Rajamani et.al (2007) Ability of microalga to adsorb and metabolize trace metals is associated with their large
surface: volume ratios, the presence of high-affinity, metal-binding groups on their cell surfaces, and efficient metal uptake
and storage systems.
• Ting Cai, Stephen Y. Park and Yebo Li (2013) stated that Microalgae have potential to remove nitrogen, phosphorus, and
toxic metals from natural wastewater under controlled environments. If key nutrients in the wastewater stream can be used
to grow microalgae for biofuel production, the nutrients can be removed, thus significantly reducing the risk of harmful
phytoplankton overgrowth.
• It has been studed by Ignacio de Godos et.al (2010) that two conventional chemical coagulants (FeCl3 and Fe2 (SO4)3)
and five commercial polymeric flocculants (Drewfloc 447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 and
Chitosan) were comparatively evaluated for their ability to remove algal–bacterial biomass from the effluent of a
photosynthetically oxygenated piggery wastewater biodegradation process.
• It has been reported by Priyadarshani, I., & Rath, B. (2012) that Microalgae are employed in agriculture as bio fertilizers
and soil conditioners. The majority of cyanobacteria are capable of fixing atmospheric nitrogen and are effectively used as
biofertilizers.
• Shields, R. J., & Lupatsch, I. (2012.) microalgae find uses as food and as live feed in aquaculture for production of
bivalve molluscs, for juvenile stages of abalone, crustaceans and some fish species and for zooplankton used in aquaculture
food chains.

Materials & Methodology
 Isolation of Algae by single
cell capillary method –
1. Product development & activation
• Micro-alga were observed under
microscope (45x resolution).
• Then single cells were isolated by using
thin hair-line capillary.
• This was conducted under sterilized
condition.
 Mass-cultivation & harvesting the micro
algae –
 Mass-cultivation was achieved by introducing isolated algae into 20
litres of R.O. water.
 Certain macro-nutrients like urea and single super phosphate were
added as a source of nutrient to alga.
 Certain micronutrients like humic acids and auxins and cytokinins
were added.
 Harvesting of live algae can be achieved by naturally evaporating the
water and scrapping out he algal biomass or it can be achieved by
applying very little centrifugal force.

 Development of Organic Green Live
(OGL) –
 OGL (Organic Green Live) is a universal product for
the treatment of industrial wastewaters.
 OGL is an algae based green product consists of
different strains of microalgae belonging to
chlorophyceae class like Chlorella, Synadusmus,
Chlorococum etc.
 After harvesting of the microalgae, each strain was
mixed in equal quantity in addition with Mixture of
Different macro nutrient nutrients like Single Super
Phosphate (S.S.P) and Ammonia.
 A specific mixture of micronutrients known as Black
and brown nutrient (commercially available) was added
to OGL.
 These nutrients provide some basic micronutrients like
Zn, Fe, Mo, Co, Ni, Cr, Mn etc. which promotes the
growth of OGL.
 This entire mixture was then preserved by the addition
of formalin.
 The minimum shelf life of this product is 6 months (can
be identified by the aroma of ammonia present in the
product.
 Activation of Organic Green Live (OGL)
–
 Different quantities like 1gm, 2gm, 3gm, 4gm, 5gm of OGL
was added in 5 consecutive bottles having 1 litre of R.O.
water and was kept for 15 days of incubation at 25-32° C.
 Cell count was monitored at 45X under haemocytometer at
every 3 conductive days.
 Principle of Hematocytometer - Hemocytometer (or
haemocytometer) is a device originally designed and
usually used for counting blood cell. Invented by Louis-
Charles Malassez and consists of a thick glass microscope
slide with a rectangular indentation that creates a chamber.
This chamber is engraved with a laser-etched grid of
perpendicular lines. The device is carefully crafted so that
the area bounded by the lines is known, and the depth of
the chamber is also known. By observing a defined area of
the grid, it is therefore possible to count the number of
cells or particles in a specific volume of fluid, and thereby
calculate the concentration of cells in the fluid overall. The
gridded area of the hemocytometer consists of nine 1 x
1 mm (1 mm2) squares. These are subdivided in 3
directions; 0.25 x 0.25 mm (0.0625 mm2), 0.25 x 0.20 mm
(0.05 mm2) and 0.20 x 0.20 mm (0.04 mm2).

 Development of OGL boost
 OGL boos consist of mixture of various vitamins and
micronutrients.
Vitamin tablets (commercially available) – tablets of 1
gram each –
B1 – Thiamine mononitrate
B2 – Riboflavin
B3 – Niacianamide
B5 – Pantothenic acid
B6 – Pyridoxine
B7 - Biotin
B9 – Folic acid
B12 – Cobalamin
Black and brown nutrients (commercially available) –
Mixture of micro nutrients like ferrous, zinc, cobalt,
manganese, molbdate, and nickel etc.
Each tablet of vitamins (1 gram) were powdered and
mixed in the R.O. water along with mixture of
micronutrients.
 Estimation of Chlorophyll content

Principle – Chlorophyll is an organic compound having a
magnesium ion in the center surrounded by 4 nitrogen atom
followed by a pyrole ring at each side. This chlorophyll can be
extracted by using certain organic solvents like methanol. The
concentration of the chlorophyll can be measured at 663nm
and 645nm in spectrophotometer.
Procedure –
 3ml algal culture was centrifuge at 5000rpm for minute at
4ºC to get the supernatant and pallet.
 Pallets were homogenized into 8% methanol by addition of
MgCO3.
 The solution is boiled for 3-4 minutes on Bunsen burner to
rapture the cell walls.
 Bring the methanol containing chlorophyll at room
temperature and take O.D. at 663nm and 645nm.
 Calculation
Chlorophyll-a = 11.47(663nm) - 0.40(645nm)
Chlorophyll-b = 20.97(645nm) - 3.94(663nm)
Total Chlorophyll = Chlorophyll-a + Chlorophyll-b

2. Possible applications of OGL to treat Industrial waste waters –
 Site selection –
 The product OGL can be used in 2 forms –
i. Free form for reduction of ammonia in fish processing
industries based at Ratnagiri Mahastra.
ii. Combined form – for reduction of C.O.D. at NCTL CETP,
Anakleshwar.
iii. For reduction of colour, C.O.D., TDS, and Hardness at
RSWM textile industry, Bhilwara, (Rajasthan).
Gadre Marine Export Limited (Ratnagiri), Maharastra
Rajasthan Spinning & Weaving Mills
(RSWM), Bhilwara (Raj.)
Narmada Clean Tech. Limited (NCTL),
Anakleshwar, Gujarat

 Development of Algal-bacterial consortium –
Algal-Bacterial
Consortium (ALBA)
Micronutrient
and growth
promoters
OGL
Acclimatized
MLSS
 ALBA was developed under
mild aeration.
 Entrapment of micro algae
into acclimatized MLSS was
checked by tracing the
Chlorophyll content in the
settled biomass.
 ALBA helps in increase in
efficiency of activated sludge
process.

 Collection of Waste waters –
• Field work was carried out at 3 different
Industries during the month of January
2018 to march 2018. Industrial waste
water samples were collected from the
Equalization tank and Aeration tank
respectively. MLSS of the industrial
waste water was collected from the
activated sludge process at an interval
of 20 days. Waste water samples were
taken to the laboratory and were kept
open to avoid the anaerobic condition.
Then these waste waters were used for
parameter analysis like Color, odor, pH,
TS, TDS, TSS, C.O.D. Ammonical
Nitrogen, Hardness, and Chlorides.
Physical and chemical properties of the
waste waters were checked by using
analysis manuals such as “Standard
Methods for the Examination of Water
and Wastewater” (APHA 20th Edition).
 Analysis of physico-chemical parameters of Waste
waters –
Parameters Method used Instrument used
pH Potentiometric
Method
pH meter
TSS Gravimetric method -
TDS Gravimetric method -
Total Hardness EDTA Titrimetric
Method
-
Chemical Oxygen
Demand
Open Reflux Methviii.
Sludge
Volume Index –od
C.O.D Digester
Colour Spectrophotometric
Method
Spectrophotometer/co
lourimeter
Ammonical Nitrogen Titrimetric Method Kjeldhal Assembly
Sludge Volume Index Gravimetric Method -

3. Applications of algal biomass as a bio-fertilizer and aqua feeds –
 Preparation of Set up to check the effect of Algal Biomass on
Seed germination and Plant growth –
 Garden Soil was collected from EDII campus Ahmedabad.
The soil collected was then cleaned and sieved to remove the
pebbles gravels and leaf litters. Four consecutive sets were
containing 10% of algal biomass, 20% of algal biomass and
3o% of algal biomass blended with soil respectively. A
control tray was set up containing 3Kg of soil in it. A plant
species Phaseolusmung.L (Moong) was selected for this
study. Ten seeds were sown in each tray which was irrigated
with 500-600ml of water daily. The trays were kept at a
place where the plant received 12hours of light period and 12
hours of dark period.
 The selected species of plant took 3-4 days for germination
and within 2 weeks it was a full grown plantlet. Phycico-
chemical parameters like dry weight, wet weight, root
length, shoot length, leaf length was monitored followed by
some biochemical parameters like protein content,
carbohydrate content etc.
 Estimation of morphological characters &
Biochemical Parameters –
Bio-chemical
Parameters
Method used Instruments
used
Total
Chlorophyll
content
Acetone as a
extracting
solution
Cenrifuge,
Spectrophoto
meter
Protien Orcenol
Method
Cenrifuge,
Spectrophoto
meter
Carbohydrates Anthron
reagent
Cenrifuge,
Spectrophoto
meter

 Application of microalga solution as aqua
feeds –
Principle – Algae are a good source of protein, omega 3 fatty acids,
phosphate and nitrates. Thus algae are used as the aqua feed
cultures in many fish and shrimp industries. The toxicity study of
the algae has to be checked to ensure the harmful effect of the algae
to the aquatic ecosystem.
Apparatus –
1m*0.5m aquarium, 10-15 different species of fishes, Aerators, 20
liters of activated OGL water,
Procedure –
To study the fish toxicity 20 liters of activated OGL was introduced
into and an aquarium containing 15 fishes. The Mortality rate of the
fishes were observed upto 96hours followed by some toxicity
symptoms like change in eye color, descaling of the skin, impaired
function of gills etc. LD50 and LC50 values were calculated using
following formula.
Calculation of LD50 and LC50 –
It can be calculated by plotting Dose and response
curve and by tracing the mortality rate.
LC50 is concentration of a toxicant that is
Lethal to a 50% to the test organisms.
The Median lethal concentration is the usual
Method of reporting acute toxicity studies.
A statistically derived expression of a single
Dose of a material that can be expected to kill
50% of the animals.
LD50 is generally utilized in case of non-
Aquatic animals.

RESULTS & DISSCUSSION
• Activation of OGL –
0
0
380,000
2,060,000
3,210,000
8,000,000
0
0
600,000
3,970,000
5200000
12,200,000
0
0
720,000
4,210,000
7200000
18,000,000
0
0
950,000
4,410,000
25,600,000
29,100,000
0
0
2,300,000
12,000,000
32,300,000
41,000,000
0
5,000,000
10,000,000
15,000,000
20,000,000
25,000,000
30,000,000
35,000,000
40,000,000
45,000,000
0 3RD 6TH 9TH 12TH 15TH
CELLCOUNT/ML
DAYS OF INCUBATION
COMPARATIVE STUDY-ACTIVATION OF OGL
1gm OGL + 1 Liter R.O. water 2gm OGL + 1 Liter R.O. water 3gm OGL + 1 Liter R.O. water
4gm OGL + 1 Liter R.O. water 5gm OGL + 1 Liter R.O. water
Figure 5 - Cumulative graph on activation period of OGL depicting the time-course study

Figure 6 – No appearance of microalgal cells on
0th day and 3rd day.
Figure 7 – Appearance of microalgal cells on day 15th
of sample containing 0.1% (w/v) of OGL in R.O. water.
Figure 8 – Appearance of microalgal cells on day 15th of
sample containing 0.2% (w/v) of OGL in R.O. water.

To check the effect of macro/micro-nutrients on the cell growth of OGL–
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
1 liter R.O. water + 5gm OGL + 100ppm urea + 50ppm
S.S.P. + 5mg Cytokinin
S.S.P. + 3mg Cytokinin + 2mg Auxin
S.S.P. + 3mg Auxin + 2mg Cytokinin
S.S.P. + 2mg cytokinin + 2mg Auxin
4330000
4450000
4570000
4820000
17900000
24200000
18900000
21900000
3650000
3850000
2410000
3120000
3590000
2900000
2500000
2500000
3530000
2860000
3120000
2200000
% Growth of cells a different incubation days
Experiments
Zero 2nd 3rd 4th 5th
Figure 12 – Effect of macro/micro nutrients on activation period of OGL.

To check the effect of nutrients and growth promoters on the cell growth of
OGL–
0% 10% 20% 30% 40% 50% 60% 70% 80% 90%100%
1 liter R.O. water + 5gm OGL + 100ppm urea +
50ppm S.S.P. + 5mg Cytokinin
50ppm S.S.P. + 3mg Cytokinin + 2mg Auxin
50ppm S.S.P. + 3mg Auxin + 2mg Cytokinin
50ppm S.S.P. + 2mg cytokinin + 2mg Auxin
4330000
4450000
4570000
4820000
17900000
24200000
18900000
21900000
3650000
3850000
2410000
3120000
3590000
2900000
2500000
2500000
3530000
2860000
3120000
2200000
% Growth of cells a different incubation days
Experiments
Zero 2nd 3rd 4th 5th
Figure 13 – Comparison of percentage growth of cells per ml by addition of growth promoters like auxins and cytokinins.

To check the effect of growth promoters on Algal-Bacterial (ALBA)
consortia development
3.55841
3.76521
5.35847
5.69825
3.0268
3.12236
3.11489
4.69728
3.78693
6.1523
8.35682
8.56289
3.92586
4.01796
4.52797
4.75264
0
1
2
3
4
5
6
7
8
9
10
0 3rd 6th 9th
Chlorophyllcontent(mg/ml)
No. of days of Incubation
150ml RSWM ALBA + 5mg Cytokinin 150ml RSWM ALBA + 5mg Auxin
150ml RSWM ALBA + 4mg Cytokinin + 1mg Auxin 150ml RSWM ALBA + 4mg Auxin + 1mg Cytokinin
Figure 14 - Comparative study on increase in entrapment of microalgae within algal bacterial consortia.

To check the effect of growth promoters on Algal-Bacterial (ALBA) consortia
development cont…
The above graph (Fig. 14) depicts the increase in the chlorophyll content was
achieved with increase in incubation time in ALBA preparation. A series of
experiments were carried out to study the increase in entrapment of algae
and bacteria by addition of various growth promoters like auxin and
cytokinins. The chlorophyll content was monitored to check the entrapment
of algae into the settled biomass of ALBA after every 3 consecutive days. The
above graph shows that maximum increase in chlorophyll after 9 days was
observed as 8.6mg/ml in sample containing 150ml of RSWM ALBA with 4mg
cytokinin and 1mg auxin (Fig. 17). Hence, increase in chlorophyll content
directly indicates the better entrapment of microalgae within ALBA consortia.
However, the similar results were observed by Rishiram Ramanan et.al.,
(2016) that Algae and bacteria shows symbiotic relationship as algae provides
oxygen to bacteria and in return algae receives carbon dioxide for
photosynthesis. Mean increase in chlorophyll content 4.30mg/ml which was
maximum in sample containing 150ml of RSWM ALBA with 4mg cytokinin and
1mg auxin (Fig. 17) and minimum chlorophyll content 3.49mg/ml was present
in sample having 150ml RSWM ALBA with 5mg Auxin (Fig. 16). This proves
that cytokinins are the growth promoters which enhances the cell division
whereas auxins will promote auxiliary growth. The mean increase in the
chlorophyll content is shown by the sample containing 150ml RSWM ALBA +
4mg cytokinin +1mg Auxin i.e. 6.71 (Fig. 17). A comparative study on
chlorophyll content signifies a positive effect of cytokinin and auxin on
increased concentration of micro-algae cells in ALBA (Fig. 17).
Figure 15 – Entrapment of micro-
algae into Bacterial floc on day 9th of
sample containing 150ml RSWM
ALBA along with 5mg Cytokinin.
Figure 16 – Entrapment of micro-algae
into Bacterial floc on day 9th of sample
containing 150ml RSWM ALBA along
with 5mg Auxin.
Figure 17 – Entrapment of micro-algae
into Bacterial floc on day 9th of sample
containing 150ml RSWM ALBA along
with 4mg Cytokinin & 1mg auxin.
Figure 18 – Entrapment of micro-algae into
Bacterial floc on day 9th of sample
containing 150ml RSWM ALBA along with
4mg auxin & 1mg Cytokinin.

Application of the Algal-bacterial (ALBA) consortium to treat industrial
Effluents
Site 1 - Rajasthan Spinning and Weaving mills (RSWM), Bhilwara (Raj.) –
i. De-colorization of Industrial effluent by anaerobic treatment using acclimatized MLSS bacterial consortia.
700 700 700 700 700 700 700
350
600
450
320
400
550
150
50%
10%
25%
40%
60%
25%
80%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
0
100
200
300
400
500
600
700
800
RSWM effulent +
Bactrial cultural 1
RSWM effulent +
Bactrial cultural 2
RSWM effulent +
Bactrial cultural 3
RSWM effulent +
Bactrial cultural 4
RSWM effulent + Alba RSWM effulent + MLSS RSWM effulent + MLSS
+ 20PPM PAC
%reductionincolor
color(pt.co.)
Experiments
colour at zero hour (Pt.Co.) Colour after 24 hours anaerobic digestion (Pt. Co.) % Reduction in Colour
Figure 19 - A comparative study on Color Reduction of Textile effluent by anaerobic treatment using various bacterial cultures
commercially available in the market and the acclimatized MLSS of Textile ETP.

Raw
effluent
Figure 20 - Color of Raw
effluent
Figure 21 - A comparative study on Color Reduction of Textile
effluent by anaerobic treatment using various bacterial
cultures commercially available in the market and
acclimatized MLSS of Textile ETP.
Figure 22 – Reduction in color by commercially available different bacterial
cultures as the outcome of 24hours of anaerobic treatment.
Figure 23 – Reduction in color by using acclimatized bacterial cultures of textile ETP as the outcome of
24hours of anaerobic treatment along with addition of 20ppm Poly Aluminum Chloride (PAC).
The image (Fig. 20, 21) depicts a comparative study
on colour reduction via anaerobic digestion using
commercially available bacterial culture and
acclimatized MLSS of textile ETP. The results
obtained are depicted below –
The images (Fig. 22, 23) gives a clear visual
interpretation of colour reduction via commercially
available bacterial cultures and the acclimatized
MLSS of textile ETP.
Bacterial
culture 1
Bacterial
culture 2
Bacterial
culture 3
Bacterial
culture 4
Acclimatiz
ed MLSS
Raw
effluent
Bacterial
culture 1
Bacterial
culture 2
Bacterial
culture 3
Bacterial
culture 4
Raw effluent
Colour reduction after 24 hrs.
of anaerobic treatment.
Raw effluent Colour reduction
after 24 hrs. of
anaerobic treatment.

ii. Evaluation of C.O.D. reduction of textile effluent by using different bacterial culture
available commercially and acclimatized MLSS after 24 hours of anaerobic digestion
1600 1600 1600 1600
1600
1600 1600
2432
2992 3024
2640
1168
1425
880
0% 0% 0% 0%
27.00%
10.90%
45.50%
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
0
500
1000
1500
2000
2500
3000
3500
RSWM effulent +
Bactrial cultural 1
RSWM effulent +
Bactrial cultural 2
RSWM effulent +
Bactrial cultural 3
RSWM effulent +
Bactrial cultural 4
RSWM effulent + Alba RSWM effulent + MLSS RSWM effulent + MLSS
+ 200PPM PAC
%ReductioninC.O.D.
C.O.D.(Mg/L)
Experiments
C.O.D. at zero hours C.O.D. at 24 hours of anaerobic digestion C.O.D. reduction
Figure 24 - A comparative study on C.O.D. Reduction of Textile effluent by anaerobic digestion using various bacterial cultures, commercially available in
the market and the acclimatized MLSS of Textile ETP.

iii. Colour Reduction by addition of acclimatized MLSS into textile effluents (Final operational
cycle at ETP)
710
760 750 740 750
120
240 230 260 240
90.47%
84.61%
88%
83.33%
84%
78.00%
80.00%
82.00%
84.00%
86.00%
88.00%
90.00%
92.00%
0
100
200
300
400
500
600
700
800
400ml RSWM effulent + 100ml
MLSS (Anaerobic treatment) +
20ppm PAC
20ppm PAC (orange shade)
MLSS (Anaerobic tretment) +
20ppm PAC (blue shade)
20ppm PAC (orange shade)
20ppm PAC (blue shade)
%reductionofcolour
Colour(Pt.Co.)
Experiments
colour zero hour (Pt.Co.) Colour after 24 hours anaerobic digestion (Pt. Co.) % Reduction in Color
Figure 25 - A comparative study on color Reduction of Textile effluent by anaerobic followed by aerobic digestion using various acclimatized
bacterial consortium of textile ETP.

iv. C.O.D. Reduction by addition of Algal-Bacterial consortium into textile effluents
(Final operational cycle at ETP)
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
400ml RSWM effulent + 100ml MLSS + 20ppm PAC (Anaerobic followed
by Aerobic digestion) + 100ml Alba in aeration
by Aerobic digestion) + 100ml Alba in aeration (orange shade)
by Aerobic digestion) + 100ml Alba in aeration (blue dye)
by Aerobic digestion) + 100ml Alba in aeration (orange shade)
by Aerobic digestion) + 100ml Alba in aeration (blue shade)
1376
1232
1184
1196
1176
780
720
576
680
590
43.33%
41.72%
51.60%
43.14%
49.90%
820
768
694
720
625
560
463
430
449
380
31.17%
39.71%
38.15%
37.60%
39.20%
% reduction in C.O.D.
Experiments
C.O.D. at zero hours C.O.D. at 24 hours of anaerobic digestion
C.O.D. reduction 24 hours aerobic digestion, C.O.D. at zero hours
24 hours aerobic digestion, C.O.D. after 24 hours hours % Reduction
Figure 26 - A comparative study on color Reduction of Textile effluent by anaerobic followed by aerobic digestion using various acclimatized

v. Reduction of total hardness by addition of Algal consortium into textile
effluents (Final operational cycle at ETP) –
1400
1600
1260
1000
1100
940
28.85% 31.25% 25.39%
0
200
400
600
800
1000
1200
1400
1600
1800
RSWM effulent (20%) + Activated
OGL (80%)
1 Liter of R.O. Reject + 5gm OGL 20% R.O. reject + 80% raw algae of
GAL
TotalHardness(mg/L)
Experiments
initial hardness Hardness after 48 hour % reductio in hardness Linear (Hardness after 48 hour)
Figure 46 - A comparative study on Reduction in total hardness of Textile effluent by using specific amount of OGL.

vi. Re-designing of water treatment process by addition of Algal-Bacterial consortium into
textile effluent Plant (Final operational cycle at ETP)
Equalization
Tank
Neutralization
Tank
Primary
Clarifier
Aeration
Tank
Secondary
Clarifier
- Lime
- FeSo4
Dosing Tank
High sludge
generation
Tube
settler
Sand carbon
filter
R.O.
Plant
R.O.
Reject
M.E.E.
Plant
(Scaling due to
hard water)
Sludge drying
Bed
Landfill
Figure 27 – The former process flow chart of RSWM textile mill illustrating the problems faced during the operation of ETP.
Raw Inlet

Equalization
Tank
(pH – 8 to 9)
Anaerobic
Digester
(pH 7-8)
20PPM Poly
Aluminium
Chloride (PAC)
Algal Bacterial
Consortium
Aeration Tank
(pH – 7)
Landfill
Colour reduction
by 25%
C.O.D. reduction
by 12%
Colour reduction –
85-90%
C.O.D. reduction - 45-
50%
C.O.D.
reduction - 45-
50%
Satability
increase
by 40%
Sand
carbon
filter
R.O.
Plant
Algae growth
tank
(Hardness
reduction by
25-31%)
M.E.E.
Plant
(reductio
n in
Scaling)
Sludge drying
Bed
Dosing
Tank
R.O.
Reject
Activated
Sludge
Process
Raw Inlet
Figure 28 - New improved process flow chart of RSWM textile mill to treat the waste water by introducing Algal-bacterial consortium and anaerobic followed by
aerobic digestion to reduce color , C.O.D. and to neutralize pH.

Site 2: Gadre Marine export limited –
i. Reduction of Ammonical Nitrogen (NH3-N) from Fish processing Industrial waste waters –
130.25 130.25 130.25 130.25130.25
103.25
91.35
80.32
0.00%
20.70%
29.86%
38.33%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
0
20
40
60
80
100
120
140
Raw Effulent sample 800ml Effulent + 200ml
Activated OGL + 150 S.S.P.
800ml Effulent + 200ml
Activated OGL + 150 S.S.P. +
5PPM Humic Acid
800ml Effulent + 200ml
Activated OGL + 150 S.S.P. +
4PPM Auxin + 20PPM
Cytokinin
%reductioninammonicalNitrogen(mg/L)
AmmonicalNitrogen(mg/L)
Experiments
Initial Ammonia 48hrs Ammonia % decrease in Ammonia
Figure 29 - A comparative study of reduction of Ammonical nitrogen by treating the waste waters with microalgae and effect of micronutrients and growth
promoters in increasing the efficiency of reduction in ammonical nitrogen (cycle 1).

Reduction of Ammonical Nitrogen (NH3-N) from Fish processing Industrial
waste waters (cycle 2 )
121 121 121 121121
106
80
70
0.00%
12.39%
33.88%
42.14%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
0
20
40
60
80
100
120
140
Raw Effulent sample 800ml Effulent + 200ml Activated
OGL + 150 S.S.P.
800ml Effulent + 200ml Activated
OGL + 150 S.S.P. + 5PPM Humic
Acid
800ml Effulent + 200ml Activated
OGL + 150 S.S.P. + 4PPM Auxin +
20PPM Cytokinin
%reductioninAmmonicalNitrogen
AmmonicalNitrogen(mg/l)
Experiments
Initial Ammonia 48hrs Ammonia % decrease in Ammonia
Figure 50 A comparative study of reduction of Ammonical nitrogen by treating the waste waters with microalgae and effect of micronutrients and growth
promoters in increasing the efficiency of reduction in ammonical nitrogen (cycle 2).

Site 3: Narmada Clean Tech. Limited, (FCETP) Anakleshwar –
i. To check the metabolic activity of the MLSS obtained from the activated sludge process of CETP –
1808
1152
3232
3584
1344
352
3056
2576
7200
5200
10000
8000
8800
6400
14800
10800
25.66%
69.44%
5.44%
28.12%
22.22% 23.00%
48%
35%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
0
2000
4000
6000
8000
10000
12000
14000
16000
400ml Tap water + 1000PPM
Glucose + 500PPM Urea +
250PPM SSP + 100ml MLSS
400ml Tap water + 1000PPM
250PPM SSP + 100ml ALBA
400ml NCTL effulent + 1000PPM
250PPM SSP + 100ml MLSS
400ml NCTL effulent + 1000PPM
250PPM SSP + 100ml ALBA
%ofC.O.D.andMLSS
C.O.D.&MLSS(mg/L)
Experiments
C.o.d (zore hours) C.O.D. (after 48 hours) M.L.S.S. (Zero hour)
M.L.S.S. (48 hours) % reduction of C.O.D. % increace in M.L.S.S
Figure 30 - A study to check the metabolic activity of acclimatized bacteria of CETP in tap water and effluent.

ii. A comparative study on reduction of C.O.D. by pure seed culture and ALBA
0
5000
10000
15000
20000
25000
400ml NCTL effluent + 100ml
pure seed clture
pure seed clture + 1ppm black
+ 1ppm brown + 1ppm
vitamin
ALBA + 150mg SPP
pure seed clture + ALBA
+1ppm black +1ppm brown +
1ppm vitamins + 1ppm humic
acids
3008 2992 3200 3184
2976 2880 2848 2912
1.06% 3.74% 11.00% 9.50%
5600 5400 6520 7280
6920 5620
7160
11200
23.57%
4.07%
36.36%
53.82%
C.O.D.&T.S.S.(mg/l)
Experiments
C.o.d (zore hours) C.O.D. (after 48 hours) % reduction of C.O.D. M.L.S.S. (Zero hour) M.L.S.S. (48 hours) % increace in M.L.S.S
Figure 31 - A comparative study on reduction of C.O.D. and increment in MLSS after 48 hours of aeration given to
CETP effluent.

iii. To check the efficiency of ALBA in C.O.D. reduction by addition of different micro-
nutrients and growth promoters –
2384
2212 22482272
2123 21262096
1856
1662
4.60%
4.60%
6.70%
12.00%
16.40% 17.90%
0.00%
2.00%
4.00%
6.00%
8.00%
10.00%
12.00%
14.00%
16.00%
18.00%
20.00%
0
500
1000
1500
2000
2500
3000
400ml NCTL effluent + 100ml MLSS +
100mg SSP
400ml NCTL effluent + 100ml ALBA +
100mg SSP + 1ppm black +1ppm brown
nutrient + 1ppm vitamine
400ml NCTL effluent + 100ml ALBA +
100mg SSP + 1ppm black +1ppm brown
nutrient + 1ppm vitamine + 0.4ppm
cytokinins + 0.1 auxins
%reductioninC.O.D.
C.O.D.(mg/l)
Experiments
C.o.d (zore hours) cycle 1 C.O.D. (after 48 hours) (cycle 1) cycle 2 C.O.D. (after 48 hours) (cycle 2)
cycle 1 % reduction of C.O.D. cycle 2 % reduction of C.O.D.
Figure 32 - A comparative study on reduction of C.O.D. by ALBA and dose of growth promoters after 48 hours of aeration given to
CETP effluent.

To study the application of algal biomass as a fertilizer and aqua
feed –
i. Study of Phsico-chemical Parameters –
1st
Week
Experiments Fresh weight of
Phaseolus moongo (gm)
Dry weight of
Phaseolus moongo
(gm)
% moisture content in
Phaseolus moongo
Control 0.178 0.051 71.34%
Soil + 10% algal
biomass
0.246 0.053 78.45%
soil + 20% algal
biomass
0.000 0.000 0.000%
soil + 30% bio mass 0.000 0.000 0.000%
2nd
Week
Experiments Fresh weight of
Phaseolus moongo
Dry weight of
Phaseolus moongo
% moisture content in
Phaseolus moongo
Control 0.439 0.063 85.56%
Soil + 10% algal
biomass
0.592 0.049 91.72%
soil + 20% algal
biomass
0.000 0.000 0.00%
soil + 30% bio mass 0.000 0.000 0.00%
Table 1 - Morphological characterization of plant Phaselous moong of sample containing control and different
concentrations of micro algal biomass to be used as a bio-fertilizer.
Root
length
(cm)
Shoot
length
(cm)
Leaf size
(cm)
Leaf no.
6 3.1 1.8 2
8 4.4 2..1 2
0 0 0 0
0 0 0 0
Root
length
(cm)
Shoot
length
(cm)
Leaf size
(cm)
Leaf no.
8 4.4 2.2 2
10.4 5.2 2.5 2
0 0 0 0
0 0 0 0

ii. Estimation of Bio-chemical Parameters of Phaselous moong –
Total chlorophyll content
Totl carbohydrate content
Total protien content
0
2
4
6
8
10
12
Control Soil + 10% algal
biomass
soil + 20% algal
biomass
soil + 30% bio
mass
4.73
5.78
0 0
5.3
7.5
0 0
7.13
10.59
0 0
BIO-CHEMICALPARAMETERS
CONCENTRATIONOFBIOMOLECULES(MG/ML)
EXPERIMENTS
Total chlorophyll content Totl carbohydrate content Total protien content
Figure 33 - An estimation of Biochemical parameters like chlorophyll, carbohydrate and protein present in
Phalous moong during its initial growth phase (week 1) to study the application of microalgal biomass to be used
as a bio fertilizer.

Total chlorophyll
Total Carbohydrate
Total Protien
0
5
10
15
20
25
Control Soil + 10% algal
biomass
soil + 20% algal
biomass
soil + 30% bio
mass
15.75
19.45
0 0
12.5
15.4
0 0
18.25
20.56
0 0
BIO-CHEMICALPARAMETERS
CONCENTRATIONOFBIOMOLECULES(MG/ML)
Experiments
Total chlorophyll Total Carbohydrate Total Protien
Figure 34 - An estimation of Biochemical parameters like chlorophyll, carbohydrate and protein present in
Phalous moong during its initial growth phase (week 2) study the application of microalgal biomass to be
used as a bio fertilizer.

To study the application of micro-algal as an aqua feed for fishes and
other organisms –
• The application of micro-algal solution to be used as an aqua feeds was studied
by designing an experiment in an aquarium containing 10 fishes of different
specie like sophore, ticto, striatus etc. along with 20 liters of micro-algal
solution. The morphological characteristic changes were observed upto 96
hours and LD50 was calculated.
• It has been studied that there was no morphological changes were observed
like change in eye color, de-scaling of fishes, impaired gill functioning etc upto
and beyond 96hours. This indicates that the algal solution incorporated in the
aquarium was nontoxic to the fishes. Thus, here was no mortality in the fish
population by consuming the micro-algal cells from the water, dose vs.
Response graph was nullified and hence, the fishes were healthy.
• It has been studied by Shields, R. J., & Lupatsch, I. (2012.) that microalgae can
be used as food and as live feed in aquaculture for production of bivalve
molluscs, for juvenile stages of abalone, crustaceans and some fish species and
for zooplankton used in aquaculture food chains.

Conclusion
• Phycoremediation is a green technology that supports the direct use of living green microalgae for in situ, or in place
removal, degradation, of contaminants in soils, sludge, sediments, surface water and ground waters by the
mechanisms of bio-transformation, bio-accumulation, bio-concentration, bio-sparging.
• It can be concluded by the current study that microalgae has a great potential for the treatment of industrial and
municipal wastewaters as compared to the chemical treatments available commercially. Biological systems are much
more efficient in cleaning the excess nutrients from the waste water followed by generation of valuable biomass which
can be applied in the food, fertilizer, energy production as use of inorganic chemicals like lime and ferrous sulphate
generates huge amount of sludge in textile industries, but on the other hand static anaerobic treatment using
acclimatized MLSS gives better colour reduction with zero sludge generation. Microalgal cells can be used in free form
to treat waste waters containing high C.O.D., high ammonical nitrogen and high TDS. It not only provides a better
reduction of chemicals from wastewaters but it also helps to reduce the operational cost of ETP. Microalgaes not only
helps to remediate industrial waste waters but also to treat sweage water and to restore natural water bodies like lakes
and ponds. As they are active in remediating the chemicals but also it shows an antagonistic effect against some
pathogenic germs like total coliforms and fecal coliforms.
• These microalgal cells can also be combined with bacterial biomass of activated sludge process to develop an Algal-
Bacterial consortium (ALBA) for better enhancement in the reduction of chemicals from the wastewaters as this
symbiotic relation of algae and bacteria provides high satiability of the microalgae along with MLSS and faceable in
terms of price and economy for instance the bacterial biomass provides carbon dioxide to algal cells for photosynthesis
and in return the bacteria acquires oxygen from algae. The harvested biomass from the ETP’s can be used as bio-
fertilizers as it consists of appropriate ratio of vital macro and micro nutrients like N,P,K etc. which enhance the growth
of plantlets. It can also be used as aqua feeds for shrimps, fishes and molluscs. Furthermore these microlgal cells are
non-toxic in the environment as it becomes a part of food chain and do not cause eutrophication. Therefore, micro-
algal based treatment is most suitable for the treating the waste waters and restoring the natural water bodies as
compared to other chemical treatments.

Scope for future study
• Micro-algae like any biological treatment requires a specific contact period and
retention time to treat waste waters which can be altered by various means. Thus,
this area of research has not been much explores. The idea is to reduce the retention
time by maintaining highest efficiency or in other words to boost the metabolic
activity of micro algae to treat waste waters with minimum time and maximum
efficiency.
• This present study shows 85-90% reduction in colour by anaerobic treatment but it
has been reported by many researchers that micro-aerophyllic conditions gives much
better reduction in colour and is also efficient in reducing C.O.D. which has to be
explored and implemented at industrial levels.
• Harvesting of the micro-algal biomass is achieved by scraping out the algae grown of
the substratum but, settling of live algal biomass has been a major challenge in the
field of Phycoremediation, which has not been much explored and has to be
discovered in near future.
• Chemicals like chloride and fluorides are the major issue in the wastewaters but no
evidence has been recorded for the reduction to these chemicals by using microalgae.
• To isolate and develop a product by using micro-algae that can work on extreme high
and low temperature conditions.

References
1. ABOU, E. M. M., & Hoda, M. A. (2005). Relationships, growth, yield of broccoli with increasing N, P or K ratio in a
mixture of NPK fertilizers.
2. Amarasinghe, U., Shah, T., Turral, H., & Anand, B. K. (2007). India's water future to 2025-2050: Business-as-usual
scenario and deviations (Vol. 123). IWMI.
3. Borde, X., Guieysse, B., Delgado, O., Muñoz, R., Hatti-Kaul, R., Nugier-Chauvin, C., ... & Mattiasson, B. (2003). Synergistic
relationships in algal–bacterial microcosms for the treatment of aromatic pollutants. Bioresource technology, 86(3),
293-300.
4. Cai, T., Park, S. Y., & Li, Y. (2013). Nutrient recovery from wastewater streams by microalgae: status and prospects.
Renewable and Sustainable Energy Reviews, 19, 360-369.
5. Christenson, L., & Sims, R. (2011). Production and harvesting of microalgae for wastewater treatment, biofuels, and
bioproducts. Biotechnology advances, 29(6), 686-702.
6. Gleick, P. H., (1996) U.S. Geological Survey Department of the Interior/USGS
7. González, L. E., Cañizares, R. O., & Baena, S. (1997). Efficiency of ammonia and phosphorus removal from a Colombian
agroindustrial wastewater by the microalgae Chlorella vulgaris and Scenedesmus dimorphus. Bioresource
Technology, 60(3), 259-262.
8. Gupta, V. K., Ali, I., Saleh, T. A., Nayak, A., & Agarwal, S. (2012). Chemical treatment technologies for waste-water
recycling—an overview. Rsc Advances, 2(16), 6380-6388.
9. Hammouda, O., Gaber, A., & Abdelraouf, N. (1995). Microalgae and wastewater treatment. Ecotoxicology and
Environmental safety, 31(3), 205-210.
10. Hanumantha Rao, P., Ranjith Kumar, R., Raghavan, B. G., Subramanian, V. V., & Sivasubramanian, V. (2011). Application
of phycoremediation technology in the treatment of wastewater from a leather-processing chemical manufacturing
facility. Water Sa, 37(1), 07-14.

Dissertation ppt biostimulation- a potential practice for wastewater treaatment by using selected species of chlorophyceae family

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Dissertation ppt biostimulation- a potential practice for wastewater treaatment by using selected species of chlorophyceae family

Similar to Dissertation ppt biostimulation- a potential practice for wastewater treaatment by using selected species of chlorophyceae family (20)

More from Sumer Pankaj

More from Sumer Pankaj (8)

Recently uploaded

Recently uploaded (20)

Dissertation ppt biostimulation- a potential practice for wastewater treaatment by using selected species of chlorophyceae family