Available online at www.sciencedirect.

com

Aquacultural Engineering 38 (2008) 2635

www.elsevier.com/locate/aqua-online

Comparisons of the growth of six diatom species between

two configurations of photobioreactors
Fernando R. Silva-Aciares *, Carlos E. Riquelme
Unidad de Microbiologa Aplicada, Departamento de Acuicultura, Universidad de Antofagasta, Casilla 170, Antofagasta, Chile
Received 18 July 2007; accepted 18 October 2007

Abstract
The present study reports on the mass culture of six benthic diatom species in two systems termed photobioreactors. One
system called bristles photobioreactor (PBB) contained polyvinyl chloride (PVC) bristles which provided attachment surface for
adhesive diatoms, and an airlift system to provide constant water movement. Results obtained using this apparatus are compared
with parallel results obtained when inoculating the same diatom species into a bubble column photobioreactor without support
bristles (PBC), and a strong column of air bubbles to provide constant agitation. The results showed high efficiency of the PBB in
terms of concentration and biomass of the adhesive diatoms Amphora spp., Amphora spp2, Navicula spp. and Nitzschia ovalis, on
the support filaments. The lesser adhesive diatoms Nitzschia sp. and Cylindrotheca closterium grew better in suspension in the PBC
system. The relation existing between the growth of the microalgae and their accompanying bacterial flora were strongly correlated,
indicating simultaneous growth of the two populations. It was also shown that higher bacterial counts were found in the systems
having the highest microalgal populations. The present results suggested positive feasibility of growing benthic diatoms of
commercial importance, particularly for aquaculture, based on the high degree of adhesiveness of the diatoms produced in the PBB
systems. Greater efficiency was obtained in the PBC systems for producing high concentrations and biomass of the lesser adhesive
diatom species. This new technology may permit optimizing yields for the mass production of microalgae of commercial
importance. Data in the literature suggest that biofilms of benthic diatoms the same as or similar to those presently cultured can aid
the settlement and growth of larvae of certain benthic marine invertebrates, some of which are important species in aquaculture.
# 2007 Elsevier B.V. All rights reserved.

Keywords: Benthic diatoms; Photobioreactor; Microalgae; Biofilm

1. Introduction exploitation. The need for high levels of production and

There exists a high diversity of photosynthetic
microorganisms which are potentially valuable for use
in the food, cosmetics, pharmaceutical, agricultural and
aquiculture industries (Grobbelaar, 2004; Richmond,
2004; Spolaore et al., 2006). Nevertheless, a small
percentage of these species have been put to industrial
use, due to the lack of culture systems available for their
* Corresponding author. Tel.: +56 55 637881; fax: +56 55 637804.
E-mail address: fsaciares@uantof.cl (F.R. Silva-Aciares).
use of monoalgal cultures has led to the development of
experimental laminar and tubular closed-circuit algal
culture units termed photobioreactors. Increase in
development of these systems intensified at the end
of the 1980s, following the general interest in
development of commercial-sized photobioreactors
(Pulz, 2001; Tredici, 2004; Krichnavaruk et al., 2005,
2007). Operating characteristics of these systems
include capacity for regulation and control of important
culture parameters including temperature, hydrody-
namics, contamination control and CO2 regulation
(Pulz, 2001). These systems have permitted the culture

0144-8609/$ see front matter # 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaeng.2007.10.005
 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635
of a variety of microalgae under reproducible culture
conditions. Some genera which have been cultured in
these systems include Chaetoceros (Krichnavaruk et al.,
2005, 2007), Haematococcus (Olaizola, 2000), Phaeo-
dactylum (Acien Fernandez et al., 2000; Molina Grima
et al., 2000), Rhodomonas (Eriksen et al., 1998),
Porphyridum (Muller-Feuga et al., 1998), Synechococ-
cus (In Soo Suh et al., 2001), Chlorella (Ogbonna et al.,
1997), Croococcus (Fischer et al., 1997), Nostoc
(Fischer et al., 1997), Chlamydomonas (Janssen
et al., 2000), Dunaliella (Janssen et al., 2000),
Chlorococcum (Masojidek et al., 2000; Zhang et al.,
1997), Nannochloropsis (Chini et al., 2000) and
Spirulina (Levert and Xia, 2001).
There are numerous microalgal species of impor-
tance for mass culture in aquaculture, including the
genera Nannochloropsis, Isochrysis, Chaetoceros, Ske-
letonema, Tetraselmis, Monochrysis, Dunaliella, Chlor-
ella, Scenedesmus, among others (Silva, 2003; Muller-
Feuga, 2004). The preceding is typically plankton in
nature; benthic diatoms, which normally live associated
with marine substrates, also can be useful for many
applications. One of the main characteristics in the
growth of benthic diatoms, is that they form biofilms on
the substrate. The typical life habits of these diatoms
make them difficult to grow in traditionally used culture
systems which are oriented toward the maintenance of
suspended microalgae. The planktonic microorganisms
play an important role as food for larval and juvenile
mollusks in mass hatchery cultures, and the benthic
microalgae together with bacteria and other micro-
organisms which form biofilms are of interest for the
coating of substrates used in attraction and settlement of
molluscan larvae (Takami et al., 1997; Avendano-
Herrera et al., 2002, 2003; Avendano-Herrera and
Riquelme, 2007). Certain biofilms are capable of
liberating chemical signals which stimulate and favor
larval settlement and metamorphosis (Kavouras and
Maki, 2000; Steinberg et al., 2002; Zhao et al., 2003).
The biofilms are a source of food for some postlarval
stages of marine invertebrates, but this characteristic
has not been exploited under controlled conditions since
the technology is not available for the massive
production of algal biofilms. Previous study in our
laboratory has shown the feasibility at the bioassay level
for the production of specific diatom-bacteria biofilms
(Avendano-Herrera and Riquelme, 2007).
The objective of the present study was to evaluate the
potential use of a bristles photobioreactor containing
contained polyvinyl chloride (PVC) bristles (PBB) for
the massive culture of benthic microalgae, and evaluating
the system in parallel with a bubble column photo-
27
bioreactor without support bristles (PBC). The latter
systems were evaluated for the mass culture of suspended
microalgae which can produce a continuous food supply
for benthic molluscan larvae in aquaculture. Both
systems represent relatively new approaches to the mass
production of potentially useful benthic microalgae
(diatoms) whose culture was previously limited by the
limited culture methods available.
2. Materials and methods
2.1. Benthic microalgae (diatoms)
Benthic microalgae including Nitzschia sp. (14 mm
 6 mm), Amphora sp. (8 mm  4 mm), Amphora sp2
(11 mm  7 mm), Navcula sp. (12 mm  7 mm), N.
ovalis arnott (7 mm  4 mm) and Cylindrotheca closter-
ium (10 mm  4 mm), were isolated from a commercial
abalone (Haliotis rufescens) hatchery at Caldera, Chile
(278030 2400 S-708510 3000 W). These algae were isolated
from the surfaces of polycarbonate plates known to be
favorable substrates for larval abalone settlement. The
microflora adhered to the plates was removed by
ultrasonic vibration provided by an Ultrasonics (Cole-
Parmer) homogenizer for 30 s. Sample volumes of 0.1
and 1.0 ml were inoculated into test tubes and
Erlenmeyer flasks containing f/2 microalgal culture
media (Guillard and Ryther, 1962), supplemented with
sodium metasilicate (F/2M, Guillard and Ryther, 1962).
These cultures were incubated for two weeks at 20 8C at a
light intensity of 100 mmol m2 s1 with a photoperiod
of 12:12 h. (light:darkness). Following incubation the
microalgal species were isolated using methods
described by Alveal et al. (1995), and subsequently
decontaminated using antibiotics following the metho-
dology proposed by Hoshaw and Rosowski (1979). The
axenic condition of the resulting microalgal cultures was
verified using epifluorescence techniques (Porter and
Feig, 1980).
2.2. Disinfection of seawater and of the
photobioreactor
In all cases the seawater employed in the experi-
ments was disinfected using electrolytic treatment
(Jorquera et al., 2002), using a model JIX-40TA
seawater electrolyzer (Hoshizaki Electric Co. Ltd.,
Japon). The photobioreactors were disinfected begin-
ning 48 h before initiating experiments by thoroughly
washing them with fresh water and then filling them
with electrolyzed seawater containing 50 mg l1
sodium hydrochlorite, and left at rest for 48 h. The
28 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635

hypochlorite solution was then removed by repeated

rinsing with sterile distilled water. The PBB and PBC
were then each filled with 30 l. electrolyzed seawater,
neutralized using 150 mg l1 of sodium thiosulfate, and
allowed to rest for a period of 2 h prior to inoculation
with microalgae.

2.3. Inoculation and culture

Microalgae for inoculating into the photobioreactors

were cultured in 1-l flasks containing 500 ml of
seawater and enriched with sodium metasilicate-
containing F/2M algae culture medium as cited above,
then autoclave-sterilized at 121 8C for 15 min. The
algae were cultured for 7 days with illumination of
100 mmol m2 s1 and a 12:12 h (light:dark) photo-
period in a culture room at 20  2 8C. Cell counts
during culture were monitored using a hemocytometer.
Algal cells were removed from the walls of the flask and
homogenized prior to cell counts and transfers using an
Ultrasonic Homogenizer 4710 Series (Cole-Palmer) for
45 s. The concentration of the inoculum of each diatom
species in the photobioreactors was 1  104 cells ml1.

2.4. Description of the PBB and PBC

The PBB (Fig. 1) are constructed of transparent acrylic

plastic 1 m in length with an inside diameter of 18 cm and
outside diameter of 19 cm. The top of the tube is closed by
a PVC cap which is 21 cm in diameter and 5 cm in height,
with a 0.5 cm diameter orifice united with a 3.5 cm PVC
connector for the introduction of an air line. The base of
the tube is closed and has a 2.5 cm port connected with
PVC tubing of the same diameter and a 2.5 cm ball valve.
Within the body of the PBB there is a structure resembling
a large bottle brush bearing PVC bristles measuring
100 cm  17.5 cm, throughout the entire length of the
tube (Fig. 1). The metal bristle support is plastic coated.
The bristles and the structure were designed as supports
for the growth of the (adherent) benthic diatoms.
Preliminary data (UNPUB.) had previously shown that
the PVC bristles were the best material for this purpose in Fig. 1. Diagram of the bristles photobioreactor with PVC bristles
comparison with a number of other types of plastic (PBB) as described in the text. (A) General View of PBB, (1) top of the
tube with a PVC cap, (2) air inlet PVC connector, (3) clear acrylic
materials tested. The circulation of the cell suspension tube, (4) base coupling, (5) ball valve, (6) PVC bristles in a bottle
throughout the photobioreactor is obtained using an airlift brush with a metal axis coated by plastic, (7) PVC bubbling
system driven by air delivered through a 0.15 cm plastic column and (8) plastic compressed airline.
compressed air line to the inside of a 1.5 cm diameter,
94 cm length PVC bubbling column enclosed within
the aparatus (Fig. 1). This PVC bubbling column is above for the PBB, except that they do not contain the
throughout the entire length of the PBB. PVC bristles, and the circulation of the suspension is
The PBC systems (Fig. 2B) have a capacity of 30 L carried out by compressed air introduced at the bottom
and the same structural characteristics as mentioned of the PBC. The preceding is done to maintain the algal
 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635
Fig. 2. Schematic diagram to show water movement and air flow in
the PBB system (A) while the PBC system (B) has simple aeration.
White arrows indicate the direction of culture water flow in the PBB
system.
cells in suspension and thus favor their growth in the
water column.
The distribution of air and flow of the culture
medium within both types of system are shown in Fig. 2.
2.5. Culture harvesting: density, microalgal
biomass, and bacterial concentration
Once the photobioreactors were filled to capacity
with sterilized seawater as described above, F/2
medium enriched with sodium metasilicate was added
(Guillard and Ryther, 1962) and the cultures were
inoculated. The cultures were carried out in triplicate
over a period of 10 days, using a light intensity of
100 mmol m2 s1 with a photoperiod of 12:12 h.
(light:dark) in a room with temperature control
maintained at 20  2 8C. The concentration and
microalgal biomass as well as the bacterial concentra-
tions were evaluated every 2 days. Microalgae were
harvested manually from the PBB, dislodging the
diatoms by vigorous agitation of all the PVC bristles in
the photobioreactor column for a period of 2 min. Then
50 ml samples from each system were taken into sterile
beakers and homogenized using an Ultrasonics (Cole-
29
Parmer) homogenizer for 30 s. Diatom counts were then
made, and each sample was filtered off on tared
0.45 mm pore filters (Millipore GS) which were dried to
constant weight and weighed to determine the biomass
in each system until the 7th day of culture at which time
all the microalgae were harvested. Diatom counts were
made using a hemacytometer, and bacterial numbers
were estimated by staining 1 ml samples of the culture
with fluorochrome 46 diamido-2 phenylindole (DAPI)
(Porter and Feig, 1980) and counted using an Olympus
BH-2 epifluorescence microscope.
Dry biomass samples recovered were weighed using
a Sartorius model TE2101 precision balance
(200  0.01 g). The rate of growth of the microalgae
on the day of harvesting was expressed as cell divisions
per day, determined following the equation proposed by
Guillard (Stein, 1979):
   
3322 N2
k log
t2  t1 N1
where k is the mean rate of duplication of the microalgal
population (duplications/day), t1 the initial time, t2 the
final time, N1 the number of cells per ml at t1 and N2 is
the number of cells per ml at t2.
The PBC systems were harvested with care to obtain
all the microalgae attached to the walls and deposited in
the bottoms of the cylinders, and cell counts and
biomass determinations are made as above. Monitoring
throughout the experiments included routine (every 2
days) measurement of temperature, pH, salinity, and
dissolved oxygen.
2.6. Statistical analysis
The values for microalgae concentration in the PBB
and PBC systems were compared using a two way
analysis of variance (ANOVA) (P < 0,05) (Sokal and
Rohlf, 1980). In cases where significant differences
were found, LSD multiple comparison test was used to
identify the most important factors responsible for the
differences (Sokal and Rohlf, 1980).
The type of correlation existing between the
microalgal concentrations of the different diatoms
and the bacterial loads was determined by submitting
the data to linear regression analysis (Zar, 1984).
3. Results and discussion
The six benthic microalgal strains produced different
maximum concentrations and biomass values (Table 1)
and differed in cell size (Table 2). The maximum
30 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635

Table 1
Mean values of (A) cell concentration (104 cells ml1) and (B) microalgal biomass (g l1) for Nitzschia spp., Amphora spp., Amphora spp2,
Navicula spp., N. ovalis and C. closterium cultured in photobioreactors containing PVC bristles (PBB) and the bubble column (PBC) system
Time (days) Nitzschia spp. Amphora spp. Amphora spp2 Navicula spp. N. ovalis C. closterium
A B A B A B A B A B A B
PBB
0 1.0 0.005 1.0 0.006 1.0 0.004 1.0 0.007 1.0 0.002 1.0 0.002
1 3.6 0.024 7.3 0.017 2.66 0.011 4.0 0.034 3.0 0.010 5.7 0.017
2 20.7 0.098 22.7 0.098 11.3 0.071 19.0 0.110 9.0 0.022 25.7 0.128
3 50.3 0.395 61.7 0.350 22.3 0.157 59.7 0.486 31.0 0.059 69.3 0.462
4 92.0 0.876 115.0 0.470 53.3 0.400 97.0 0.825 80.7 0.310 129.0 0.713
5 151.7 1.443 163.3 0.863 90.0 0.650 157.3 1.190 126.0 0.605 215.7 1.090
6 186.7 1.778 122.7 1.180 107.3 0.950 252.0 1.390 156.7 0.740 266.7 1.280
7 210.0 2.020 216.0 1.276 115,4 1.166 277.0 1.563 167.6 0.833 281.7 1.410
PBC
0 1.0 0.005 1.0 0.006 1.0 0.004 1.0 0.007 1.0 0.002 1.0 0.002
1 11.7 0.111 3.3 0.012 2.6 0.011 2.3 0.010 2.3 0.008 7.7 0.025
2 22.4 0.163 18.0 0.083 7.3 0.054 8.7 0.098 6.7 0.017 33.0 0.185
3 75.7 0.623 36.3 0.223 16.4 0.130 23.0 0.414 24.7 0.044 89.0 0.600
4 120.1 1.153 74.3 0.372 37.8 0.342 52.7 0.632 63.7 0.240 170.3 0.823
5 185.0 1.579 115.2 0.662 59.0 0.764 88.6 0.895 109.0 0.458 276.6 1.241
6 220.0 1.942 142.7 0.891 87.7 0.935 126.0 1.120 135.7 0.612 331.0 1.382
7 259.3 2.266 157.6 1.080 95.3 0.973 155.3 1.200 143.0 0.690 350.0 1.600

Table 2
Data on the growth conditions of the microalgae Nitzschia spp., Amphora spp., Amphora spp2, Navicula spp., N. ovalis and C. closterium in the PBB
(A) and PBC (B) systems every 2 days and at harvest (7 days)
Time/parameter (days) Nitzschia spp. Amphora spp. Amphora spp2 Navicula spp. N. ovalis C. closterium
A B A B A B A B A B A B
Day 0 (beginning)
S% 35.5 35.5 35.4 35.5 35.6 35.5 35.5 35.4 35.3 35.5 35.3 35.3
pH 7.5 7.5 7.6 7.7 7.3 7.5 7.4 7.4 7.3 7.3 7.3 7.5
T (8C) 19.4 19.3 19.2 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.4 19.3
O2 (mg l1) 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4
Day 2
S% 35.5 35.5 35.6 35.6 35.6 35.6 35.5 35.5 35.4 35.6 35.4 35.4
pH 7.6 7.6 7.6 7.7 7.4 7.6 7.5 7.5 7.5 7.4 7.4 7.5
T (8C) 19.8 19.5 19.8 19.7 19.7 19.8 19.8 19.6 19.5 19.6 19.5 19.5
O2 (mg l1) 8.5 8.7 8.6 8.5 8.6 8.5 8.6 8.5 8.6 8.4 8.6 8.5
Day 4
S% 35.6 35.6 35.6 35.6 35.7 35.7 35.6 35.5 35.5 35.6 35.5 35.5
pH 7.6 7.6 7.7 7.7 7.5 7.7 7.6 7.6 7.6 7.5 7.5 7.6
T (8C) 19.5 19.6 19.4 19.5 19,5 19.7 19.74 19.7 19.6 19.7 19.7 19.6
O2 (mg l1) 8.8 9.1 8.9 8.7 8.7 8.6 8.9 8.6 8.8 8.6 8.8 8.9
Day 6
S% 35.7 35.7 35.7 35.7 35.8 35.7 35.7 35.6 35.5 35.6 35.5 35.7
pH 7.8 7.9 7.9 7.8 7.7 7.9 7.8 7.8 7.8 7.9 7.7 7.8
T (8C) 19.6 19.5 19.5 19.7 19.8 19.6 19.5 19.4 19.7 19.6 19.4 19.5
O2 (mg l1) 9.1 9.4 9.2 9.0 8.9 8.7 9.2 8.9 9.0 8.9 9.1 9.5
Day 7 (harvest)
S% 35.8 35.8 35.8 35.8 35.8 35.8 35.8 35.7 35.7 35.8 35.8 35.7
pH 7.5 7.5 7.6 7.7 7.3 7.5 7.4 7.4 7.3 7.3 7.3 7.5
T (8C) 19.5 19.3 19.6 19.6 19.5 19.5 19.4 19.4 19.6 19.5 19.4 19.5
O2 (mg l1) 9.2 9.5 9.2 9.1 9.0 8.8 9.2 9.0 9.1 8.9 9.2 9.6
 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635
concentrations and microalgal biomasses of all the
benthic diatoms were produced on the 7th day of culture
(day of harvest) in both the PBB and PBC systems.
Significant differences were observed in the cell
concentrations between the two different culture systems
over these culture periods (Table 1). The maximum cells
concentrations and biomass for Nitzschia spp.
(F = 36.26, P = 0.004) were obtained in the PBC system,
reaching values of 259.33  104 cells ml1 and a
biomass of 2.26 g l1. For Amphora spp. (F = 70.63,
P = 0.001) the maximum concentration and microalgal
biomass were obtained in the PBB, reaching values of
210.00  104 cells ml1 and 1.27 g l1, respectively.
For Amphora spp2 (F = 39.13; P = 0.003) the maximum
concentration and microalgal biomass were obtained in
the PBB, reaching values of 115.33  104 cells ml1 and
1.16 g l1, respectively. For Navicula spp. (F = 882.28,
P = 0.000) the maximum concentration and microalgal
biomass were obtained in the PBB, reaching values of
277.00  104 cells ml1 and 1.56 g l1, respectively.
For N. ovalis (F = 14.56, P = 0.019) the maximum
concentration and microalgal biomass were obtained in
the PBB, reaching values of 167.67  104 celulas ml1
and 0.83 g l1, respectively. For C. closterium (F =
88.47, P = 0.001) the maximum concentration and
microalgal biomass were obtained in the PBC, reaching
values of 350.00  104 cells ml1 and 1.60 g l1,
respectively.
The results showed a greater efficiency of production
in terms of concentration and biomass of the benthic
diatoms Amphora sp., Amphora sp2, Navicula spp. and
N. ovalis, in the PBB than in the PBC systems.
Laboratory observations showed that these diatoms
grew only on the PVC bristles provided as substrates, as
well as on the internal walls of the photobioreactor. No
growth of these diatoms was observed in the water
column in PBC systems. The ability of these benthic
diatoms to adhere to a substrate was reported by
Kawamura (1996) who indicated that diatoms typically
used at least seven modes of attachment, in solitary or
colonial arrangement. Their motility and adhesive force
allow them to grow in mono-(bidimensional) or
multiple (tri-dimensional) layers. Based on the preced-
ing reference, these microalgae had type B growth,
with slow movement and a high degree of adhesion,
explaining the better growth of these types of diatom on
the PVC bristles and not in the water column. In contrast
Nitzschia spp. and C. closterium, which produced
higher concentrations and biomass in the PBC systems,
grew well in the water column, forming biofilm micro-
aggregates of only 3050 cells. These diatoms had
typically rapid movement and weak substrate adhesion
31
type A growth (Kawamura, 1996). These factors
explain the higher degree of production of these two
species in the PBC systems as compared with the PBB
systems, due to the lack of adhesion to the walls of the
systems and growth in the water column. Besides, other
factor that explains the higher microalgae production in
PBC is the turbulence created by the air bubbles that
produce the detachment of microalgae from the wall of
photobioreactors.
The differences in yields between the two types of
photobioreactores may also be explained based on their
functioning. It has been reported that cultures of the
diatom Chaetoceros calcitrans in bubbled column
photobioreactors similar to those presently used had
superposed aeration which produced a random flow of
water and turbulence within the photobioreactor, were
the microalgal cells were not appropriately circulated in
the water column, as compared with the airlift systems
which provided homogeneous water circulation pro-
moting good microalgal growth due to more efficient
utilization of the nutrients and light (Merchuk et al.,
1998; Krichnavaruk et al., 2005). Nevertheless, these
considerations are more applicable to planktonic
microalgae rather than benthic microalgae which need
a substrate in order to produce optimal growth. It should
be noted that very few studies have been made on
photobioreactors in which production of benthic
diatoms has been attempted (Lebeau and Robert,
2003; Avendano-Herrera and Riquelme, 2007). Systems
most commonly studied have included those for the
production of small-sized planktonic microalgae, used
for the feeding of marine invertebrate larvae important
in aquaculture (Becker, 1994; Krichnavaruk et al., 2005,
2007). In one of the first studies on production of
benthic biofilms, Avendano-Herrera and Riquelme
(2007), reported the production of a diatom-bacterial
microfilm of the microalga Navicula veneta and the
bacterium Halomonas sp. in a tubular photobioreactor
provided with bristles as a substrate.
In the present study, significant differences in cell
concentrations were observed between the six diatom
species during the culture period, as follows: (Nitzschia
spp.; F = 85.45, P = 0.000, Amphora spp.; F = 60.13,
P = 0.000, Amphora spp2; F = 15.23, P = 0.000, Navi-
cula spp.; F = 40.77, P = 0.000, N. ovalis; F = 42.05,
P = 0.000, C. closterium, F = 125.88, P = 0.000).
The relation existing between microalgae and their
associated bacterial load during culture trials in the two
photobioreactor systems are shown in Fig. 3. The
results suggested a strong positive correlation between
the microalgal concentration and their bacterial load.
The correlations coefficients between microalgal
32 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635

Fig. 3. Yield in microalgae concentration and bacterial load in the PBB and PBC systems by the diatoms Nitzschia spp. (A), Amphora spp. (B),
Amphora spp2 (C), Navicula spp. (D), N. ovalis (E) and C. closterium (F) during the period of culture. Vertical lines represent S.D.

concentration and bacterial load were high for all
species tested, ranging 0.9466340.991574. It was also
shown that the largest bacterial loading occurred in
photobioreactors containing the highest microalgal
concentrations. There have been studies reporting on
the strong positive correlation between bacterial
abundance and chlorophyll a concentration, variables
which are intimately linked with numbers of phyto-
planktonic cells (Abarzua et al., 1995; Bulyon and
Paveleiva, 1998).
With all the species of benthic diatoms cultured in
this study, as the diatom cells increased there was an
increasing bacterial loading of the culture which
reached the highest concentrations at the end of the
experiment (day 7) representing the microalgal sta-
tionary growth phase. Similar results have been
obtained in batch cultures of six different species of
microalgae important in aquaculture, with high con-
centrations of bacteria associated with the microalgae in
their stationary phase rather than during the exponential
growth phase (Salvensen et al., 2000). Recent studies
reported that the number of bacteria associated with the
culture of the microalgae Tetraselmis chuii and
Chlorella minutissima showed a maximum between
 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635
10 and 16 days in culture, which represented the
stationary phase of the microalgal culture (Makridis
et al., 2006). This indicated that the habitat of the
bacteria associated with the microalgae could be termed
the phycosphere, which identified and area around
the microalgal cell within which the bacteria obtain
nutrition from algal extracellular products (Bell and
Mitchell, 1972).
Consumption of extracellular products from the
benthic diatom N. veneta by associated bacteria was
demonstrated by Avendano-Herrera and Riquelme
(2007), suggesting a role of the exudates as a carbon
source for the bacteria. Microalgal cells are well-known
for their excretion of organic compounds including high
proportions of carbohydrates (Myklestad, 1995) which
contribute nutrients to heterotrophic microorganisms
(Lancelot, 1983). From a different perspective, numer-
ous studies have indicated that bacterial-microalgal
interactions also favor the growth of the microalgae
from organic compounds secreted by numerous species
of bacteria in the environment (Avendano and
Riquelme, 1999; Avendano-Herrera and Riquelme,
2007). Transformation of some of these organic
compounds into carbon dioxide probably occurs,
promoting phototrophic growth of the microalgae as
well as nitrogen fixation (Pisman et al., 2005).
Table 2 presents a record of culture conditions
observed during the entire experiment and to show that
there are not differences between the two photobior-
eactor systems. However, throughout the experiment,
the values of pH, oxygen and salinity increased, and
only the temperature remained constant. Studies by
Laurenco et al. (1997) on Tetraselmis gracilis showed
an increase in the pH after 6 days of culture, with
variations between 8.2 and 9.8. The same authors
suggested that the increase in pH of the microalgal
culture was produced by the alcalizing of the
nitrogenous organic components present in the culture
medium. On the other hand, Camacho et al. (1999)
indicate that in the cultivation of microalgae in
photobioreactors, the pH increase due to the CO2
consumption demanded by the microalgae growth and
the volumetric CO2 demand by growing culture shifted
the CO2-bicarbonate equilibrium to higher pH. These
observations agree with those of Brewer and Goldman
(1976), Spectorova et al. (1982) and Fabregas et al.
(1986), who detected a similar behavior in microalgal
cultures of Dunaliella tertiolecta, Phaeodactylum
tricornutum and Monochrysis lutheri. The increase in
oxygen was probably due to the increase in microalgal
concentration, due to its release to the culture medium
as part of the photosynthetic process. The increase in
33
salinity occurred throughout the culture period
undoubtedly due to loss of water by aeration of the
cultures.
It is of importance to note that the benthic diatoms
produced en masse in the photobioreactors (in axenic
form) could potentially be used for obtaining nutri-
tionally valuable polyunsaturated fatty acids, (PUFA).
These include a-linoleic and g-linoleic acids, and
arachidonic acid (AA), eicosapentaenoic acid (EPA)
and docohexadecanoic acid (DHA) (Spolaore et al.,
2006). The high capacity of these diatoms for
attachment to substrates makes them of great impor-
tance as a potential food for advanced larvae and
juveniles of abalone (Haliotidae) (Kawamura, 1996;
Kawamura et al., 1998; Roberts et al., 1999; Searcy-
Bernal et al., 2001; Uriarte et al., 2006) and scallop
juvenile stages (Avendano-Herrera et al., 2003) and/or
to colonize the substrata that are used for adherence,
improving the settlement and attachment of larvae,
reducing the time in the operation culture, which takes
13 weeks to grow to a density suitable for the settling
larvae (Hahn, 1989; Avendano-Herrera et al., 2003;
Lebeau and Robert, 2003). On the other hand, the
microalgae biomass obtained from bristles photobior-
eactors could be used as inocula for larger culture
systems.
In conclusion our results suggest the feasibility of
culturing commercially important benthic diatoms in
the new systems, especially for aquaculture activities.
The PBB were particularly well adapted for producing
highly adhesive diatoms. The PBC systems were best
adapted for producing high concentrations and biomass
of benthic diatoms of low adhesive capacity. This
potentially valuable technology should allow optimiza-
tion of yields in the mass production of commercially
important microalgae.
Acknowledgments
This study was made possible thanks to financing
from project FONDEF No. D03I1132. We thank Yery
Luza of the University of Antofagasta, Chile for the
technical support.
References
Abarzua, M., Basualto, R., Urrutia, H., 1995. Relacion entre la
abundancia y biomasa de fitoplancton y bacterioplancton hetero-
trofico en aguas superficiales del Golfo de Arauco, Chile. Invest.
Mar. Valparaso 23, 6774.
Acien Fernandez, F.G., Garca Camacho, F., Sanchez Perez, J.A.,
Fernandez Sevilla, J.M., Molina Grima, E., 2000. Modeling of
Eicosapentaenoic acid (EPA) production from Phaeodactylum
34 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635
tricornutum cultures in tubular photobioreactors. Effects of dilu-
tion rate, tube diameter and solar irradiance. Biotechnol. Bioeng.
68 (2), 173183.
Alveal, K., Ferrano, M.,Olivieira, E., Sar, E., 1995. Manual de
Metodos Ficologicos. Editorial Anibal Pinto S.A. Concepcion,
Chile, 863 p.
Avendano, R.E., Riquelme, C., 1999. Establishment of mixed-culture
probiotics and microalgae as food for bivalve larvae. Aquacult.
Res. 30, 893900.
Avendano-Herrera, R.E., Riquelme, C., 2007. Production of diatom-
bacteria biofilm in a photobioreactor for aquaculture applications.
Aquacult. Eng. 36, 97104.
Avendano-Herrera, R.E., Riquelme, C., Silva, F., 2002. Utilizacion de
biopelculas en el asentamiento de larvas de Argopecten purpur-
atus (Lamarck 1819) en un hatchery comercial. Rev. Biol. Mar.
Ocean. 37, 3541.
Avendano-Herrera, R.E., Riquelme, C., Silva, F., Avendano, M.,
Irgang, R., 2003. Optimization of settlement of larval Argopecten
purpuratus using natural diatom biofilms. J. Shellfish Res. 22,
393399.
Becker, E.W., 1994. Microalgae. Biotechnology and Microbiology.
Cambridge University Press, Cambridge, NY, p. 291.
Bell, W., Mitchell, R., 1972. Chemotactic and growth responses of
marine bacteria to algal extracellular product. Biol. Bull. 143,
265277.
Brewer, P.G., Goldman, J.G., 1976. Alkalinity changes generated by
phytoplankton growth. Limnol. Oceanogr. 21, 108117.
Bulyon, V.V., Paveleiva, E.B., 1998. Interrelationship between abun-
dance of bacteria and chlorophyll content of freshwater phyto-
plankton. Microbiology 67 (2), 261266.
Camacho, F., Acien, F.G., Sanchez, J.A., Garcia, F., Molina, E., 1999.
Prediction of dissolved oxygen and carbon dioxide concentration
profiles in tubular photobioreactors for microalgae culture. Bio-
technol. Bioeng. 62, 7185.
Chini, G., Pastorelli, R., Tredici, M., 2000. A modular flat panel
photobioreactor (MFPP) for indoor mass cultivation of Nanno-
chloropsis sp. Under artificial illumination. J. Appl. Phycol. 12,
525526.
Eriksen, N., Poulsen, B., Lonsmann, J., 1998. Dual sparging labora-
tory-scale photobioreactor for continuos production of microal-
gae. J. Appl. Phycol. 10, 377382.
Fabregas, J., Herrero, C., Abalde, J., Liano, R., Cabezas, B., 1986.
Biomasa production and biochemical variability of the marine
microalga Dunaliella tertiolecta (Butchner) with high nutrient
concentrations. Aquaculture 53, 187199.
Fischer, D., Schlosser, F., Pohl, P., 1997. Exopolysaccharide produc-
tion by cyanobacteria grown in closed photobioreactors and
inmobilized using white cotton towelling. J. Appl. Phycol. 9,
205213.
Grobbelaar, J.U., 2004. Algal biotechnology: real opportunities for
Africa. S. Afr. J. Bot. 70, 140144.
Guillard, R., Ryther, J., 1962. Studies of marine planktonic diatoms.
Cyclotella nana (Hustedt) and Detonula confervacea (Cleve).
Can. J. Microbiol. 8, 229237.
Hahn, K.O., 1989. Induction of settlement in competent abalone
larvae. In: Hahn, K.O. (Ed.), Handbook of Culture of Abalone
and Other Marine Gastropods. CRC Press Inc., Boca Raton,
Florida, pp. 101112.
Hoshaw, R.W., Rosowski, J.R., 1979. Methods for microscopic algae.
In: Stein, J. (Ed.), Handbook of Phycologycal Methods. Culture
and Growth Measurement. Cambridge University Press, Cam-
bridge, 446 p.
In Soo Suh., Sun Bok, L., 2001. Cultivation of cyanobacteriun in an
internally radiating air-lift photobioreactor. J. Appl. Phycol., 13,
381388.
Janssen, M., Bresser, L., Bajens, T., Mur, L., Snel, J., Wijffels, R.,
2000. Scale-up aspects of photobioreactors: effects of mixing-
induced light/dark cycles. J. Appl. Phycol. 12, 225237.
Jorquera, M., Valencia, G., Eguchi, M., Katayose, M., Riquelme, C.,
2002. Desinfection of seawater for hatchery aquaculure using
electrolytic water treatment. Aquaculture 207, 213224.
Kavouras, J.H., Maki, J.S., 2000. Biofilm effects on the attachment
behavior of zebra mussels. In: Proceedings of the ASM Work-
shop: Biofilm 2000, July, Big Sky, Montana USA, pp. 1620.
Kawamura, T., 1996. The role of benthic diatoms in the early life
stages of the Japanese abalone (Haliotis discus hannai). In:
Watanabe, Y., Yamashita, Y., Oozeki, Y. (Eds.), Survival Strategies
in Early Life Stages of Marine Resources. A.A. Balkema,
Brookfield, pp. 355367.
Kawamura, T., Roberts, R.D., Nicholon, C., 1998. Factors affecting
the food value of diatom strains for post-larval abalone Haliotis
iris. Aquaculture 160, 8188.
Krichnavaruk, S., Loataweesup, W., Powtongsook, S., Pavasant, P.,
2005. Optimal growth conditions and the cultivation of Chaeto-
ceros calcitrans in airlift photobioreactors. J. Chem. Eng. 105,
9198.
Krichnavaruk, S., Powtongsook, S., Pavasant, P., 2007. Enhanced
productivity of Chaetoceros calcitrans in airlift photobioreactors.
Bioresour. Technol. 98, 21232130.
Lancelot, C., 1983. Factors affecting phytoplankton extracellular
release in the Southern Bight of the North Sea. Mar. Ecol. Prog.
Ser. 12, 115121.
Laurenco, S.O., Lanfer, U.M., Mancini-Filho, J., Barbarino, E., Aidar,
E., 1997. Changes in biochemical profile of Tetraselmis gracilis I.
Comparison culture media. Aquaculture 148, 153168.
Lebeau, T., Robert, M.R., 2003. Diatom cultivation and biotechno-
logically relevants products. Part I. Cultivation at various scales.
Appl. Microbiol. Biotechnol. 60, 612623.
Levert, J., Xia, J., 2001. Modeling the growth curve for Spirulina
(Arthosphira) maxima, a versatile microalga for producing uni-
formly labelled compounds with stable isotopes. J. Appl. Phycol.
13, 359367.
Makridis, P., Alves Costa, R., Dinis, M.T., 2006. Microbial conditions
and antimicrobial activity in cultures of two microalgae species,
Tetraselmis chuii and Chlorella minutissima, and effect on bacter-
ial load of enriched Artemia metanauplii. Aquaculture 255, 7681.
Masojidek, J., Torzillo, G., Kopecky, J., Koblzek, M., Nidiaci, L.,
Komenda, J., Lukavska, A., Sacchi, A., 2000. Changes in chlor-
ophyll fluorescence quenching and pigment composition in the
green alga Chlorococcum sp. Grown under nitrogen deficiency and
salinity stress. J. Appl. Phycol. 12, 417426.
Merchuk, J.C., Ronen, M., Geris, S., Arad, S., 1998. Light/dark cycles
in the growth of the red microalga Phorphylidium sp. Biotech.
Bioeng. 59, 705713.
Molina Grima, E., Acien Fernandez, F.G., Garca Camacho, F.,
Camacho Rubio, F., Chisti, Y., 2000. Scale-up of tubular photo-
bioreactors. J. Appl. Phycol. 12, 355368.
Muller-Feuga, A., 2004. Microalgae for aquaculture. In: Richmond,
A. (Ed.), Handbook of Microalgal Culture: Biotechnology and
Applied Phycology. Blackwell Science, Oxford, pp. 352364.
Muller-Feuga, A., Le Guedes, R., Herve, A., Durand, P., 1998.
Comparison of artificial light photobioreactors and other produc-
tion systems using Porphyridium cruentum. J. Appl. Phycol. 10,
8390.
 F.R. Silva-Aciares, C.E. Riquelme / Aquacultural Engineering 38 (2008) 2635
Myklestad, S., 1995. Release of extracellular products by phytoplank-
ton with special emphasis on polysaccharides. Sci. Total Environ.
165, 155164.
Ogbonna, J.C., Masui, H., Tanaka, H., 1997. Sequential heterotrophic/
autotrophic cultivation. An efficient methods of producing Chlor-
ella biomass for health food and animal feed. J. Appl. Phycol. 9
(4), 359366.
Olaizola, M., 2000. Commercial production of astaxanthin form
Haematococcus pluvialis using 25000-liter outdoor photobioreac-
tors. J. Appl. Phycol. 12, 499506.
Pisman, T.I., Galayda, Y.V., Loginova, N.S., 2005. Population
dynamics of an algal-bacterial cenosis in closed ecological system.
Adv. Space Res. 35, 15791583.
Porter, K.G., Feig, Y.S., 1980. The use of DAPI for identifying and
counting aquatic microflora. Limnol. Oceanogr. 25, 943948.
Pulz, O., 2001. Photobioreactors: Production Systems for Photo-
trophic Microorganisms. Springer-Verlag.
Richmond, A., 2004. Handbook of Microalgal Culture: Biotechnology
and Applied Phycology. Blackwell Science, Oxford, p. 566.
Roberts, R.D., Kawamura, T., Nicholson, C., 1999. Growth and
survival of post-larval abalone (Haliotis iris) in relation to devel-
opment and diatom diet. J. Shellfish Res. 18, 243250.
Salvensen, I., Reitan, K.I., Skejermo, J., Oie, G., 2000. Microbial
environments in merine larviculture: impacts of algal growth on
the bacterial load in six microalgae. Aquat. Int. 8, 275287.
Searcy-Bernal, R., Velez-Espino, L.A., Anguiano-Beltran, C., 2001.
Effects of biofilm density on grazing and growth rates of Haliotis
fulgens post larvae. J. Shellfish Res. 20, 587591.
Silva, F.C., 2003. Cultivo de microalgas marinhas. in: Poli, C.R. et al.
(Orgs). Aquicultura: experiencias brasileiras. Florianopolis, Mul-
titarefa, 93120.
Sokal, R., Rohlf, J., 1980. Introduccion a la Bioestadistica. de reverte
S.A., Barcelona, 362 p.
35
Spectorova, L.V., Goronkova, O.I., Nosova, L.P., Albitskaya, O.N.,
1982. High density culture of marine microalgae-promisisng items
for mariculture. I. Mineral feeding regime and installations for
culturing Dunaliella tertiolecta Butch. Aquaculture 26, 289302.
Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., 2006.
Commercial applications of microalgae. J. Biosci. Bioeng. 101,
8796.
Stein, J., 1979. Handbook of Phycological Methods, Culture and
Growth Measurement. Cambridge University Press, Cambridge,
Massachusetts, 446p.
Steinberg, P.D., de Nys, R., Kjelleberg, S., 2002. Chemical cues
for surface colonization. Minirev. J. Chem. Ecol. 28 (10),
19351951.
Takami, H., Kawamura, T., Yamashita, Y., 1997. Contribution
of diatoms as food sources for post-larval abalone Haliotis
discus hannai on a crustose coralline alga. Molluscan Res.
18, 143151.
Tredici, M.R., 2004. Mass production of microalgae: photobioreac-
tors. In: Richmond, A. (Ed.), Handbook of Microalgal Culture:
Biotechnology and Applied Phycology. Blackwell Science,
Oxford, pp. 178214.
Uriarte, I., Roberts, R., Farias, A., 2006. The effects of nitrate
supplementation on biochemical composition of benthics diatoms
and the growth and survival of post-larval abalone. Aquaculture
261, 423429.
Zar, J., 1984. Biostatistical Analysis, Segunda Edicion. Prentice-Hall
International, p. 718.
Zhang, D., Ng, Y., Phang, P., 1997. Composition and accumulation of
secondary carotenoids in Chlorococcum sp. J. Appl. Phycol. 12,
355368.
Zhao, B., Zhang, S., Qian, P., 2003. Larval settlement of the silver-or
goldlip pearl oyster Pinctada maxima (Jamenson) in response to
natural biofilm and chemical cues. Aquaculture 220, 883901.