Bioresource Technology 155 (2014) 136143

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Design of algal lm photobioreactors: Material surface energy effects

on algal lm productivity, colonization and lipid content
Scott N. Genin a, J. Stewart Aitchison b, D. Grant Allen a,
a
b

Department of Chemical Engineering and Applied Chemistry at the University of Toronto, 200 College St, Toronto, Ontario M5S 3E5, Canada
The Edward S. Rogers Sr. Department of Electrical and Computer Engineering at the University of Toronto, 10 Kings 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 biolms on different materials.
 Algal biolm growth kinetics are linear.
 Algal biolm 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 biolms
Photobioreactor
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 biolms
grown on various materials (acrylic, glass, polycarbonate, polystyrene and cellulose acetate). The growth
kinetics of the algal biolms were non-linear overall and their overall productivities ranged from
1.102.08 g/m2 day, with those grown on cellulose acetate having the highest productivity. Overall algal
biolm 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 signicantly different at approximately 2 g/m2/day. Lipid content of the algal biolms
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 lm
photobioreactors.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Microalgae are a potential feedstock for biofuels and bioproducts
and can be used to treat wastewater by removing and xing nitrogen
and phosphorous (Chisti, 2007; Mulbry et al., 2008). Production of
biodiesel and green diesel from algae is possible, but large scale commercialization of this process remains unproven (Chisti and Yan,
2011). There are many challenges that the production of algal biofuels face, such as insufcient 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 2030% of the total production costs of algal biomass
(Gudin and Therpenier, 1986; Uduman et al., 2010).

Corresponding author. Tel.: +1 416 978 8517; fax: +1 416 978 8605.
E-mail addresses: scottnicholas.genin@mail.utoronto.ca (S.N. Genin), stewart.
aitchison@utoronto.ca (J. Stewart Aitchison), dgrant.allen@utoronto.ca (D. Grant
Allen).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.12.060

Algal biolms present an opportunity to reduce the cost

of dewatering since the biomass can be more concentrated
(90150 g/L) (Ozkan et al., 2012), compared to the typical suspended algae concentrations found in photobioreactors and
raceway ponds (0.54 g/L) (Chisti, 2007). Current research on algal
biolms has mainly focused on ecological studies (Burns and Walker, 2000), with only a few studies investigating the use of algal
biolms 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 biolm photobioreactors most of which have been developed to remove nitrogen and phosphorous from waste streams. In previous
studies on algal lm photobioreactors, the algal lms 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
lm photo bioreactor for waste treatment and algal biomass production to be used in conjunction with raceway ponds; the land
productivity of the algal lm photobioreactor, was in the range

S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

of 2031 g/m2 day which was signicantly higher than the raceway pond (7.4 g/m2 day).
An algal biolm 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, glycoproteins, glycolipids, and extracellular DNA produced by the microorganisms that are imbedded in the biolm (Flemming et al., 2007).
Biolm composition varies between different lms depending on
the microorganisms present, shear forces, temperature, and availability of nutrients (Flemming and Wingender, 2010). The EPS matrix bonds the cells to each other and the attachment material
which immobilizes the cells. The cells within these biolms 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 biolm 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 algal biolm 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 lm growth kinetics in a reactor environment. Johnson
and Wen (2010) reported signicant 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 biolms grown on a rotating biolm 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 attachment of bacteria. Holmes (1986) found that in mixed algal biolm
cultures, bacterial attachment preceded algal attachment and
Hodoki (2005) showed that higher initial bacterial colonization
density led to higher algal attachment. Orientation of the biolm
may also be important in the development of algal biolms, but
has 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 biolm growth.
Irving and Allen (2011) reported that water-material contact angle
did not affect algal biolm 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 algae 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 inuence
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 interactions between algae and surface were the dominating
mechanism.

137

In previous studies of algal lm photobioreactors, suitable

materials for algal lm 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 measurement can determine the overall the productivity of algal biolms,
the colonization and conditioning phases of biolm 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 biolm productivity on different materials 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 biolm photobioreactors by determining which intrinsic
material surface energy properties affect algal biolm productivity,
colonization and lipid content. To complete this objective, a parallel 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 biolms 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 to
provide vertically grown algal biolms 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  25 cm3. 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 approximately 2  8 cm2. The materials were weighed before the experiments and then again after being cleaned and dried.
The coupon materials tested were: glass, cellulose acetate,
acrylic, polystyrene, polycarbonate and silicone rubber. The selection criteria for the materials were based on transparency, toxicity
towards algae, and water-material contact angle. The material coupons were placed in the reactor in a random order 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 biolm. In order to introduce the bacteria and EPS required to form the biolms, unsterile
wastewater from Ashbridges Bay Wastewater Treatment Facility,
Toronto, ON, was blended with Fortied Bolds 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: NaNO3
250 mg/L, CaCl2
2H2O 25 mg/L, MgSO4
7H2O 75 mg/L, K2HPO4
75 mg/L, KH2PO4 175 mg/L, NaCl 25 mg/L, Na2EDTA 10 mg/L,
FeSO4
7H2O 4.98 mg/L, H3BO3 11.42 mg/L, Na2SiO3
7H2O 58 mg/L,
ZnSO4
7H2O 8.82 mg/L, MnCl2
4H2O 1.44 mg/L, Na2MoO3
0.70 mg/L, CuSO4
5H2O 1.57, and Co(NO3)2
6H2O 0.49 mg/L. The
solution was sparged with air at 1 L/min in the reactor for 24 h

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S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

Fig. 1. Reactor conguration and setup.

before being inoculated. The inoculum contained seven algal species 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), Nannochloris
sp. (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 mL Erlenmeyer asks on an orbital shaker
set to 110 rpm before being used to inoculate the reactor. The reactor operated in batch mode for 48 h before the pumps were started
to introduce fresh FBBM. The day the pumps were started was considered day zero, and three samples of each material were removed
from the reactor on days 0, 3, 5, 7, and 10. Suspended algae samples 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 owing at 10 mL/min to give a
total CO2 content of 1% by volume. A peristaltic pump (Cole Parmer
Masterex, Model #7520-35) was used to add and remove media
from the reactor at a dilution rate of 0.96 day 1. 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 lms and to ensure there is
enough nutrients provided to the biolm. 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 Lifshitzvan der Waals components of
the surface energy, sessile drop tests were conducted using reverse
osmosis (RO) water, glycerol (Sigma Aldrich #G5516), and hexadecane (Sigma Aldrich #H6703) on the following materials: glass, cellulose 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 Lifshitzvan 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 biolms
were suspended in RO water. A vacuum ltration unit was used

to lter the suspended algal mass through Supor-450 47 mm lters with a pore size of 0.45 lm. The lters were baked at 103 C
for 3 h and weighed before and after ltration to measure the difference in dry mass.
A minimum of three coupons on day 10 were harvested, the
algal lm biomass was then freeze dried and the lipids were extracted 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 Identication System, Microbial ID Inc. (MIDI Method) (Smid and Salnger,
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 
0.25 lm) and a ame 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 biolm. Samples
of live biolm 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
cells electron density. The samples were then loaded into a Hitachi
S-3400N scanning electron microscope, frozen to
20 C to
preserve the biolm structure at a pressure of 220 Pa. At these conditions, the algal biolm remains hydrated and biolm structures
can be seen. Backscattering electron (BSE) mode was used to
observe microbes which had accumulated signicant quantities
of osmium tetroxide, which were predominantly algae cells, while
secondary electron (SE) mode was used to image the entire biolm
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 biolms predominantly contained algae, particularly S. obliquus, Ooscystis sp., C. vulgaris, and N. palea. Algal biolms grown on cellulose acetate tended
to slough off the material when harvested on day 10, but the biolm remained intact in a detached state. The overall growth kinet-

139

Glass

25
Run 1

20

Run 2

Run 3

15
10
5
0

10

12

Algal Film Biomass (g/m2)

Algal Film Biomass (g/m2)

S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

Silicone Rubber

25
20
15
10
5
0
0

Polycarbonate

20
15
10
5
0
0

10

12

Algal Film Biomass (g/m2)

Algal Film Biomass (g/m2)

Time (days)

25

Algal Film Biomass (g/m2)

Algal Film Biomass (g/m2)

20
15
10
5
0
2

10

12

10

12

10

12

20
15
10
5
0
0

Time (days)

Cellulose Acetate

Acrylic

25

Time (days)

25

Time (days)

10

12

Time (days)

Polystyrene

25
20
15
10
5
0
0

Time (days)

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

ics of the algal biolms 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 increased linear growth.
Linear growth curves for microorganisms in conventional bioprocessing systems suggest chemical or mass transport limitations
to growth. This implies there is either a nutrient diffusion or light
limitation within the biolm. Modeling of algal biolms by Flora
et al. (1995) showed the CO2 concentration within an algal biolm
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 biolm which would suggest algal biolms thicker than
200 lm would exhibit linear growth kinetics. The same concept
could also be applied to light limitations.
3.2. Algal biolm productivity
Past studies calculated overall algal biolm 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 the
initial colonization time (Schnurr et al., 2013). Fig. 3a shows the al-

gal biolm productivities for this study based on linear regressions

conducted over the entire growth period. The productivity of the
algal biolms 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 biolms grown on cellulose acetate had the highest overall productivity of 2.08 g/m2 day. The productivities of the algal biolms grown
on cellulose acetate were statistically signicantly higher than
those grown on all other materials except for silicone rubber at
the 95% condence level. The overall productivity of the algal biolms 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 period 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 lm biomass yields
above 1 g/m2 were used. This 1 g/m2 cut off point is the approximate algal lm biomass yield on a surface if the lm was 10 lm

140

S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

Algal Biofilm Productivity (g/m2 day)

(a)2.5
2
1.5
1
0.5
0

Glass (33.7 )

Cellulose Acetate
(63.1)

Glass (33.7 )

Cellulose Acetate
(63.1)

Algal BIofilm Productivity (g/m2 day)

(b) 2.5

Acrylic (66.8)

Polystyrene (72.8)

Polycarbonate
(79.1)

Silicone Rubber
(93.6)

2
1.5
1
0.5
0

Acrylic (66.8)

Polystyrene (72.8)

Polycarbonate
(79.1)

Silicone Rubber
(93.6)

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.

thick. This is the approximate thickness of an algal biolm 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 biolm and not the material, and wed expect this could reduce the effect of the material surface properties
on algal biolm growth and attachment. The new calculated productivity is shown in Fig. 3b.
The revised productivities of the algal biolms grown materials
are not statistically different at the 95% condence 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 cellulose acetate, 1.21 g/m2 day for acrylic, 1.96 g/m2 day for polystyrene, 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 biolm productivity values are on par with those obtained by Johnson and Wen (2010) (2.570.58 g/m2 day) Schnurr
et al. (2013) (2.12.8 g/m2 day), and Gross et al. (2013) (11.5 g/
m2 day). The algae cells in the PPAL reactor are not able to settle
on the materials unlike the reactor congurations 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 difcult to conclude whether algal biolms grown in a vertical orientation have any disadvantage over algal biolms grown in horizontal
congurations.
The highest reported overall algal biolm 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 reactor of 0.12 g/day if all 40 coupons were cellulose acetate. The suspended algae productivity from the reactor is calculated to be
0.72 g/day which is about 6 times greater than the algal biolm

productivity for this reactor. The higher suspended algae productivity is due to the fact that the reactors design and operation
are not optimized for algal biolm growth, but for the rapid testing
of many different material coupons.
3.3. Colonization time analysis
Conceptually, colonization time is dened as the point at which
at least one cell layer covers the entire material, which corresponds
to 1 g/m2. This point can be determined from the linear regressions
described above using only algal biolm yields greater than 1 g/m2,
then by determining the time when the algal biolm 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 polar surface energy (Fig. 4a and b, respectively). The negative colonization 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 colonization time for the cellulose acetate implies that bacteria are able to
colonize the material rapidly. Hodoki (2005) demonstrated that algal biolms had a higher growth rate on materials which were initially 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 biolm
growth rate and lower colonization time.
Colonization time as dened in this study is poorly correlated to
the water-material contact angle (P = 0.12), but is strongly correlated to the polar surface energy of the material (P = 0.0001) as
shown in Fig. 4a and b, respectively. This is counter to what Christenson and Sims (2012) claimed regarding algal biolm preferential attachment towards materials with a high surface energy,
but is in agreement with the results from Finlay et al. (2002) and
those of Ozkan and Berberoglu (2013). Ozkan and Berberoglu

S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

(a)

Colonization Time (days)

Cellulose Acetate

Acrylic

Glass

Polycarbonate

Silicone Rubber

Polystyrene

5
4
3
y = -0.04x + 5.37
R2 = 0.16
P = 0.12

2
1
0
-1
-2

30

40

50

60

70

80

90

100

Water-Material Contact Angle (degrees)

(b) 6

Colonization Time (days)

5
4
3
2

y = 1.16x - 7.62
R2 = 0.69
P = 0.0001

1
0
-1
-2

10

11

12

Polar Surface Energy (mJ/m2)

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

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

acid-base interactions between algae cells and surfaces, with
charge and Lifshitzvan der Waals forces being less important.
Since the colonization time is a signicant fraction of the algal biolm 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 interactions
still play an important role in the growth of algal biolms and can
be observed in bench scale reactors. Cellulose acetate is known to
degrade and undergo hydrolysis (Buchanan et al., 1993). The
acetate lm used in this experiment is a mixture of cellulose diacetate 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 acetate 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 biolm and
therefore is not purely a surface interaction, there is a correlation
between water-material contact angle and colonization time

141

(P = 0.002, R2 = 0.67) and the correlation between polar surface energy and colonization time remains strongly correlated (P = 0.0001,
R2 = 0.80).
The overall algal biolm productivity is correlated to the colonization time (P = 0.0001), which implies differences in productivity
of algal biolms 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 algal biolm formation on materials. It can be used to assess the rate
at which algal biolms are capable of establishing a foundation and
at which material affects will have limited impact growth. The colonization time of the algal biolm represents the initial phases of
microbial colonization and surface conditioning. Palmer (2007) described the conditioning of a surface as the accumulation of molecules at the solidliquid 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 biolms grown on the
various materials was 68% (w/w) and was not statistically significantly different between materials at the 95% condence level, as
shown in Fig. 5a. These results are consistent with the ndings of
Johnson and Wen (2010) (69% w/w) and Schnurr et al. (2013)
(510% w/w). The neutral lipid content in algal biolms was lower
than those reported for suspended algae cultures, which is typically reported to be between 10% and 50% depending on growing
conditions and species (Chisti, 2007). The neutral lipid content
for algal biolms is likely lower compared to algae grown in a suspended culture due to either the presence of bacteria and/or EPS.
Bacteria and EPS could add to the total mass of the biolm while
not signicantly contributing to the overall lipid content.
The similarity of lipid content between algal biolms grown on
different materials, suggests that algal species composition did not
vary signicantly between the biolms and that material of attachment does not affect algal biolm lipid content. Seven different
species of algae with a range of lipid contents were used as the
inoculum. If the species composition of the algal biolms grown
on different materials varied signicantly, it could cause the lipid
content of the algal biolms to be different between materials. Results from Schnurr et al. (2013) demonstrated that for algal biolms inoculated with S. obliquus the neutral lipid content of the
biolm (5% w/w) was signicantly lower than those inoculated
with N. palea (10% w/w).
Using the neutral lipid content, the lipid productivity was calculated (Fig. 5b). The lipid productivity ranges from 0.06 to 0.13 g/
m2 day and the differences in lipid productivity between algal biolms grown on different materials can be attributed to differences
in algal biolm productivity. The surface area lipid productivities
are lower than those of terrestrial crops (0.25 g/m2 day (Mata
et al., 2010)), likely because the operating conditions for the reactor have not yet been optimized and the algae in the suspended
phase has been ushed out of the reactor and not accounted for.
The lipid content of the algal biolms 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 biolms 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 biolms to the material and not affect the internal lipid content of the algae. It may not be possible to select or design materials which can improve the lipid productivity of algal biolms and
therefore other factors such as species or conditions inuence algal
lipid content should be investigated instead.

142

S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

12

Lipid Content (%w/w)

10
8
6
4
2
0

Glass

Cellulose Acetate

Acrylic

Polystyrene

Polycarbonate

Silicone Rubber

Glass

Cellulose Acetate

Acrylic

Polystyrene

Polycarbonate

Silicone Rubber

Lipid Productivity (g/m2 day)

b 0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0

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

Algae species can have a preference in initial attachment

depending on the material properties as shown by results from Sekar 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 conrm
this. The results from this study imply that while it is possible that
different algae species may initially (over 2448 h) colonize different materials at different rates, the algal biolms have a tendency
to become more or less uniform in species composition irrespective of the material properties.
Cellulose acetate appears to be an ideal material to grow algal
biolms but there was notable difculty in harvesting. Occasionally, on days 7 and 10, the entire lm would fall off the material
and fall into the reactor; this likely indicates that the biolm became too thick and the adhesion between the material and the algal biolm was not strong enough to maintain the algal biolm
when disturbed.
While algal biolms have a higher biomass concentration than
suspended algae, they tend to have lower lipids concentration
compared 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 requirements to meet the same demand. This could be improved with
reactor designs that maximize surface area to land area ratios.
Based on the algal biolm productivity and lipid results from this
experiment and the reactor conguration presented, an algal
biolm 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 conguration presented by
Christenson and Sims (2012), which has an aerial biomass productivity of 2130 g/m2 and lipid productivity of 2.22.5 g/m2, would
require 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 biolms can be grown in an airlift
reactor has signicant implications for the scaling of algal biolm
photobioreactors. The design considerations, operating parameters
and scaling of airlift reactors is well known and documented (Chisti and Moo-Young, 1987). The hydrodynamic ow in the PPAL reactor is similar to those which would be on a pilot or commercial
scale. The observations of algal biolm growth kinetics and colonization time found in the PPAL have potential to be translated to pilot or commercial scale airlift algal lm photobioreactors.
The results on how colonization time affects algal biolm productivity have signicant impacts on future design considerations
for algal lm photobioreactors with respect to the material selection and reactor conguration. Understanding that polar surface
energy is strongly correlated to the colonization time of algal biolms, it is possible to select or engineer materials which have low
polar surface energies to reduce colonization time or select materials with high polar surface energies to prevent the initial colonization of algal biolms. It is possible to grow algal biolms in a
vertical air lift reactor and get algal biolm surface area productivities comparable to those with a horizontal conguration. Growing
algal biolms in a vertical orientation will enable more efcient
land use for algal biomass and lipid production.

4. Conclusion
The substrate material affects the overall algal biolm productivity; with biolms 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 biolms
grown on different materials were largely explained by differences
the colonization time; after the colonization time, biolm growth

S.N. Genin et al. / Bioresource Technology 155 (2014) 136143

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 biolms grown on different materials
was not statistically different among the materials tested.
Acknowledgements
The authors are grateful to the Natural Sciences and Engineering 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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