Application of photosynthetic microalgae as efficient pH bio-stabilizers and bio-purifiers in sustainable aquaculture of Clarias gariepinus (African catfish) fry

Chlorella lewinii LC172265 and Scenedesmus dimorphus NIES-93 were grown with BG-11 medium and then transferred to fish seedlings' farm and the effects on changes in the pH, nitrite, total ammonia nitrogen and toxic ammonia were studied. Inoculation of the ponds with C. lewinii, S. dimorphus and the combined culture of C. lewinii and S. dimorphus significantly reduced the rise in the pH of the ponds (p<0.05). With these three cultures, the pond pH increased from 6.3±0.03 to only 6.5±0.2, 6.7±0.6 and 6.4±0.1 respectively within a period of 240 hours, as against control pond's pH which increased from 6.3±0.03 to 9.0±0.1 within the same period. Furthermore, inoculation with S. dimorphus reduced the nitrite concentration in the ponds to zero on the 144th hour and the concentration remained zero throughout the experiment. This was closely followed by the combined culture of C. lewinii and S. dimorphus which reduced the nitrite concentration to zero at 240th hour while C. lewinii was the least in nitrite removal. Toxic ammonia was also zeroed by the combined culture of C. lewinii and S. dimorphus at 144th hour of experiment which was followed by C. lewinii (at 192nd hour). Aquaculture ponds co-cultured with microalgae witnessed a maximum fry death rate of 40% which was much lower than 80% death rate observed in the control ponds. These results show that C. lewinii and S. dimorphus are very efficient in sustaining the quality of aquaculture water, and thus prolonging the length of time water can be used before changing.


Introduction
consumption provides protein and a range of Fisheries and aquaculture play a critical other nutrients, particularly essential fatty acids role in food and nutrition security and in (such as the long-chain polyunsaturated fatty providing for the livelihood of several millions of acids (LCPUFA)), minerals and vitamins people.Aquaculture supplies more than half of (Beveridge et al., 2013;Kareem and Olanrewaju, all the fish for human consumption (FAO, 2016) 2015; FAO, 2016).Long-chain omega-3 fatty and also generates employment opportunities acids, such as eicosapentaenoic acid (EPA) and which have grown faster than employment in docosahexaenoic acid (DHA), are the main traditional agriculture with about 56 million building blocks of the neural system (Nelson, people directly engaged in the fisheries sector 2006) and are very important for optimal brain (FAO, 2014;FAO Committee on Fisheries, 2014; and neurodevelopment in young children FAO, 2016;Umaru et al., 2016).Fish (Thilsted et al., 2014;USAID, 2016).
Despite the breakthrough with the use of approximately 70% of global nitrogen assimilation with about 65% consumed as hormone in induced spawning of Clarias reduced nitrogen (ammonia and organic gariepinus, fry survival is still beset with a nitrogen), approximately 10% through nitrogen number of challenges (Adewumi, 2015).A major fixation and the remaining as nitrate (Taziki et abiotic challenge stems from depletion in water al., 2015).Microalgae have been widely used for quality partly resulting from the accumulation of nutrient removal in waste water both as free and poisonous nitrogenous wastes such as ammonia immobilized cells (Ogbonna et al., 2000; and nitrite and the consequent increase in water Banerjee et al., 2015; Kaparapu and Geddada, pH in aquaculture ponds (Kroupova et al., 2005(Kroupova et al., ). 2016)).Therefore algae growth in fish ponds can Ammonia is the basic end product in the aid in reducing the concentrations of ammonia breakdown of feed proteins in fish (Ebeling et al., (Durborow et al., 1997) and other nitrogenous 2006; Lazur 2007;Chen et al., 2012).It is waste (Taziki et al., 2015) and at the same time excreted either through their gills or feces produce microalgae biomass which have various (Durborow et al., 1997;YSI Environmental, applications (Chen et al., 2012).2010; Hii et al., 2011).Ammonia is present in two The aim of this study was therefore to forms in biological fluids or water, namely the + develop a method of prolonging the life span of nontoxic ionized or dissociated form (NH ) and 4 water, and thus reduce the frequency of water the toxic un-ionized or molecular (nonchanges in fish ponds by regulating and/or dissociated) form (NH ) (Lazur, 2007;Ogbonna 3 stabilizing pond water pH, and maintaining low and Chinomso, 2010;Somerville, et al., 2014).
concentrations of poisonous nitrogenous Both of them are always in equilibrium and the wastes.relative concentrations depend on the pH and temperature of the water (Crab et al., 2007; - Lazur, 2007;Somerville et al. 2014).Nitrite (NO 2

Materials and methods
Preparation of media and culture collection ) is the most ephemeral and intermediate BG-11 medium was prepared according nitrogenous waste substance in aquaculture to the modified methods described by Rippka et (Lazur, 2007;Bhatnagar and Devi, 2013;Taziki al. (1979), Bittencourt-Oliveira et al. (2011) and et al., 2015).It is produced when autotrophic Bortolia et al. (2014).It was composed of the Nitrosomonas bacteria oxidize ammonia (Lazur, following major nutrients; KNO , 1.5g/L; 3 2007; Bhatnagar and Devi, 2013) (Svobodova et al., 1993;Kroupova et al., 2005).Ferric ammonium salt, 0.006g/L; Disodium Both of them are very toxic to fish (Lazur, 2007; magnesium EDTA, 0.001g/L; and Na CO ,  (Bhatnagar and Devi, 2013) concentrations of these nitrogen species cultures of Chlorella lewinii LC172265 and increase above their critical values, a process Scenedesmus dimorphus NIES-93 (10 mL each) that is expensive and limit aquaculture to areas were inoculated into two different 250 mL with readily available sources of water.conical flasks containing 100 mL of BG-11 Microalgae are microscopic unicellular medium.The conical flasks were incubated at organisms (Priyadarshani and Rath, 2012; room temperature under fluorescent lamps of Kiepper, 2013;Bleeke et al., 2014) or 1,000 lux intensity with occasional shaking for 21 pluricellular organisms (Mimouni et al., 2012) days.The microalgae were sub-cultured into 20 with a simple structure, ranging from three to 80 conical flasks containing BG 11 medium (10 for µm in size (Scharff, 2015) hand opening of the Colour Comparator and the Artificial spawning and hatching of the tube of untreated sample was also inserted into experimental fry were done according to the the left-hand opening of the Comparator.The methods of Potangham and Miller, (2006); and Comparator was held up to light and viewed Bosworth et al. (2011).Hatched fry were through the two openings in the front while maintained for 13 days in the fish seedling's rotating the colour disc until a colour match was facility by initially feeding them with artemia and obtained and the pH was read through the scale then with 0.20 mm Coppens floating feed.
window and recorded.Transparent plastic ponds with dimensions of 50cm x 30cm x 35cm were filled with water to 20 Ammonia-nitrogen cm depth and setup as nursery for fry at the fish The concentration of ammonia-nitrogen farm of the Department of Zoology and ® was determined with Hach 's water ammonia-Environmental Biology, University of Nigeria, nitrogen testing kit according to the Nsukka, Nigeria.The experimental ponds were manufacturer's manual.One viewing tube was partially covered to accommodate both the filled to the 5 mL mark with deionized water photophobic fry and photophilic photosynthetic which served as the reagent blank while the microalgae.Ten (10) fourteen-day old fry were second viewing tube was also filled to the 5 mL introduced into each set of the partially covered and that with the co-culture of C. lewinii and S.

Mortality rate of fries
The control ponds without microalgae The mortality rate of the experimental fry experienced a drastic increase in the pH from was calculated thus: 6.3±0.03(at zero hour) to 9.0±0.06(at 240 h) as shown in Figure 1. at 144 h and 240 h respectively in the control Changes in the nitrite concentrations during the ponds.Also, the ponds inoculated with both C. culture lewinii and S. dimorphus experienced a gradual There was significant nitrite reduction in the ponds with microalgae compared to the drop in the nitrite concentration until it was control ponds without microalgae (Table 1).The completely exhausted at the 240th hour of the experiment.In the case of the ponds inoculated nitrite concentration in the ponds with S.
with C. lewinii, the nitrite concentration dimorphus decreased to zero mg/L within 144 decreased to 0.0440±0.0762mg/L at the 240th hours and remained zero until the end of the hour of the experiment (Table 1).experiment (240 h).This compared to 0.2310±0.1143mg/L and 0.0770±0.0381mg/LTable 1: Changes in toxic nitrite concentration (mg/L) in the aquaculture ponds the concentrations of the toxic ammonia were Changes in ammonia concentration during the successfully reduced to zero mg/L at 192nd h of culture the experiment and the concentration remained T h e r e w a s o s c i l l a t i o n i n t h e zero all through the 240 h of the experiment concentrations of the total ammonia-nitrogen in (Table 3).In the experimental ponds inoculated the ponds inoculated with microalgae but the with the co-culture of C. lewinii and S. dimorphus, oscillation was not apparent in the control ponds.
the concentration of the toxic ammonia was (Table 2).On the whole, the concentrations of reduced to zero mg/L within 144 h of the toxic ammonia were significantly lower in the experiment (48 h earlier than in ponds ponds containing the microalgae than those in separately inoculated with C. lewinii or S. the control ponds.The differences became very th pronounced from the 196 hours of the dimorphus) and remained zero all through the experiment.There were drastic reductions in the 240th hour of the experiment.On the other concentrations of toxic unionized ammonia (NH ) hand, the control ponds witnessed rise in the 3 in the experimental ponds inoculated with concentration of the toxic ammonia, rising to microalgae.In the experimental ponds 0.4832±0.0711mg/L on the 240th hour of the experiment (Table 3).inoculated with only C. lewinii or S. dimorphus, Table 2: Changes in total ammonia-nitrogen concentrations (mg/L) in the aquaculture ponds Table 3: Changes in the toxic ammonia concentration in the aquaculture ponds There was a strong correlation between Mortality rate of fry the pH and toxic ammonia concentration in the The average mortality of the fry was 40% control aquaculture ponds.The pH of the in the ponds inoculated with C. lewinii, 37% in aquaculture pond increased as the concentration the ponds inoculated with co-culture of C. lewinii of the toxic ammonia increased (Figure 2).The and S. dimorphus, 35% in the ponds inoculated pH reached a peak of 9.0±0.06 as the toxic with S. dimorphus but 80% in the control ponds ammonia rose to a peak of 0.4832±0.0711mg/L (without microalgae) .
as shown in Figure 2.This direct proportional relationship was not very apparent in the Relationship between pH and toxic ammonia in experimental ponds inoculated with microalgae the aquaculture ponds (Figures 3, 4 and 5).et al., 2015).The control ponds without efficient bio-stabilizer of pH in aquaculture microalgae experienced a drastic pH increase ponds.This is mainly attributed to the fact that from 6.3±0.03(at 0 h) to 9.0±0.06(at the 240th microalgae use ammonia as their primary source hour).This is deleterious to fry growth and of nitrogen (Artorn et al., 2013;Liu et al., 2017).survival.This drastic pH increase is as a result of Therefore, ammonia (NH ) which is produced accumulation of toxic unionized ammonia (NH ) 3 3 from fish excretal and excess uneaten fish feed in the control ponds (without microalgae) (Table (Hii et al., 2011;Chen et al., 2012

Gendel and Lahav, 2013) was assimilated by
The combined co-culture of C. lewinii and S. these microalgae thereby stabilizing the ponds' dimorphus gave the least overall fluctuation in pH (Hii et al., 2011;Artorn et al., 2013).The pH pH and therefore regarded as the best pH bioof the experimental ponds with C. lewinii, S.
stabilizer, followed by C. lewinii and then S. dimorphus and that with the combination of C.
dimorphus.The use of microalgae as a pH biolewinii and S. dimorphus increased slightly from stabilizer in aquaculture in this research work is 6.3±0.03 to 6.5±0.2,6.7±0.6 and 6.4±0.1 quite novel as there is currently no published respectively during the 240 h of the experiment work on it.(Figure 1).These concentrations are all within NH (aq) + H O (l) ?NH H O (aq)……(i)  Liu et al., 2017) probably because less energy is ammonia.This is why microalgae are used in sewage treatment scheme (Muylaert et al., required for its uptake and assimilation (Chen et 2015; Taziki et al., 2015;Ge et al., 2016;al., 2012).It therefore implies that toxic Kaparapu and Geddada, 2016).In this very toxic nitrogenous waste (Bhatnagar and for it to be assimilated by these microalgae (C. Devi, 2013;Taziki et al., 2015), was efficiently and effectively removed by microalgae.There lewinii and S. dimorphus, which are all eukaryotic was significant nitrite-nitrogen reduction in the cells) (Hii et al., 2011).The aquaculture ponds ponds with microalgae compared to the control ponds without microalgae as represented in with combined cultures of C. lewinii and S. Table 1.This reduction may be attributed to the dimorphus achieved the best toxic ammonia bioproduction of the enzyme ferredoxin-nitrite reductase (by the experimental microalgae) purification as it reduced ammonia to zero level which converts nitrite directly to ammonia which within 144 hours of the experiment.This was are then assimilated by microalgae since their direct source of nitrogen is ammonia (Hii et al., followed by C. lewinii (in 192nd hour) and then S. 2011;Liu et al., 2017).S. dimorphus proved to be dimorphus (Table 3) as against the control ponds the best nitrite-remover as it zeroed toxic nitrite in the aquaculture ponds from the 144th hour of where the ammonia concentration increased to a the experiment followed by the combined peak of 0.4832±0.0711mg/L in the 240th hour culture of C. lewinii and S. dimorphus (in 240th of the experiment.These results are consistent hour) and C. lewinii, which was the least nitrite removal (Table 1).There are several works on with those of Kim et al. (2010) in which Chlorella the use of microalgae in waste water treatment vulgaris was used to efficiently remove ammonia (Ogbonna et al., 2000;Yusoff et al., 2011;Abdel-Raouf et al., 2012;Sriram and Seenivasan, 2012; from wastewater effluent.Also, Al-Balushi et al.Martins et al., 2013;Wahid et al., 2013;Banerjee (2012) demonstrated the effectiveness of et al., 2015;Muylaert et al., 2015;Nasir et al., 2015;Taziki et al., 2015;Kaparapu and Trentepohlia aurea microalgae to remove nitrite Geddada, 2016;Ramsundar et al., 2017) but from wastewater.However, there seems to be no there is currently none on the use of photosynthetic microalgae in the removal of reports on the use of photosynthetic microalgae nitrite from fish seedlings' aquaculture ponds.
to remove toxic ammonia from fish seedlings' However, similar pattern of nitrite assimilation was obtained in shrimp's aquaculture by Ge et aquaculture ponds.The oscillatory or trajectory al., (2016) when they co-cultured Platymonas patterns of total ammonia nitrogen (TAN) helgolandica, Chlorella vulgaris and Chaetoceros observed in the aquaculture ponds with mulleri separately with shrimps for 84 days.Nitrite was kept within the recommended levels microalgae (Table 2) may be attributed to the of = 0.55 mg/L as against the control which conversion of other forms of nitrogen (especially peaked at 3.5 mg/L on the 84th day of nitrite and nitrate) to ammonia, which is the experiment.
primary source of nitrogen to microalgae (Hii et Toxic ammonia was also effectively al. 2011;Artorn et al., 2013).Microalgae release reduced in the ponds with photosynthetic ferredoxin-nitrite reductase which converts microalgae compared to the control ponds.This nitrite to ammonia, and then NADH -nitrate may be attributed to several reasons.Firstly, the 2 primary source of microalgal nitrogen is reductase which converts nitrate to nitrite and ammonia (Hii et al., 2011;Artorn et al., 2013; Liu then to ammonia with the help of ferredoxin- et al., 2017).Secondly, eukaryotic microalgae nitrite reductase (Hii et al., 2011;Liu et al., generally frugal in their metabolism, they because when the concentration of the therefore do not produce enzymes when the microalgae increased to a significant level, they enzymes are not needed except in basal or assimilated ammonia as soon as it is formed and house-keeping level (Willey et al., 2009).Thus, thus prevented increase in the pH of the ponds.The reduction in fry's mortality rate in the as TAN drops, these enzymes are secreted to experimental ponds with microalgae may be produce more ammonia from either nitrite attributed to a multiple of factors.Primarily, it and/or nitrate, and then as ammonia increases, may be due to the removal of, and/or reduction in the poisonous nitrogenous waste (i.e. the enzymes production is inhibited or drops ammonia and nitrite) (Tables 1 and 3) from the drastically until TAN concentration drops again ponds and the bio-stabilization of the ponds' pH similar to the illustrations by Artorn et al. (2013).
(Figure 1) by the photosynthetic microalgae; as well as the ample supply of dissolved oxygen into Hii et al. (2011) stated that this temporal the aquaculture ponds by the photosynthesizing inhibition of these enzymes in microalgae may microalgae (data not included).Secondarily, the either be due to the inactivation of the enzyme physical presence of microalgae in ponds also system by ammonia or by the by-product of protects fish from direct solar radiations (Hii et al., 2011); and the good nutritive value (such as ammonia assimilation.These therefore resulted protein, vitamins and polyunsaturated fatty acids to the oscillation in the concentration of TAN in (PUFA)) (Tibbetts et al., 2014;Ge et al., 2016) the experimental ponds with microalgae as that the fry may have accumulated from the consumption of microalgae may have added to against the control ponds without microalgae.
their health status and the consequent reduction There was also a direct proportional in the mortality of the fry in the ponds corelationship between toxic ammonia and the pH cultured with microalgae (40%, 37% and 35% for C. lewinii, C. lewinii and S. dimorphus, and S. of the aquaculture ponds (mostly pronounced in dimorphus co-cultured ponds) as against the the control ponds without microalgae).This is control ponds that doubled to 80%.consistent with the report by Hurtado and C. lewinii and S. dimorphus have proven Cancino-Madariaga (2014); Li and Boyd (2016).
to be effective and efficient pH bio-stabilizers This relation is due to the fact that toxic and bio-purifiers in fish fry's aquaculture.The combined culture of C. lewinii and S. dimorphus unionized ammonia (NH ) which is very soluble in 3 were the best pH bio-stabilizer and toxic water gives hydroxyl ion when it dissolves (as ammonia bio-purifier followed by S. dimorphus.indicated in equations i and ii above) that S. dimorphus is the best nitrite bio-purifier closely consequently results in a corresponding increase followed by the combined culture of C. lewinii and S. dimorphus.Therefore, more research is in the pH of the pond (Ababio, 2011;Hii et al., needed to determine the best proportions of C. 2011; Artorn et al., 2013).In the control ponds, a lewinii and S. dimorphus that may be used as mini and major peaks of 7.6±0.06and 9.0±0.06efficient bio-stabilizer and bio-purifier.These values in pH were obtained respectively as a photosynthetic microalgae can therefore be result of the corresponding mini and major peaks used in fish seedlings' sustainable aquaculture as replacement to constant water changing or the in toxic ammonia of 0.0637±0.0152mg/L and water re-circulatory system currently in practice, 0.4832±0.0711mg/L respectively (Figure 2).which are very expensive and add to the cost of Ponds with microalgae only witnessed this direct fish seedlings' production.proportional relationship within the first 96 h of

9
mark with the sample from the pond.A drop of experimental ponds.Initial inoculum of 1x10 Rochelle Salt Solution was added to each viewing 9 cells of C. lewinii and 1x10 cells of S. dimorphus tube and swirled to mix after which three drops were inoculated into separate sets of the of Nessler Reagent was added to each viewing experimental ponds while combined inoculum of tube, stoppered and swirled to mix.The viewing 8 8 5 x10 cells of C. lewinii and 5 x10 cells of S. tubes were allowed to stand for 10 minutes for dimorphus were inoculated into another set of colour development.Thereafter, the prepared the experimental ponds.The control ponds also sample was inserted into the right hand opening contained 10 fry each, were partially shaded of the Colour Comparator while the reagent under sunlight but without photosynthetic blank was inserted into the left hand opening.microalgae.All the aquaculture ponds were The Comparator was held up to a light source covered with transparent plastic roofing to allow and the disc rotated until the colours in the left only sunlight and not rain into the systems.The and right windows match.The concentration of fry were feed with 0.20 to 0.50 mm Coppens ammonia nitrogen in mg/L (N) was read through floating feed with daily feeding ration of 6% the scale window.The Toxic ammonia (NH ) Mortality rate = F1-F2 x 100 Nitrite-nitrogen F1 The concentration of nitrite-nitrogen was ® Where: F = Initial number of fry and F = Final determined with Hach 's water nitrite-nitrogen 1 2number of fry.testing kit.One viewing tube was rinsed several times with the water sample and then filled to the 5 mL mark.The content of one NitriVer® 3Statistical analysis Powder Pillow was then added to the 5 mLThe results were reported as mean ± sample after which the viewing tube was standard deviations of triplicate experiments.stoppered and shook vigorously for exactly one Data were subjected to One-Way Analysis of minute.The prepared sample was allowed to sit Variance (ANOVA) and considered significant at undisturbed for 10 to 15 minutes and the tube P < 0.05.was placed into the right opening of the Colour Comparator.A control tube was filled to the 5 mL Results mark with untreated water sample and placedStabilization of pond pH into the left opening of the Colour Comparator.There was only slight increase in the pH The Comparator was held up to a light source of the ponds inoculated with microalgae during while the colour disc was rotated until the the 10 days (240 h) of experiment.However, in colours in the left and right windows match.The the control ponds (without microalgae) there nitrite-nitrogen concentration (mg/L) was read was significant increase in the pH.The pH of the through the scale window and Nitrite (NO ) 2 experimental ponds with C. lewinii, S. dimorphus concentration was calculated as:

Figure 1 :
Figure 1: Changes in the pH of aquaculture ponds during the co-culture of fry and microalgae.

Figure 2 :
Figure 2: Relationship between pH and toxic ammonia concentration in control ponds

Figure 3 :Figure 4 :
Figure 3: Relationship between pH and toxic ammonia concentration in the experimental ponds with C. lewinii