Received 26 February 2016; accepted 24 April 2016; published 27 April 2016
Cyanobacteria are photosynthetic microorganisms with morphological diversity and metabolic versatility. They are included in a wide range of microorganisms that are able to synthesize and secrete extracellular polymeric substances (EPS) mainly of polysaccharidic nature, which can remain covalently linked or loosely bound to the cell surface, or be liberated to the surrounding medium  .
Due to their interesting physicochemical properties, the EPS have found applications in many industries like textiles, adhesives, paint, food, and beverage, among others  . The EPS released into the culture medium can be easily recovered making cyanobacteria one of the most attractive sources of new polymers  . Besides, the monosaccharide composition of cyanobacterial EPS has some peculiar characteristics when compared with polymers produced by other microorganisms, such as the presence of one or two uronic acids, constituents rarely found in the EPS produced by other microbial groups  . The factors affecting EPS production during micro- organism growth have been studied previously. Several works are available that report the effects of light inten- sity both in diazotrophic and non-diazotrophic conditions. In general, the EPS production showed a positive linear effect with light intensity especially in the presence of combined nitrogen for different Nostoc species  , Arthro- spira platensis  and Cyanothece sp. CCY 0110  . However, the different Nostoc species yielded contra- dictory results indicating that EPS production is affected both by nutritional and environmental parameters even to a species-specific level  . Additionally, the synthesis of increased amounts of EPS could be part of a stress response. Some of the major stress factors are the presence of metal ions  , the availability of carbon substrates and the balance between carbon and other limiting nutrients   . The cyanobacterium Cyanospira capsu- lata responded to changes in metabolic carbon flux, showing an improvement in the synthesis of EPS with enhanced carbon flux  . The aim of this work was to study the influence of different laboratory growth conditions on the biomass and EPS production by Nostoc minutum, a diazothophic cyanobacterium locally isolated.
2. Materials and Methods
2.1. Microorganism and Culture Media
The cyanobacterium Nostoc minutum  was used in this study. Stock cyanobacterial cultures were maintained in the BW3 medium with the following composition (g/L): K2HPO4 0.25; NaHCO3 0.5; MgSO4 0.7 H2O 0.375; CaCl2 0.4; NaCl 4; NaNO3 0.5; FeCl3∙6H2O 0.024; EDTA.2H2O 0.0625; micronutrients 6.25 ml  and BG11 medium with the following composition (g/L): NaNO3 1.5; K2HPO4∙3H2O 0.04; MgSO4∙7H2O 0.075; CaCl2∙2H2O 0.036; C6H8O7 0.06; C6H5+4yFexNyO7 0.06; Na2EDTA 0.0005; Na2CO3∙(H2O) 0.010; micronutrients 1 ml  . All strains were unicyanobacterial and non-axenic, but the number of observed associated bacteria was very low. All reagents used were of analytical grade, obtained from Merck.
2.2. Culturing Procedures
All culturing procedures were performed aseptically. Stockcultures, 10 mL (0.15 g/L), were used to inoculate either erlenmeyers flasks (still cultures) or glass columns (stirred cultures). Cultivation was carried out in sterilised photobiorectors consisting of 250 mL Erlenmeyer flasks equipped with a device for aseptic removal of samples (still cultures) or 250 ml glass columns 37 mm i.d., containing 200 ml of BW3 or BG11 medium with or without the addition of NaNO3 and mixed through air injection with an aeration flow of 6.13 mL/seg (stirred cultures). The aeration was performed with filter-sterilized air. The cultures were run for 2 weeks (14 days) at 30˚C under permanent lighting of 4530 (low intensity), 7300 luxes (intermediate intensity) and 9860 luxes (high intensity).
2.3. Analytical Determinations
Cyanobacterial growth was estimated by optical density (OD) at 580 nm every 48 h. EPS production was determinated after cultures reached the stationary phase, qualitatively by Alcian blue tintion and India ink, and quantitatively by dry weight determinations. The experiment was conducted in triplicates and values were expressed as their mean.
EPS was quantified by the method of Mondal et al. modified  , culture broth were decanted into 50 ml centrifuge tubes, vortexed for 5 min and centrifuged at 10,000 rpm for 30 min at 4˚C to remove cells. The supernatants were collected and 2.0 volume of acetone 80% was added and kept overnight at 4˚C for precipitation. The pellets, collected by centrifugation at 12,000 rpm for 10 min, were dissolved in deionized distilled water and dialyzed overnight at 4˚C against deionized distilled water. Dialyzed materials were lyophilized and weighed (AdventurerTM OHAUS Corp. USA). Experiments were performed three times to ensure reproducibility.
For physical characterization of the EPS, it was determined the electric charge by precipitation with cetylpe- ridium chloride (CPC)  . The dried EPS (5 mg) was dissolved in 5 ml of 0.05 M NaCl; thereafter, 10% CPC solution was added into it until no more precipitate of EPS-CPC complex was formed. The rheological behaviour was studied using a Brookfield programmable rheometer at 25˚C following a standard method with some modification  . The EPS solution (2.0%) was prepared in deionized double distilled water. The pH of the EPS solution was adjusted to 7.0 using 1N HCl and 1M NaOH.
3. Results and Discussion
3.1. Growth and Biomass Production
An increase in biomass production and specific growth rate with the increment of light intensity were observed. However, for still cultures the light intensity showed a slight incidence. Different authors reported the impor- tance to consider this parameter because it is often found as a limiting factor in culture systems   . The efficient air-lift stirring of the glass columns plays a crucial rol for reducing shading between cells, thus allowing to obtain higher values of biomass than in still cultures. BW3 medium was better than BG11 for N. minutum growth, with maximal values of biomass concentration (4.98 DO) and the highest specific growth rate (0.019 h−1) at 9860 lux of light intensity. The BW3 medium have 50 times NaHCO3 concentration and 5 times MgSO4∙7H2O and K2HPO4 than BG11, composition that was optimal for N. minutum growth. The presence of combined nitrogen also had a positive effect on growth and specific growth rate (µ) under all the conditions assayed. The lowest values of biomass and specific growth rate were obtained for diazotrophic conditions in BG110 medium. In fact, biomass production was more affected by the availability of combined nitrogen than by light intensity (Table 1).
N. minutum cells are enclosed in a fibrous matrix at the cell wall surface that has a structural coherence suffi- cient to exclude particles (e.g. India ink, see Figure 1(A)). The observation of the preparations stained with Al- cian Blue and India ink under the light microscope, showed an EPS production according to that determined by dry weight technique. For non diazotrophic cultures, it was observed a gradual increase in EPS production with the days that was coinciding with a remarkable increase in the culture viscosity. The viscosity of the cultures was maximal at 14 days in BG11 medium (Figure 1(B) and Figure 1(C)). Besides, the Alcian Blue stain is
Table 1. Influence of N, light intensity and stirring on N. minutum biomass production.
BG110, BW30: culture media without NaNO3 added. Mean values ±SD are shown. Máximum values are in bold letter. nd = not determined.
specific to acid mucopolysaccharide  . The EPS produced by N. minutum was stainable with Alcian Blue (Figure 1(B)), which indicate that it is mainly composed of polysaccharides.
3.2. EPS Production
Figure 2 shows EPS production by N. minutum. Both the still and stirred cultures followed a similar pattern, with best yields of EPS for non diazotrophic BG11 and diazotrophic BW3 cultures. The maximal concentration of EPS (2.87 g/L) was obtained with BG11 medium under stirred conditions and high irradiance (Figure 2(B)).
Figure 1. Microphotograph of: negatively stained preparation (India ink) showing the thickness of the capsule which surrounds the trichomes of N. minutum strain (A), EPS stained with Alcian blue (B) and India ink (C) in BG11 medium (14 days).
Figure 2. EPS production by N. Minutum that were grown: in still (A) or stirred (B) cultures, diazotrophic () and non- diazotrophic () in low light (L), intermediate light (I) and high light (H), all data are means of three replicates.
There is a marked difference between the main components of the two culture media used. While, both BG110 and BW30 have no NaNO3 added, BG11 have three times NaNO3 concentration than BW3. The absence of NaNO3 could be a stress factor favourable for EPS synthesis. In fact, a number of diazotrophic strains of Nostoc have shown to produce EPS under N2-fixing conditions, with reduce production when grown on an exogenous N source  . However, the EPS production by N. minutum was maximal when the NaNO3 concentration was tripled. Similar results were informed by Nicolaus et al. for Anabaena WSAF with 56 mg/L in BG11 and 22 mg/L in BG110 media  . The yields of EPS were in agreement with other values reported for cyanobacteria by Otero and Vincenzini  with 3.5 g/L of total carbohidrates and 1.8 g/L for soluble ones. On the other hand, the EPS production by N. minutum growing in BW3 medium was maximal in diazotrophic conditions, both for still (1.66 g/L) and stirred (2.56 g/L) cultures showing that EPS production varies with nutrient availability  and environmental conditions  . The progressive increase in culture viscosity of N. minutum stirred cultures observed when growing non diazotrophically in BG11 medium, may be due to the release of large amounts of EPS. On the contrary, this phenomenon was reported for the cyanobacteria Cyanospira capsulata growing diazotrophically   . N. minutum released the EPS while the capsule that surrounds the cells became minimal, in contrast, the unicellular cyanobacterium Cyanothece sp showed the solubilization of the external part while the thickness of the capsule remained almost constant  . EPS produced by N. minutum showed precipitation with CPC, indicating the presence of acidic groups. The EPS include different classes of macro- molecules such as proteins, polysaccharides, uronic acids and other compounds not yet fully characterised  . The precipitation of EPS with CPC allow to confirm the anionic nature of the EPS due to interaction of qua- ternary ammonium ions of the CPC with the acidic groups to form a polysaccharide-CPC complex  , showing interesting properties for metal uptake in bioremediation field  . The viscosity of N. minutum EPS showed a decrease with an increase in the shear rate. Such rheological behaviour is the characteristic of a typical pseu- doplastic non-Newtonian ﬂuid  and agrees with results reported by Khattar et al.  .
This work was supported by the Secretaría de Ciencia y Técnica, UNSL, Argentina.