versión On-line ISSN 0718-560X
Lat. Am. J. Aquat. Res. vol.40 no.SpecIssue Valparaíso set. 2012
Lat. Am. J. Aquat. Res., 40(3): 553-561, 2012
Control of Branchionus sp. and Amoeba sp. in cultures of Arthrospira sp.
Control de Branchionus sp. y Amoeba sp. en cultivos de Arthrospira sp.
Carlos Méndez1 & Eduardo Uribe2
1Programa de Magíster en Acuicultura, Departamento de Acuicultura Facultad de Ciencias del Mar, Universidad Católica del Norte, P.O. Box 117, Coquimbo, Chile.
2Departamento de Acuicultura, Facultad de Ciencias del Mar Universidad Católica del Norte, P.O. Box 117, Coquimbo, Chile.
ABSTRACT. Cultivation of cyanobacterium Arthrospira sp. has been developed in many countries for the production of proteins, pigments and other compounds. Outdoor mass cultures are often affected by biological contamination, drastically reducing productivity as far as bringing death. This study evaluates the control of Branchionus sp. and Amoeba sp. with two chemical compounds: urea (U) and ammonium bicarbonate (AB), in laboratory conditions and outdoor mass culture of Arthrospira sp. The lethal concentration 100 (LC100) at 24 h for Branchionus sp. and Amoeba sp. determined was of 60-80 mg L-1 (U) and 100-150 mg L-1 (AB). The average effective inhibition concentration for 50% of the population (IC50) in Arthrospira sp., after 72 h, was 80 mg L-1 (U) and 150 mg L-1 (AB). The application of doses of 60 mg L-1 (U) or 100 mg L-1 (AB) in the outdoor mass culture of this contaminated microalga, completely inhibited grazing and did not affect the growth of Arthrospira sp. but rather promoted rapid recovery of algal density at levels prior to infestation. These compounds provided an economical and effective control of predators in cultures of Arthrospira sp.
Keywords: Arthrospira, Branchionus, Amoeba, predator control, ammonium, bicarbonate, urea, Chile.
RESUMEN. El cultivo de la cianobacteria Arthrospira sp. ha sido desarrollado en muchos países para la obtención de proteínas, pigmentos y otros compuestos. Cultivo que a nivel industrial se ve afectado frecuentemente por contaminación biológica, reduciendo drásticamente la productividad hasta causar la muerte. Este estudio evalúa el control de Branchionus sp. y de Amoeba sp. con dos compuestos químicos, la urea (U) y bicarbonato de amonio (AB) en cultivos de Arthrospira sp. La concentración letal 100 (LC100) determinada a las 24 h para Branchionus sp. y Amoeba sp. fue de 60-80 mg L-1 (U) y 100-150 mg L-1 (AB). La concentración media de inhibición efectiva, después de 72 h, para el 50% de la población (IC50) en Arthrospira fue de 80 mg L-1 (U) y 150 mg L-1 (AB). La aplicación de dosis de 60 mg L-1 (U) ó 100 mg L-1 (AB) en los cultivos abiertos contaminados, inhibió completamente el pastoreo y no afectó el crecimiento de Arthrospira sp., permitiendo una rápida recuperación de la densidad algal a niveles anteriores a la infestación. Estos compuestos proporcionan un medio económico y efectivo de control de estos depredadores en cultivos de Arthrospira sp.
Palabras clave: Arthrospira, Branchionus, Amoeba, control depredadores, bicarbonato de amonio, urea, Chile.
The microalgae in their natural habitat, such as extensive and intensive cultures developed by aquaculture, are affected by different types of biological contaminants, among which are viruses, bacteria, protists, fungi and several predators (Hoffman et al., 2008). Microorganisms influence negatively the microalgal growth and the quality of the product (Richmond, 2000), being most significant in intensive outdoor mass cultures, where the microorganisms can compete for nutrients, and graze upon the microalgae, reducing drastically the commercial production (Richmond & Becker, 1986; Contreras et al., 2003; Ugwu et al., 2008).
The colonizing organisms of the microalgae culture, can reach very high densities, generating operation problems, increasing the costs of production as well as inputs and workforce. This can reduce the production up 90% and in some cases the total loss of the culture (Lincoln et al., 1983; Richmond, 2004). For these reasons, it is important to identify the type of contamination in order to implement control measures to mitigate or eliminate the contaminant, to maintain a healthy culture, highly productive, to obtain specific products such as vitamins, proteins, fatty acids, polyunsaturated, pigments, antibiotics, hydrogen, hydrocarbons, and bioactive compounds (Borowitzka, 1992; Metting, 1996; Cohen, 1999; Pulz et al., 2001; Morales et al., 2002; Domínguez et al., 2003).
The culture of Arthrospira sp. outdoors can be contaminated with microalgae, as Chlorella sp., Oocystis sp. or by other species of cyanobacteria and protozoans such as Amoeba sp. and Paramecium sp. (Vonshak et al., 1983; Vonshak & Richmond, 1988; Pedraza, 1989; Belay, 1997; Vonshak, 1997). In some cases the Chlorella spp. can even displace the Arthrospira sp. and turn rapidly into a dominant algae in the culture if there is not a suitable control of it (Richmond et al., 1990). The estimated annual losses caused by the biological contamination in the culture of Arthrospira sp. is around 15 to 20% (Belay, 1997). The outdoor mass cultures of Arthospira sp. conducted in the north of Chile are being contaminated with amoebae (Amoeba sp.) and rotifers (Branchionus sp.). These organisms generate stress, cellular breaking, detriment and death of the culture. Described contaminations by amoebae suggest that cultures die within days if invasion is not treated quickly (Wagener & de Luca, 1987; Duerr et al., 1997). Rotifers have always been seen as very useful organisms in aquaculture as live food (Planas & Cunha, 1999), however these organisms can cause significant biological contamination in algal cultures (Oswald, 1980). Currently there are no mass cultures of Arthrospira sp. reports of contamination by species of this genus; therefore, this one would be the first case of infestation control in culture of Arthrospira sp. in Chile.
A number of methods have been investigated for predator control in the cultures of microalgae, with limited success. Early attempts of control have been performed by means of heating, centrifugation, changes in pH, salinity and the use of chemicals such as chlorine, formaldehyde, hydrogen peroxide, ivermectin, metronidazole, quinine sulfate, ammonium and ammonia (Lincoln et al., 1983; Vonshak et al., 1983; Vonshak & Richmond, 1988; Borowitzka, 1999; Moreno-Garrido & Canavate, 2001; Richmond, 2004; Zmora & Richmond, 2004, Garric et al., 2007).
This study was aimed to control the biological contamination of Amoeba sp. and Branchionus sp. present in the culture of Arthrospira sp. using two chemical agents such as urea and ammonium bicarbonate. These compounds can reduce to ammonium and ammonia (Trenkel, 1997; Carpio & Moran, 2005), which are toxic for aquatic organisms (Zhao et al., 1997). Ammonium toxicity for aquatic organisms is related to the non-ionized form (ammonia), mainly due to its high lipid solubility, which facilitates their passage through biological membranes, causing damage to the respiratory structures (Jensen, 2003). The urea and ammonia have been used to control a type of amoeba in the culture of Arthrospira sp., giving good results (Vonshak & Richmond, 1988). As for rotifers, it is known that ammonia is one of the main compounds that inhibit growth and cause death of these organisms (Yoshimura et al., 1996).
From the economic point of view, the use of ammonium compounds and/or urea is particularly attractive, since they are inexpensive (Corverti et al., 2006). The urea and some ammonium have been used as sources of nitrogen in culture of Arthrospira sp. (Stanca & Popovici, 1996; Vieira et al., 2001; Danesi et al., 2002; Costa et al., 2004; Rangel-Yagui et al., 2004; Sanchez-Luna et al., 2004; Soletto et al., 2005; Sassano et al., 2007, 2010), however, there are no reports on the use of ammonium bicarbonate in the culture of this microalgae.
MATERIALS AND METHODS
Arthrospira sp., inoculums were grown at 30 ± 1°C and fluorescent light intensity of 117 μmol m2 s-1 in 250 mL Erlenmeyer flasks containing the medium Zarrouk (1966). The work was developed in several phases. The first phase in the laboratory involved toxic tests in Arthrospira sp., Amoebas sp., and Bran-chionus sp. with different concentrations of urea [CO(NH2)2], and ammonium bicarbonate [NH4HCO3] (industrial grade) to determine the average concentration of inhibition (IC50) and lethal concentration (LC100) for microalgae and predators respectively. The second phase was performed outdoors, using the outcome of the toxicity test in microalgae and predators in series of 1,000 m2 raceway contaminated.
Toxicity test in Arthrospira sp.
The bioassays were carried out in 5 L plastic bottles with a final volume of 4 L using medium SSM (Ayala & Bravo, 1982) modified by Ayala & Ayala (2009), which was maintained through the daily addition of distilled water to replace lost by evaporation. Cultures were performed in triplicate with constant aeration and a photoperiod of 12:12 illumination with white fluorescent light intensity of 117 μmol m2 s-1. In the experiments, two different concentrations of ammonium bicarbonate (mg L-1) 0, 50, 100, 150 and of urea (mg L-1) were used, respectively. Evaluations were performed at 24, 48, 72 y 96 h, to observe the effect on microalgae growth. Biomass of Arthrospira sp. was determined daily by sample optical density at 560 nm in a Smart LMC202 colorimeter. These values were compared with previously prepared calibration curves of optical density versus biomass dry weight as described by Leduy & Therien (1977) and applied by Vonshak et al. (1988). The specific growth rate (μ) was calculated by the method of Leduy & Zajic (1973).
Toxicity test in Branchionus sp. and of Amoeba sp.
For toxicity experiments, microorganisms were isolated from contaminated outdoor cultures. They were carried out in 5 L plastic bottles with volume of 4 L, with an initial density of 400 rotifers mL-1 and 600 amoeba mL-1 in each of the bottles. The culture conditions of these microorganisms were the same used for toxicity tests with microalgae Arthrospira sp.: concentrations of ammonium bicarbonate (mg L-1) 0, 50, 100, 150 and of urea (mg L-1) 0, 40, 60, 80. Predator's mortality was counted by Olympus CX21 light microscopy at 20x magnification, with a Sedgewick-Rafter counting chamber. In every culture there were 10 subsamples of 1 mL; each concentration experiment was performed in triplicate. After 24 h, survival of predators was checked. Death of Amoeba sp. was defined as breaking of the cytoplasm and for Branchionus sp. when the lorica was empty.
After the toxicity test in microalgae and predators, results were applied in 1,000 m2 outdoor contaminated mass cultures of Amoeba sp. and Branchionus sp.
Prior to statistical analysis, data was tested for normality (Kolmogorov-Smirnov test) and homogeneity of variances (Levene test). Comparison between concentrations of urea and ammonium in specific growth rate and algae biomass was done by one-way ANOVA. Where significant main effects were found, multiple comparisons Dunnett test was used to determine significant differences among means (Zar, 1984). All statistical analyses were performed with Statistica 10 at a significance level of P < 0.05.
The effective inhibition concentration for 50% of the population (IC50) in Arthrospira sp., after 72 h and lethal concentration 100 (LC100) at 24 h for Branchionus sp. and Amoeba sp. were calculated according to protocols for toxicity testing in microorganisms OECD (1984), USEPA (1996), and ASTM (2004).
The biomass curves of Arthrospira sp. under different concentrations of ammonium bicarbonate and urea in laboratory are shown in Fig. 1. Arthrospira sp. showed a maximum dry weight biomass of 1339 ± 9.815 mg L-1 and 1255 ± 9.238 mg L-1 after 72 h, with a concentration of 100 mg L-1 ammonium bicarbonate and 60 mg L-1 urea, respectively. At 80 mg L-1 urea and 150 mg L-1 ammonium bicarbonate showed decreasing of biomass with increasing incubation time. Statistical analysis revealed that there was significant difference in the growth of this microalga (P < 0.05). The curves of Figure 1 with ammonium bicarbonate 0, 50, 100 mg L-1 and of urea 0, 40, 60 mg L-1 did not show any lag phase, notwithstanding the use of a different nitrogen source. The specific growth rates responses at each of the selected chemicals are presented in Table 1.
The outcomes of the laboratory tests of toxicity for urea and ammonium bicarbonate for Amoeba sp. and Branchionus sp., (24 h LC100) are shown in Table 2. IC50 values and LC100 for Arthrospira sp. and predators for each of the compounds used are shown in Table 2. In toxicity tests for predators of the culture, it was observed that concentrations of 60 and 80 mg L-1 and 100 and 150 mg L-1 urea and ammonium bicarbonate, respectively, exterminated between 99 and 100% of these microorganisms in 24 h. These results suggest that urea and ammonium bicarbonate could be highly toxic to Amoeba sp. and Branchionus sp. and slightly toxic to Arthrospira sp.
Considering the laboratory results, the selected dose of urea was 60 mg L-1 and 100 mg L-1 ammonium bicarbonate for predator control in the culture of Arthrospira sp. open reactor of 1,000 m2. The effect of the application of these compounds in the reactors contaminated with Amoeba sp. and Branchionus sp. is described in Figures 2a and 2b, showing that the cultures of these microalgae have a fast recovery.
Recommendations by different authors for the treatment against grazers' contamination in micro algal cultures are limited to maintaining optimal values of pH, light and temperature, in order to increase algal growth, and therefore surpass the damage caused by grazing (Richmond & Becker, 1986). Such equilibrium was not possible in the present case, since the high grazing capacity of the rotifer and amoebae involved leads to a complete elimination of the micro algal population within two days. Upon adding urea or ammonium bicarbonate to culture, a fast increment is produced in the concentration of ammonium and ammonia which is regulated by pH (Alonso, 2006). These compounds themselves become the main factors that inhibit or limit the growth of the rotifer (Yoshimura et al., 1996). In the culture of Arthrospira sp. the pH is maintained in a range of 9.5 to 10.5, that favors the predominance of NH3 over NH4 (Laliberté et al., 1997). The rotifers have been more affected with the high levels of NH3 than other groups of aquatic invertebrates (Arauzo, 2003). It has been reported that these compounds affect other rotifers like Bran-chionus rotundiformis and B. plicatilis causing their collapse in the culture, diminishing swimming and causing death of the culture (Snell et al., 1987; De Araujo et al., 2000, 2001; Yoshimura et al., 2003). In natural populations B. calyciflorus and B. rubens are inhibited with concentrations of around 2.5 mg L-1 (Arauzo, 2003). In a study carried out by Lincoln et al. (1983), they utilized ammonium hydroxide of to control the contamination by the rotifer (Brachionus rubens) in 24 h. LC100 with an approximate concentration of 17 mg L-1 of ammonium without inhibiting the algal growth. Duerr et al. (1997) supplied urea in a daily concentration of 50 mg L-1 to the culture of Arthrospira sp., contaminated with Amoeba sp., giving as a result the control of these protozoa, besides providing an entrance of nitrogen to the culture. With similar concentrations used in this work (60 mg L-1 of urea and 100 mg L-1 ammonium bicarbonate) this microorganism was controlled.
As it can be seen, the inhibition concentrations of these reduced nitrogen compounds is lower for Branchionus sp., and Amoeba sp., than to inhibit the growth of these microalgae, which corroborates the results of this study. The nitrogen source has a great importance because it is the second most abundant element of Arthrospira sp. biomass, representing up to about 10% of its total content (Soletto et al., 2005). After carbon, nitrogen is the nutrient which provides more algal biomass (Grobbelaar, 2004). The conventional nitrogen source for Arthrospira sp. is nitrate (Zarrouk, 1966). The microalgae having used nitrate needs to reduce it to nitrite and later to ammonia (Hatori & Myers, 1966). Since this process needs energy, the algae prefer using reduced nitrogen in the forms of ammonium and urea that are toxic in high concentrations (Belkin & Boussiba, 1991). Nevertheless, urea and ammonium bicarbonate are easily assimilated by Arthrospira sp. probably due to its spontaneous hydrolysis to ammonia under alkaline conditions, which is the preferential form of nitrogen uptake by this cyanobacterium (Danesi et al., 2002; Coverti et al., 2006). This process of assimilation is realized by the stirrup-strap of the ureasa and by the glutamine synthetase (Carvajal et al., 1980; Herrero et al., 2001). Stanca & Popovici (1996) observed that using reduced nitrogen improves the production of biomass of Arthrospira sp., a result obtained in the present work with concentrations of 60 mg L-1 urea and 100 mg L-1 ammonium bicarbonate. On the contrary, in the processing of 80 mg L-1 urea and of 150 mg L-1 ammonium bicarbonate, a decrease in the biomass of this microalgae was observed, this observation is in agreement with the results reported by Sassano et al. (2004) that also determined that the excess of urea or ammonium may cause the inhibition of the growth of Arthrospira sp. Belkin & Boussiba (1991), indicate that ammonia in high concentrations inhibits the growth and is responsible for cell death of this microalga. The ammonia present in the cultivations could enter the cells without energy expenditure, according to an extracellular/intracellular gradient of pH ((Boussiba, 1989). The pH in culture is among 9.5 to 10.5 and the cell cytoplasm is 8.5, this difference of pH suggests that there exists a movement of ammonia towards inside the cell with an intracellular accumulation that can arrive to a toxic level. The inhibition levels of ammonium are among 30-36 mg L-1, and toxic 180 mg L-1 for this microalga (Carvalho et al., 2004; Soleto et al., 2005; Coverti et al., 2006). These authors concentrations are high compared to given compounds in the present study.
The Arthrospira sp. used in the present study achieved higher biomass concentrations compared to previous studies concerning Arthrospira sp. (Lodi et al., 2005; Narayan et al., 2005; Radmann et al., 2007). This species had higher growth rate (μ) than in previous studies for Arthrospira sp. (Lodi et al., 2005; Andrade & Costa, 2007; Colla et al., 2007). It is a consequence of species having different response to different environmental conditions and demonstrates a quick adaptation to the new medium.
The production average of Arthrospira sp., in biomass drought of a reactor is of 9 kg m-2 day-1 and it drops drastically when the culture presents a biological contamination. If the necessary controls to the culture are not applied, a strong economic loss is presented since, upon trying a new culture, this will begin its production after 15 to 20 days, that costs around US$ 1,500 by reactor and a business handles near 20 reactors; the total loss would be of US$ 30,000 without including the nutrients. Besides the economic losses, also a delay in the planning and the goals of production of the business is generated.
The nutrients in the culture of Arthrospira sp., have a cost about 15 to 20% (Vonshak, 1997). It is therefore important to use low-cost nutrients to reduce the production costs. The use of urea and/or ammonium bicarbonate as nutrients in the culture of these microalgae offers great advantages, since the cost of these inputs in comparison with other nutrients is cheaper. The urea has a cost of approx. US$ 0.25 kg, the ammonium bicarbonate approx. US$ 0.2 kg, in comparison with the main source of nitrogen, the sodium nitrate, which is approx. US$ 0.5 kg (these are prices in Chile). The decrease of costs is reflected in industrial cultures, in which large quantities of nutrients are employed, in the case of a reactor of 1,000 m2 an approximate quantity of 600 kg of sodium nitrate is used for the preparation of the initial broth. If using urea as nitrogen source, the company would save US$ 20, this reduction is reflected as more culture reactors are used, considering a company that has 20 reactors it will be saving US$ 400 only at the planting and this reduction in the costs of the nutrients would be of US$ 28,000, besides a control on the present predators is generated and finally would be done an entrance of carbon to the culture, because a mol of urea contributes a mol of carbon (Levasseur et al., 1993).
The application of urea as of ammonium bicarbonate in the selected concentrations eliminates these two grazing organisms as Branchionus sp. and Amoeba sp. in a short period of time (24 h), helping to recover the cultures with the supply of nutrients and thus avoiding economic losses for the companies that cultivate Arthrospira sp.
This study formed part of a Master's degree thesis by CAMR. We are grateful to Dr. Francisco Ayala for his help and constructive comments for this paper. The authors also thank anonymous reviewers and editor for critically reviewing the manuscript.
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Received: 23 February 2011; Accepted: 30 March 2012.
Corresponding author: Carlos Méndez (firstname.lastname@example.org)