Synthesis and Characterization of Starch-based Cationic Flocculants for

Harvesting Microalgae

A THESIS
SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL
OF THE UNIVERSITY OF MINNESOTA
BY

Liang Li

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF
MASTER OF SCIENCE

Advisor: Roger Ruan

August, 2012
© Liang Li 2012
 i
Acknowledgements

This program was supported by Legislative-Citizen Commission on Minnesota

Resources (LCCMR),University of Minnesota Initiative for Renewable Energy and the
Environment (IREE), and University of Minnesota Center for biorefining.

Principal Investigator: Dr. Roger Ruan, Director, Professor, Center for Biorefining,
Department of Bioproducts and Biosystems Engineering and Department of Food Science
and Nutrition, University of Minnesota
Co-investigator: Paul Chen, Program Director, Associate Research Professor, Center for
Biorefining, Department of Bioproducts and Biosystems Engineering, of Minnesota

Committee Members: Bo Hu , Bioproducts and Biosystems Engineering, Robert Morrison,

Veterinary Population Medicine

Lab Members: Yanling Cheng

Laboratory Support: Imaging center, University of Minnesota

 ii
Dedication

This dissertation is dedicated to my dear grandpa. You have lightened my thirst for
knowledge, that’s why I can pursue so much in these 25 years. Hope you live in peace
in heaven.
 iii
Abstract

Harvest of tiny microalgae cells is a technically and economically challenging step

in algal biomass production and utilization. Many techniques have been developed and
tested, and some of them are used with limited success. However none of these
techniques has broad commercial applications, especially in the area of algae based
fuels production. The purpose of this thesis project was to develop a novel flocculation
process which would be able to concentrate algal biomass, allowing efficient separation
and collection of algal biomass from culture broth. The core of the research was to
develop a procedure to synthesize starch-based cationic flocculants which would be
renewable, biodegradable, and non-toxic with harvest performance comparable with or
better than commercial flocculants. The procedure involves cationization reaction
between starch and glycidyltrimethylammonium chloride (GTAC), where the hydroxyl
groups on starch are substituted by quaternary ammonium cations with the help of
alkaline catalyst. In this project, experiments were designed and carried out to study the
effects of key reaction variables, namely temperature, time, GTAC dosage, water
content, and catalyst dosage on the degree of substitution (DS), an indication of how
well GTAC was utilized in the reaction, and reaction efficiency (RE). The DS and RE
generally increased and then decreased with increasing temperature, time, water content,
and catalyst dosage. An increase in GTAC dosage increased DS but decrease RE.
Pretreatments of starch using acid, alkaline, and microwave did not significantly affect
the DS and RE. The resultant cationic starch flocculants and cationic polyacrylamide
(CPAM), a commercial flocculant, were tested in harvesting experiments involving live
algae grown in fresh water or animal manure. The harvest efficiency was affected by
DS of the cationic starch, flocculant dosage, pH, and flocculation time. The cationic
starch based flocculant performed better than CPAM in all conditions. Pilot scale
cationic starch production and harvest tests were conducted. The results agreed well
with those obtained from the lab scale experiments. Based on these results, an
optimized cationic starch synthesis procedure was proposed. The novel procedure has
great potential for commercial production of renewable, biodegrable, and non-toxic
starch based flocculants for cost effective and eco-friendly harvest of algal biomass.
 iv
Table of Contents

List of Tables...................................................................................................................... vi
List of Figures.................................................................................................................... vii
Background...........................................................................................................................1
Harvesting method of microalgae.................................................................................1
Flocculants....................................................................................................................8
Hypothesis.......................................................................................................................... 12
Goal ans objectives.............................................................................................................13
Method and materials..........................................................................................................13
Lab scale cationic starch synthesis...............................................................................13
Pilot scale cationic starch synthesis under optimized condition...................................14
Pretreatment of corn starch effect on DS and RE..........................................................15
Measurement of degree of substitution.........................................................................16
Algae culture..................................................................................................................17
Flocculation experiment ...............................................................................................18
Scanning electron spectroscopy.....................................................................................19
Result and Discussion............................................................................................................19
Effect of process variables on DS and RE....................................................................19
Effects of pretreatment on DS and RE.........................................................................30
Flocculation of microalgae with cationic starch...........................................................31
Pilot scale microalgae harvesting experiment..............................................................38
Scanning electron microscope......................................................................................39
Summary and conclusions.....................................................................................................43
Reference...............................................................................................................................45
 v

List of Tables

Table 1. Table of the most three significantly influence factors of cationic starch

synthesis..........................................................................................................................29

Table 2. Forms of fit details of the most significant affect synthesis condition.............30

Table 3. Reaction effected by different pre-treating ways.............................................31

Table 4. Comparison of cationic starch and CPAM on large scale algae

harvesting........................................................................................................................39
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List of Figures

Fig 1. AlgaeVenture microalgae harvesting and drying system......................................4

Fig 2. Comparison of harvested, original, and clarified liquid of froth flotation (from left

to right) ...........................................................................................................................5

Fig 3. Process of cationic starch synthesis......................................................................11

Fig 4. Degree of substitution of cationic starch and reaction efficiency affected by

temperature......................................................................................................................21

Fig 5. Degree of substitution of cationic starch and reaction efficiency affected by

synthesis time..................................................................................................................22

Fig 6. Degree of substitution of cationic starch and reaction efficiency affected by

catalyst dosage................................................................................................................24

Fig 7. Degree of substitution of cationic starch and reaction efficiency affected by slurry

water content...................................................................................................................25

Fig 8. DS affected by the relationship of both synthesis temperature and reaction

time..................................................................................................................................27

Fig 9. DS and RE affected by dosage of cationization reagent.......................................28

Fig 10. Harvesting efficiency affected by degree of substitution of cationic

starch................................................................................................................................32

Fig 11. Flocculation ability affected by pH value of the algae broth..............................33

Fig 12. Microalgae harvesting efficiency by industrial cationic starch affected by pH of

algal culturing broth........................................................................................................34

Fig 13. Harvesting rate influenced by flocculation time.................................................35

 vii
Fig 14. Dry weight ratio of algae harvesting of cationic starch and cationic

polyacrylamide................................................................................................................36

Fig 15. Comparison of cationic starch and CPAM of swine cultured algae

harvesting........................................................................................................................38

Fig 16. Cationic starch structure under SEM..................................................................40

Fig 17. The shape of centrifugation harvested algae under SEM...................................41

Fig 18. The shape of cationic starch flocculated algae under SEM................................42
 1
Background

There is increasing attention to microalgae which are a potential feedstock for

production of renewable energy and products to substitute for the fossil fuel (Chisti

2007). The production of algal biomass can be coupled with flue gas CO2 mitigation

(Sawayama, Minowa et al. 1999), wastewater treatment (Wang, Min et al. 2010), and

recovery of valuable resources in the form of the co-products such as biofuels, animal

feeds (He, Hollwich et al. 2002), fertilizers, foods and high value chemicals and nutrient

(Barclay, Meager et al. 1994) (Becker 2007). Developments in microalgae cultivation,

harvesting, and downstream processing are expected to further enhance the cost

effectiveness of biofuel and other products from microalgae (Li, Du et al. 2008). Hence,

every step in the algae to fuel approach is considered to be important to the

commercialization and sustainability of an algae-based fuel technology. The present

study was focused on the development of cost effective harvesting techniques.

Harvesting Methods of Microalgae

Microalgae harvesting is thought to be one of the most difficult steps in microalgae

cultivation and utilization, due to extremely small size (around 5-40µm) and low

concentration (<1-3%) of algae cells in the culture broth. A number of harvesting

techniques have been tested. They include sedimentation, filtration, flotation,

flocculation, and electric method, as well as combinations of the above. Some of these

traditional techniques are simple and easy to operate. For example, gravity
 2
sedimentation and simple filtration are good choice due to the low cost and ease of

manipulation.

Flocculation is a process in which dispersed particles are aggregated together to

form large particles for settling, and is considered one of the most convenient method.

Flotation, chemical flocculation, centrifugation, some combined processes, and

new methods are being used or developed nowadays. Selection of a method must

consider the shape, size, motility of algae cells and target products (Shelef, Sukenik et

al. 1984; Vlaški, Van Breemen et al. 1997). Therefore it is very difficult to find one

technique that fits all situations. Following is a brief discussion of the common

harvesting techniques.

Sedimentation

Sedimentation is considered as a low cost simple technique because no chemicals

and extra force other than gravity is used. This technique is limited to large cell strains

which are capable of self-aggregating through a process called auto-flocculation

(Demirbas 2010), forming ample size particles that settle down without any additives.

However, in most cases, sedimentation is not usually used for algae cells harvesting but

for algae suspension enrichment in industry (Mohn 1980). For small sized algae,

gravity sedimentation must be combined with chemical flocculation to be useful for

algae harvest (Shelef, Sukenik et al. 1984).

Filtration

Membrane is the key component in filtration method, and in some systems, extra

force is added (pump in most case) in addition to gravity (Jaouen, Vandanjon et al. 1999)

which could accelerate the water removal. Previous research on microbial collection
 3
also mentioned “drum filter” (Ludwig and Lochmann 2000) and “disc filter” (Pessoa

and Vitolo 1998). The key issues with filtration are that the membrane is easily fouled

by algal cells (Wilde, Benemann et al. 1991) and the residual water in the biomass is

often high. Magnetic filtration was tested for algal mass removal in many lakes near

Gainesville, Florida, and for some algae species/strains, the removal efficiency could

reach 94% (Bitton, Fox et al. 1975). The tangential flow filtration was developed to

remove microalgae of mixed strains from very low algae concentration liquid

(Petrusevski, Bolier et al. 1995). The membrane with an average pore size of 0.45/m

was found to minimize membrane fouling, and was able to remove 70-89% of algae

from the culture broth. However, in most cases, filtration is inefficient (Papazi,

Makridis et al. 2010).

For the large-scale and high-density algae cultivation, cell packing is another

serious problem (Dodson and Thomas 1964). Stirring or shaking the suspension was

proven to be helpful for reducing cells accumulation in the pores of the membrane

(Morris and Yentsch 1972). However, stirring a large amount of liquid will consume a

lot of energy, and it is therefore not cost effective, and time consuming too. That is why

filtration can only be used to harvest large cell species/strains in small quantities. As to

small sized algae in large scale, the algae cells can be chemically agglomerated to form

larger sized flocs (Petrusevski, Van Breemen et al. 1996), to reduce cell packing and

membrane fouling.

AlgaeVenture, a private company in Ohio, developed a harvesting and dewatering

system which utilizes a special fabric membrane with strong capillary water absorption
 4
capacity to remove water from algae paste while it travels on the moving belt (Fig. 1).

The company claimed that the system is very energy and cost effective.

Fig. 1 AlgaeVenture microalgae harvesting and dewatering system

Flotation

Flotation was developed for clarifying drinking water (Edzwald 1993). In this

method, suspended solid particles adhere to tiny bubbles created in the water through air

injection, and float to the surface and form a particle-air-water foam which can be easily

removed and collected. This principle should be applicable to algae removal.

Early research found that air flotation is a very effective way of concentrating or

removing microalgae. In Fig. 2, the middle column is the original culture broth, the left

one is harvested algae, and the right column is clarified water. They also reported that

lowering the pH of the culture broth helped stabilize the foam, resulted in an improved

algae recovery through froth flotation. However, the acidic condition could erode the

algae growth (Levin, Clendenning et al. 1962). Acid condition may also corrode

facilities and release inclusions into culture broth which might have some negative
 5
effect on algae growth and later applications. Flotation alone is still considered

inefficient because of low removal rate and high residual water content of the harvested

algal biomass. There is also serious water loss through evaporation during aeration

(Levin, Clendenning et al. 1962).

Fig. 2 Comparison of harvested, original, and clarified liquid of froth flotation

(from left to right) (Levin, Clendenning et al. 1962)

Harvesting efficiency can be improved by combining flotation with other methods

such as flocculation (Sandbank and Shelef 1987) and filtration, or by adding some other

chemicals like coagulants (Chen, Liu et al. 1998) and ozone (Betzer, Argaman et al.

1980). Coagulant and surfactants were used in flotation process to greatly facilitate

generation of fine air bubbles, effectively reduce the equipment size and energy

consumption while improving harvest rate (Wiley, Brenneman et al. 2009). Sim et al.
 6
designed an algae harvesting system which consists of flocculation, dissolved air

flotation and continuous filtration, addition of chitosan. The harvesting efficiency could

achieve 95%, and the harvesting cost was proven to be much cheaper than

centrifugation.(Sim, Goh et al. 1988). Ozone is commonly used in treatment of

drinking water and was found to also help remove algal cells and produce relatively low

water content algae froth (8.5% solid content) because ozone can react with algae cell

wall to result in a hydrophobic suspension amenable separation (Betzer, Argaman et al.

1980).

Centrifugation

High-speed centrifugation is a widely used method with reasonable algae recovery

rate although it may collapse some algal cells during the process (Moraine, Shelef et al.

1979). Compared with other methods, algal pastes harvested using centrifugation have

higher solid contents, usually above 15% (Sim, Goh et al. 1988). It was reported that a

cream separator at a centrifuge force of 13000g, which is very high, could recover over

95% of algae cells with relatively low cell damage percentage (0-3%). Lower speed

centrifugation 6000g was proven much less efficient (60% recovery) (Heasman, Diemar

et al. 2000).

Similarly, centrifugation was used in combination with other processes in both

pilot and bench scales. For example, centrifugal force was used to help the filtration to

dewater algal mass. For large scale algae production, centrifugation is not practical

because of the high costs needed to treat large volumes of culture broth, especially

when the suspended algal mass content is low (Sim, Goh et al. 1988). However,
 7
centrifugation is an option for further removal of water from algal pastes which were

harvested using other methods such as sedimentation, floatation, and flocculation.

Flocculation

Microalgae cells usually carry negative charges on their surface which keep the

cells separated due to repulsion (Ives 1959). This property is important to the

suspension of the cells in culture media for healthy growth but also can be utilized for

harvest. Positively charged electrodes and cations containing polymers will attract algal

cells to form flocs which can be easily collected. Both the electrode and polymer based

methods have been studied (Sridhar, Namasivayam et al. 1988), (Alfafara, Nakano et al.

2002). It is widely believed that chemical flocculation is more convenient, more

practical and faster than electrode and other conventional methods. In chemical

flocculation, algae cells are agglomerated together and form larger particles which can

be separated from water easily. Many researchers suggested that flocculation is the best

way for algae harvesting in large scale.

Algae flocculation may occur if the pH of the culture medium is changed, and the

degree of flocculation varies wth algal species and strains. Lime and Mg(OH)2 were

used to raise the pH to as high as 10-11 to cause flocculation (Folkman and Wachs

1973). Lowering pH can also be used to flocculate algae. One down side of pH based

flocculation is that costly neutralization of the remaining water must be carried out

before the water can be reused for cultivation. Furthermore, the extreme pH

environment may result in algae cell lysis. In addition to pH adjustment, inorganic and

organic flocculants are commonly used in wastewater treatment, and their uses in algae

harvest have also been reported (Van Vuuren and Van Duuren 1965), (Tenney,
 8
Echelberger Jr et al. 1969). The fact that chemical flocculants are used in the

wastewater treatment industry indicates the great potential of using chemical flocculants

for algae harvest. The next section is therefore dedicated to the discussion of flocculants.

Flocculants

Inorganic Flocculants

Inorganic flocculants like aluminum and ferric salt as well as their polymeric

compounds are being used in many places like sewage treatment and drinking water

clarification (Yu, Fang et al. 2001). In theory, the positive metal ions capture the

negatively charged algae cells, prompting many researchers to investigate the use of

these flocculants for algae harvest (Chow, House et al. 1998) (McGarry 1970). Chow et

al reported that ferric chloride keeps algae cells intact so that cell inclusion could be

locked in the algae, which is good for subsequent recovery of valuable components

from the harvested algae cells and avoiding water pollution (Chow, House et al. 1998).

Use of a mixture of aluminum sulfate and polyelectrolyte in wastewater treatment ponds

was shown to yield better flocculation than when they were used individually,

suggesting that there were some sorts of synergetic effects between the two flocculants

(McGarry 1970). The same study also showed that the flocculant concentration, stirring

speed of the mix, and flocculation time were very important (McGarry 1970). Poly-

aluminum chloride (PAC) was found to be more effective than monomeric aluminum

chloride for flocculation (Gao, Chu et al. 2005). However, some reports indicate that the

required dosage of inorganic flocculants is too high (Hejzlar, Dolejš et al. 1998), and
 9
the metal ions may adversely affect the downstream processing and utilization of the

harvested algal biomass (Becker 1994).

Organic Flocculants

Organic flocculants can be divided to two groups: synthetic polymers such as

polyacrylamide and polyethylene imines, and nature macromolecules such as chitosan

and modified starch. These flocculants could be cationic, neutral, and anionic. Some

studies indicated that only cationic polymers could produce satisfactory microalgae

harvesting (Tilton, Murphy et al. 1972).

Industrial organic flocculants

Cationic polyacrylamide (CPAM) is commonly used due to its strong capability of

agglomerating algae and low price (Salehizadeh and Shojaosadati 2001). However,

though the polymer itself is nontoxic, its monomer is acrylamide, which has been

proven to be a peripheral nerve toxin and a cancerogenic agent (Smith, Prues et al. 1996)

(Rudén 2004) causing large bowel, kidney and bladder cancers in human (Mucci,

Dickman et al. 2003). Commercial CPAM may contain residual monomer acrylamide

due to incomplete polymerization. Some monomer acrylamide molecules may be

released from CPAM due to degradation and depolymerization to a certain extent in

application (Vers 1999) posing a potential risk to public health (Smith, Prues et al.

1996). CPAM will be banned for use in sludge treatment in Germany by the end of

2013 (Krentz, Lohmann et al. 2006).

Biobased flocculants

To address the issues with industrial polymeric flocculants such as CPAM,

researchers looked into biobased materials and their derivates, such as chitosan, guar
 10
gum and starch (Salehizadeh, Vossoughi et al. 2000). Compared with CPAM, these

biodegradable materials are usually toxin free, environmental-friendly, effective at low

dosage, and not so susceptible to shearing force in liquid (Karmakar and Singh 1998;

Singh, Karmakar et al. 2000).

Chitosan is derived from chitin of crustaceans’ exoskeletons. The amino groups

on the molecular chain backbone of chitosan are able to coagulate negatively charged

suspended matters. It has been tested to harvest both freshwater algae and marine algae.

It works very efficiently under appropriate temperature, mixing speed and time, and pH

(Lavoie and Noüe 1983). Even in saline condition, chitosan is able to achieve

satisfactory flocculation efficiency (Divakaran and Sivasankara Pillai 2002) (Morales,

De La Noüe et al. 1985). Due to its natural and completely non-poisonous characteristic

(Hirano, Itakura et al. 1990), the algae harvested using chitosan is nontoxic too.

However, chitosan cost is considered to be too high for large scale algae harvesting

(Ravi Kumar 2000).

Starch is an abundant biopolymer, which can be a cheaper alternative to chitosan.

The –OH groups on the backbones of starch molecules can be substituted by positively

charged groups such as quaternary ammonium (Pal, Mal et al. 2005), effectively

turning starch into a cationic polymer which can attract negatively particles such as

algae cells. A good choice of cationization reagents is possible to render cationic starch

non-toxic and eco-friendly (Dencs, Nos et al. 2004). N-(3-chloro-2-hydroxypropyl)-

N,N,N-trimethylammonium chloride (CHPTAC) and N-(glycidyl)-N,N,N-

trimethylammonium chloride (GTAC) are two common etherifying agents (Nichifor,

 11
Stanciu et al. 2010). During the reaction, alkaline is catalyst (Fig. 3) (Wei, Cheng et al.

2008).

Fig. 3 Process of cationic starch synthesis

Cationic starch is used in municipal and mineral industry wastewater treatment

and paper making industry to absorb the negatively charged cellulose fibers in white

water (Nachtergaele 1989), and increase the paper strength (Eriksson, Pettersson et al.

2005). Cationic starch has the potential to replace expensive and hazardous inorganic

and synthetic organic flocculants in wastewater treatments.

Cationic starch can also be used to collect negatively charged algae cells.

Vandamme et al. reported that cationic starch was very effective in fresh water

microalgae harvesting (Vandamme, Foubert et al. 2010). Cationic starch works under a

wider range of pH value than those inorganic ones (Kato, Gyoten et al. 2004).

A parameter important to the flocculation performance of cationic starch is the

degree of substitution (DS), a measure of the extent of cationization reaction and the

molecular electro-strength that determines the absorption/flocculation capability. Each

anhydroglucose unit has three OH groups, and hence the maximum DS is 3. Molecular

weight (MW) is another important parameter that may influence the size of the
 12
flocculated particles and settlement velocity. The cationic starch currently available on

the market has a relatively low DS and low harvesting rate for microalgae.

In this thesis project, improvements on cationic starch synthesis process were

sought to enhance the performance cationic starch in harvesting microalgae.

Hypothesis

Microalgae have negative charges on the surface of cell wall, which can be

attracted by positive charged substance. A cationic flocculant could react against a

negatively charged suspension. In this project, the cationic starch flocculant will be

synthesized by treating granules of starch with a reactive cationic regent which contains

a positive charge. Bridging of segments of cationic starch chain attracting negative

particles (microalgae) would help them aggregate. Meanwhile cationic starch will

counterbalance the negative charge of microalgae particles. So cationic starch could be

used to harvest microalgae. Theoretically, the higher the charges strength, the stronger

the attraction force between particles. The charge strength of cationic starch is relative

to the DS which will have direct influence on microalgae harvesting effiency. So

appropriate DS of cationic starch should be obtained to achive better microalgae harvest

effect.

During the synthesis process, many conditions may affect the degree of

subsititution of cationic starch, and influence significance is different. We need to

determine an “optimum” conditions combination of cationic starch synthesis.

Cationic polyacrylamide is considered to be an effective algae harvesting agent,

and was applied widely in labs and greenhouses. However, its monomer is toxic so it is
 13
hypothetical that cationic starch could replace the cationic polyacrylamide in

microalgae harvesting.

Goal and Objectives

The goal of this thesis project was to develop a novel flocculation process which

would be able to concentrate algal biomass, allowing efficient separation and collection

of algal biomass from culture broth by other techniques. The core of the research was

to develop a procedure to synthesize starch-based cationic flocculants which would be

renewable, biodegradable, and non-toxic with harvest performance comparable with

commercial flocculants. The specific research objectives were:

1. to quantify the effects of key reaction variables on the degree of

substation and reaction efficiency during synthesis,

2. to optimize the synthesis procedure,

3. to evaluate the harvest performance of the resultant cationic starch

against CPAM, a commercial flocculant,

4. to test the synthesis procedure and flocculation in pilot scale.

Methods and Materials

Lab Scale Cationic Starch Synthesis

In this study, cationic starch was prepared by attaching positively charged groups

onto the H of hydroxyl groups of the starch backbone with the help of base catalyst
 14
sodium hydroxide. Reaction variables studied included dosage of catalyst, water content,

temperature, and polymerization time. Baseline procedure was used in making cationic

starch. Five grams of corn starch (National starch, Bridgewater) were placed in a flask,

1 mol/L NaOH solution was prepared by NaOH powder (Fisher, Fair Lawn). A solution

containing glycidyltrimethylammonium chloride (GTAC) 80% solution (Sigma, USA),

0.5 ml - 2ml 1 mol/L NaOH solution and 0.5ml -2ml distilled water was added. All the

reactants in the flask were well mixed and allowed to react for different times ranging

from 2 to 7 hours at temperature at 45-65 ℃ in a water bath. After the reaction

completed, an excess amount of 95% alcohol (Sigma) was poured into the flask to stop

the reaction and allow the modified starch to settle down. The obtained modified starch

slurry was placed on a filter on a vacuum filtration funnel and washed with 70% alcohol

twice and 95% alcohol once under vacuum to remove the cationization reagent and

NaOH. The cake was dried under 60℃ for 6 hours. The dried modified starch cake was

milled to fine powder which was the final starch based flocculant product.

Pilot scale Cationic Starch Synthesis under Optimized Conditions

Corn starch (National starch, Bridgewater), GTAC solid powder (Guofeng, China),

NaOH (Fisher, Fair Lawn) and distilled water were mixed homogeneously as the

optimized synthesis ratio at larger scale. Firstly, starch powder and solid etherifying

agent were mixed and preheated at 90 ℃, then the liquids mixture was added, allowed

to react at 90℃ for 160 min.

 15
Pretreatment of Corn Starch Effect on DS and Harvest Rate

The reaction between starch and cationization agents depends very much on

properties of functional groups on the starch molecules. Modifications of starch

molecules prior to the reaction may affect the reaction and ultimately the substation rate

and flocculation performance. In this study, we tested a number of treatments such as

acid, alkaline and microwave heating. The total moisture content of the reaction

mixtures in all pretreatment experiments was set to 32.7% which was found to be

optimal in experiments described in “Lab scale cationic starch synthesis”. .

Acid Pretreatment

Dilute acid treatment could hydrolyze the glycosidic bonds between

anhydroglucose units and change the composition ratio of amylose and amylopectin

(Hoover 2000; Wang, Truong et al. 2003). This may alter the starch granules structure,

viscosity, and texture and expose more reaction sites for cationization and hence

increase the chance of substitution.

In this experiment, 0.5 ml 1 mol/L HCl solution was uniformly mixed with 5

grams starch powder for 5 minutes as pretreatment, followed by addition of a mixture of

2.0 ml 1 mol/L NaOH solution, 0.5 ml distilled water and 3 grams GTAC 80% solution

and other synthesis steps as described in “Lab scale cationic starch synthesis”.

Alkaline Pretreatment

Alkaline functions similarly to acid in partially breaking down polysaccharide

chains, which exposes more reaction sites (Gugliemelli, Weaver et al. 1969). NaOH was

chosen for alkaline pretreatment because it was used as the catalyst in synthesis process.
 16
In this experiment, 0.5 ml 1 mol/L NaOH solution was firstly mixed with 5 grams

of corn starch powder and allowed to react for 5 minutes, followed by addition of a

mixture of 1 ml 1 mol/L NaOH solution, 1.5 ml distilled water and 3 grams of GTAC

80% solution.

Microwave Pretreatment

Singh et al (Singh, Sethi et al. 2003) reported that microwave energy can cause

breakage of starch chains. Adebowale and Lawal (Adebowale and Lawal 2003) found

that the heat provided by microwave could also help swell the moist starch granules.

We speculate that the structural and conformational changes caused by microwave

heating may affect the cationization reaction.

In this experiment, 1.5 ml distilled water was added into flask as the microwave

absorbent and then mixed with 5 grams of corn starch powder for 5 minutes. The starch

slurry was heated in a microwave oven for 10 to 20 seconds at power level 5. The

microwave heated corn starch was then subjected to the synthesis steps described in

“Lab scale cationic starch synthesis”.

Measurement of Degree of Substitution

Zero point one gram of cationic starch powder was dissolved in 100ml deionized

water (80C) in a 250ml beaker and stirred with a heated magnetic stirrer (11-498-7SH,

Fisher, USA) at speed 3, till the solution became uniform (about 30 minutes). The final

concentration of cationic starch solution was 1 gram per liter. The solution was allowed

to cool to the room temperature prior to test for the nitrogen content "n" (mg/L) which
 17
was tested using total nitrogen kit (HACH Company, Germany) and spectrophotometer

(Spectronic Instruments Genesys 5, USA).

The degree of substitution (DS) was calculated using the equation below:

n(mg / L)
162( g / mol ) 
500(mg / L)
DS 
n(mg / L)
100(14 g / mol )  152.5( g / mol ) 
500(mg / L)

The coefficient 162 g/mol is the molecular weight of the residue of each

anhydroglucose unit (AGU)monomer of starch, 14 g/mol is the molecular weight of

nitrogen, and 152.5 g/mol is the molecular weight of GTAC group that was connected

on to the starch back bone.

The reaction efficiency (RE) was calculated using following equation:

RE= DS / (Mass of GTAC / Mass of AGU) × 100%

Algae Culture

Algal biomass was produced in lab scale for flocculation tests. A locally isolated

microalgae strain 10B (Chlorella) was cultured on TAP-Medium (Tris-Acetate-

Phosphate) (Harris, Stern et al. 1989) contained (all quantities of the ingredients were in

g/L for solid and ml/L for liquid): NH4Cl (Fisher, Fair Lawn), 0.4; MgSO4·7H2O

(Fisher, Fair Lawn), 0.1; CaCl2·2H2O (Fisher, Fair Lawn), 0.05; K2HPO4 (Fisher, Fair

Lawn), 0.108; KH2PO4 (Fisher, Fair Lawn), 0.056; Tris (hydroxymethyl aminomethane)

(Fisher, Fair Lawn), 2.42; glacial acetic acid (Fisher, Fair Lawn), 1; trace elements

solution, 1; consisted of the components listed below (grams per liter) Na2EDTA, 50;

ZnSO4·7H2O, 22; CaCl2·2H2O, 0.05; H3BO3, 11.4; MnCl2·4H2O, 5.06; FeSO4·7H2O,

 18
4.99; CoCl2·6H2O, 1.61; CuSO4·5H2O, 1.57; (NH4)6Mo7O24·4H2O, 1.10; and KOH 16.

Algae seeds were cultured on TAP medium in three 250ml flasks on shaker. To produce

algal biomass for the flocculation experiments, 100ml of the seed broth was inoculated

on 1L TAP medium in 2L flasks on magnetic stirrers. All cultures were maintained at

20C with artificial lighting at an illumination intensity of 80μmol/(㎡/s). The algae

density of the culture broth was monitored using a spectrophotometer at 550 nm

according to a method by S Ahmed (Ahmed, Gilerson et al. 2004). The culture broth

was considered ready for flocculation tests when its algae density reached 1g/L (dry

weight), which is close to the normal algae density in industrial photobioreactor

cultivation.

Flocculation Experiment

The original algae suspension (concentration over 1g/L) was adjusted using

distilled water to 1g/L. The pH was adjusted to different values using 1 mol/L HCl or

NaOH solution. The adjusted algae suspension was mixed with cationic starch solution

in a 50 ml centrifugal tube at a ratio of 1:12,1:14, 1:16, 1:18, 1:20 (dry mass)

respectively, and mixed on vortex (G-560, Fisher, Bohemia) at speed 10 for 10 seconds.

After 30 minutes settling, the algae flocs formed a satisfied sedimentation (show a

picture here). The sample in the middle part of liquid phase in the tube was taken out

for test. The concentration of algae in the sample was measured using the

spectrophotometer method described above. The harvest rate was calculated as:

HR (%)= (1-CH (g/L)) / 1 (g/L) x 100%

 19
Here HR is the harvest rate, an indication of flocculation efficiency, and CH is the

concentration of algae in supernatant after flocculation.

Scanning Electron Spectroscopy

SEM was used to observe delicate morphological features of algae and starch

granules. Original corn starch, cationic starch, micro algae and flocculated micro algae

were studied using a variable Pressure Scanning Electron M, S3500N, Hitachi, located

in the Imaging Center in Department of Biological Science at University of Minnesota.

Results and Discussion

Effects of Process Variables on Degree of Substition and Reaction

Efficiency

We first studied the effects of four process variables, namely temperature, reaction

time, catalyst dosage, and water content on the reaction rate and degree of substitution

(DS). The optimal reaction conditions were determined.

Temperature

High temperature can accelerate the reaction not only by increasing the contacts

and collisions between starch and GTAC molecules, but also by enhancing starch

gelatinization and swelling. Starch granule’s crystalline structure is so hard and compact

that etherifying agents can hardly access the OH groups inside the particles in normal

condition. In the presence of water and heat (at 60℃) starch granules swell (Bagley and

Christianson 1982), and under higher temperature (between 70 and 82℃) they
 20
gelatinize (Lelievre 1974), which breaks down the intermolecular bonds of starch

molecules, resulting in mobilized and more reactive starch molecules. However, too

high temperature may lead to the positive groups (quaternary ammonium groups) of

GTAC decomposition and conversion(Kavaliauskaite, Klimaviciute et al. 2008).

Therefore we must find an optimal temperature range.

Figure 2 shows the changes in DS and RE as a function of temperature ranging

from 45℃ to 65℃. DS and RE increased with increasing temperature in the range of

45 to 60 C and declined with further increase in temperature. The steep increase in DS

and RE observed when temperatures were between 55 and 60℃ may be attributed to the

onset of starch swelling (Bagley and Christianson 1982). At temperature above 60℃,

thermal side reactions of quaternary ammonium groups resulted in decreased RE and

DS. We therefore concluded that 60℃ is the optimal temperature under our reaction

conditions.
 21

Fig. 4. Change in degree of substitution of cationic starch and reaction efficiency as a

function of temperature while water content (50%), catalyst dosage (0.5 ml 1mol/L

NaOH solution), reaction time (300 minutes), cationization reagent (GTAC) were kept

constant.

Reaction Time

Starch cationization is a lengthy process. It usually requires several hours reaction

time to reach a satisfactory utilization rate of the oxidation reagent and in some cases,

several days at low temperatures are needed to reach high DS (e.g., close to 1) which

may involve reactions between starch and epoxides (Kavaliauskaite, Klimaviciute et al.

2008).
 22
To determine an appropriate reaction time, we studied the changes in RE and DS

as a function of reaction time while keeping reaction temperature (60 , water content

(50%), and NaOH dosage (0.0005 mol) constant. Figure 5 shows that both RE and DS

increased with increasing time up to 360 min and decreased thereafter. For prolonged

reaction time, the reduced DS and RE is due to side reactions involving decomposition

of positive charged groups on cationized starch.

Fig. 5 Change in degree of substitution of cationic starch and reaction efficiency as a

function of reaction time while water content (50%), catalyst dosage (0.5 ml 1mol/L
 23
NaOH solution), reaction temperature (60 ºC cationization reagent (GTAC) were kept

constant.

Catalyst Dosage

Both DS and RE increased rapidly with increasing catalyst dosage up to 0.001 mol

NaOH and decreased gradually when the catalyst dosage was above 0.0015 mol NaOH

(Figure 6). It is believed that NaOH provides additional hydroxyl ions in the reaction

system so that the chemical reaction equilibrium shifts towards products (cationic starch)

(Khalil and Farag 1998) . However, too much NaOH may cause many other side

reactions of cationic starch (Carr 1994) due to enhanced hydrolysis of epoxy groups,

and produce the N-(2,3-dihydroxy)propyl-N,N,N-trimethylammonium chloride under

alkaline condition (Goclik, Stach et al. 2004). De-etherification reaction become more

serious under high pH circumstances (Khalil and Farag 1998). These may be the main

reasons for the decrease in DS and RE at NaOH concentration above 0.0015 mol.

Furthermore, a high NaOH concentration may trigger side reactions between NaOH and

GTAC too(Kavaliauskaite, Klimaviciute et al. 2008).

 24

Fig. 6. Change in degree of substitution of cationic starch and reaction efficiency as a

function of catalyst dosage while water content (50%), d reaction time (300 minutes),

reaction temperature (60 C), and cationization reagent (GTAC) were kept constant.

Water Content

Water acts as a dispersant and medium in many chemical reactions which involve

diffusion of reactants for efficient molecular collisions. Further to the reaction leading

to starch cationization, water is required for starch swelling. It is therefore necessary to

study the effect of water content on the RE and DS in our study.

Figure 7 shows that when the system water content was below 33%, the reaction

efficiency increases with increasing water content, which may be attributed to the better

mixing, lowered viscosity, increased reactant diffusion, and starch gelatinization. .

 25
Further increase in moisture content led to decrease in RE and DS. Too much water

may result in reactant dilution, and is etherifying agent GTAC hydrolysis(Khalil and

Farag 1998) and hence reduction in overall RE and DS.

Fig. 7. Change in degree of substitution of cationic starch and reaction efficiency as a

function of water content while reaction time (300 minutes), catalyst dosage (0.0015

mol), cationization reagent (GTAC), and reaction temperature (60 ºC) were kept

constant.

Interaction of Temperature and Reaction Time

There is usually an inverse relationship between reaction rate and temperature,

i.e., a higher reaction rate is expected at a higher temperature. However, as

 26
demonstrated earlier RE and DS decreased when temperature was above 60C,

indicating that a high temperature is not always suitable for our case. Figure 8 shows a

series of reaction curves (reaction time vs DS) at different temperatures. All the curves

share the same parabola shape with one maximum. The curves shift from right to left

when temperature was increased, indicating that the reaction reached its maximum

quicker when temperatures were higher. Lower temperatures appear to give rise to

higher maxima at long reaction time. Highest DS values were observed when

temperatures were at 50-60C. However, the fact that a short reaction time of 160 min

with DS value of 0.38 at 90C compared with a long reaction time of 360 min with DS

value of 0.42 obtained at 60C suggests that it is worth considering high temperature

short time process in large scale industry production of cationic starch for high

productivity. Further study to assess the energy consumption between high temperature

short time and low temperature long time process schemes is necessary.
 27

Fig 8. DS affected by the relationship of both synthesis temperature and reaction time.

Cationization Reagent Dosage

Figure 9 shows that the DS increased and RE decreased with increasing

cationization reagent. As expected, an increase in the concentration of reactants will

shift the reaction equilibrium towards products but on the other hand may lower the

efficiency of reactants utilization. For example, the utilization efficiency was about 97%

when the GTAC was only 1 gram but dropped down to 71.8% when the GTAC was

increased to 4.5 grams.

 28

Fig 9. DS and RE affected by dosage of cationization reagent

The Most Significant Factor Influencing Cationic Starch Synthesis

Three factors and 27 groups of synthesis samples were set in a full arrest test.

Calculation of variance was applied to analysis these data.

Water
Reaction
content Catalyst Reaction
# time DS/3
(mass (mol) rate
(min)
ratio)
1 0.37 0.0015 320 0.46 0.672
2 0.41 0.0015 320 0.446 0.651
3 0.45 0.0015 320 0.432 0.631
4 0.37 0.002 320 0.499 0.729
5 0.41 0.002 320 0.519 0.759
6 0.45 0.002 320 0.524 0.766
7 0.37 0.0025 320 0.524 0.766
 29
8 0.41 0.0025 320 0.489 0.715
9 0.45 0.0025 320 0.479 0.7
10 0.37 0.0015 360 0.437 0.638
11 0.41 0.0015 360 0.432 0.632
12 0.45 0.0015 360 0.504 0.736
13 0.37 0.002 360 0.509 0.744
14 0.41 0.002 360 0.54 0.789
15 0.45 0.002 360 0.535 0.781
16 0.37 0.0025 360 0.479 0.7
17 0.41 0.0025 360 0.489 0.715
18 0.45 0.0025 360 0.484 0.707
19 0.37 0.0015 400 0.428 0.625
20 0.41 0.0015 400 0.428 0.625
21 0.45 0.0015 400 0.455 0.665
22 0.37 0.002 400 0.479 0.7
23 0.41 0.002 400 0.494 0.722
24 0.45 0.002 400 0.489 0.715
25 0.37 0.0025 400 0.465 0.679
26 0.41 0.0025 400 0.46 0.672
27 0.45 0.0025 400 0.46 0.672
Table 1. Table of the most three significantly influence factors of cationic starch

synthesis
 30

Table 2. Forms of fit details of the most significant affect condition

Data was analyzed through ANOVA using the master model in SAS software.

The usage of catalyst significantly influenced the DS (P<0.05) while the other two

factor including water content and reaction time didn't show obivous effect on DS

(P>0.05). The order significance was: catalyst dosage > reaction temperature > system

water content.

Effects of Pretreatments on RE and DS

Acid, alkaline and microwave pretreatment may cause structural change of starch

chains, which may influence molecular weight and viscosity alternation and other

effects on flocculation experiment, so it needs to be discussed separately.

 31
We tried three methods of corn starch pretreatments to test improvement of degree

of substitution and reaction efficiency of cationic starch. Firstly, the acid and base

pretreatment showed only a slight improvement of reaction efficiency but was not

significant. Acid pretreatment was better than base (Table. 1). In the table, DS is the

dimensionless value, and all the values in this form are average data from 3 parallel

experiments.

Method DS/3 RE

Control 0.423 82.4%

Acid 0.437 85.1%

Alkaline 0.428 83.3%

Microwave 10s 0.410 79.8%

Microwave 15s 0.416 81.0%

Microwave 20s 0.414 80.2%

Table 3. Reaction effected by different pre-treating ways

Flocculation of Microalgae with Cationic Starch

Relationship Between Harvesting Efficiency and Degree of Substitution (DS)

Flocculation using the cationic starch was carried out with a dry mass ratio of algae

to cationic starch at 16:1, under the pH of 6.67, which is the original pH of culture broth,

and flocculation time 40 minutes. Figure 10 shows that the harvesting efficiency

increased with increasing DS up to 0.41 where 94% of algae cells were removed and

then decreased. A higher DS gave rise to higher amount of positively charged groups

which are capable of binding negatively charged algae cells, resulting in a higher
 32
harvesting efficiency. When the DS is too high, starch chains may become too stiff

(Bratskaya, Schwarz et al. 2005), due to the mutual exclusion between positive charge

as well as the steric clash on ammonia groups, which will reduce their ability of

capturing the negative charged algae cells.

Fig. 10. Harvesting efficiency affected by degree of substitution of cationic starch

Since DS of 0.41 is the best for microalgae harvesting, an optimized reaction

condition (reaction time: 360 minutes, catalyst dosage: 0.0015 mol, GTAC: 2.85g, and

reaction temperature: 60C) was used to produce cationic starch with DS value of 0.41,

which was used for the rest of algae flocculation and harvest experiments.

Effect pH of Culturing Medium on Flocculation and Harvest

 33
Cationic starch can be used in a wide range of pH values but perform better at

weak alkaline conditions than neutral and acid condition because the quaternary

ammonium group is stable under high pH value condition. In our study, the harvesting

efficiency was above 90% at around the natural pH value (6.67) of the algae culture

broth and the highest harvesting efficiency was obtained at pH around 8.3-8.5 (Figure

11). A solution’s pH is known to affect the surface charges of the particles in the

solution. It is quite possible that the changes in the electrical properties of the surfaces

of algae cells and cationic starch at different pH values affect the interactions between

algae cells and cationic starch, and hence the flocculation efficiency.

Fig. 11 Flocculation ability affected by pH value of the algae broth

 34
For the pH higher than 10, the high HR is mainly because the algae surface charge

has been destroyed under strong base environment. The repulsion force between algae

fade away and then algal cell could agglograte together.

Compared to a flocculation experiment with industrial cationic starch (Greenfloc

120) in figue 12, algal biomass and cationic starch at ratio of 6:1 dry base and yielded

highest harvest rate at about 80% under mild alkaline condition. Our cationic starch can

get much higher harvest rate (95%) at the ratio of 16:1, and the dosage is about 200%

lower than the Greenfloc 120.

Fig 12. Microalgae harvest efficiency by industrial cationic starch affected by pH

of algal culturing broth (Vandamme, Foubert et al. 2010)

Effect of Flocculation Time on Harvest Efficiency

 35
After the cationic starch was added to an algae culture broth, the algae cells settled

quickly to the bottom. The data in Fig. 13 illustrates that harvest efficiency increased

rapidly in the first 15 min of flocculation and rose only slightly afterwards. With a dry

mass ratio of cationic starch: algae at 1:18, a flocculation time in the range of 15-20 min

should be sufficient to achieve over 90% harvest rate in industrial setting.

Fig 13. Microalgae harvest efficiency influenced by flocculation time

Comparison Between CPAM and Cationic Starch for Harvesting Fresh Water

Grown Algae

To provide a meaningful assessment of our cationic starch in perspective, we

compared the performance of our cationic starch with that of CPAM (cationic

polyacrylamide), a commercial flocculant which is widely used in wastewater treatment

and being tested for algae harvest. The flocculation time was set to 30 min, and pH
 36
broth was set to 8.5. The ratio of algae to flocculant varied from 10 to 45 (a higher ratio

indicates a lower usage of flocculant). Figure 14 shows that our cationic starch

performed better (about 10%-15% higher) than CPAM at all ratios. The algae:flocculant

ratios in the range from 20:1 to 13:1 are best. The decline in harvest efficiency with

increasing flocculant at the high flocculant concentration end is probably due to

increase in viscosity and charges which slowed down the sedimentation of algae flocs.

Too high a concentration of flocculant may adversely affect the recycle of water for

algae cultivation. Our data suggest that a ratio of 15:1 is suitable for harvest of algae

grown in fresh water in lab scale.

Fig 14. Harvest efficiency as a function of algae to flocculant ratio.

Comparison Between CPAM and CS for Harvesting Manure Grown Microalgae

 37
Growing algal biomass on wastewater is considered an economically and

environmentally viable approach. While we have demonstrated the excellent

performance of our cationic starch in harvesting fresh water grown algae, this cationic

starch’s performance in wastewater grown algae is unknown. Due to the variation in

manure nutrient profile, it was difficult to measure the component ratio of algae and

manure, so we can not use algae concentration and dry weight of biomass here to reflect

the harvest efficiency. So we only OD value was used to show the clarification

capability of flocculants: the lower the OD stands for the higher biomass clarification

efficiency.

The result (Figure 15) shows that the cationic starch has a better ability than

CPAM to clarify the manure cultured algal suspension, which agrees with the result

obtained with fresh water grown algae. Considering the excellent performance and

biodegradability and low toxicity of the cationic starch, we concluded that our cationic

starch is an excellent replacement for CPAM.

 38

Fig 15. Comparison of cationic starch and CPAM of swine cultured algae harvesting.

Pilot scale Microalgae Harvesting Experiment

We further compared the performance of cationic starch and CPAM in pilot scale.

The algae cultivation was carried out with a multilayer photobrioreactor housed within a

greenhouse. The reactor had a culture volume of 500 liters to flocculate, flocculants

were added to the 500 liter algae culture broth (0.9g dry algae/L) at a ratio of algae to

flocculant of 13:1. Experimental variables, observations and harvesting efficiency data

are shown in Table 4. The cationic starch performed slightly better than CPAM. The

cationic starch required slower but longer mixing and produced smaller flocs compared

with CPAM. Mixing in large scale algae production is energy intensive. Further
 39
research and development efforts should aim to reduce the mixing requirement for the

cationic starch.

Large scale flocculation comparison Cationic Starch CPAM

Stable time 30 min 15-20 min

Clumps Size 3-10mm 8-30mm

Average OD of supernatant liquid (Å) 0.252 0.306

Table 4. Comparison of cationic starch and CPAM on large scale algae harvesting

Scanning Electron Microscope

SEM (Scanning electron microscope) is an excellent tool for study of

morphological features of microorganisms (Collins, Pope et al. 1993) and starch

granules (Jane, Kasemsuwan et al. 1994). In this project, SEM was used to study the

morphological features of original corn starch, cationic starch, naturally suspended

algae, and microalgae flocculated by the cationic starch. The SEM micrographs are

shown in Figures 16-18. The algae cells harvested through centrifugation are around 3-4

m with smooth cell wall surface and disperse individually (Figure 17). However, the

cationic starch flocculated algae are covered by a coat of starch and appear to be

bridged together by starch to form some large flocs of 2mm to 3cm in size which are

visible the macroscopically (Figure 18).

 40

Fig 16. Cationic starch structure under SEM

 41

Fig 17. The shape of centrifugation harvested algae under SEM

 42

Fig 18. Cationic starch flocculated algae under SEM

 43
Summary and Conclusions

Flocculation for microalgae harvest was studied. Specific emphasis on this

research was on cationic starch flocculant synthesis. The behavior of cationic starch

flocculant was evaluated in term of microalgae harvest effiency. The effects of cationic

starch flocculant with different DS on microalgae harvest were investigated. The

influence of synthesizing variables such as reaction time, reaction temperature, and

catalyst content for cationic starch flocculant on harvest effiency were studied. The

specific conclusions of this study are:

1. The optimized cationic starch synthesis condition in our lab are: synthesis

temperature at 60℃, the reaction time is 360 min, with total system moisture

content at 32.7%, and catalyst is 0.0015 mol when the corn starch is 5 grams.

And the order of effect significancy is: catalyst dosage > reaction temperature >

water content;

2. Acid and alkaline pretreatment could bring some slight improvement to the

reaction rate but not dramatically. However, microwave pretreatment shows no

improvement to DS;

3. The best DS for chlorella harvesting is 0.41 and the optimal flocculation pH is

8.0-9.0. And 95% of the algal biomass in culturing broth could be flocculated

which is a very effective harvesting rate;

4. After cationic starch was added, the separation of algal broth could reach a

relatively stable situation in 20 minutes;

5. By direct comparison, CS performed better than CPAM on fresh water

microalgae harvesting, as well as manure cultured algae. This development is

 44
expected to further enhance the cost effectiveness of biofuel and other products

from microalgae;

For future studies, more bio-molecules will be investigated for the development

of biodegradable flocculants. Microalgae harvesting by cationic starch will be applied

into larger scale microalgae production and water will be recycled in actual algae

culture operation. The influence of cationic starch to further downstream algae

utilization process will be explored also.

 45
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 50
microalgae. Small microalgae should be flocculated into larger bodies that can
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 51
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