Aquaculture biological waste as culture medium to cultivation of Ankistrodesmus gracilis ( Reinsch ) Korshikov

Current study investigated the effectiveness of different macrophytes as culture media for Ankistrodesmus gracilis in laboratory conditions. Significant difference (p < 0.05) was reported in cell density with regard to conventional culture medium (CHU12) and macrophytes culture media. Mean cell density in NPK, Eichhornia crassipes and E. azurea media was higher (p < 0.05) than in conventional culture medium. Chlorophyll-a was higher than 1 g.L-1, except in CHU12 (0.7 ± 0.4 g.L -1) and T. domingensis (0.8 ± 0.3 g.L-1) media. Nitrate decreased sharply as from the 7th-day of the experiment. Ammonium and total phosphorus were highest in culture media and ranged between 0.4 g.L-1 (P. cordata medium) and 1.7 g.L-1 (CHU12 medium) for ammonium, and between 0.8 g.L -1 (CHU12 medium) and 1.9 g.L -1 (T. domingensis medium) for total phosphorus. Results revealed inorganic fertilizer and macrophytes combined with vitamins may be effective as culture media and strongly supports the growth of Ankistrodesmus gracilis, since cell density and biochemical composition are similar to or higher than conventional culture medium (CHU12). Macrophyte is a tool for aquaculture since biological wastes may be used with nutrients to improve the cultivation of microalgae.


Introduction
Aquaculture produces large quantities of aquatic organisms with the primary purpose of consumption.In fact, great amounts of organic and inorganic matter generate wastes rich in nitrogen and phosphorus that enhance the growth and invasion of aquatic macrophytes in fishponds.This type of invasion is common in the southeastern region of Brazil due to large fishponds, with macrophytes forming beds of aquatic plants.The use of biological wastes has been carried out mainly in effluent treatment (wetlands) and as a culture medium for microalgae.Macrophyte culture medium with Eichhornia crassipes has shown high growth and protein content of Ankistrodesmus gracilis when compared to conventional culture medium (Sipaúba-Tavares et al., 2009, 2011).
The production of natural food, such as microalgae, is commonly accompanied by high financial investments, often attributed to the high costs of the culture medium.Moreover, these culture media are highly specialized and sometimes difficult to obtain on the local markets.However, efforts have been made to find alternative media for microalgae culture which include macrophytes (Sipaúba-Tavares et al., 2009, 2011, 2015) and inorganic fertilizers (Campaña-Torres et al., 2012;El-Nabris, 2012;Martinez-Córdova et al., 2012;Ramaraj et al., 2015;Sipaúba-Tavares and Pereira, 2008).Macrophytes have dominant and supporting roles in nitrogen and phosphorus recovery.Besides the direct assimilation of nitrogen and phosphorus, their roots provide colonizing sites and exude carbohydrates, sugar, amino acids, enzymes and many other compounds.In fact, they exert considerable influence dynamics of nutrients (Polomski et al., 2007).
Nitrogen compounds (ammonia, nitrate and nitrite) are major contaminants in aquaculture waste water.Toxic effects in the water include impairment of physiological parameters, such as, growth rate, oxygen consumption and disease resistance in fish species.However, macrophytes contribute towards nutrient transformation by removing these compounds for their own growth (Sirakov et al., 2015).It is well-known that nitrogen and phosphorus are essential compounds to increment the cultivation of microalgae, and when the medium is obtained for culturing microalgae, it has a direct influence on biological activity for high cell density; and on the metabolic process related to carbon fixation and energy transfer (Grabski and Tukaj, 2008;Singh et al., 2015).
There has recently an increasing interest in the culture of microalgae because it represents a valuable source of various natural products with diverse applications, such as, food industry, biodiesel sources, cosmetic industry, vegetal oils, fish feed, vitamin E, pigments and others.All these factors are directly correlated with the culture medium, because it supports the growth and development of microalgae (Arumugam et al., 2013).Microalgae media, which depend on trace minerals, vitamins (mainly B 12 , biotin and thiamin), nutrient composition, nitrogen and phosphorus, are compounds that may be added to the culture medium (Shah et al., 2003).
High costs in microalgae cultivation have triggered efforts to find a cheaper and economically feasible approach for microalgae culture.Alternative strategies, aimed at decreasing costs without affecting production should analysed.Current study investigates the effectiveness different macrophytes as culture media for Ankistrodesmus gracilis in laboratory conditions.

Microalgae culture and laboratory conditions
Ankistrodesmus gracilis strain was obtained from culture collection n. 005CH, originally from Broa Reservoir, Brazil at 22º15' S and 47º19' W. The algae were batch cultured at 22±2°C, exposed to light at 60 µmol.m -2 .s - on a 24-h light cycle, and bubbled with air.So that the effects of different media on the growth of A. gracilis could be analyzed, six culture media were employed: a conventional CHU 12 medium microalgae cultured in pure nutritive medium was the control, and five alternative media, or rather, one with inorganic fertilizer (NPK 20-5-20) (Sipaúba-Tavares and Rocha, 1993) and four macrophytes culture media.Macrophytes comprised: Eichhornia crassipes, E. azurea, Typha domingensis and Pontederia coradata, with great available on the site.Table 1 shows ingredients and composition of nutrients in the culture media.The effects of six different culture media on the growth of A. gracilis were simultaneously investigated during a 28-days cultivation period with experiments performed under the same growth conditions.The culture media with different macrophytes were based in Sipaúba-Tavares et al. (2009) that used E. crassipes with aquatic plant.Approximately 5 kg of each plant were ground and boiled in distilled water for one hour.The hot extract was filtered and autoclaved at 120°C during 20 minutes.A 70 mL sample was collected and, after cooling it was diluted with distilled water up to 1.4-L when 2.5 mL NPK were added.The experiment started with 10 mL at a density of 3.5 x 10 5 cells.mL - containing NPK medium.When cultures reached the last exponential growth phase (7-days), approximately 10 mL with density 1.9 x 10 5 cells.mL - were added to the 2-L NPK medium.After the 7-day exponential growth phase, the culture density 0.75 x 10 5 cells.mL - was transferred to 2-L sterilized recipients containing the six different media (NPK, CHU 12 and macrophytes: Ec= Eichhornia crassipes; Ea= Eichhornia azurea; Td= Typha domingensis; Pc= Pontederia cordata).Vitamin B complex was added to the alternative culture media at the rate of 0.02 g.L -1 plus biotin (0.01 mg.L -1 ).The samples were analysed weekly (1, 7, 14, 21, 28 days) during the study period, for growth performance and other physiological parameters by the analytical method, except plants biomass.

Growth performance
Cell growth was monitored for 28-days.Triplicate 1 mL aliquots were removed daily from the microalgae culture and a minimum of 2 x 1 μL sub-samples were used for cell quantification by a Neubauer hemocytometer.Growth rate (k) was calculated by the formula: k = (3.322/t 2 -t 1 x log N 2 /N 1 ) (t = time; N = number of cells; subscripts denote values at different times) (Guillard, 1973).Doubling time (cell division time or generation time) was calculated from results obtained from growth rate, by the formula: Td = 1k -1 (Td = duplication time, 1k -1 = days per division) (Guillard, 1973).Total length of 50 specimens was determined with microscope Leica DFC 295 with image analysis system LAS core (LAS V3.8), and 40X micrometric objective.Cell volume was calculated by mean cell size with the use of the most appropriate geometric form, or rather two coupled cones formula (Hillebrand et al., 1999).Total organic carbon (TOC) was calculated by C = 0.1204.

Parameters of culture media
Physical and chemical parameters of culture media were performed weekly.Dissolved oxygen, pH and conductivity of culture media were measured with YSI 556 MPS multi-sensor.Total phosphorous and nitrogen compounds were quantified spectrophotometry, following Golterman et al. (1978) and Koroleff (1976).Chlorophyll-a was extracted with alcohol 90% and quantified at 663nm and 750nm (Nusch, 1980).Analyses were performed immediately after sampling or samples were duly stored under refrigeration.

Biomass of Ankistrodesmus gracilis
The biomass of microalgae was harvested, centrifuged and lyophilized for the analysis of proteins and lipids (AOAC, 1990).Analyses of macro-minerals (N, P, K, Ca, Mg and S) and micro-nutrients (B, Cu, Fe, Mn, and Zn) of microalgae biomass were performed weekly according to methodology by Bataglia et al. (1983).

Composition of plant's nutrients
Dry and wet mass of macrophytes was retrieved and dried at 60 °C until constant weight and weighted.The composition of the nutrients of aquatic plants was analysed following Bataglia et al. (1983).

Data analysis
All data underwent one-way analysis of variance (ANOVA) with Statistica 8.0 package, to test the effects of the culture media (Statsoft, 2007).When differences between culture media occurred Tukey's test was applied.Differences were considered significant at p < 0.05.All results were expressed as means ± SD (standard deviation) and all experiments were carried out in triplicate.

Results
Cell concentration of Ankistrodesmus gracilis in alternative media reached maximum cell density at the end of the experiment with 43 x 10 6 cell.mL - on the 27 th day in the culture medium NPK, with 43 x 10 6 cell.mL - on the 28 th day in Eichhornia crassipes medium, with 32 x 10 6 cell.mL - on the 28 th day in E. azurea medium, with 29 x 10 6 cell.
mL -1 on the 27 th day in Typha domingensis medium, with 28 x 10 6 cell.mL - on the 27 th day in Pontederia cordata medium (Figure 1).In the case of the conventional medium (CHU 12 ), maximum cell density of A. graclis in the culture medium occurred on the 18 th day with 30 x 10 6 cell.mL - .Lowest cell density (p < 0.05) was reached between the macrophyte media Pontederia cordata and Typha domingensis, albeit similar (p > 0.05) to conventional medium (Figure 1, Table 2).Mean cell density in E. crassipes and NPK media reached a higher   mean cell density (p < 0.05) than conventional medium (Figure 1, Table 2).Constant speed for A. gracilis was highest (p < 0.05) in CHU 12 (K= 0.31), whilst between alternative media were highest in T. domingensis (K= 0.22) and E. crassipes (K= 0.20) and lowest in NPK (K= 0.11) medium (Table 2).
Protein content was similar (p > 0.05) between culture media, whilst protein contents of macrophyte media decreased throughout the analyzed period with highest contents on the 7 th day ranging between 48% (T.domingensis and NPK media) and 43% (P.cordata medium) of dry biomass.Lipids were below 8.2% of dry biomass, but similar (p > 0.05) between culture media.Ash content was higher in CHU 12 medium (8.4 pg.cell -1 dry biomass) until the 14 th day and similar among E. crassipes, E. azurea and T. domingensis media.However, the lowest contents were reached in NPK and P. cordata media, ranging between 1.1 and 5.6 pg.cell -1 of dry biomass (Figure 2).
High nitrogen compounds and total phosphorus in the culture media are due to concentrations of nutrients in the inorganic fertilizer and aquatic plants.
The composition of nutrients mainly nitrogen and potassium, in the plants was high ranged between 6.5g.kg -1 (P.cordata medium) and 24g.kg -1 (E.crassipes medium).However, calcium was above 5.4 g.kg -1 .The highest plant nutrients occurred in Eichhornia.In general, the plants nutrients composition was over 0.1 g.kg -1 , whilst boron and copper concentrations in the plants were below 0.05 g.kg -1 .Wet mass was similar between plants and dry mass ranging between 996 g.kg -1 in Typha domingensis medium and 1,148 g.kg -1 in E. crassipes medium (Table 3).

Discussion
The use of inorganic fertilizer NPK (20-5-20) and floating macrophytes provide more efficient growth rate and nutritional values than conventional medium (CHU 12 ).These alternative culture media provide a better possibility to replace the conventional culture medium, more expensive and less accessible to consumers when compared to inorganic fertilizer (NPK) and to macrophytes.Therefore it may be hypothesized that the use of floating macrophytes and inorganic fertilizer (NPK, 20-5-20) could provide similar or better results at lower costs.Martinez-Córdova et al. ( 2012) used agricultural fertilizer and obtained similar proximate composition of Chaetoceros muelleri when compared to conventional culture medium (Guillard F/2).These results are consistent with the A. gracilis experiment where mean cell density in NPK medium at 23 x 10 6 cell.mL -1 and maximum cell density at 43 x 10 6 cell.mL - were higher than conventional culture medium (CHU 12 ).
Results of current study indicated great variations in cell density during the study period.Since nitrogen is a major nutrient for microalgae cultivation, difference may be due to availability and variability of nitrogen sources in the media.It may be due to nitrogen sources ammonium and nitrate in the former media.El Nabris (2012) indicated a positive correlation between nitrogen concentration and cell density of Nannochloropsis sp cultured in agricultural fertilizer, with ammonium as the main source of nitrogen.According to the author, when ammonium is highly concentrated it is consumed faster than nitrate due to  (Arumugam et al., 2013).
In current study, the culture media with inorganic fertilizer and macrophytes media had the highest contents of nitrogen and potassium, and nitrate contents were consumed fast.These nutrients in the alternative culture media improve mean cell density, chlorophyll-a and maximum cell density that were similar or higher than the conventional culture medium.Chemical proximate composition was similar among all culture media used.
Phosphorus is one of the major nutrients required for the normal growth of microalgae.High total phosphorus contents in the water media directly affect plants due concentration above 1.8 g.kg -1 and consequently improve microalgae growth.In general, culture media have enough amount of phosphorus for microalgae growth with special reactions associated to cell division (Ramaraj et al., 2015).
Nitrogen deprivation is a severe stress condition for all organisms, since nitrogen is a major constituent of proteins and nucleic acids and a highly abundant atomic species in living cells (Huang et al., 2013).Nitrogen also influences chlorophyll-a high content above 0.3 g.L -1 were obtained for A. gracilis mainly in the alternative media, except Typha domingensis medium which was similar to the conventional culture medium (CHU 12 ).Mutlu et al. (2011) reported a decrease in chlorophyll-a content of Chlorella vulgaris when treated with nitrogen-starvation, featuring a yellowish color in the culture.
Lipid contents lower than 8.2% of total dry biomass of microalgae in current study is in accordance with the well documented fact that lipid accumulation occurs under limited nitrogen conditions and high iron supplementation.Nitrogen compounds current analysis had higher contents mainly due to inorganic fertilizer and aquatic plants.On the other hand, iron contents were very low in the culture media used and in the composition of macrophytes.Singh et al. (2015) contained the highest lipid contents (59.6%) for Ankistrodesmus falcatus under moderate nitrogen (750 mg.L -1 ) and high iron (9 mg.L -1 ) supplementation.The same authors demonstrated that N and Fe have a significant effect on lipid production when compared to phosphorus.Low lipid content in A. gracilis biomass using different alternative media was due to low Fe (below 0.8 mg.L -1 ), whilst high nitrogen compounds (above 10 g.L -1 ) in the culture media influenced protein contents and high A. gracilis biomass.Msanne et al. (2012) observed that reduction in proteins levels affects photosynthesis and amino acid synthesis which resulted in slow growth and low biomass yield of Chlamydomonas reinhardtii and Coccomyxa sp.A combination of nitrogen deficiency, moderately high light intensity and high Fe level improved lipid accumulation in four strains of Botryococcus sp.(Yeesang and Cheirsilp, 2011).Mata et al. (2013) reported that Dunaliella tertiolecta cultured in medium with Fe showed a higher biomass concentration than with Mn and Zn.Iron contents in the culture media used in this study were lower but K, N, and P contents had the highest nutrient contents in the culture media and influenced the growth of A. gracilis in macrophytes media.Sevaral factors, such as pH, temperature, nitrogen deficiency and phosphorus limitation of culture medium, are known to influence the lipid content of microalgae (Huang et al., 2013).
Water quality of culture media in current study was totally under control and free from pollution.This setup provided an ideal environment for microalgae culture media.According to Shah et al. (2003) alternative media with organic matter have their own limitations and cannot compete with pure nutrients.Further, pH, dissolved oxygen and conductivity were in satisfactory conditions for the growth of A. gracilis.In general, pH was alkaline and the constant mixing of water kept the dissolved oxygen above 5.4 mg.L -1 .The increase of pH during photosynthesis, due to nutrients uptake to consume bi-carbonate; and in the culture media with ideal mixing might optimize microalgae access nutrients and uptake easily the dissolved inorganic carbon (Ramaraj et al., 2015) Results show that the use of inorganic fertilizer and macrophytes with vitamin solutions may be effective as culture media and strongly support the growth of Ankistrodesmus gracilis, since cell density and biochemical composition were similar to or higher than conventional culture medium (CHU 12 ).Further research is necessary to verify the real potential of macrophytes and which plants are more appropriated for cultivation of microalgae.The use of these aquatic plants as culture medium should always be associated with NPK and vitamins.In our case, besides the inorganic fertilizer the floating macrophytes Eichhornia crassipes and E. azzurea showed had the best results for the growth of A. gracilis.To balance adverse conditions (appearance of macrophytes) in aquaculture transformation into a high quality product for the growth of microalgae is a tool that may be employed in aquaculture since biological wastes may be used with nutrients to improve microalgae cultivation.