Appl Biochem Biotechnol

DOI 10.1007/s12010-016-2215-4

Characterization of Co-Cultivation of Cyanobacteria

on Growth, Productions of Polysaccharides
and Extracellular Proteins, Nitrogenase Activity,
and Photosynthetic Activity

Chuizhao Xue 1,2,3 & Libo Wang 1,2 & Tong Wu 1 &
Shiping Zhang 1 & Tao Tang 1 & Liang Wang 1 &
Quanyu Zhao 1,3 & Yuhan Sun 1,3

Received: 23 June 2016 / Accepted: 12 August 2016

# Springer Science+Business Media New York 2016

Abstract Cyanobacteria as biofertilizers are benefit to reduce the use of chemical fertilizers
and reestablish the ecological system in soil. In general, several strains of cyanobacteria were
involved in the biofertilizers. The co-cultivation of cyanobacteria were characterized on
growth profile, production of polysaccharides and extracellular proteins, nitrogenase activity,
and photosynthetic activity for three selected N2-fixing cyanobacteria, Anabaena cylindrica
(B1611 and F243) and Nostoc sp. (F280). After eight-day culture, the highest dry weights were
obtained in F280 pure culture and co-cultivation of B1611 and F280. Higher production of
extracellular proteins and cell-bonding polysaccharides (CPS) were observed in co-cultivations
compared with pure culture. The highest released polysaccharides (RPS) contents were
obtained in pure culture of F280 and co-cultivation of F280 and F243. Galactose and glucose
were major components of CPS and RPS in all samples. Trehalose was a specific component
of RPS in F280 pure culture. Based on the monosaccharide contents of CPS and RPS, F280
was the dominant species in the related treatments of co-cultivation. The nitrogenase activities
in all treatments exhibited a sharp rise at the late stage while a significant decrease existed
when three cyanobacteria strains were mixed. Photosynthetic activities for all treatments were
determined with rapid light curve, and the related parameters were estimated.

* Quanyu Zhao
zhaoqy@sari.ac.cn
* Yuhan Sun
sunyh@sari.ac.cn

1
CAS Key lab of Low-Carbon Conversion Science & Engineering, Shanghai Advanced Research
Institute, Chinese Academy of Sciences, 99 Haike Road, Shanghai, China
2
University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
3
ShanghaiTech University, 319 Yueyang Road, Shanghai 200031, China
 Appl Biochem Biotechnol

Keywords Co-cultivation . Cyanobacteria . Polysaccharides . Extracellular proteins .

Nitrogenase activities . Rapid light curve

Introduction

Agriculture supports the increasing populations in this world, and much more synthetic
fertilizers are produced by chemical industries in the last decades [1]. Excessive utilizations
of synthetic fertilizers lead to serious problems for the sustainable development of human
society. The application efficiency of synthetic fertilizers is very low if they exceed the growth
need of crops. It is widely considered that it also leads to soil degradation and water
contamination. Then, the yields of rice, wheat, and other crops will be decreased in these
regions because the microenvironment in soil is destroyed. Microalgae attract lots of attentions
because they could be applied for biofuel production [2], CO2 capture [3], wastewater
treatment [4], and biofertilizer development [5]. It has a long history in India and other
countries using cyanobacteria as biofertilizers [6]. Utilization of biofertilizers is of great benefit
to reestablish the ecological systems and increases soil fertilities [7]. There are several possible
mechanisms of microalgal biofertilizer for improving the crop yield. Firstly, nitrogen could be
fixed from air into soil by cyanobacteria and the available nitrogen in soil will be increased [8].
Secondly, the algal extracts are considered as the growth-promoting substances [9, 10]. Some
of the exopolysaccharides (EPS) also have plant growth promotion activity [11]. The interac-
tions of cyanobacteria and bacteria in soil are important for the plant growth [12–14]. In
general, the microalgae biofertilizers are mixtures of cyanobacteria [15] or cyanobacteria with
bacteria [16]. Combinations of two or three cyanobacteria strains could obtain similar yields of
wheat compared with that using full dose of synthetic fertilizer [15]. It should be mentioned
that combination with much more kinds of cyanobacteria will not always lead to higher
fertility. Therefore, it is necessary to make investigations to obtain a better combination of
cyanobacteria for biofertilizer.
The development of cyanobacteria biofertilizer is a long-term work from screening in
laboratory, pot experiment to field test. There are lots of experimental studies for growth
profiles of cyanobacteria, the productions of extracellular polysaccharides (EPS), and extra-
cellular proteins in the co-cultivation of cyanobacteria [17–19]. Much more characterizations
will be helpful to develop a biofertilzier. The co-cultivation of two strains of cyanobacteria
may show positive effect for their growth profiles and EPS productions, but it is possible that
the related metabolic phenotypes are inhibited. In this study, two strains of Anabaena
cylindrica and one strain of Nostoc were selected to investigate the interactions during co-
cultivations. The growth profile, productions of EPS and extracellular proteins, and N2 fixation
capabilities of these N2-fixing cyanobacteria were characterized.

Material and Methods

Microorganisms

In this study, three strains of N2-fixing cyanobacteria were used. A. cylindrica B1611 (denoted
as B1611) was obtained from the UTEX Culture Collection of Algae (Austin, USA). Both
A. cylindrica (FACHB-243, denoted as F243) and Nostoc sp. (FACHB-280, denoted as F280)
Appl Biochem Biotechnol

were purchased from the Freshwater Algae Culture Collection at the Institute of Hydrobiology
(Wuhan, China).

Co-Cultivation of Cyanobacteria

The single strain and the mixed ones of B1611, F243, and F280 were cultivated in 400-mL
bubble column photobioreactor with BG11-N/2 liquid medium for 8 days [20]. The cultivation
temperature was 28 ± 0.5 °C; the light intensity was 133.34 ± 9.14 μmol/m2/s during the
whole process of cultivation, and the flow rate of 1 % CO2 was 1.5 vvm (volume/volume/min).
The experiment design of the co-cultivation was shown in Table 1. All arrangements were
made in triplicate.
After 8 days cultivation, the biomass concentration in each tube was characterized by the
dry weight of cyanobacteria according to the method described previously [3]. The concen-
tration of extracellular proteins was measured with the reported method [21]. The cell-bonding
polysaccharides (CPS) and the released polysaccharides (RPS) were separated by Singh’s
method [18]. The concentrations of CPS and RPS were determined by the phenol-sulfuric acid
method [22]. The released polysaccharides and the cell-bonding polysaccharides after 8 days
cultivation were isolated, and their monosaccharide components were analyzed using HPLC
(Shimadzu, Japan). The chromatographic conditions are as follows: 0.65 mL/min flow rate,
50 °C, 10 mM sulfuric acid solution used as mobile phase, using HPX-87H ion exclusion
column (300 mm × 7.8 mm) and differential detector to separate the components and detect the
monosaccharides. Nitrogenase activity of each culture was determined by acetylene reduction
activity (ARA) method [23]. Rapid light curve (RLC) of each treatment was determined using
fluorescence monitoring system (FMS2, Hansatech instruments). The calculated relative
electron transport rates and photosynthetic active radiations (PAR) were correlated with
Eq. (1) [24].
P ¼ Pm •ð1−expð−α•PAR=Pm ÞÞ•expð−β•PAR=Pm Þ ð1Þ

Where P is relative electron transport rate, Pm is the photosynthetic capacity at saturation, α

is photosynthetic rate in light-limited region of RLC, and β is photoinhibition parameter.
Based on Eq. (1), the half-saturating irradiance (Ik) could be estimated by equation (2).

I k ¼ Pm=α ð2Þ

All of the parameters were estimated by Matlab (R2013a, USA).

Table 1 Initial cell density in all

treatment of co-cultivation of N2 No. B1611 F280 F243
fixing cyanobacteria (g/L)
T1 0.150 – –
T2 – 0.150 –
T3 – – 0.150
T4 0.075 0.075 –
T5 0.075 – 0.075
T6 – 0.075 0.075
T7 0.050 0.050 0.050
– no strain in this treatment
 Appl Biochem Biotechnol

Results and Discussion

Microalgae Growth Profile

The interactions between different cyanobacteria strains could affect their metabolic pheno-
types. The growth profiles of all treatments are shown in Fig. 1. The strain F280 (T2) and
B1611 + F280 (T4) had the highest biomass concentration at day 8 (Fig. 1). There was no
significant influence when F280 was co-cultivated with B1611 (T4) while the negative effect
was shown when it was mixed cultivated with F243 in T6. A slight decrease was also proposed
when B1611 was cultivated with F243 (T5) compared with pure culture of B1611 (T1). There
was no significant different between the biomass of algae in F243 pure culture (T3) and
B1611 + F243 mixed culture (T5), and the phenomenon is also observed in the comparison of
F280 + F243 co-cultivation (T6) and B1611 + F280 + F243 co-cultivation (T7).

Extracellular Protein Content

Just as shown in Fig. 2, the extracellular proteins of almost all of the treatments except T4
reached stationary phase at day 4. The extracellular protein content of F280 was higher than
that of B1611 or F243 under pure culture conditions. After 8 days co-cultivation, the
extracellular protein content in mixed cultivation treatment of B1611 and F280 (T4) were
significantly higher than those of pure culture treatment of B1611 (T1) and F280 (T2). It was
also the same pattern for the co-culture treatment of B1611 and F243 (T5), and the extracel-
lular protein content was increased compared with those in pure culture of B1611 (T1) and
F243 (T3). The extracellular protein content of three strains (T7) was closed to that of T2 and
T3 which a little higher than that of T1.

Fig. 1 Growth profiles of co-cultivation of three N2 fixation cyanobacteria for 8 days

Appl Biochem Biotechnol

Fig. 2 Productions of extracellular proteins of co-cultivation of three N2 fixation cyanobacteria for 8 days

Cell-Bonding Polysaccharides (CPS) Content

The CPS contents are shown in Fig. 3 for all treatments. At the 8th day after inoculation, the
CPS content of cyanobacteria mixed culture was significantly higher than those of the pure
culture treatments of B1611, F280, and F243, indicating that the co-culture of cyanobacteria
could increase the CPS content compared with pure culture. The highest CPS content was
obtained when B1611 was mixed cultivated with F280 (T4). The CPS contents of the other
treatment of co-cultivation were a little lower than 150 mg/L.

Released Polysaccharides (RPS) Content

Just as shown in Fig. 4, the highest RPS were observed in F280 + F243 mixed culture
treatment (T6) which was slightly higher than that of F280 pure culture treatment (T2). The
lowest RPS content was from BG11 pure culture treatment (T1). Although the RPS content in
B1611 + F280 set (T4) is lower than F280 pure culture treatment (T2), it is higher than the
average of B1611 pure culture treatment (T1).

Monosaccharide Component Analysis of CPS and RPS

Glucose (Glc), galactose (Gal), arabinose, rhamnose, and trehalose were identified in the CPS
and RPS of these treatments. The monosaccharide components are shown in Fig. 5. Glucose
and galactose are the major components of the CPS and RPS, and the total content of Glc and
Gal is higher in CPS than in RPS. Glucose is the dominant monosaccharide of CPS in B1611
pure culture treatment (T1) and B1611 + F243 mixed culture treatment (T5) shown in Fig. 5a.
 Appl Biochem Biotechnol

Fig. 3 Productions of cell-bonding polysaccharides (CPS) of co-cultivation of three N2 fixation cyanobacteria

for 8 days

An equal content of Glc and Gal was observed in RPS of F280 pure culture treatment (T2) and
F280 + F243 co-cultivation treatment (T6) shown in Fig. 5b, and Gal is the dominant
monosaccharide of others (Fig. 5). Trehalose is an important and specific component of

Fig. 4 Productions of the released polysaccharides (RPS) of co-cultivation of three N2 fixation cyanobacteria for
8 days
Appl Biochem Biotechnol

Fig. 5 Monosaccharide compositions of CPS (a) and RPS (b) in all treatments of three N2 fixation cyanobacteria

RPS in F280 pure culture treatment (T2), and arabinose (Ara) appears only in the treatments
containing F280.

Nitrogenase Activity Assay

The nitrogenase activities of all treatments during 0–6 days were much lower than those at the
end of the cultivation shown in Fig. 6. The nitrogenase activities were improved due to the lack
of nitrogen in culture medium. The nitrogenase activity at the 8th day in the mixed cultivation

Fig. 6 Nitrogenase activities (ARA) in all treatments of three N2 fixation cyanobacteria

 Appl Biochem Biotechnol

of B1611 and F280 (T4) was lower than that of the pure culture treatment of B1611 (T1) or
F280 (T2). The nitrogenase activities of T6 (F280 and F243) and T7 (B1611, F280, and F243
mixture) were higher than that of F280 pure culture (T2), illustrating that the addition of strain
F243 and B1611 + F243 mixture could raise the nitrogenase activity of the co-culture systems.
A slight difference exists between T5 (B1611 and F243 mixture), T6 (F280 and F243 mixture),
and T3 (F243 pure culture). A prominent decrease of the nitrogenase activity was observed in
T7 (three strains mixture) compared with B1611 (T1) and F243 pure culture (T3). It means that
the addition of B1611 and F280 mixture into F243 was not a good choice to obtain a higher
nitrogenase activity compared with F243 pure culture.

Rapid Light Curve (RLC) Parameters

Rapid light curve (RLC) parameters, Pm, α, β, and Ik, represent photosynthetic capacity [25].
These RLC parameters varied for different treatments during the cultivation process. The
estimated results are shown in Table 2. At the beginning of the cultivation, the performance of
the parameters Pm, α, β, and Ik of three pure strains of cyanobacteria B1611, F280, and F243
(T1, T2, and T3) is similar. At the same time, Pm, β, and Ik of T4 (B1611 and F280) and T7
(B1611, F280, and F243 co-cultivation) were significantly higher than those of the pure
strains. The maximum photosynthetic efficiency (Pm) and the light energy utilization efficien-
cy (α) of T5 (B1611 and F243 mixed culture) and T6 (F280 and F243 mixture) were
decreased, but the light suppression parameters (β) and the light semi-saturation intensity
(Ik) changed little in T5 (B1611 and F243 mixed culture), and the parameters β and Ik
increased in T6 (F280 and F243 mixture) instead. After 8 days cultivation, the maximum
photosynthetic efficiency (Pm) and the light energy use efficiency (α) were significantly
increased in all treatments, while the light suppression parameter (β) was significantly
decreased in all treatments, and the light semi-saturation intensity (Ik) was increased in T1
(B1611 pure culture), T3 (F243 pure culture), T5 (B1611 and F243 mixed culture), and T6
(F280 and F243 mixture) but was decreased in T2 (F280 pure culture), T4 (B1611 and F280
mixed culture), and T7 (B1611, F280, and F243 co-cultivation).
The interaction of difference species in co-cultivation leads to varied metabolic phenotypes.
The contributions of these species are also distinct. It is difficult to estimate or determine the
algal concentration of each species in co-cultivation process of this study. A possible evidence

Table 2 Estimated RLC parameters of N2-fixing cyanobacteria in co-cultivation at the beginning of the
cultivation (0 day) and the 8th day

Treatments Pm α β Ik

0 day 8 days 0 day 8 days 0 day 8 days 0 day 8 days

T1 25.38 41.81 0.12 0.18 0.01 0.01 216.80 235.85

T2 23.57 45.63 0.11 0.22 0.01 0.02 210.70 205.59
T3 23.17 52.79 0.12 0.14 0.01 0.01 191.20 365.54
T4 34.97 49.67 0.09 0.19 0.02 0.03 379.12 261.08
T5 19.20 61.27 0.10 0.18 0.01 0.02 194.13 335.85
T6 19.31 62.10 0.07 0.21 0.01 0.03 272.05 295.94
T7 37.60 45.84 0.10 0.19 0.05 0.02 374.02 241.75
Appl Biochem Biotechnol

of the dominant species of the treatments of co-cultivation is shown through the monosaccha-
rides contents shown in Fig. 5. Arabinose is only contributed by F280 in pure culture. It is also
found in T4, T6, and T7. The monosaccharide contents of these three treatments are similar to
T2 which indicates F280 is the dominant species in the co-cultivation. The similar result is also
shown in Fig. 5b.

Conclusions

The co-cultivation of cyanobacteria were characterized on growth profile, production of

polysaccharides and extracellular proteins, nitrogenase activity, and photosynthetic activity
for three selected N2-fixing cyanobacteria, A. cylindrica (B1611 and F243) and Nostoc sp.
(F280). After the cultivation for 8 days, the highest dry weight exist in the F280 pure culture
and the co-cultivation of B1611 and F280 strains and the lowest dry weight exist in the F243
pure culture. In this co-cultivation experiment, a higher production of the extracellular proteins
was observed in mixed culture compared with the pure cultivation and the highest output of the
extracellular proteins appeared in B1611 + F280 mixed culture, as the behavior of the cell-
bonding polysaccharides contents. The highest RPS contents were observed in F280 pure
culture and F280 + F243 mixed culture; the lowest RPS content existed in the pure culture of
B1611. It could be mentioned the contribution of nitrogen fixation by cyanobacteria will not
propose the dominant contributions if the usage amount of cyanobacteria is very low. A field
test of lettuce was performed (data not shown). The higher lettuce yield were obtained when
F280 or F280 + F243 was used as biofertilizers. The possible contribution of cyanobacteria as
biofertilizer will be evaluated in the further study.

Acknowledgments This study was supported by the Shanghai Municipal Agricultural Commission Program
(2014, No. 5–7).

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