Biotechnol Lett (2015) 37:18191827

DOI 10.1007/s10529-015-1864-7

ORIGINAL RESEARCH PAPER

The water footprint of biofilm cultivation of Haematococcus

pluvialis is greatly decreased by using sealed narrow
chambers combined with slow aeration rate
Shuchao Yin . Junfeng Wang .
Lin Chen . Tianzhong Liu

Received: 14 April 2015 / Accepted: 7 May 2015 / Published online: 21 May 2015
Springer Science+Business Media Dordrecht 2015

Abstract
Objective Biofilm cultivation of microalgae has
great potential in many applications. However, the
water footprint for this method has not been well
assessed. This issue was explored with the microalga
Haematococcus pluvialis.
Results Only 1.25 l water is sufficient to support
1 m2 biofilm cultivation surface. To produce 1 kg
Haematococcus biomass and astaxanthin, the water
footprint could be as low as 35.7 and 1440 l,
respectively, by sealing the biofilm in a narrow

chamber and supplying the proper amount of nutrients

if the evaporation water loss was not considered.
However, when loss of water by evaporation was
considered, the water footprint was as low as 66.9 and
2700 l, respectively, if the chamber was aerated with
CO2 at 0.014 vvm. These water footprint values are
much lower than values obtained in other research
work.
Conclusions The water footprint of biofilm microalgal cultivation can be potentially reduced by more
than 90 % if the biofilm is sealed in a narrow chamber
and supplied with a slow aeration of CO2 as carbon
source.

Electronic supplementary material The online version of

this article (doi:10.1007/s10529-015-1864-7) contains supplementary material, which is available to authorized users.

Keywords Algal culture  Astaxanthin  Biofilm

cultivation  Haematococcus pluvialis 
Photobioreactor  Water footprint

S. Yin  J. Wang (&)  L. Chen  T. Liu (&)

Key Laboratory of Biofuels, Qingdao Institute of
Bioenergy and Bioprocess Technology, Chinese Academy
of Sciences, Qingdao 266101, Shandong, Peoples
Republic of China
e-mail: wangjf@qibebt.ac.cn
T. Liu
e-mail: liutz@qibebt.ac.cn
S. Yin
e-mail: yinsc@qibebt.ac.cn
L. Chen
e-mail: chenlin@qibebt.ac.cn
S. Yin
University of Chinese Academy of Sciences,
Beijing 100049, Peoples Republic of China

List of symbols
0.1 9 N-BG11 Modified BG11 medium in which
the concentration of NaNO3 is 10 %
of the original value
CCO2
Optimized CO2 concentration for
aeration
Wt
Total biomass of the algal disk
sample at day t
DWt
Biomass concentration of the
biofilm at day t
DW0
Biomass concentration of the
biofilm at day 0

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P
T

Biotechnol Lett (2015) 37:18191827

The biomass productivity

Cultivation time

Materials and methods

Microalgal strain and inoculum preparation

Introduction
Microalgae have attracted the interest of researchers
due to their abilities to accumulate high value bioproducts. Microalgae are being investigated as promising candidates for CO2 bio-mitigation and for sustainable biofuel production due to their high productivities
and less competition with arable land (Wijffels and
Barbosa 2010). Currently, microalgae are mainly
cultivated in open ponds or enclosed photobioreactors
(PBRs). Only the open pond method is considered
financially viable for large scale processes (Jorquera
et al. 2010). The water footprints for both of these two
methods are extremely huge (Yang et al. 2011).
Biofilm cultivation method is a different method in
which dense algal cells are immobilized and attached
onto artificial supporting material(s), and liquid
medium is supplied to the biofilm to keep the algal
cells in wet conditions. By combining this immobilized biofilm with light dilution PBR structures, this
method is highly efficient in photo-phytomass conversion (Liu et al. 2013; Schultze et al. 2015) and can
be widely applied to different microalgal species
(Cheng et al. 2013; Liu et al. 2013; Zhang et al. 2014).
However, the water footprint for this method has not
been well assessed. In this paper, we proposed a tactic
to minimize the water footprint of biofilm cultivation,
which is based on the following rules: (1) the biofilm is
inoculated with a minimum volume of a modified
medium that contained enough nutrients for cultivation, (2) the biofilm is sealed in a moisture saturated
chamber, and (3) the photobioreactor is aerated with a
CO2-enriched air stream and the aeration speed is as
slow as possible.
To verify the effectiveness of the proposed tactic, a
photobioreactor was created and a serial of experiments were carried out to estimate the minimum
water footprint required by Haematococcus pluvialis.
Results indicate that the proposed water saving tactic
is very effective. To produce 1 kg Haematococcus
biomass and astaxanthin, the water footprint is as low
as 35.7 and 1440 l, respectively, which are much
lower than values obtained in other research work.

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Haematococcus pluvialis SAG 34/1b was purchased

from the Culture Collection of Algae at University of
Gottingen (SAG, Gottingen, Germany) and maintained
in BG-11 medium (Stanier et al. 1971). The algal cells
were inoculated into glass columns under continuous
illumination of 30 5 lmol photons m-2 s-1 and was
harvested at late growth phase (ca. 10 days after
inoculation) to inoculate the biofilm PBRs.
The biofilm cultivation system
Algal biofilm disks (Fig. 1b) were placed in a sealed
glass chamber. The inner space of the chamber was
moisture saturated by the evaporation of medium
during the cultivation. The height of the chamber was
only 10 mm in order to reduce the water requirement
of moistening the chamber space. The upper surface
was 210 mm 9 260 mm and the algal biofilm was
placed under the illuminating surface of 210 mm 9
210 mm. Two small ventilation holes (d = 2 mm)
located on the vertical sides of the chamber. To
inoculate the photobioreactor, modified medium was
firstly poured into the cultivation section to make a
filter paper (200 mm 9 200 mm) fully and evenly
wetted then 16 pieces of algal disks were put on the
filter paper in a 4 9 4 style. The algal disks were made

Fig. 1 The schematic diagram of the biofilm photobioreactor

adopted in this research

Biotechnol Lett (2015) 37:18191827

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by filtering some 10 mg algal biomass on a cellulose

acetate/nitrate membrane (pore size = 0.45 lm,
d = 50 mm) to make a footprint area of 10
0.5 cm2 so that the initial biomass concentration for
the cultivation surface was ca. 10 g m-2 and the
effective cultivation area is 160 cm2 for each bioreactor. The chamber was then covered and sealed with
Vaseline and finally illuminated.

Dierent amount of
nutrients

Biolm culvaon

Dierent CO2

Opmal amount

concentraons

of nutrients

Experimental design
The water footprint of the biofilm attached cultivation
is mainly composed with two parts, medium water and
evaporation loss due to aeration. A serial of experiments were arranged to determine the minimum
medium water and the minimum aeration water loss
for biofilm cultivation (Fig. 2).
First, the optimal culture medium was determined
by treating the algal disks with two sets of medium
gradients. During cultivation, the water loss due to
aeration was compensated per 24 h by adding 5 ml deionized water. Three pieces of algal disks were
sampled randomly from each chamber after 7 days
cultivation to estimate the biomass concentration and
six other algal disks were sample for analyzing
astaxanthin. Treatment led to the highest astaxanthin
productivity and was selected as the optimal culture
medium for the following studies.
To determine the aeration strategy, the optimum
CO2 concentration (CCO2 ) was estimated by cultivating the algal biofilm under air flow that enriched with
CO2 concentration gradients of 1.5, 5, 10, 15, 20, 50 %
(v/v). The CO2 concentration that resulted in the
highest astaxanthin productivity was selected as CCO2 .
The slowest aeration speed was then determined by
aerating the chamber with speed gradients of 6, 10, 20,
40, 75 ml min-1. (Supporting information gives the
evidence why 6 and 75 ml min-1 were selected as the
lower and upper limits of aeration speed).
Finally, the minimum medium water and minimum
water loss were determined to estimate the water
footprint of the biofilm cultivation. The optimal
nutrients determined earlier were dissolved in 10, 20,
30 and 40 ml water, and then were used to inoculate the
biofilm. During cultivation, the chambers were aerated
with optimized aeration strategy as determined earlier.
The water losses for different treatments were recorded
and compensated per 8 h. The water footprint for

Biolm culvaon

Dierent

Opmal CO2

aeraon speed

concentraon

Biolm culvaon

Dierent medium

Opmal

volume

aeraon speed

Biolm culvaon

Water loss due

Opmal medium

to aeraon

water volume

Water footprint for Biolm culvaon

Fig. 2 The roadmap to determine the water footprint of biofilm
cultivation for Haematococcus pluvialis

biofilm cultivation was defined as the total volume of

medium plus volume loss during aeration.
Analysis
Biomass density and biomass productivity for biofilm
cultivation
Biomass concentration and productivity were measured
with gravimetric method according to Liu et al. (2013).
For each of the algal disks, the attached algal cells were

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Biotechnol Lett (2015) 37:18191827

Fig. 3 Effects of nutrient amount on the growth and astaxanthin accumulation of biofilm cultivation of H. pluvialis. The
chamber was continuously illuminated with white fluorescent
lamps at 100 5 lmol m-2 s-1. The temperature of the algal
biofilm was 29 1 C measured by an infrared thermometer.
Compressed air (0.1 MPa) enriched with 1.5 % of CO2 (v/v)

was injected into the chamber at 40 ml min-1. Biomass

productivity results were average SD of 9 replications (3
measurements for each of 3 independent experiments); Astaxanthin content results were average SD of 3 replications (1
measurement for each of 3 independent experiments)

totally detached by de-ionized water and then filtered

onto pre-weighted 0.45 lm GF/C filter membrane
(Whatman, England). The membrane with biomass
was dried to constant weight at 105 C for 12 h. The
weight difference of the GF/C membrane was taken as
the total biomass of the algal disk sample (Wt, g) and the
biomass concentration (DWt, g m-2) was calculated as:

in which DW0 was the biomass concentration

of day 0 and t represented the cultivation time, viz.
7 days.

DWt Wt =0:001

in which 0.001 represented the footprint area (m2)

of the algal biofilm on algal disk.
The biomass productivity (P, g m-2 d-1) was
calculated as:
P DWt  DW0 =t

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Astaxanthin content analysis

The astaxanthin content was analyzed according to the
method as adapted by Zhang et al. (2014).
Analysis of data
The data were processed with Spss 11.0 and Sigmaplot
8.0 ((SPSS, Chicago, IL, US). The differences among
treatments were analyzed by ANOVA at p level of
0.05.

Biotechnol Lett (2015) 37:18191827

Fig. 4 Effects of CO2 concentration on the growth and

astaxanthin accumulation of biofilm cultivation of H. pluvialis.
The culture medium was 40 ml 10 9 0.1 N-BG11. The
chamber was continuously illuminated with white fluorescent
lamps at 100 5 lmol m-2 s-1. The temperature of the algal
biofilm was 29 1 C measured by an infrared thermometer.
Compressed air (0.1 MPa) enriched with different concentration
of CO2 was injected into the chamber at 40 ml min-1. Biomass
productivity results were average SD of 9 replications (3
measurements for each of 3 independent experiments); astaxanthin content results were average SD of 3 replications (1
measurement for each of 3 independent experiments)

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Fig. 5 Effects of aeration speed on the growth and astaxanthin

accumulation of biofilm cultivation of H. pluvialis. The culture
medium was 40 ml 10 9 0.1 N-BG11. The chamber was
continuously illuminated with white fluorescent lamps at
100 5 lmol m-2 s-1. The temperature of the algal biofilm
was 29 1 C measured by an infrared thermometer. Compressed air (0.1 MPa) enriched with 1.5 % of CO2 (v/v) was
injected into the chamber at different rates. Biomass productivity results were average SD of 9 replications (3 measurements for each of 3 independent experiments); astaxanthin
content results were average SD of 3 replications (1
measurement for each of 3 independent experiments)

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Biotechnol Lett (2015) 37:18191827

Results and discussion

The determination of culture medium for watersaving biofilm photobioreactor
When cultivated with 40 ml of 19, 59, 10 9 BG11
mediums, the biomass productivity of H. pluvialis
kept almost constant at ca. 6 g m-2 d-1 (Fig. 3a),
while the astaxanthin content and productivity decreased (Fig. 3b, c). When the initial nutrient concentration was further increased to 15 and 20 9 BG11,
the biomass productivity decreased while the astaxanthin content increased (Fig. 3a, b). For these two
treatments, the effects of salinity stress should be
considered (Kobayashi et al. 1997; Sarada et al. 2002).
In experiments with concentrated 0.1 N-BG11,
salinity and nitrogen content were decreased to a tenth,
biomass productivity increased with the increase of
medium concentration finally reaching the same level
of ca. 6 g m-2 d-1 as that in the experiment with
concentrated BG11 (Fig. 3a, d). The astaxanthin concentration was higher when algal biofilm was treated
with lower nutrient levels (Fig. 3e), so that the peak
value of astaxanthin productivity of ca. 120 mg m-2 d-1 was reached at a moderate concentration of
10 9 0.1 N-BG11 (Fig. 3f). The 10 9 0.1 N-BG11
was selected as the optimal nutrient condition for this
water-saving type biofilm photobioreactor because
under this condition growth, astaxanthin concentration
as well as astaxanthin productivity reached their
maximum values simultaneously (Fig. 3). The aim of
this research is to condense the nutrient salt in 40 ml of
such medium (10 9 0.1 N-BG11) to a water volume of
as small as possible.
The determination of aeration strategy for watersaving biofilm photobioreactor
The biomass productivity and astaxanthin concentration decreased gradually with the increase in CO2
concentration. When the CO2 concentration exceeded
20 %, the biomass and astaxanthin accumulation
almost ceased (Fig. 4a) and the algal cells were white
by 24 h if pure CO2 was injected into the chamber
(data not shown). Accordingly, 1.5 % was considered
as optimal CO2 concentration.

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Fig. 6 Effects of medium volume on the growth and astaxanthin accumulation of attached cultivation of H. pluvialis. The
nutrients in 40 ml 10 9 0.1 N-BG11 were dissolved in different
volumes of water. The chamber was continuously illuminated
with white fluorescent lamps at 100 5 lmol m-2 s-1. The
temperature of the algal biofilm was 29 1 C measured by an
infrared thermometer. Compressed air (0.1 MPa) enriched with
1.5 % of CO2 (v/v) was injected into the chamber at
6 ml min-1. Biomass productivity results were average SD
of 9 replications (3 measurements for each of 3 independent
experiments); astaxanthin content results were average SD of
3 replications (1 measurement for each of 3 independent
experiments)

Biotechnol Lett (2015) 37:18191827

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As shown in Fig. 5, the growth and astaxanthin

accumulation for different aeration speeds were close
to each other and no significant differences were found
(ANOVA, p [ 0.05).
Based on the results of CO2 concentration experiment and aeration speed experiment, aeration at
6 ml min-1 with 1.5 % CO2 was the optimal strategy
for the biofilm cultivation with this PBR. This aeration
speed could be converted into an aeration rate of
0.014 vvm (the spare volume above the cultivation
surface of this bioreactor is 441 cm3). This aeration
rate was a much lower value than that of conventional
cultivation for Haematococcus. According to our
experience, the aeration rate should be at least
0.1 vvm for this type of photobioreactor in order to
prevent the sedimentation of the cells.
The slow aeration rate provides additional benefits
such as reducing energy consumption and water
footprints of the biofilm cultivation system. During
cultivation, the slow aeration speed of 6 ml min-1
(0.014 vvm) still removed 2.5 ml water out of the
chamber per day: a total of 17.5 ml during the 7 days
cultivation. This is a comparable value with the
medium water during cultivation and it will add a
considerable portion to the total water footprint.
Can this aeration speed of 6 ml min-1 (0.014 vvm)
be further decreased? There are at least two possible
ways to meet this aim. The first one is to change

continuous aeration style to batch mode, which means

the entire required carbon element is injected into the
chamber in the form of CO2 at the commencement of
cultivation and then the bioreactor is totally sealed so
that the water loss due to aeration is significantly saved
because there is no gas flow at all. Another way to
make a compromise between continuous and batch
mode, means that CO2 is intermittently injected into
the chamber so that the total aeration time as well as
water loss is significantly decreased. For example, if
the chamber is aerated at 1 min/h, the water loss would
be reduced to approx. 1/60 of the continuous aeration
value. However, for both of these two possible water
saving ways, the CO2 concentration as well as O2
build-up, should be handled carefully because of
significant effects on growth (Fig. 4).
Estimation of the water footprint for water-saving
biofilm photobioreactor
The minimum amount of water to initialize the biofilm
cultivation was determined by monitoring the effects
of water volume on the growth and astaxanthin
production. Results showed that with values of 20,
30 and 40 ml, the growth, astaxanthin content and
productivity were at high levels and close to each
other; however, when the water volume was decreased
to 10 ml, both of the biomass and astaxanthin

Table 1 Comparison of water footprint during biomass and astaxanthin production with Haematococcus by different cultivation
systems a
Strain

Outdoor/indoor

PBRs

Water footprint
for biomass (l kg-1)

Water footprint for

astaxanthin (l kg-1)

References

AQSE002

Outdoor

Pond

3846b

1,28,200b

Olaizola (2000)

K-0084

Outdoor

Tubular

952

13,513b

Aflalo et al. (2007)

K-0084

Outdoor

Column

172b

5882b

Wang et al. (2013a)

K-0084

Outdoor

Column

285b

6250b

Wang et al. (2013b)

26

Outdoor

Pond

547b

19,584b
b

Zhang et al. (2009)

b

CCAP 34/8

Indoor

Column

1429

178,571

Del Rio et al. (2005)

CCAP 34/7

Indoor

Column

675b

25,000b

Harker et al. (1996)

NIES-144

Indoor

Column

465b

15,873b

Yoo et al. (2012)

SAG 34-1b

Indoor

biofilm

714b

26,785b

Zhang et al. (2014)

SAG 34-1b

Indoor

biofilm

35.7 (66.9)c

1440 (2700)c

This research

The water footprint was briefly estimated with only the culture broth volume. The water loss due to evaporation was not considered
because no reliable data were reported for most of these listed researches

The data were calculated by the authors of this article according to the original reported results

The number in the brackets were water footprint that included the evaporation losses

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1826

accumulations were also decreased (Fig. 6). This

might be due to the high salinity stress in the 10 ml
treatment. Ten ml was considered insufficient to
inoculate the chamber so 20 ml was used as the
minimum volume to start this biofilm cultivation. In
this bioreactor, the total cultivation area is 160 cm2,
and, consequently, 1.25 l water is required to inoculate
each m2 of cultivation surface. As shown in Fig. 6, the
biomass productivity, astaxanthin content as well as
astaxanthin productivity for 20 ml treatment was ca.
5 g m-2 d-1, 2.6 % and 124 mg m-2 d-1, respectively. Accordingly, the water footprint for producing
1 kg Haematococcus dry mass (that contains more
than 2.5 % of astaxanthin) and 1 kg pure astaxanthin
were 35.7 and 1440 l, respectively. A total of 17.5 ml
water was removed out of the chamber due to aeration.
If this part of water loss was included, the water
footprint will increase 87.5 %, viz. 66.9 l for 1 kg dry
mass and 2700 l for 1 kg astaxanthin.
Water footprint comparison for different
cultivation system
The water footprint for biomass and astaxanthin
production with Haematococcus is summarized in
Table 1.
During this research, we found that 0.5 l water was
sufficient to maintain 1 m2 biofilm. This finding
indicates that the current water footprint might be
further decreased. In this research, all the nutrient salts
were supplied to the biofilm at one time at the
beginning of cultivation. This may, however, bring
about a high salt shock to the microalga. A precise and
sophisticated nutrient supply technology might be
very helpful to improve the growth and reduce the
water footprint. Moreover, the water footprint might
be related to the algal species, strains and cultivation
time. A high salt stress-toleratant species might have
an even smaller water footprint, while, species that
secrete products might require more water to relieve
any possible feedback inhibition effects. In this
research, cultivation lasted for 7 days for each batch
because, according to our previous research (Zhang
et al. 2014), both shorter or longer cultivation times
would decrease astaxanthin productivity. Other species or strains though may need different times and
conditions to reach the best output. The current
narrow-chamber, water-saving biofilm photobioreactor is only suitable for indoor conditions. If applied

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Biotechnol Lett (2015) 37:18191827

outdoors, temperature control must be considered. For

large scale applications, other aspects, including
inoculation, harvesting, supporting material, etc.,
should also be carefully evaluated.
Acknowledgments This work was supported by the National
Natural Science Foundation of China (41276144), the Director
Innovation Foundation of Qingdao Institute of Bioenergy and
Bioprocess Technology, CAS and Science and Technology
Development Planning of Shandong Province (2013GHY11520).
Supporting information Additional materials and methods.
Supplementary Table 1Stoichiometric calculation of carbon element that deposited in algal biofilm (the carbon source
in medium, e.g. Na2CO3, was not considered in calculation).

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