Introduction

Organisms of the genus Geitlerinema exist in marine, terrestrial, and hypersaline environments; their presence is associated with low ammonia and orthophosphate concentrations (Major et al. 2005). Geitlerinema is also linked to ecosystems that have anthropogenic impacts (Vicente and Miracle 1992) and artificial environments, such as secondary wastewater plants that treat effluent from the paper industry, in which Geilterinema, Phormidium, and Pseudoanabaena predominate over heterotrophic bacteria and are the only photoautotrophic organisms (Kirkwood et al. 2001). The first report on the existence of Geitlerinema in Baja California, México, detected and isolated it from thermal ponds in 2001 (López-Cortés et al. 2001).

The species Geitlerinema lemmermannii (Woloszyńska) Anagnostidis was described as a freshwater planktonic cyanobacterium that is common to tropical swamps and marsh environments (Komárek and Anagnostidis 2005). In 2006, cells of this species were isolated from shrimp cultures that were grown in highly carbonated water in the Mexicali Valley, Baja California, México, where this species was the predominant species in winter at 26.3 °C, 8.8 mg oxygen L−1, 0.08 mg ammonium L−1, 0.001 mg nitrites L−1, and undetectable nitrate levels (Fierro 2006).

There is little data on the biology and physiology of G. lemmermannii. Thus, we examined its growth characteristics, proximal composition, and capacity for phosphorus and nitrogen uptake.

Materials and methods

Geitlerinema lemmermannii, isolated from the tanks and effluent of shrimp farms in the Mexicali Valley, Baja California, Mexico (Fierro 2006), was identified using the taxonomic keys of Komárek and Anagnostidis (2005).

G. lemmermannii was maintained in triplicate, monospecific, nonaxenic batch cultures in 2.8-L Fernbach flasks that contained 2-L F Guillard medium or the 2 F variation with double the concentration of nutrients (Guillard and Ryther 1962; Table 1). These media preparations had the same nitrogen and phosphate concentrations as the aquaculture wastewaters in Baja California (Aguilar-May and Sánchez-Saavedra 2009). The original nitrogen source of F Guillard medium was replaced by ammonium nitrate. The cultures were kept at 24 ± 0.5 °C under continuous light at 100 μmol photons m−2 s−1 (Daylight fluorescent light F40D.EX, General Electric Company, USA). pH was not controlled and was pH 7 ± 0.5 at the start of the cell culture and pH 9.3 ± 0.2 at the end.

Table 1 Chemical composition of culture medium based on 1-L Guillard and Ryther (1962) medium
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Growth assessment

For cultivation of G. lemmermannii, 81.3 g of glass pearls (2-mm diameter) was added in order to avoid the formation of films or flocks on the walls and bottoms of the flasks. The increase in cell density was measured using a hemocytomer. Clumps of cells were dislodged by ultrasonication for 2 min at 100 kHz with an L&R Solid State/Ultrasonic model T−9B (L&R Manufacturing Company, USA), as proposed by Voltolina (1985). The growth rate ( \( \mu =\frac{ \log {}_2\left({\mathrm{N}}_2\right)- \log {}_1\left({\mathrm{N}}_1\right)}{t_2-{t}_1} \), defined by the number of cells N 2 at a second day t 2 after growing from initial values N 1 and t 1) and maximum of cell divisions (DP max as the higher value of DP = N 2 − N 1) were calculated according to Fogg and Thake (1987). The daily growth rate was added to that of the previous day to obtain the accumulated growth rate ( \( \varSigma \mu ={\displaystyle \underset{i\le t}{\overset{t}{\varSigma }}}={\mu}_1+{\mu}_2+\dots +{\mu}_t \), according to Nieves et al. 1998).

Proximal composition and nutrient removal

The cell density and proximal composition were measured at 0, 48, 120, and 168 h. Total ash-free dry weight and ash content were measured per Sorokin (1973). Proteins, carbohydrates, and lipids were measured in 30-mL samples that had been concentrated by filtration through 2.5-cm GF/C glass fiber filters with a nominal 1-μm pore size (Whatman, England). Proteins were extracted with 0.1 N NaOH at 100 °C for 15 min and quantified according to Lowry et al. (1951). Carbohydrates were extracted according to Whyte (1987) and quantified according to Dubois et al. (1956). Lipids were analyzed using the method of Bligh and Dyer (1959). Chlorophyll and carotenoids were measured according to Parsons et al. (1984).

To assess the nutrient levels in the culture medium, 50-mL samples were filtered through a 4.7-mm GF/C glass fiber filter to remove cells. Nutrient concentrations were spectrophotometrically measured at various times on a Hach DR/400 UV (Hach Company, USA)—nitrates were evaluated by cadmium reduction, nitrites by the diazotization method, ammonium by the silicate method, and phosphates by the ascorbic acid using the standard reagents and techniques of Hach (1997). As a control treatment, culture medium that lacked G. lemmermannii was used at similar nutrient concentrations as in the experimental treatments. The nutrient consumption was expressed as medium percentage concentrations, measured to each nutrient through time, as well as uptake rate in pg cell−1 h−1, which resulted from dividing the concentration of the nutrient consumed at a certain time, by the number of cells at the same period of time.

Statistical analysis

Differences in growth, nutrient removal, chlorophyll a and carotenoid content, and proximal composition were evaluated by analysis of covariance (ANCOVA). The assumptions of the parametric statistics were verified, and Tukey’s comparison test was used when significance was observed. When the data were nonparametric, a Kruskal-Wallis test was used. All statistical analyses were performed with Statistica®, version 6, and the significance level was α = 0.05. Student’ t test and Kruskal-Wallis test were performed using MINITAB 15.

Results

Growth

The growth of G. lemmermannii in either medium (F and 2 F) was not modified by the availability of nutrients. Its daily cellular density was similar between media (t cal = 0.551, t crit = 2.101; Fig. 1). The acclimation phase occurred during the first 9 h and was followed by an exponential phase at 96 h in both media (Fig. 1). The growth rate (μ) was similar between treatments (p = 0.610) and its maximum value was that of the first day. Also, the accumulated growth rate (Σμ) (t cal = 1.898, t crit = 2.024) and daily production (p = 0.598) were statistically similar (Table 2).

Fig. 1
figure 1

Mean values of log10 numbers of Geitlerinema lemmermannii (cell number mL−1). Cultures in F medium (open circles) and 2 F medium (filled circles). Bars indicate standard deviation (n = 3)

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Table 2 Mean values and standard deviation of cell density (cells mL−1), growth rate during exponential phase (μ as divisions by day), accumulated growth rate (Σμ as divisions by day), and maximum daily production (DP max as cells mL−1 day−1) of Geitlerinema lemmermannii cells in F and 2 F media cultures
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Proximate composition

The total percentages of proteins (p = 0.323), carbohydrates (p = 0.957), and lipids (p = 0.414) were similar between treatments, increasing over time in both media, with a higher protein and lipid percentage at 120 h in F medium (Fig. 2). The percentage of ash was similar between treatments (p = 0.947) and decreased over time (Fig. 2). Chlorophyll a (p = 0.159) and carotenoid (p = 0.195) content increased over time and did not differ between treatments (Fig. 3).

Fig. 2
figure 2

Mean percentage of ash and biomolecules in Geitlerinema lemmermannii relative to organic dry weight obtained in F (dotted bars) and 2 F (diagonal lined bars) media cultures: a ash, b proteins, c carbohydrates, d lipids. Lines indicate standard deviation, and a > b > c > d > e > f > g > h (n = 3)

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Fig. 3
figure 3

Mean chlorophyll a (a Cha) and total carotenoid contnet (b Ct) in Geitlerinema lemmermannii cultures growth in F (dotted bars) and 2 F (diagonal lined bars) media. Lines indicate standard deviation (n = 3)

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Nutrient removal

G. lemmermannii showed selective nitrogen consumption as species-specific ammonia consumer. The pH in this experiment was not controlled and ranged from pH 7.5 at the beginning to 9.5 at the end of both cultures.

The nitrate uptake rate was similar between treatments (p = 0.738; Fig. 4b). Nitrite concentrations were similar between treatments (p = 0.668) and increased with age in both media (F, p = 0.001, and 2 F, p = 0.002; Fig. 5). Ammonia uptake in F medium differed over time (p = 0.001), decreasing steadily until 96 h (Fig. 4a). The total ammonia removal rate was 80.21 % in F medium and 61.81 % in 2 F (Table 4).

Fig. 4
figure 4

Percentages of mean values of nutrient concentration in Geitlerinema lemmermannii cultures for ammonia (a), nitrate (b), and phosphate (c) (n = 3). Cells were maintained in F (open circles) and 2 F (filled circles) media. 2 F data in a missing due to technical problems

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Fig. 5
figure 5

Nitrite production in Geitlerinema lemmermannii cultures in F (open circles) and 2 F (filled circles) media (n=3)

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Phosphorous uptake differed between treatments (p = 0.039) and over time (p = 0.11; Fig. 4d). Phosphorous consumption by cells was favored in the first several hours in 2 F medium. The total orthophosphate removal was 90.99 % in F medium and 63.79 % in 2 F (Table 4).

Discussion

To the best of our knowledge, this study represents the first report on G. lemmermannii growth in culture, and of its proximate composition and macronutrient consumption rates (Table 2). These values were lower than those for Synechococcus elongatus (Aguilar-May and Sánchez-Saavedra 2009) and similar to those for Arthrospira maxima (Cruz-Fraga 2003) which are two well-known cyanobacteria, when cultured under similar conditions. The growth rate calculated for G. lemmermannii was higher than the rate calculated for other microalgae that are commonly used in aquaculture (Table 3).

Table 3 Growth rate (μ as division by day) and percentage (%) mean values of proximal composition in Geitlerinema lemmermannii cultures compared with other microalgae
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Chlorophyll a and carotenoid synthesis in G. lemmermannii is linked to their ability to perform oxygenic and nonoxygenic photosynthesis (Whitton and Potts 2000). Certain cyanobacteria synthesize pigments and proteins proportionally to the amount of nitrogen available (Loreto et al. 2003). Generally, the carotenoid content declines because of limited nitrogen availability (Rüker et al. 1995). In G. lemmermannii, chlorophyll a values were decreased at 168 h in F medium, but carotenoids continued to be synthesized until the experiment was terminated. In S. elongatus, the formation of chlorophyll-protein complexes increases as a consequence of a self-shading effect, regardless of the concentration of available nutrients in the media; this is a common response that occurs in high-density cyanobacteria cultures (Aguilar-May and Sánchez-Saavedra 2009). In this experiment, G. lemmermannii cells grew to form both dense conglomerates and free trichomes. Therefore, light limitation could occur in the cell conglomerates, producing a response similar to that observed for S. elongatus.

Although protein synthesis in G. lemmermannii increased in correlation with chlorophyll a content, the percentage of protein was stabilized at the stationary growth phase, as was the removal of ammonia and its incorporation into nitrogenous compounds. These trends might also be attributable to the high nitrite concentrations and limited CO2 availability that were presented at the end of the experiment.

Cyanobacteria are commonly known to be high protein producers (Table 3) and, based on our present study, so is G. lemmermannii. Certain species of Chlorella spp., which are maintained in batch cultures, show increased lipid content and reduced protein content over time (Pratt and Johnson 2006). However, for G. lemmermannii, the protein content remained threefold higher than the lipid content under both culture conditions (media F and 2 F), but the difference was smaller in the initial hours of culture. Limited carbon availability and low nitrogen assimilation at the stationary growth phase might have influenced biomolecules synthesis, which may explain the concurrent reduction in the assimilation of ammonia.

The high ash content present during the first days of G. lemmermannii culture could be attributed to the accumulation of minerals as the consequence of the high specific requirements of this group (Vonshak 1986). This observation could also be ascribed to salts present in the media that was used, which can accumulate in the intracellular space within the cyanobacteria (Zhu and Lee 1997). Ash content is one of the cell components that shows high variation, which is correlated with the age of the cyanobacteria cultures—i.e., 15 % at day 1 and 50 % at day 30 (Torres-Ariño and Mora-Heredia 2010). When cyanobacteria cells develop blooms, they produce low dry matter (0.2–14 %) and high ash content (31–71 %) (Roger et al. 1986; Roger 2005).

Inorganic nitrogen is a nutritional requirement of microalgae; nitrate is the most common dissolved form of nitrogen (Herrero et al. 1985). However, in certain conditions, microalgae prefer to use ammonia to conserve the energy of eight electrons from the photosynthetically reduced ferrodoxin, which is needed to reduce nitrate (Manzano et al. 1976; Miller and Castenholz 2001). It is also known that concentrations of ammonia greater than 0.5 to 1.0 μM can inhibit nitrate absorption (Darley 1987). Based on these findings, the nitrogen consumption pattern observed in G. lemmermannii cultures could have resulted from ammonia-dependent repression of gene expression including genes encoding functional enzymes, such as nitrate reductase (Nar) and nitrate permeases (Wang et al. 2000). Even the lowest concentration of ammonia detected in F medium, 22 μM (0.41 mg L−1), can inhibit nitrate metabolism (Darley 1987). This finding is supported by a report that the Geitlerinema genus contains no nitrate consumers, as a consequence of nonfunctional reductases enzymes and no functional NtcA transcription factor that enhances the expression of glnA, a glutamine synthetase (GS) (Luque et al. 1994; Miller and Castenholz 2001). In freshwater ecosystem soil samples in which Geitlerinema sp. has been isolated, the nitrate and nitrite concentrations are high and vary more widely (250 to 600 μM) than ammonia concentrations (4 to 166 μM) (Major et al. 2005) (Fig. 5). Those observations suggest a more direct link of Geitlerinema spp. with ammonia than with nitrate.

Furthermore, G. lemmermannii produced an abundance of blue pigment, which was not measured but was evident visually. Blue pigment production has been implicated in an evolutionary inability to use nitrates in certain cyanobacteria (Miller and Castenholz 2001).

Nitrite excretion has been attributed to limited CO2 availability that is induced by high intracellular ammonia content and the inhibition of nitrite reductase (Nir) when ammonia or nitrates are available in the medium (Reyes et al. 1993; Suzuki et al. 1995). The decrease in medium dissolved ammonia and the nitrite excretion observed for G. lemmermannii cultures indicate a biological effect of moving nitrogen from the extracellular to intracellular environment.

Regarding the phosphate depletion observed in this study, there are some reports for Geitlerinema in natural environments that suggest some members of the genus to be biological drivers of phosphate removal: They inhabit environments with an important dynamic of phosphates and become predominant species when these ions had decreased at the lowest levels, 0.04 and 1 μM (Major et al. 2005). This is similar to what occurs in a mesohaline hypereutrophic environment, in which appears when phosphorus starts to be depleted and becomes dominant when nutrient concentrations decline. In contrast, when G. amphibium appeared but was replaced by another genus, such as Planktothrix and Pseudoanabaena, the phosphorus content did not decline (Chomérat et al. 2007). One of the most limiting factors for photosynthetic growth is low phosphate availability. Therefore, when phosphate is available, cells tend to store it intracellularly in the cytoplasm, to use it directly for energy, or to form cellular structures.

The nutrient removal measured for G. lemmermannii was similar to that described for S. elongatus, for which phosphorus removal is more efficient than nitrogen removal (Aguilar-May and Sánchez-Saavedra 2009). In this study, the total phosphorus uptake by G. lemmermannii was nearly equivalent in both media (F = 1.79 mg L−1 and 2 F = 1.80 mg L−1), although 2 F media had a higher concentration of phosphorus. Nevertheless, a higher rate of uptake was observed in 2 F media, which indicates that a rapid environmental response occurs during the first 3 h. Additionally, lower phosphorus concentrations and equivalent rates of phosphorus uptake were observed at the end of the experiment, indicating species-specific consumption of phosphorus (Table 4). As mentioned previously, this pattern suggests a rapid intracellular storage response triggered by the presence of phosphorus rather than cellular assimilation.

Table 4 Phosphorus and nitrogen consumption of Geitlerinema lemmermannii in F and 2 F media
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It is well known that two of the most water quality hazards are the N and P, which are introduced into water bodies worldwide directly or by poorly treated discharges. Releases of N and P are predicted to increase by about 60 % by 2050 (MEA 2005). For the first time, our study shows that the cyanobacterium G. lemmermannii, cultured in medium with high nitrogen and phosphorus content, drives a biological process that results in the removal of at least 60 % of N and P. This effect was achieved without any sophisticated engineered method, using a single-batch cycle of 168 h. The use of microalgae in water treatment is not new and is still in a research level in most cases. G. lemmermannii, similar to many microalgae, represents a candidate for secondary or tertiary treatment processes aimed at the removal of N and P (Metcalf et al. 2004), in addition to oxygenation and CO2 fixation. Notably, because of biomass quality production measured in this study for G. lemmermannii, there are additional secondary metabolites of biotechnological interest.

In conclusion, a rapid phosphorus uptake was observed by G. lemmermannii that was isolated from an anthropogenic environment. In response to the culture conditions, it changed its proximal composition over time but caused no difference by the end of the culture period. However, the population and growth rate did not change, indicating a high nutrient storage capacity for this cyanobacterium. G. lemmermannii, at 120 h of culture, shows a high proximal composition (58.56 % proteins, 13.99 % carbohydrates, and 15.24 % lipids) when grown in F medium. Based on the removal of N and P observed in this study, G. lemmermannii represents a good candidate for secondary or tertiary water treatment processes.