Domestic Wastewater Treatment Using Microalgae

1.0 Introduction
“Thousands have lived without love, not one without water” said W.H.Auden, a British
poet, who witnessed the accounts of the Basra water crisis in 2018. The brackish water pouring
from the taps of homes in Basra has caused stomach ailments and skin rashes for thousands in the
southern Iraqi city once famous for its network of freshwater canals that gave it the nickname the
"Venice of the East." Wastewater pollution is a global environmental crisis as 80% of the world’s
wastewater is dumped back into the environment untreated,1 polluting the ecosystem while
drastically reducing the gross total suitable water to sustain human life and activity. According to
the United Nations, lack of awareness for proper wastewater treatment globally will cause
detrimental effects to the global clean water supply with estimations that total available clean water
will reduce to one-third by 2050.1 Wastewater is characterized as a by-product of human activities
like domestic and industrial usage which produces water discharges containing organic and
inorganic substances. Most places employ the primary and secondary treatment process which
filters easily settled materials and degrade the organic material present in wastewater. However,
water that undergoes the primary and secondary treatment still contains inorganic nitrogen and
phosphorus which causes eutrophication if discharged without further removal. Therefore,
researchers have sought the usage of microalgae culture as it provides a tertiary biotreatment
coupled with the production of biofuels. In this proposal, we would like to propose a research to
investigate the suitable strain and operating conditions for effective wastewater treatment using
microalgae.

2.0 Literature Review

Modern wastewater treatment process consists of three stages with the aim of removing
biochemical oxygen demand (B.O.D), suspended solids, nitrogen and phosphorus nutrients,
bacteria and toxic waste. Of the three stages present in wastewater treatment, tertiary treatment is
designed to remove all organic ions with the use of microalgae system as the discharge of these
nutrients into sensitive water bodies will lead to eutrophication as well as poisoning of aquatic life
caused by non-ionized ammonia. The success of microalgae system in modern wastewater
treatment is highly attributed to the ability of microalgae to take up nitrogen and phosphorus
nutrients from wastewater for their growth and production of valuable biofuel, making the
implementation of the system highly attractive as it resolves the problem of wastewater pollution
and fuel depletion. Not only that, compared to physical and chemical treatment processes, algae
based treatment can potentially achieve nutrient removal in a less expensive and ecologically safer
way with the added benefits of resource recovery and recycling.2 The nutrient removal efficiency
is further enhanced as microalgal growth intensifies leading to increased pH thus stripping toxic
ammonia.3

As domestic wastewater is unique in its variation of chemical composition and physical

properties, the identification of a dominant strain and factors that affect microalgae growth is
crucial to the successful treatment of domestic wastewater. It is reported that there are currently
240 genera and 725 species of microalgae present in domestic wastewater around the world and
several studies have been carried out to narrow down the most tolerant genera which currently
includes eight green algae, five blue greens, six flagellates and six diatoms.4 In Palmer’s
investigation of the American sewage stabilization ponds, he determined that microalgae strain
species like Chlorella, Ankistrodesmus, Scenedesmus, Euglena, Chlamydomonas, Oscillatoria,
Micractinium and Golenkinia were shown to have high tolerance towards diversified chemical
profiles as well as high growth rate.5 On the other hand, analysis by Erganshev and Tajiev
concluded that Chlorophyta, Cyanophyta, Bacillariophyta and Euglenophyta occurred in larger
variety and quantity from sewage ponds in Central Asia.6

Microalgae can grow under a broad range of temperatures ranging from 15 to 40 °C

depending upon strain, region, and season.7 Below optimal growth temperatures, growth rate
increases with increasing temperature but declines markedly above the specific optimum.7 Optimal
temperature allows efficient nutrient utilization, photosynthesis and minimal cell size.8 Low
temperatures decreases cell membrane fluidity which results in inefficient utilization of carbon and
nitrogen nutrient causing photoinhibition. To sustain its photosynthetic activities, cells secret
unsaturated fatty acids to increase fluidity but this reduces its growth rate as the membranes more
susceptible to damage by free radicals.7 Increasing the temperature beyond the optimum reduces
protein synthesis of microalgae which decreases growth rate.7 A study involving the growth rate
of Botryococcus braunii at three different temperatures (18°C, 25°C, and 32°C) has shown that at
32°C there was no growth as lipid synthesis was inhibited while sizes of cells at 18°C were smaller
than that at 25°C.9 Similar effects were observed in Nannochloropsis oculata and Chlorella
vulgaris, both of which have an optimum growth temperature of 25 °C. Increasing the growth
temperature from 20 to 25 °C doubled the growth rate while an increase of temperature from 25
to 30 °C saw a decrease in growth rate by three times.10

Microalgae attains maximum growth rate around neutral pH (7.0-7.6). CO2 solubility is
limited at high pH, which reduces the photosynthetic activity of microalgae required for growth
while nutrient utilization decreases and metal toxification might occur at low pH.7 A study by
Visviki and Santikul on the growth rate of Chlamydomonas applanata within a pH range 1.4 to
8.4, demonstrated that no growth was observed at low pH (1.4-3.4) while growth rate increased
when pH was increased from 5.4 to 8.4, achieving maximum growth at pH 7.4.11 Some research
suggests that despite neutral pH being the ideal optimal pH, some microalgae can adapt to different
pH conditions and grow optimally as long as pH does not change once the microalgae has adapted
to it. Examples are seen in studies like placing Chlamydomonas sp. in highly acidic conditions
(pH=1) where Tatsuzawa observed that Chlamydomonas sp. was able to adapt and maintain
cellular metabolism12 while Fontes found that Anabaena variabilis thrive in slightly alkaline
conditions (8.2-8.4) but could not survive beyond pH 9.13 Therefore, the optimal pH for each
microalgae strain species might vary and research has to be carried out to determine the optimal
pH for a particular strain which is required for efficient wastewater treatment.

3.0 Proposal of Method

To determine the dominant strain of microalgae to grow in domestic wastewater,
wastewater samples will be collected from five areas to provide seven microalgae strain species.
The microalgae strains will be cultivated in a lab before being placed into 5 different sewage ponds
for 1 month. Samples will be collected from each pond on a weekly basis and the total of each
microalgae from the five ponds will be tabulated and compared. The species with the highest
proportion in all five ponds is the dominant strain of microalgae. To determine optimum
temperature for removal of nitrogen from domestic wastewater, the microalgal strain will be
cultivated and transferred into five bioreactor with domestic wastewater. Temperatures will be set
at 10°C, 15°C, 20°C, 25°C and 30°C respectively and samples will be collected every hour from
each reactor to analyze and quantify nitrogen removal. The temperature which results in the lowest
nitrogen content will be the optimal temperature for nitrogen removal by microalgae. To determine
optimum pH for removal of nitrogen from domestic wastewater, the microalgal strain will be
cultivated and transferred into five bioreactor with domestic wastewater. The pH will be set at 7,
8, 9, 10, and 11 respectively and samples will be collected every hour from each reactor to analyze
and quantify nitrogen removal. The pH which results in the lowest nitrogen content will be the
optimal pH for nitrogen removal by microalgae.

4.0 Problem Statement

The determination of a suitable microalgal strain for domestic wastewater treatment is
difficult as most strains are generally sensitive to the imbalance in nutrient profile, deficiency of
some important trace elements and presence of inhibiting/toxic compounds in wastewater streams,
and only a limited number of strains within a few species could adapt well in different domestic
wastewater environments. Therefore, the selection of a dominant strain which can adapt well and
grow in domestic wastewater is crucial as an increase in total amount of microalgae increases the
nutrient removal efficiency. The growth rate of microalgae and its efficiency in removing nitrogen
nutrient from wastewater is tightly linked to environmental conditions like temperature and pH.
Optimal temperature is crucial for microalgal to grow and remove nitrogen as low temperatures
result in photoinhibition and low efficiency of nitrogen utilization while at high temperatures
reduces protein synthesis. At low pH, a microalgal nutrient uptake varies and metal toxicity is
induced. At high pH, the dissolved CO2 concentration in wastewater is too low to maintain
photosynthetic activity required for microalgal growth. Therefore, the optimal operating
parameters like temperature and pH has to be determined as its plays an important role in nitrogen
removal efficiency from domestic wastewater.

5.0 Research Objectives

1. To determine dominant strain of microalgae to grow in domestic wastewater.
2. To determine optimum temperature for removal of nitrogen from domestic wastewater.
3. To determine optimum pH for removal of nitrogen from domestic wastewater.

6.0 Materials and Methodologies

6.1 Determination of dominant microalgae strain
 Different wastewater samples were collected from several areas, such as ponds, aqueduct,
roadside pits, water blooms, and deep cuts to enrich the composition. After analyzing the samples
ecologically and systematically, specific algal phylum will be selected as materials. These algal
species are Cyanophyta, Chrysophyta, Bacillariophyta, Xanthophyta, Pyrrhopytha, Chlorophyta,
and Euglenophyta. 30 cell/mL-1 of each type of microalgae will be extracted from the algal mixture
and placed into seven 50 Litre bottles containing 40% water and 60% domestic wastewater to be
cultivated for 3 days. The cultivation conditions are in room temperature and atmospheric pressure.
Throughout the cultivation, the pH will not be altered. 12 hours of light intensity will be provided
per day. After 3 days of cultivation, these microalgae will be applied to 5 sewage ponds for 1
month. 10 Litre of each algal inoculum will be used in each pond. Every week, 1 Litre of sewage
sample will be collected from each sewage pond. The algal biomass will be systematically
determined based on cell number and volume. The cell number of volume of each microalgae will
be counted in a Goryaev counting chamber. The determination will be based on the classical
studies of A. Pascher (1915, 1925) and W. Heering (1914). The total of each microalgae from the
5 ponds will be tabulated and compared.

6.2 Determination of optimal temperature

Pure algal stock culture will be grown in Bristol medium and settled down for 1 hour before
being harvested.14 Later, the microalgae will be washed with sterilized deionized water and
transferred into five 20 Litre laboratory-scale stirred tank photobioreactor. Constant stirring is
provided to ensure no algal sedimentation. 2 compact fluorescent lamps were used to provide
sufficient light intensity for 12 hours a day for each bioreactor. The stirrer will be stopped for 1
hour and the 5L domestic wastewater will be fed into each reactor. Reactor 1 to 5 will be heated
or cooled, and maintained at 10°C, 15°C, 20°C, 25°C, and 30°C. After 10-days cycle, 10 Litre of
suspension will be removed and replaced by 10 Litre of fresh wastewater. There will be 4 cycles
in total. Every 1 hour, the temperature will be measured by using a temperature sensor. After that,
300 mL samples will be collected from each reactor to analyze and quantify the various forms of
nitrogen using Tecator Kjeltec Auto 1030 Analyzer. The data will be plotted in a line graph and
compared. The optimum temperature will be determined based on which reactor has the lowest
amount of nitrogen content.
6.3 Determination of optimal pH
The pure algal inoculum will be transferred into five 20 Litre laboratory-scale stirred tank
photobioreactor. Constant mixing is provided to ensure no algal sedimentation. 2 compact
fluorescent lamps were used to provide sufficient light intensity for 12 hours a day for each
bioreactor. The pH values of Reactor 1 to 5 will be maintained at 7, 8, 9, 10, and 11. After 10-days
cycle, 10 Litre of suspension will be removed and replaced by 10 Litre of fresh wastewater. There
will be 4 cycles in total. Every 1 hour, the pH will be measured by using a Crison pH electrode.
After that, 300 mL samples will be collected from each reactor to analyze and quantify the various
forms of nitrogen using Tecator Kjeltec Auto 1030 Analyzer. The data will be plotted in a line
graph and compared. The optimum pH will be determined based on which reactor has the lowest
amount of nitrogen content.

7.0 Research Hypothesis

In Erganshev and Tajiev (1986) study on seasonal variation of number of algal species,
Chlorophyta was dominant in both quantity and variety, in Spring, Summer, and Winter, except in
Autumn where Cyanophyta was dominant.6 In tropical country like Malaysia, which has weather
in between Spring and Summer, we predicted that after 3 months, Chlorophyta will be dominant
among these 7 algal phylum. According to Yanyan Su, et. al. (2011), who is able to achieve nearly
100% nitrogen removal, the temperature of each batch stopped at room temperature (20°C - 23°C)
at the end of each test. The pH also increased until 8.4, and had no sign of dropping. According to
K. Larsdotter (2006), when the temperatures near optimum microalgae can tolerate high light
intensities better before being inhibited, and the temperatures are around 15°C to 25°C. Besides,
at higher pH values, such as pH greater than 9, most of the inorganic carbon is in the form of
carbonates.15 Carbonates cannot be assimilated by the microalgae, which in return, will halt the
growth of microalgae. Thus, we predict that, to achieve full nitrogen removal, the optimum
temperature and pH is 25°C and 9.

8.0 Expected Outcome

Based on our literature review and research hypothesis, we predict that the Chlorophyta
will be dominant and amount to at least 45% of the total number of algal phylum, the optimum
temperature will be 25°C and the optimum pH will be 9.
9.0 Research Scope
9.1 Determination of dominant microalgae strain
This research aims to determine the dominant strain of microalgae out of seven species,
namely Cyanophyta, Chrysophyta, Bacillariophyta, Xanthophyta, Pyrrhopytha, Chlorophyta, and
Euglenophyta which will be retrieved from 5 areas (ponds, aqueduct, roadside pits, water blooms,
and deep cuts). Each strain will be cultivated for 3 days in the same manner as stated in part 6.1.
The strains will be placed into 5 different sewage ponds and 1L samples will be collected on a
weekly basis to measure cell number and volume. The whole research will take 2 months to
complete.

9.2 Determination of optimal temperature

This research aims to determine the optimal temperature for maximum nitrogen removal
within temperature ranges of 10°C to 30°C. The dominant strain determined from the previous
research will be cultivated in a lab and transferred into five 20L bioreactors with continuous mixing
and exposure to 12 hours of light a day. 300mL of samples are collected every hour to be analyzed
using Tecator Kjeltec Auto 1030 Analyzer. The whole research will take 2 months to complete.

9.3 Determination of optimal pH

This research aims to determine the optimal pH for maximum nitrogen removal within pH
range of 7 to 11. The dominant strain determined from the previous research will be cultivated in
a lab and transferred into five 20L bioreactors with continuous mixing and exposure to 12 hours
of light a day. 300mL of samples are collected every hour to be analyzed using Tecator Kjeltec
Auto 1030 Analyzer. The whole research will take 1 month and 3 weeks to complete.

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11.0 Appendix