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Algaebasedwastewatertreatment

TECHNICALREPORT·JANUARY2015

DOI:10.13140/2.1.1241.8885

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HarshadRathod

IndianInstituteofTechnologyRoorkee

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ALGAE BASED WASTEWATER TREATMENT

A SEMINAR REPORT

of

MASTER OF TECHNOLOGY

in

CIVIL ENGINEERING

(With specialization in Environmental Engineering)

By

HARSHAD RATHOD

(Enrolment No: 13519016)

ENVIRONMENTAL ENGINEERING GROUP

DEPARTMENT OF CIVIL ENGINEERING

INDIAN INSTITUTE OF TECHNOLOGY ROORKEE

ROORKEE – 247 667, UTTARAKHAND, INDIA

AUGUST, 2014

i

CANDIDATE’S DECLARATION

I hereby declare that the work carried out in this seminar report entitled, “ALGAE BASED

WASTEWATER TREATMENT”, is being submitted in partial fulfillment of the

requirements for the award of degree of “Master of Technology” in Civil Engineering with

specialization in Environmental Engineering submitted to the Department of Civil Engineering,

Indian Institute of Technology, Roorkee, under the supervision of Dr.Raja chowdhury and

Dr.B.R.Gurjar, Department of Civil Engineering, IIT Roorkee.

I have not submitted the record embodied in this seminar report for the award of any other

degree or diploma.

Date: 29 August, 2014 (Harshad Rathod)

Place: Roorkee

CERTIFICATE

This is to certify that the above statement made by the candidate is correct to the best of our

knowledge and belief.

(Dr. Raja Chowdhury) (Dr. B.R.Gurjar)

Assistant Professor Professor

Department of Civil Engineering Department of Civil Engineering

Indian Institute of Technology, Roorkee Indian Institute of Technology,Roorkee

ii

ACKNOWLEDGEMENT

I would like to take this opportunity to express my profound sense of gratitude and sincere

thanks to my guides Dr.Raja Chowdhury and Dr.B.R.Gurjar, Department of Civil

Engineering, IIT Roorkee, for being helpful and a great source of inspiration. Their keen

interest and constant encouragement gave me the confidence to complete my work. I wish

to extend my sincere thanks for their excellent guidance and suggestions for the successful

completion of my seminar work.

Date: 29 August, 2014

Place: Roorkee

(Harshad Rathod)

iii

ABSTRACT

Organic and inorganic substances which were released into the environment as a result of

domestic, agricultural and industrial water activities lead to pollution. The normal primary and

secondary treatment processes of these wastewaters have been introduced in a growing number

of places, in order to eliminate the easily settled materials and to degrade the organic material

present in wastewater. The final result is a clear, apparently clean effluent which is discharged

into natural water bodies. This secondary effluent is, however, loaded with inorganic nitrogen

and phosphorus and causes eutrophication and long-term problems because of refractory

organics and heavy metals that are being discharged. Microalgae culture offers an interesting

step for wastewater treatments, because they provide a tertiary biotreatment coupled with the

production of potentially valuable biomass, which can be used for biofuel production.

Microalgae cultures offer an elegant solution to tertiary treatments due to the ability of

microalgae to use inorganic nitrogen and phosphorus for their growth. And also, for their

capacity to remove heavy metals, as well as some toxic organic compounds, In the current

review we highlighted the role of micro-algae in the treatment of wastewater and growth

parameters to be affected for the cultivation.

Key words: Microalgae, Wastewater, Inorganic carbon, Treatment

iv

CONTENTS

TITLE PAGE NO.

Candidate’s declaration i

Acknowledgement ii

Abstract iii

Contents iv

List of figures v

Abbreviations v

1. Introduction 1

2. Algal growth affecting parameters 2

2.1 Carbon and nutrients 2

2.1.1. (A) Cellular biochemistry towards lipid production 3

2.2 Light 6

2.3 Temperature 7

2.4 pH 7

2.5 Salinity 8

2.6 Inhibitory substances 8

2.7 Other Parameters 9

3. Cultivation Methods 9

3.1 Open Pond cultivation 9

3.2 closed photobioreactor 10

3.3 Immobilized cultivation 10

4. Harvesting Techniques 11

4.1 Filtration 11

4.2 Sedimentation and Floatation 11

4.3 Centrifugation 12

4.4 Biological Filtration 12

5. Recommendation on operation 12

6. Reference 13

v

LIST OF FIGURES

Photoautotrophic mode of nutrition (Page No.4 )

ABBREVIATIONS

1. TAG- Triacylglycerol

2. Co- A- Co enzyme

3. FAS- Fatty acid synthesis

4. ATP-Adenosine triphosphate

5. NADPH- Nicotinamide adenine dinucleotide phosphate

1

1. INTRODUCTION

Since hundreds of years, macroalgae have been one of the eminent sources as food, fodder,

nutraceuticals, and fertilizers. Ancient record shows that people collected macroalgae for food as

long as 500 B.C. in China and one thousands of years later in Europe. Currently, there are 42

countries in the world with reports of commercial macroalgae cultivation activity. China holds

first rank in macroalgae production, with Laminaria sp. accounting for most of its production

followed by North Korea, South Korea, Japan, Philippines, Chile, Norway, Indonesia, US and

India. These top ten countries contribute about 95% of the world’s commercial macroalgae based

products. About 90% macroalgae comes from culture based practices. The most cultivated

macroalgae is the kelp Laminaria japonica, which alone accounts for 60% of the total cultivated

macroalgae production (Laura and Paolo, 2006). The main use of kelp Laminaria japonica is to

prevent obesity and diabetes (Miyuki and Tomoyuki, 2011). A Laminaria stick can be used as an

osmotic dilator of the cervix when induction of pregnancy is necessary.

The main difference between macroalgae and microalgae is the size and cell binding formation.

Normally, macroalgae called as “seaweed” and found in sea water. It is multicellular compound

made of holdfast, blade, frond, stipe, thallus, mid rib, air bladder etc. Microalgae are normally

unicellular called as phytoplankton. We cannot see by naked eye due to microscopic in size

(oilgae report, 2009).

If we consider present scenario, microalgae has been in news since last ten years in terms of the

capability of producing biofuels as well as treating or polishing municipal and dairy wastewater.

Extreme focus to be given on microalgae based treatment.

The term microalga refers to all algae too small to be seen properly without microscope, and

often includes both eukaryotic microalgae and the prokaryotic cyanobacteria (Soeder and C.J.,

1981). The most important common feature of all eukaryotic microalgae and cyanobacteria is

that they have oxygen evolving photosynthesis and that they use inorganic nutrients and carbon

as their source of nutrients. Research shows that wastewater that has been exposed with

microalgae shows a rapid decrease in level of metals, nitrates and phosphate. This shows the

possibility of microalgae to be commercialized for tertiary wastewater treatment solution.

Normally, wastewater comes from activated sludge effluent or anaerobic wastewater treatment,

can be effectively treated by means of microalgae. Before starting any kind of treatment by

algae, primary treatment must be required.

Wastewater that contains of carbon, nitrogen, phosphorous and other metals can be effectively

used by microalgae. The centrate, which is generated from activated sludge, is one of the viable

alternatives for the cultivation media of the microalgae (Esther et al., 2013). First, the

concentrations of carbon, nitrogen and phosphorus are higher in the centrate than in any other

wastewater streams obtained from a wastewater treatment plant, which can provide sufficient

nutrients for algae growth. Second, centrate contains a variety of minerals such as K, Ca, Mg, Fe,

Cu, and Mn, which are essential micronutrients for algae growth and metabolism. Third, the volume of the centrate produced daily is extremely large with no availability problem all year

2

round, and the centrate need to be recycled to the activated sludge process for further treatment

to avoid environmental contamination, which adds extra load for the treatment process,

especially the high concentration of phosphorus. Thus the use of the centrate for algae cultivation

could serve the dual role of waste reduction and biomass/bioenergy production.

After primary and secondary treatment of wastewater, effluent is discharged to the nearby water

bodies. However, the effluent still contains considerable nitrogen, phosphorus and pathogens.

Especially, in anaerobic wastewater treatment, nitrogen, and phosphorus are not removed much,

and raised concern in several places in India. These nutrients leads to eutrophication in lakes and

causes harmful microalgal blooms and have considered P and N to be the key elements behind

the eutrophication. In this regard polishing of anaerobically treated wastewater using algae can

absorb nitrogen and phosphorus from wastewater, increases the dissolved oxygen content and

helps to reduce pathogens present in the treated wastewater. Symbiotic association of algae and

microorganisms present in the effluent could satiate the mutual need of CO2 and O2 of algae and

microorganisms and thus reduces the need of external sources of CO2 or O2 for polishing the

wastewater. Alkaline pH of the algal culture due to limitation of CO2 transfer from air and from

microbial respiration could help to kill the pathogens. However, algal growth in the wastewater

is severally restricted due to light penetration (colored wastewater absorbs a part of the incident

light).

The main objective of this literature review with respect to algae based wastewater treatment is:

1. Synthesizing the current knowledge and applications.

2. Evaluating nutrient removal performance.

3. Characterizing environmental variables affecting growth.

2. ALGAL GROWTH PARAMETERS

Successful treatment with the microalgae requires a thorough knowledge of various parameters

that affect the growth. If one of the factors varies drastically then the whole process can go

wrong in terms of algal productivity and treatment efficiency. The growth rate of the algae

depends on the various factors such as physical, chemical as well as the biological. Abiotic

factors such as light are one of the crucial parameter that algae survive. Examples of physical

factors are light and temperature. Chemical factors can be availability of nutrients and carbon

dioxide, and biological factors are e.g. competition between species, and virus infections.

Operational factors such as mixing, dilution rate, depth, addition of bicarbonate, harvesting

frequency also affect the growth.

2.1 Carbon and nutrients

Algae are autotrophs, i.e. they can synthesize organic molecules themselves from inorganic

nutrients. Some algae may be heterotroph as well as mixotroph depending upon nutrient and

light availability. A stoichiometric formula for the most common elements in an average algal

cell is C106H181O45N16P, and the elements should be present in these proportions in the medium

for optimal growth. Scenedesmus was grown in chemostat and found that optimal cell N: P 30,

growth was determined solely by N limitation and above 30, by P limitation (G-Yull Rhee,

1978). According to the ratios most often found in wastewater, phosphorus is rarely limiting

3

nutrient for algal growth. Though, since wastewater often exposes the algae to nutrient

concentrations of up to three orders of magnitude higher than under natural conditions, growth is

more likely limited by carbon and light (Joel et al., 1992)

The rate at which an algal cell takes up a specific nutrient depends on the difference between the

concentration inside and outside the cell, and also on the diffusion rates through the cell wall.

The thickness of the unstirred layer of water just outside the cell wall also plays a role, where

thicker layers give slower diffusion rates. To avoid such thick boundary layers in order to

enhance mass transfer rates of nutrients and metabolites, turbulence in the water is essential.

2.1.1 Carbon

Carbon is one of the important sources for microalgae for cell growth as inorganic carbon

dioxide, in autotrophic mode of cultivation. It would be organic carbon source for heterotrophic

and mixotrophic mode of cultivation. Heterotrophic algal cultivation has been reported to

provide not only a high algal biomass productivity, but high cellular oil content as well (Miao

and Wu 2004; Xu et al. 2006; Li et al. 2007). In the case of Chlorella protothecoides,

heterotrophic growth on corn powder hydrolysate results in a 3.4 times higher biomass yield than

that from autotrophic growth while the lipid content is increased by 4.2 times (Yanna et al.,

2009). Microalgae are considered as more photosynthetically efficient than terrestrial plants for

fixing CO2. In the process of fixation, microalgae use CO2 as an inorganic carbon source, while

water acts as an electron donor for production of glucose, which is further transformed to various

complex sugar forms such as carbohydrate, starch etc. (Figure 1). Many microalgae species are

able to utilize carbonates such as Na2CO3 and NaHCO3 for cell growth (Wang et al., 2009).

Some studies have indicated that about 25–50 % of the algal carbon in high rate algal ponds is

derived from heterotrophic utilization of organic carbon. The organic carbon sources can be

assimilated either chemo- or photoheterotrophically. In the first case, the organic substrate is

mused both as the source of energy (through respiration) and as carbon source, while in the

second case, light is the energy source. In several algal species, the mode of carbon nutrition can

be shifted from autotrophy to heterotrophy when the carbon source is changed; this is the case

with e.g. the green algae Chlorella and Scenedesmus (Becker and E.W., 1994)

2.1.1(A) Cellular biochemistry towards lipid production

Algae are diverse group of organisms that inhabit a vast range of ecosystems, from the extremely

cold (Antarctic) to extremely hot (desert) regions of the Earth (Guschina and Harwood, 2006;

Round, 1984). Algae account for more than half the primary productivity at the base of the food

chain (Hoek et al., 1995). Lipid metabolism (the biosynthetic pathways of fatty acids and

triacylglycerol, or TAG synthesis), particularly in algae, has been less studied than in higher

plants (Fan et al., 2011). It is generally believed that the basic pathways of fatty acid and TAG

biosynthesis in algae are directly analogous to higher plants (Fan et al., 2011). Algae fix CO2

during the day via photophosphorylation (thylakoid) and produce carbohydrate during the Calvin

cycle (in stroma), later carbohydrates converts into various products, including TAGs, depending

on the species of algae or specific conditions pertaining to cytoplasm and plastid (Liu and

Benning, 2012). Microalgae are proficient at surviving and functioning under phototrophic or

heterotrophic conditions or both.

4

The biosynthetic pathway of lipid in algae occurs through four steps: carbohydrates

accumulating inside the cell, formation of acetyl- CoA followed by malony-CoA, synthesis of

palmitic acid, and finally, synthesis of higher fatty acid by chain elongation (Figure 1.)

Figure 1. Photoautotrophic mode of nutrition (source: S.Venkata et al., 2013)

Glucose accumulation inside the cell

Accumulation of energy-rich compounds is the primary step for microalgal lipid biosynthesis.

However, this carbon accumulation varies with both autotrophic and heterotrophic organisms.

Autotrophs synthesize their own carbon through photosynthesis, whereas heterotrophic

organisms assimilate it from outside the cell. In photoautotrophs, the chloroplast is the site of

photosynthesis where, light reaction takes place at the thylakoid followed by CO2 fixation to

carbohydrates in the stroma of the chloroplast. Heterotrophic nutrition is again light-dependent

and light-independent. (Ewald et al., 1983). However, carbon assimilation is more favorable in

the case of light-independent processes (dark heterotrophic) over light-dependent ones

(photoheterotroph). In dark heterotrophic algae, light inhibits the expression of the hexose/H+

symport system (Perez-Garcia et al., 2011), which decreases glucose transport inside the cell.

Algae can also accumulate carbon in the presence of light through photoheterotrophic nutrition.

CO2

Glucose

3PG

Pyruvate

Acetyl Co-A

Malony Co-A

Lipids

Calvin

Cycle

RR.

Fatty

acid

synth.

5

Once carbon enters the cytosol, it follows cytosolic conversion of glucose to pyruvate through

glycolysis and leads to the generation of acetyl-CoA, similar to photoautotrophs, followed by the

pathway of lipid biosynthesis. In mixotrophic nutrition, both the biochemical process of

autotrophs and heterotrophs occur simultaneously, and the preference of substrate uptake

depends on the substrate availability in addition to other environmental conditions.

Formation of Acetyl Co-A/Malony Co-A

CO2 fixation provides an endogenous source of acetyl-CoA by activated acetyl-CoA synthesize

in the stroma, from free acetate, or from the cytosolic conversion of glucose to pyruvate during

glycolysis (Somerville et al., 2000). This acetyl-CoA is preferentially transported from the

cytosol to the plastid, where it is converted to the fatty acid and subsequently to TAG, which

again is transported to the cytosol and forms the lipid bodies. The first reaction of the fatty acid

biosynthetic pathway towards the formation of malonyl-CoA from acetyl- CoA and CO2 is

catalyzed by the enzyme Acetyl-CoA carboxylase. During this process, seven molecules of

acetyl-CoA and seven molecules of CO2 form seven molecules of malonyl-CoA. This malonyl

Co-A undergoes synthesis of long carbon-chain fatty acids through repeating multistep

sequences. A saturated acyl group produced by this set of reactions becomes the substrate for

subsequent condensation with an activated malonyl group

7 Acetyl Co-A + 7CO2 + 7ATP → 7 Malony Co-A +7ADP + 7P

↓

Seven cycle condensation and reduction

Acetyl Co-A + 7 Malony Co-A + 14 NADPH + 14H+ → Palmitate + 7CO2 + 8Co-A+

14NADPH+ 6H2O

Overall reaction

8Acetyl Co-A+ 7ATP+14NADPH+14H+ → Palmitate+ 8Co-A+7ADP+

7P+14NADP

Synthesis of Palmitic acid

After the formation of seven malonyl-CoA molecules, a four-step repeating cycle (extension by

two carbons/cycle), i.e., condensation, reduction, dehydration, and reduction, takes place for

seven cycles and forms the principal product of the fatty acid synthase systems, i.e., palmitic

acid, which is the precursor of other long-chain fatty acids (Fan et al., 2011) With each course of

the cycle, the fatty acyl chain is extended by two carbons. When the chain length reaches 16

carbons, the product (palmitate) leaves the cycle (Liu and Benning, 2012). All the reactions in

the synthetic process are catalyzed by a multienzyme complex, i.e., fatty acid synthase (FAS).

6

2.1.2 Nitrogen and Phosphorous

After consideration of the Carbon, Nitrogen and Phosphorous are key components for the algae

for nutrient assimilation. The nitrogen mainly comes from the sewage by the metabolic

conversion and more than 50% phosphorous comes from the detergents. The principal forms in

which they occur in wastewater are NH4 (ammonia), NO2 (nitrite), NO3 (nitrate) and PO4

(orthophosphate). Together these two elements are known as nutrients and their removal is

known as nutrient stripping (Horan 1990).

Microalgal culture offers a cost-effective approach for removing nutrients from wastewater

(tertiary wastewater treatment) (Evonne et al., 1997). Microalgae have a high capacity for

inorganic nutrient uptake and they can be grown in mass culture in outdoor solar bio-reactors

(Joel de et al., 1992). Biological processes appear to perform well compared to the chemical and

physical processes, which are in general, too costly to be implemented in most places and which

may lead to secondary pollution (Joel de et al., 1992).

The widely used microalgae cultures for nutrient removal are species of Chlorella (Lee and Lee,

2001; Gonzales et al., 1997), Scenedesmus (Martinez et al., 1999, 2000), and Spirulina (Olgun et

al., 2003). It was pointed out through research that Scenedesmus sp. is very common in all kinds

of fresh water bodies, which play an important role as primary producers and contributes to the

purification of eutrophic waters (Mohamed, 1994).

The interest in microalgal cultures stems from the fact that conventional treatment processes

suffer from some important disadvantages: (a) variable efficiency depending upon the nutrient to

be removed; (b) costly to operate; (c) the chemical processes often lead to secondary pollution;

and (d) loss of valuable potential nutrients (N, P) (De la.,1992). The last disadvantage is

especially serious, because conventional treatment processes lead to incomplete utilization of

natural resources (Guterstan and Todd, 1990).

2.2 Light

Microalgae are phototrophes, which mean that they obtain energy from light. However, some

algae are able to grow in the dark using simple organic compounds as energy and carbon source.

The light energy is converted to chemical energy in the photosynthesis, but large parts are lost as

heat. It was reported that in outdoor ponds, more than 90 % of the total incident solar energy can

be converted into heat and less than 10 % into chemical energy (Oswald and W.J.,1988). It was

also reported that the conversion efficiency of sunlight energy into chemical energy of only 2 %

(Fontes et al., 1987).

Light conditions affect directly the growth and photosynthesis of microalgae (Duration and

intensity). Microalgae needs a light/dark regime for productive photosynthesis, it needs light for

a photochemical phase to produce (ATP) Adenosine triphosphate (NADPH) Nicotinamide

adenine dinucleotide phosphate-oxidase) and also needs dark for biochemical phase synthesize

essential molecules for growth (Benjamas and Salwa, 2012). However, it is foremost important

to reach light for algae in mass culture production like raceway pond and algal pond. There are

several strategies used by microalgae to remain near the water surface in order to catch sufficient

7

light. These strategies aim to decrease the specific gravity and thereby minimize the sinking rate

(Fogg and G.E., 1975). Moreover, in dense cultures the algae themselves can decrease the light

availability due to internal shading (Borowitzka and M.A., 1998).

The easiest way to prevent algal cultures from light limitation is to decrease the depth of the

culture vessel. The productivity in light limited ponds is inversely correlated to the depth

.Generally, depths of between 15 and 50 cm are recommended. However, during winter

shallower depths are recommended due to the lower light conditions, and depths greater than 20

cm markedly decreases production (Fontes et al., 1987). However, even though light is most

often limiting the growth of microalgae, intense light may also cause lowered photosynthetic

efficiency, which is known as photoinhibition (Bo-Ping et al., 1999).

2.3 Temperature

Temperature is also one of the crucial parameter for the growth of algae in countries where

fluctuation of the temperature is high. Increased temperature is good for the growth of algae up

to certain range, after that critical temperature growth is ceased (Monique and Jean,2013).The

temperature at which culture are maintained should ideally be close as possible to the

temperature at which the organism were collected. Most commonly cultured species of

microalgae tolerate temperature between 16 and 27 degree celsius (Laura and Paolo, 2006).

Overheating of the algae can reduce growth rate especially in the humid country where

evaporation is inhibited. The effects of temperatures which exceed thresholds for optimal growth

are described as more deleterious than lower temperatures. Indeed, this is visualized by the

asymmetrical growth curve versus temperature. Beyond optimal temperatures, growth rate

decrease is linear and reaches critical temperatures more or less abruptly depending on the

species (Butter Wick et al., 2005). The occurrence of mortality when temperatures exceed

optimal is a reality, but the time over which these changes are experienced is an important factor

in order to define the extent of mortality.

The occurrence of mortality when temperatures exceed optimal is a reality, but the time over

which these changes are experienced is an important factor in order to define the extent of

mortality.

2.4 pH

pH is also an important factor that affects the growth of microalgae. In microalgae cultivation,

pH value usually increases because of the photosynthetic CO2 assimilation. pH value will affect

the availability of inorganic carbon (Y Azov, 1982). Absorption of nitrogen by microalgae also

increases the pH value of the medium, as every nitrate ion reduces to ammonia produces one

OH- ion (Xu et al., 2012). Higher pH value may cause precipitation of phosphate in the medium

but this can be avoided by reducing the pH value by the process of respiration where CO2 assimilation is not happen.it is studied the influence of pH on the microalgae in wastewater and

the result shows that the highest growth rate of microalgae when the medium at a constant pH

value of 7.0 (Gassan et al., 2009) Normally, microalgae use inorganic carbon and HCO3- for the

growth of the cell productivity. Depending on these parameters, pH may vary from low to high

8

in the alkaline region. Depending on the various forms of carbon available, pH variation can be

studied.

The pH range for most cultured algal species is between 7 and 9, with the optimum range being

8.2-8.7. Complete culture collapse due to the disruption of many cellular processes can result

from a failure to maintain an acceptable pH. The latter is accomplished by aerating or mixing the

culture. In the case of high-density algal culture, the addition of carbon dioxide allows to correct

for increased pH, which may reach limiting values of up to pH 9 during algal growth.

CO2 is provided by the buffer system of bicarbonate-carbonate for photosynthesis according to

the following reactions;

1. 2HCO3- ‹ › CO3

-2 + H2O + CO2

2. HCO3- ‹ › CO2 + OH

-

3. CO3-2

+ H2O ‹ › CO2 + OH-

2.5 salinity

Marine phytoplankton is extremely tolerant to changes in salinity. The best algae growing

conditions for most species is at a salinity level that is slightly lower than that of their native

habitat, which is obtained by diluting sea water with tap water. Salinities of 20-24 g/l have been

found to be optimum (Laura and Paolo, 2006). Lipids are vital to cell function as structural

component of cell membrane and storage products. The lipid content and composition of

microalgae have been shown to change in response to environmental variables such as light,

temperature and salinity. It is noted that the increase in salinity can increase the lipid content of

microalgae, but lowers the growth rate of a species (Asulabh et al., 2012). All species responded

to change in salinity by modifying their cellular fatty acid compositions. Significant positive

correlations were observed between increase in salinity and increase in the percentage of

hexadecenoic acid [16: 1(n-7)]( S. M. Renaud and D. L. Parry, 1994). The percentage of

saturated fatty acid in microalgae decreased as the concentration of NaCl increased, while the

percentage of highly unsaturated fatty acid increased (Kirrolia et al, 2011).The decrease in

photosynthetic activity commonly observed under salt stress may be due to limitations in

photosynthetic electron transport and partial stomatal closure (Zhang et al, 2010).

2.6 Inhibitory substance

Many substances behave like inhibitory in cell growth as well as photosynthetic efficiency.

Examples of such substances are heavy metals, herbicides, pesticides, substances in detergents,

some microbes, household cleaning products and personal care products. High concentrations of

ammonia act inhibitory on algal growth at high pH, and this toxicity is intensified at higher

temperatures when a higher proportion of the ammonia occurs as free ammonia which may

freely diffuse over membranes into the cells (Aharon and Yosef, 1976). As already mentioned,

total ammonium concentration should not Exceed 20 mg NH4-N. Too high levels of organic

compounds can inhibit the nutrient uptake by microalgae, and acetate can be toxic to some

species, due to the un-ionized molecule, which can penetrate the cell membrane and damage the

9

cell interior by ionization (Wood et al., 1998). Some algae also produce substances toxic to

themselves in the course of their metabolism. These eventually accumulate to concentrations

high enough to inhibit growth; a phenomenon called autoinhibition.

Some species inhibit the growth of others in mixed culture; e.g., some cyanobacteria can produce

inhibitory substances against the growth of eukaryotic algae and some eukaryotic algae can

produce antibacterial substances. The later may indirectly affect competition among algal species

by their effects on associated bacteria.

In pure monocultures, infections by parasites, predators or competing species can be deleterious,

and protozoa and rotifers present the greatest threat.

2.7 Other parameters

Apart from above mentioned growth parameters, appropriate mixing of cell, dilution rate of the

cell for the optimum growth, depth of the microalgae in water, distance between algae and light

source, optimum dilution ratio for the cell growth, addition of bicarbonate for the optimum pH

adjustment, harvesting frequency for the fatty acid production are all very crucial parameters

when we culturing microalgae on lab based as well as commercial level. So before starting any

types of algal culture straining, we have to think these hidden parameters for the growth of algae

as well.

3. CULTIVATION METHODS

There are three main groups of system for cultivation of microalgae. They are open system,

closed system and immobilized system. Open is simpler to conduct and cheaper. However, open

system is expose to the environmental factors such as temperature and light intensity. Closed

system cultivation is more complex to conduct but it allows condition control for cultivation. The

third is immobilized system where algae are trapped in a solid medium.

3.1 open pond culture

Open system cultivation is the more preferable due to its low cost and can be done in large scale

cultivation and it is easier to manage. Moreover it is more durable than large closed reactors.

Open system cultivation can be carried out in natural or artificial lake and ponds. They are many

types of ponds that had been designed and experimented before for the optimum cultivation of

microalgae. Despite many types of open system (ponds) have been proposed before, only three

major designs have been developed and operated at relatively large scale. The three major

designs are raceway ponds, inclined system and circular ponds. Normally we prefer the entire

culture pond depending upon the culture condition. Due to high cell growth in raceway pond, we

discussed here more on this aspect.

10

3.1.1 Raceway pond

For commercial cultivation of algae, shallow raceway ponds and circular ponds with a rotating

arm to mix the cultures are usually used. The raceway pond is set in a meandering configuration

with paddle wheel mixers that exert low shearing forces. In this system, water level is kept no

less than 15 cm, and algae are cultured according to the optimum growth condition by supplying

needed nutrient to the medium. The raceways are typically made from poured concrete to

prevent the ground from soaking up the liquid. The fresh feed (containing nutrient including

nitrogen, phosphorus and inorganic salt) is added in front of the paddle wheel and algal broth is

harvested behind the paddle wheel. Although the open system is cost effective, it has some

disadvantages. Among the disadvantages are it requires large land areas for a considerable

biomass yield. Moreover, because this cultivation technology is carried out in the open air, the

water level can be effected by from evaporation and rainfall. Besides that, biomass productivity

is limited by contamination with unwanted algal species and organism that feed on algae.

Studied by Lee showed that some algal species like Dunalilla sp., Spirulina sp. Chlorella sp.are

resistance to the culture in open pond system (Lee Y.K., 2001).

3.2 closed photobioreactor

Closed photobioreactors can be grouped into two major classes: covered raceways and tubular

reactors (Richmond and A., 1990). Closed photobioreactors usually have better light penetrating

characteristics than open ponds; the light path is usually less than 30 mm, which make it possible

to sustain high biomass and productivity with less retention time than is possible in ponds

(Borowitza and M.A., 1998). However, since they are more technically complicated, often need

expert personnel and require more energy than open systems; the operating cost is higher

(Benjamin et al., 1997). Basically, these reactors are a closed system consisting of a clear tube

within which the algae grow. The algae are circulated by means of a pump and the system also

has a gas exchange unit where CO2 can be added and photosynthetically produced O2 is stripped

from the medium. If necessary, a heat exchanger is also added to either cool (in tropical areas) or

heat (in temperate areas) the culture.

3.3 Immobilized cultivation system

Immobilized system can be defined as the cell of the algae is trapped in a solid medium and is

prevented from moving independently. This system can solve the harvesting problem (P.S.Lau et

al., 1998). To immobilize the algae, algae cells are trapped in substances in wastewater diffuse

through the cell. The algae-medium mixture is often shaped as beads, but it can even cover

screens or surfaces. Immobilized system has been tested for several wastewater treatments and it

is proved that entrapped algae are able to efficiently remove nitrogen and phosphorus from

secondary effluent and be considered a tertiary wastewater treatment (Chevalier and de la.,

1985). It should be noted that almost of immobilization of algal cell have been done only in

small scale in the laboratory which will hinder our knowledge of how it will perform in the large

scale. It will be interesting to confirm the feasibility of using immobilized microalgae and

cyanobacteria for removing nitrate, ammonium, and phosphate from high volume effluent

discharges. It has been reported that Phormidium laminosum immobilized on polymer foam has

the potential to remove nitrate in a continuous-flow system with uptake efficiencies above 90%.

11

4. HARVESTING TECHNIQUES

Algae growing in open waste ponds can reach biomass levels of up to 300 mg dry weight per

liter. Harvesting of microalgae is therefore crucial for wastewater treatment in order to separate

both nutrients and BOD from the water. However, this is not easily achieved, and is

consequently a cost expensive part of the cultivating process. Even though harvesting effectively

can be accomplished by e.g. filtration or centrifugation, such methods may be too difficult or

costly to implement. Some studies have suggested that chemical flocculation with e.g. the

polysaccharide chitosan, biological filtration (see below) and even to use immobilized systems

should be more advantageous. There are some methods that are used for the harvesting of

microalgae as sedimentation, floatation, filtration, centrifugation, biological filtration etc.

4.1 filtration

Filtration is the most simple and cost effective harvesting method. Filtration can be carried out

either in small or large scale. Filtration can be done by using filter paper in laboratory scale or

using coarse screening in a large scale harvesting of microalgae. Besides that, large colonial

algae such as Spirulina can be harvested by using basic principal of filtration called

microstraining. This process consist a rotating fine-mesh screen and backwash to harvest

microalgae. It is proved that, by using microstraining for harvesting microalgae, almost 20-fold

of concentration, or higher can be achieved (Benemann J.R., 1979). For the filtration harvesting

method, rotary vacuum and the chamber filter appears to be commonly employed type of filter in

fairly large size of microalgae (Mohn, 1980). The advantages of these filters are they can be used

in continuous operation and useful when sterility and contaminant is not reviewed. However,

filtration is only suitable for harvesting fairly large microalgae (e.g spirulina platensis) and not

succeeds to separate bacteria size microalgae like scenedesmus, dunaliella or chlorella species.

4.2 sedimentation and floatation

Using sedimentation or flotation, the biomass can be concentrated already in the water, which in

turn can be decanted. Sedimentation without addition of chemicals is the most common method

in full-scale facilities .Flotation processes operate more efficiently and rapidly than

sedimentation and achieve a higher solids fraction (up to 7 %) in the concentrate, but these on the

other hand can be more expensive (Borowitza and M.A.,1988).

To facilitate sedimentation or flotation, previous flocculation is desirable. Many algal species are

particularly difficult to sediment without treatment due to their natural tendency to float in order

to catch enough light. Flotation of unicellular algae without flocculation may also be very

difficult due to the hydrophilic cell surface on which air bubbles will not attach (personal

unpublished experiences). Algae can be flocculated by addition of various chemical flocculants

such as alum, lime, FeCl3, cationic polyelectrolytes, and Ca (OH)2 (de la et al.,1992) . A major

disadvantage of adding these chemicals, however, is that they can cause secondary pollution.

Some toxically safe flocculating agents recognized are e.g. potato starch derivatives and

chitosans, and these are suitable for initiating sedimentation.

12

Some microalgae may flocculate naturally, so-called bioflocculation. The process is often

induced by turbulence stress and results in formation of biopolymers by extracellular enzymes.

The polymers can also be produced by bacteria associated with the algal cells, but in both cases

this leads to changes in the surface charge that in turn causes the cells to aggregate (Lincoln et

al., 1986). Especially, some species of cyanobacteria forms flocs spontaneously. This allows

easy harvesting of the biomass. Some cyanobacteria are also able to form gas vacuoles that

makes them accumulate at the surface, and some tend to settle under certain circumstances, and

both these features makes them easy to harvest.

4.3 centrifugation

Centrifugation harvesting method can be applied to almost every type of microalgae.

Centrifugation is using the same sedimentation principal but with addition with enhanced

gravitational force to increase the sedimentation rate (Knuckey et al., 1986) .There is some types

of centrifugation harvesting method and it is depending on the particle size ranges. Tubular bowl

centrifugation provides the most efficient result in harvesting but the capacity is very limited.

This type of method is more preferably done in small laboratory scale. However, there are some

disadvantages of centrifugation which is microalgal cells structure can damage due to the

exposure to high gravitational and shear forces.

4.4 biological filtration

Biological filtration means feeding of easily harvested filter feeders with algae, and is

consequently a form of aquaculture. Well known filter feeders are mussels and cladocerans like

Daphnia spp. (de la, 1992). Complete food chains starting with wastewater have been studied in

order to develop integrated systems able to generate useful biomass simultaneously with effluent

purification (Etnier and B., 1991). Pathogen safety of such biomass does not appear to be of

major concern although more complete and systematic monitoring of pathogens should be made

before the final edible biomass (fish in general) is available for human consumption (de

la.,1992).

5. RECOMMENDATION ON OPERATION

Wastewater is naturally abundance in nutrient that can be used for algal growth. From all the

factor that effecting algal growth and treatment efficiency, it is likely that the major factors is

carbon and light. Light is the important parameter to be consider and hence in open culture the

depth of culture and turbulence are major factors to obtain optimum performance. For the humid

climate like India, where rain may affect the performance of wastewater treatment and algal

growth, greenhouse would be recommended. By observing the properties and ability of the

microalgae in the uptake of carbon, nitrogen, phosphorus and heavy metal, microalgae show a

potential in wastewater treatment for various types of effluent.

It is recommended for starting a microalgae wastewater treatment by inoculating large variety of

algae because this will create a mixture of algae where the best suited species will strive and

grow faster and dominate the treatment steps. This approach requires less supervision and

operation than if a particular algal is chosen to be cultivated for any purpose.

13

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