design of algal film photobioreactors material surface energy effects (2)

8/21/2019 Design of Algal Film Photobioreactors Material Surface Energy Effects (2)

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Design of algal film photobioreactors: Material surface energy effects

on algal film productivity, colonization and lipid content

Scott N. Genin a, J. Stewart Aitchison b, D. Grant Allen a,⇑

a Department of Chemical Engineering and Applied Chemistry at the University of Toronto, 200 College St, Toronto, Ontario M5S 3E5, Canadab The Edward S. Rogers Sr. Department of Electrical and Computer Engineering at the University of Toronto, 10 King’s College Road, Toronto, Ontario M5S 3G4, Canada

h i g h l i g h t s

Designed and built a parallel plate airlift reactor for growing algal biofilms on different materials.

Algal biofilm growth kinetics are linear.

Algal biofilm productivity is dependent on material type.

Colonization time is strongly correlated to polar surface energy.

a r t i c l e i n f o

Article history:

Received 10 October 2013

Received in revised form 11 December 2013

Accepted 14 December 2013

Available online 22 December 2013

Keywords:

Algal biofilmsPhotobioreactor

Material properties

a b s t r a c t

A parallel plate air lift reactor was used to examine the growth kinetics of mixed culture algal biofilms

grown on various materials (acrylic, glass, polycarbonate, polystyrene and cellulose acetate). The growth

kinetics of the algal biofilms were non-linear overall and their overall productivities ranged from

1.10–2.08 g/m2 day, with those grown on cellulose acetate having the highest productivity. Overall algal

biofilm productivity was largely explained by differences in the colonization time which in turn was

strongly correlated to the polar surface energy of the material, but weakly correlated to water-material

contact angle. When colonization time was taken into account, the productivity for all materials except

acrylic was not significantly different at approximately 2 g/m2/day. Lipid content of the algal biofilms

ranged from 6% to 8% (w/w) and was not correlated to water-material contact angle or polar surface

energy. The results have potential application for selecting appropriate materials for algal film

photobioreactors.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Microalgae are a potentialfeedstock for biofuels and bioproducts

andcan be usedto treat wastewater by removing andfixingnitrogen

and phosphorous (Chisti, 2007; Mulbry et al., 2008). Production of

biodiesel andgreendieselfrom algae ispossible, butlarge scale com-

mercialization of this process remains unproven (Chisti and Yan,

2011). There are many challenges that theproductionof algal biofu-

els face, such as insufficient supply of low-cost concentrated CO2,

high capital costs of photobioreactors (Pate et al., 2011) and low

concentration of algal biomass from photobioreactors and raceway

ponds (Chisti, 2007). In addition, the cost of dewatering suspended

algae can be 20–30% of the total production costs of algal biomass

(Gudin and Therpenier, 1986; Uduman et al., 2010).

Algal biofilms present an opportunity to reduce the cost

of dewatering since the biomass can be more concentrated

(90–150 g/L) (Ozkan et al., 2012), compared to the typical sus-

pended algae concentrations found in photobioreactors and

raceway ponds (0.5–4 g/L) (Chisti, 2007). Current research on algal

biofilms has mainly focused on ecological studies (Burns and Walk-

er, 2000), with only a few studies investigating the use of algal

biofilms for biodiesel production ( Johnson and Wen, 2010;

Christenson and Sims, 2012; Ozkan et al., 2012).

There is limited research on the design aspects of algal bio-

film photobioreactors most of which have been developed to re-

move nitrogen and phosphorous from waste streams. In previous

studies on algal film photobioreactors, the algal films were

grown horizontally (Craggs et al., 1996; Kebede-Westhead

et al., 2006; Mulbry et al., 2008; Johnson and Wen, 2010; Ozkan

et al., 2012) or on paddles and spools (Christenson and Sims,

2012). Christenson and Sims (2012) developed a rotating algal

film photo bioreactor for waste treatment and algal biomass pro-

duction to be used in conjunction with raceway ponds; the land

productivity of the algal film photobioreactor, was in the range

0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.biortech.2013.12.060

⇑ Corresponding author. Tel.: +1 416 978 8517; fax: +1 416 978 8605.

E-mail addresses: [email protected] (S.N. Genin), stewart.

[email protected] (J. Stewart Aitchison), [email protected] (D. Grant

Allen).

Bioresource Technology 155 (2014) 136–143

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of 20–31 g/m2 day which was significantly higher than the race-

way pond (7.4 g/m2 day).

An algal biofilm is a mixed community of many different algae

and bacteria species within a matrix of extracellular polymeric

substances (EPS) (Hodoki, 2005; Johnson and Wen, 2010; Lawrence

et al., 1998). EPS is a matrix of polysaccharides, proteins, glycopro-

teins, glycolipids, and extracellular DNA produced by the microor-

ganisms that are imbedded in the biofilm (Flemming et al., 2007).

Biofilm composition varies between different films depending on

the microorganisms present, shear forces, temperature, and avail-

ability of nutrients (Flemming and Wingender, 2010). The EPS ma-

trix bonds the cells to each other and the attachment material

which immobilizes the cells. The cells within these biofilms often

exist in a symbiotic relationship with other species in the matrix,

where metabolites from one species can serve as nutrients for

other organisms (Hodoki, 2005; Flemming et al., 2007).

Algal biofilm growth kinetics have not been studied in depth,

but previous work on growth kinetics has shown a linear trend

(Christenson and Sims, 2012; Schnurr et al., 2013). Single point al-

gal biofilm productivity measurements on various substrates has

been reported and is dependent on the material of attachment

( Johnson and Wen, 2010; Irving and Allen, 2011; Christenson and

Sims, 2012), but there has been limited research on how materials

affect algal film growth kinetics in a reactor environment. Johnson

and Wen (2010) reported significant differences in algal biomass

productivity between polystyrene foam (2.57 g/m2day), cardboard

(1.47 g/m2day), polyethylene fabric (0.58 g/m2day), and Loofah

sponge (1.28 g/m2day). Christenson and Sims (2012) demonstrated

that algal biofilms grown on a rotating biofilm reactor have a

preference for growth on cotton rope when compared to polyester,

jute and acrylic. They concluded that the differences in substrata

performance were likely due to the differences in initial attach-

ment of bacteria. Holmes (1986) found that in mixed algal biofilm

cultures, bacterial attachment preceded algal attachment and

Hodoki (2005) showed that higher initial bacterial colonization

density led to higher algal attachment. Orientation of the biofilm

may also be important in the development of algal biofilms, buthas not been discussed or researched in depth.

Attachment of cells onto surfaces is widely attributed to the

hydrophobicity of the surface (Sekar et al., 2004; Palmer et al.,

2007), but there are other factors that affect cell attachment onto

surfaces such as the pH of the bulk liquid, surface charge and cell

charge (Palmer et al., 2007). The literature is inconclusive about

the material surface properties which impact algal biofilm growth.

Irving and Allen (2011) reported that water-material contact angle

did not affect algal biofilm growth on materials for the species

Chlorella vulgaris and Senedescumus obliquus, but Sekar et al.

(2004) reported that hydrophobicity was important for the initial

attachment for C. vulgaris when comparing metals and glass. The

disparity may be due to differences in time scales of the

experiments.There has been research into the surface and wettability effects

of materials on bacteria and algae, particularly from a biofouling

perspective (Finlay et al., 2002; Palmer et al., 2007). These studies,

such as the one conducted by Finlay et al. (2002) on two marine al-

gae species, Entermorpha and Amphora, set out to determine which

surface properties affect attachment and adhesion of microbes.

They found that primary adhesion and settling of the spores of

Enteromorpha were promoted by hydrophobic surfaces, while the

adhesion strength of the settled spores was greatest on hydrophilic

surfaces. For the species Amphora, hydrophobicity did not influence

the initial settling, but the cells were more strongly adhered

to hydrophobic surfaces. Work by Ozkan and Berberoglu (2013)

on algal cell attachment onto surfaces showed that acid-base inter-

actions between algae and surface were the dominatingmechanism.

In previous studies of algal film photobioreactors, suitable

materials for algal film growth were chosen based on high single

point productivities of many different materials ( Johnson and

Wen, 2010) or high surface energies or high-water material contact

angles (Christenson and Sims, 2012). Each of these methods has

disadvantages: it is costly and impractical to measure single point

productivity for all potential materials; there are multiple types of

surface energies; and water-material contact angle is a course

measurement which accounts for multiple types of surface and

material interactions. While single point productivity measure-

ment can determine the overall the productivity of algal biofilms,

the colonization and conditioning phases of biofilm formation,

which are expected to have lower productivities, are aggregated

into these values. It has been shown by Sekar et al. (2004) and

Finlay et al. (2002) that during the initial attachment phase,

different algal species show preferential attachment to different

materials based on the intrinsic material properties, therefore

differences in overall algal biofilm productivity on different mate-

rials reported by Johnson and Wen (2010), Irving and Allen (2011)

and Christenson and Sims (2012) could be the result of different

colonization times.

The objective of this study is to improve material selection for

algal biofilm photobioreactors by determining which intrinsic

material surface energy properties affect algal biofilm productivity,

colonization and lipid content. To complete this objective, a paral-

lel plate air lift (PPAL) reactor was designed and built. The reactor

consists of a glass case with two internal plates to which various

material coupons can be attached to rapidly test the productivity

of algal biofilms grown on various materials. The parameters

measured in these experiments were biomass production, fatty

acid methyl ester (FAME) content, temperature and pH.

2. Methods

2.1. Reactor operation

The PPAL reactor (Fig. 1) used in this study was designed toprovide vertically grown algal biofilms with consistent lighting,

nutrients, and shear. The reactor case was constructed of glass,

with two vertical internal plates of cast acrylic which were secured

by a silicone adhesive. The reactor can hold 15 L of media and has

the following dimensions: 41 20 25cm3. Up to 20 coupons

each with two different materials were placed in the reactor for

each run, giving a total of 40 samples. The materials were clipped

to the internal plates with each material coupon size approxi-

mately 2 8 cm2. The materials were weighed before the experi-

ments and then again after being cleaned and dried.

The coupon materials tested were: glass, cellulose acetate,

acrylic, polystyrene, polycarbonate and silicone rubber. The selec-

tion criteria for the materials were based on transparency, toxicity

towards algae, and water-material contact angle. The material cou-pons were placed in the reactor in a randomorder determined by a

random number generator. Past work by Irving and Allen (2011)

determined that the presence of wastewater is an important factor

in enhancing the formation of an algae biofilm. In order to intro-

duce the bacteria and EPS required to form the biofilms, unsterile

wastewater from Ashbridge’s Bay Wastewater Treatment Facility,

Toronto, ON, was blended with Fortified Bold’s Basal Media (FBBM)

(Bold, 1949) in a ratio of 1:2. FBBM was buffered to pH 6.8 and

prepared to have the following concentration of nutrients: NaNO3250 mg/L, CaCl22H2O 25mg/L, MgSO47H2O 75 mg/L, K2HPO475 mg/L, KH2PO4 175 mg/L, NaCl 25 mg/L, Na2EDTA 10 mg/L,

FeSO47H2O 4.98 mg/L, H3BO3 11.42 mg/L, Na2SiO37H2O 58 mg/L,

ZnSO47H2O 8.82 mg/L, MnCl24H2O 1.44 mg/L, Na2MoO3

0.70 mg/L, CuSO45H2O 1.57, and Co(NO3)26H2O 0.49 mg/L. Thesolution was sparged with air at 1 L/min in the reactor for 24 h

S.N. Genin et al. / Bioresource Technology 155 (2014) 136–143 137


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before being inoculated. The inoculum contained seven algal spe-

cies which were purchased from the Canadian Phycological Culture

Centre (CPCC) or the Culture Collection of Algae and Protizoa

(CCAP). The species used in this experiment were: S. obliquss (CPCC

157), C. vulgaris (CPCC 147), Coccomyxa sp.(CPCC 508), Nannochlorissp. (CCAP 251/2), Nitschia palea (CPCC 160), Oocystis sp. (CPCC 9)

and Oocystis polymorpha. The cultures were cultivated in a light

incubator at 25 C in 250 mLErlenmeyer flasks on an orbital shaker

set to 110 rpm before being used to inoculate the reactor. The reac-

tor operated in batch mode for 48 h before the pumps were started

to introduce fresh FBBM. The day the pumps were started was con-

sidered day zero, and three samples of each material were removed

from the reactor on days 0, 3, 5, 7, and 10. Suspended algae sam-

ples were also taken from the reactor in triplicate. Each experiment

was repeated three times.

Compressed air was provided at a constant rate of 0.990 L/min

STP, where it was mixed with CO2 flowing at 10 mL/min to give a

total CO2 content of 1% by volume. A peristaltic pump (Cole Parmer

Masterflex, Model #7520-35) was used to add and remove media

from the reactor at a dilution rate of 0.96 day1. The dilution rate

was set to be higher than the growth rate of the suspended algae

in order to wash out suspended algae which would otherwise

obstruct light from reaching the films and to ensure there is

enough nutrients provided to the biofilm. White light is provided

by four 8 Watt Light Emitting Diodes (LEDs) which are positioned

outside of the reactor. A Variner Tris-buffer pH probe and stainless

steel temperature probe were used to record pH and temperature

data continuously over the course of the experiment.

2.2. Sessile drop tests

To determine the polar and Lifshitz–van der Waals components of

the surface energy, sessile drop tests were conducted using reverse

osmosis (RO) water, glycerol (Sigma Aldrich #G5516), and hexadec-

ane (Sigma Aldrich #H6703) on the following materials: glass, cellu-

lose acetate, acrylic, polystyrene, polycarbonate, and silicone rubber.

5 lL of each liquid was pipetted onto each material using a 10 lL

pipette and a picture was taken using a Nikon D3000 camera with

a macro lens (model number: AF-S DX Micro Nikon 40 mmf/256).

This process was repeated three times for each material and the

resulting pictures were processed using imageJ v. 1.46. The Lif-

shitz–van der Waals and polar surface energies were then calculated

using the Good Van Oss model (Van Oss et al., 1988).

2.3. Sampling and analysis

The harvested coupons were scraped clean and the biofilmswere suspended in RO water. A vacuum filtration unit was used

to filter the suspended algal mass through Supor-450 47 mm fil-

ters with a pore size of 0.45 lm. The filters were baked at 103 C

for 3 h and weighed before and after filtration to measure the dif-

ference in dry mass.

A minimum of three coupons on day 10 were harvested, thealgal film biomass was then freeze dried and the lipids were ex-

tracted using the Folch method (Folch et al., 1956) using 1:2 (v/

v) chloroform to methanol as the solvent. The methanol phase

and polar lipids solution was discarded to target the neutral lipids.

The neutral lipids were then methylated using the Fatty Acid

Methyl Ester (FAME) technique based on the Microbial Identifica-

tion System, Microbial ID Inc. (MIDI Method) (Smid and Salfinger,

1994). The samples were analyzed using gas chromatography

(Perkin Elmer Clarus 680 GC) with a special performance capillary

column (Hewlett Packard model #HP-5 MS, 30 m 0.25 mm

0.25lm) and a flame ionization detector. Hexadecane (Sigma

Aldrich #H6703) was used as the internal standard and olive oil

was used as a calibration standard.

Scanning electron microscopy (SEM) was used to observe the

presence of microbes and EPS within the algae biofilm. Samples

of live biofilm were taken while still attached to the substrate

and were submersed in a 1% (v/v) solution of osmium tetroxide

for 10 min. Osmium tetroxide bonds to lipids and increases the

cell’s electron density. The samples were then loaded into a Hitachi

S-3400N scanning electron microscope, frozen to 20 C to

preserve the biofilm structure at a pressure of 220 Pa. At these con-

ditions, the algal biofilm remains hydrated and biofilm structures

can be seen. Backscattering electron (BSE) mode was used to

observe microbes which had accumulated significant quantities

of osmium tetroxide, which were predominantly algae cells, while

secondary electron (SE) mode was used to image the entire biofilm

including bacterial cells, EPS and inert solids.

3. Results and discussion

3.1. Growth kinetics

The pH and temperature of the reactor was stable at 6.9 ± 0.2

and 23 ± 1 C respectively throughout the experiment. The stability

of the pH in the system is likely due to the continuous addition of

fresh media into the PPAL system. The total suspended solids in the

PPAL were below 0.050 g/L, which represents an algal productivity

in the suspended phase of 0.048 g/L day or 0.72 g/day. Qualitative

observations of the SEM images showed the biofilms predomi-

nantly contained algae, particularly S. obliquus, Ooscystis sp., C. vul-

garis, and N. palea. Algal biofilms grown on cellulose acetate tended

to slough off the material when harvested on day 10, but the bio-film remained intact in a detached state. The overall growth kinet-

Fig. 1. Reactor configuration and setup.

138 S.N. Genin et al. / Bioresource Technology 155 (2014) 136–143


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ics of the algal biofilms grown on the various materials appear to

be initially non-linear with lower productivities followed by linear

regions of growth with higher productivities (Fig. 2). This trend is

observable in results by Schnurr et al. (2013) and Gross et al.

(2013) which show periods of initial slow growth followed by in-

creased linear growth.

Linear growth curves for microorganisms in conventional bio-

processing systems suggest chemical or mass transport limitations

to growth. This implies there is either a nutrient diffusion or lightlimitation within the biofilm. Modeling of algal biofilms by Flora

et al. (1995) showed the CO2 concentration within an algal biofilm

dropped to zero by 200 lm depth. If this were the case, the algae

up to the depth of 200 lm would be providing the bulk of growth

of the algal biofilmwhich would suggest algal biofilms thicker than

200lm would exhibit linear growth kinetics. The same concept

could also be applied to light limitations.

3.2. Algal biofilm productivity

Past studies calculated overall algal biofilm productivity based

on single point measurements ( Johnson and Wen, 2010; Irving

and Allen, 2011; Christenson and Sims, 2012; Gross et al., 2013)

or linear regressions over the entire growth period including theinitial colonization time (Schnurr et al., 2013). Fig. 3a shows the al-

gal biofilm productivities for this study based on linear regressions

conducted over the entire growth period. The productivity of the

algal biofilms on the materials were for 1.12 g/m2 day for glass,

0.97 g/m2 day for acrylic, 1.25 g/m2 day for polycarbonate, 1.34 g/

m2 day polystyrene and 1.52 g/m2 day silicone rubber. Algal bio-

films grown on cellulose acetate had the highest overall productiv-

ity of 2.08 g/m2 day. The productivities of the algal biofilms grown

on cellulose acetate were statistically significantly higher than

those grown on all other materials except for silicone rubber atthe 95% confidence level. The overall productivity of the algal bio-

films does not correlate to the water-material contact angle

(P = 0.33) which is consistent with results obtained by Irving and

Allen (2011), but it does correlate to the polar surface energy of

the material (P = 0.01).

In order to take into account the potential differences due to

colonization, we subsequently considered the overall growth peri-

od to consist of two phases: the initial colonization during which

the cells attach to the coupon material; and, the subsequent

growth phase after the material is covered by at least one layer

of cells. To test whether the impact of the material persisted after

the initial colonization phase, a linear regression was conducted on

the data from the kinetic study where algal film biomass yields

above 1 g/m2

were used. This 1 g/m2

cut off point is the approxi-mate algal film biomass yield on a surface if the film was 10 lm

0

5

10

15

20

25

0 2 4 6 8 10 12 A l g a l F

i l m B

i o m a s s ( g / m 2 )

Time (days)

Glass

Run 1 Run 2 Run 3

0

5

10

15

20

25

0 2 4 6 8 10 12

A l g a l F

i l m B

i o m a s s ( g / m 2 )

Time (days)

Silicone Rubber

0

5

10

15

20

25

0 2 4 6 8 10 12

A l g a

l F i l m B

i o m a s s ( g / m 2 )

Time (days)

Polycarbonate

0

5

10

15

20

25

0 2 4 6 8 10 12

A l g a

l F i l m B

i o m a s s ( g / m 2 )

Time (days)

Acrylic

0

5

10

15

20

25

0 2 4 6 8 10 12 A

l g a l F i l m B

i o m a s s ( g / m 2 )

Time (days)

Cellulose Acetate

0

5

10

15

20

25

0 2 4 6 8 10 12 A

l g a l F i l m B

i o m a s s ( g / m 2 )

Time (days)

Polystyrene

Fig. 2. Growth kinetics curves for algal biofilms grown on the various materials.


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thick. This is the approximate thickness of an algal biofilm that is a

single algae cell thick. Once a layer of cells is established on the

material of attachment, the cells growing or being recruited will

interact with the algal biofilm and not the material, and we’d ex-

pect this could reduce the effect of the material surface propertieson algal biofilm growth and attachment. The new calculated pro-

ductivity is shown in Fig. 3b.

The revised productivities of the algal biofilms grown materials

are not statistically different at the 95% confidence level from each

other when data points below 1 g/m2 are removed with the

exception of those grown on acrylic. The revised productivities

for the materials are: 1.96 g/m2 day for glass, 1.92 g/m2 day for cel-

lulose acetate, 1.21 g/m2 day for acrylic, 1.96 g/m2 day for polysty-

rene, 1.58 g/m2 day for polycarbonate and1.79 g/m2 day for

silicone rubber. The revised productivities do not correlate to the

water-material contact angle (P = 0.32) or the polar surface energy

(P = 0.45).

The algal biofilm productivity values are on par with those ob-

tained by Johnson and Wen (2010) (2.57–0.58 g/m2

day) Schnurret al. (2013) (2.1–2.8 g/m2 day), and Gross et al. (2013) (1–1.5 g/

m2 day). The algae cells in the PPAL reactor are not able to settle

on the materials unlike the reactor configurations presented by

Schnurr et al. (2013), Irving and Allen (2011) and Johnson and

Wen (2010). Since the conditions between reactor operations are

so different between this experiment and the literature, it is diffi-

cult to conclude whether algal biofilms grown in a vertical orienta-

tion have any disadvantage over algal biofilms grown in horizontal

configurations.

The highest reported overall algal biofilm productivity in this

study was 2.08 g/m2 day for those grown on cellulose acetate

which would result in an estimated total productivity for the reac-

tor of 0.12 g/day if all 40 coupons were cellulose acetate. The sus-

pended algae productivity from the reactor is calculated to be0.72 g/day which is about 6 times greater than the algal biofilm

productivity for this reactor. The higher suspended algae produc-

tivity is due to the fact that the reactor’s design and operation

are not optimized for algal biofilmgrowth, but for the rapid testing

of many different material coupons.

3.3. Colonization time analysis

Conceptually, colonization time is defined as the point at which

at least one cell layer covers the entire material, which corresponds

to1 g/m2. This point can be determined from the linear regressions

described above using only algal biofilm yields greater than 1 g/m2,

then by determining the time when the algal biofilm yield of the

regression is equal to 1 g/m2. The colonization time is plotted for

each material against the water-material contact angle and the po-

lar surface energy (Fig. 4a and b, respectively). The negative coloni-

zation time for cellulose acetate is a result of the fact that some

colonization may have occurred during the two days the algae

are given to acclimatize to the reactor before the pumps are

started, which was chosen to be time zero. The negative coloniza-tion time for the cellulose acetate implies that bacteria are able to

colonize the material rapidly. Hodoki (2005) demonstrated that al-

gal biofilms had a higher growth rate on materials which were ini-

tially colonized with bacteria, thus bacteria may be attracted to

cellulose acetate and will attach and grow faster than the other

materials, which in turn would lead to a higher algal biofilm

growth rate and lower colonization time.

Colonization time as defined in this study is poorly correlated to

the water-material contact angle (P = 0.12), but is strongly corre-

lated to the polar surface energy of the material (P = 0.0001) as

shown in Fig. 4a and b, respectively. This is counter to what Chris-

tenson and Sims (2012) claimed regarding algal biofilm preferen-

tial attachment towards materials with a high surface energy,

but is in agreement with the results from Finlay et al. (2002) andthose of Ozkan and Berberoglu (2013). Ozkan and Berberoglu

0

0.5

1

1.5

2

2.5

Glass (33.7 ) Cel lulose Acetate(63.1)

Acrylic (66.8) Polystyrene (72.8) Polycarbonate(79.1)

Silicone Rubber(93.6)

A l g a l B i o f i l m P

r o d u c t i v i t y ( g / m 2 d a y )

(a)

(b)

0

0.5

1

1.5

2

2.5

Glass (33.7 ) Cellulose Acetate(63.1)

Acrylic (66.8) Polystyrene (72.8) Polycarbonate(79.1)

Silicone Rubber(93.6)

A l g a l B I o f i l m P r o d u c t i v i t y ( g / m 2 d a y )

Fig. 3. (a and b) Productivity analysis linear regression conducted over the entire time and excluding points below 1 g/m2. The water-material contact angles are presented

with each material. The error bars represent the standard deviation.



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(2013) showed that green algae attachment is very dependent on

acid-base interactions between algae cells and surfaces, with

charge and Lifshitz–van der Waals forces being less important.

Since the colonization time is a significant fraction of the algal bio-

film growth period for some materials, therefore it has an impact

on the overall productivity. The correlation between colonization

time and polar surface energy suggests that acid-base interactionsstill play an important role in the growth of algal biofilms and can

be observed in bench scale reactors. Cellulose acetate is known to

degrade and undergo hydrolysis (Buchanan et al., 1993). The

acetate film used in this experiment is a mixture of cellulose di-

acetate and cellulose tri-acetate. It was observed that cellulose

acetate coupons lost 5 ± 1% of their mass over the course of the

experiment, which suggests that microorganisms may be using

cellulose acetate as a carbon source. Buchanan et al. (1993)

reported that cellulose di-acetate degrades to 20% of mass in a

wastewater treatment system within 4-12 days depending on ace-

tate substitution, but cellulose tri-acetate did not have any notable

degradation after 28 days. If the data point for cellulose acetate is

removed on the basis that it maybe feeding the algal biofilm and

therefore is not purely a surface interaction, there is a correlationbetween water-material contact angle and colonization time

(P = 0.002, R2 = 0.67) and the correlation between polar surface en-

ergy and colonization time remains strongly correlated (P = 0.0001,

R2 = 0.80).

The overall algal biofilmproductivity is correlated to the coloni-

zation time (P = 0.0001), which implies differences in productivity

of algal biofilms grown on the materials used in this experiment

are caused by differences in colonization time. Colonization time,

as described in this study, is a useful measurement for assessing al-

gal biofilm formation on materials. It can be used to assess the rate

at which algal biofilms are capable of establishing a foundation and

at which material affects will have limited impact growth. The col-

onization time of the algal biofilm represents the initial phases of

microbial colonization and surface conditioning. Palmer (2007) de-

scribed the conditioning of a surface as the accumulation of mole-

cules at the solid–liquid interface on surfaces. Colonization time

takes into account of surface conditioning and that of the cell

attachment.

3.4. Lipid analysis

The neutral lipid content for the algal biofilms grown on the

various materials was 6–8% (w/w) and was not statistically signif-

icantly different between materials at the 95% confidence level, as

shown in Fig. 5a. These results are consistent with the findings of

Johnson and Wen (2010) (6–9% w/w) and Schnurr et al. (2013)

(5–10% w/w). The neutral lipid content in algal biofilms was lower

than those reported for suspended algae cultures, which is typi-

cally reported to be between 10% and 50% depending on growing

conditions and species (Chisti, 2007). The neutral lipid content

for algal biofilms is likely lower compared to algae grown in a sus-

pended culture due to either the presence of bacteria and/or EPS.

Bacteria and EPS could add to the total mass of the biofilm while

not significantly contributing to the overall lipid content.

The similarity of lipid content between algal biofilms grown on

different materials, suggests that algal species composition did not

vary significantly between the biofilms and that material of attach-

ment does not affect algal biofilm lipid content. Seven differentspecies of algae with a range of lipid contents were used as the

inoculum. If the species composition of the algal biofilms grown

on different materials varied significantly, it could cause the lipid

content of the algal biofilms to be different between materials. Re-

sults from Schnurr et al. (2013) demonstrated that for algal bio-

films inoculated with S. obliquus the neutral lipid content of the

biofilm (5% w/w) was significantly lower than those inoculated

with N. palea (10% w/w).

Using the neutral lipid content, the lipid productivity was calcu-

lated (Fig. 5b). The lipid productivity ranges from 0.06 to 0.13 g/

m2 day and the differences in lipid productivity between algal bio-

films grown on different materials can be attributed to differences

in algal biofilm productivity. The surface area lipid productivities

are lower than those of terrestrial crops (0.25 g/m2

day (Mataet al., 2010)), likely because the operating conditions for the reac-

tor have not yet been optimized and the algae in the suspended

phase has been flushed out of the reactor and not accounted for.

The lipid content of the algal biofilms grown did not correlate

with the water-material contact angle (P = 0.19) for each material

and nor did it correlate with the polar surface energy (P = 0.68).

Lipid productivity of the algal biofilms does not correlate to the

water-material contact angle (P = 0.74) nor to the polar surface

energy of the material (P = 0.85). This is expected since material

properties should affect the rate of attachment and adhesion of

algal biofilms to the material and not affect the internal lipid con-

tent of the algae. It may not be possible to select or design materi-

als which can improve the lipid productivity of algal biofilms and

therefore other factors such as species or conditions influence algallipid content should be investigated instead.

y = -0.04x + 5.37R2 = 0.16P = 0.12

-2

-1

0

1

2

3

4

5

6

30 40 50 60 70 80 90 100

C o l o n i z a t i o n T i m e

( d a y s )

Water-Material Contact Angle (degrees)

Cellulose Acetate Acrylic

Glass Polycarbonate

Silicone Rubber Polystyrene

y = 1.16x - 7.62R2 = 0.69

P = 0.0001

-2

-1

0

1

2

3

4

5

6

6 7 8 9 10 11 12 C

o l o n i z a t i o n T i m e ( d a y s )

Polar Surface Energy (mJ/m2)

(a)

(b)

Fig. 4. (a and b) Colonization time vs. water-material contact angle and material

polar surface energy.


http://-/?-


7/8

Algae species can have a preference in initial attachment

depending on the material properties as shown by results from Se-

kar et al. (2004), Finlay et al. (2002) and Ozkan and Berberoglu

(2013). Since the biomass on the coupons was too low to conduct

a lipid analysis at day zero, results presented here cannot confirmthis. The results from this study imply that while it is possible that

different algae species may initially (over 24–48 h) colonize differ-

ent materials at different rates, the algal biofilms have a tendency

to become more or less uniform in species composition irrespec-

tive of the material properties.

Cellulose acetate appears to be an ideal material to grow algal

biofilms but there was notable difficulty in harvesting. Occasion-

ally, on days 7 and 10, the entire film would fall off the material

and fall into the reactor; this likely indicates that the biofilm be-

came too thick and the adhesion between the material and the al-

gal biofilm was not strong enough to maintain the algal biofilm

when disturbed.

While algal biofilms have a higher biomass concentration than

suspended algae, they tend to have lower lipids concentrationcompared to suspended algae as shown in this study and by others

( Johnson and Wen, 2010; Schnurr et al., 2013). This is problematic

for fuels derived from lipids as it increases surface area require-

ments to meet the same demand. This could be improved with

reactor designs that maximize surface area to land area ratios.

Based on the algal biofilm productivity and lipid results from this

experiment and the reactor configuration presented, an algal

biofilm grown on cellulose acetate, it would require 521 m2 of

surface area or 347 m2 of land area to produce 1 kg of algal biomass

on cellulose acetate. Assuming 6% neutral lipid content, it would

require 8680 m2 of surface area or 5710 m2 of land area to produce

1 kg of neutral lipids. With the reactor configuration presented by

Christenson and Sims (2012), which has an aerial biomass produc-

tivity of 21–30 g/m2

and lipid productivity of 2.2–2.5 g/m2

, wouldrequire 400 m2 of land to produce 1 kg/day of algae oil. This land

use is better than the lipid productivity of open ponds, but there

is potential to improve these ratios.

Demonstrating that algal biofilms can be grown in an airlift

reactor has significant implications for the scaling of algal biofilm

photobioreactors. The design considerations, operating parametersand scaling of airlift reactors is well known and documented (Chis-

ti and Moo-Young, 1987). The hydrodynamic flow in the PPAL reac-

tor is similar to those which would be on a pilot or commercial

scale. The observations of algal biofilm growth kinetics and coloni-

zation time found in the PPAL have potential to be translated to pi-

lot or commercial scale airlift algal film photobioreactors.

The results on how colonization time affects algal biofilm pro-

ductivity have significant impacts on future design considerations

for algal film photobioreactors with respect to the material selec-

tion and reactor configuration. Understanding that polar surface

energy is strongly correlated to the colonization time of algal bio-

films, it is possible to select or engineer materials which have low

polar surface energies to reduce colonization time or select mate-

rials with high polar surface energies to prevent the initial coloni-zation of algal biofilms. It is possible to grow algal biofilms in a

vertical air lift reactor and get algal biofilm surface area productiv-

ities comparable to those with a horizontal configuration. Growing

algal biofilms in a vertical orientation will enable more efficient

land use for algal biomass and lipid production.

4. Conclusion

The substrate material affects the overall algal biofilm produc-

tivity; with biofilms grown on cellulose acetate had the highest

overall productivity (2.08 g/m2/day) among the materials tested.

Differences in the overall productivities between algal biofilms

grown on different materials were largely explained by differencesthe colonization time; after the colonization time, biofilm growth

0

2

4

6

8

10

12

Glass Cellulose Acetate Acrylic Polystyrene Polycarbonate Silicone Rubber

L i p i d C o

n t e n t ( % w / w )

a

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Glass Cellulose Acetate Acrylic Polystyrene Polycarbonate Silicone Rubber

L i p i d P r o d u

c t i v i t y ( g / m 2 d a y )

b

Fig. 5. (a and b) Lipid content of algal biofilms grown on the different materials and their lipid productivity.



8/8

rate was independent of material at 2 g/m2/day for all materials

except acrylic at 1.2 g/m2/day. The colonization time was

positively correlated to the polar surface energy of the material.

The lipid content of algal biofilms grown on different materials

was not statistically different among the materials tested.

Acknowledgements

The authors are grateful to the Natural Sciences and Engineer-

ing Research Council strategic grant and for Ontario Graduate

Scholarships for helping fund the research. Special thanks to

Margaret Pittman for her contributions for the lipid extraction

and analysis.

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