Abstract: Using of ecofriendly biofertilizers instead of chemical ones in fish aquacultures represents an important aim to minimize water pollution, improve water quality and enhance fish growth rate. The impact of inoculation of two strains of Azospirillum brasilense and/or Azotobacter chroococcum on bacterial count, chemical characteristics of water, Specific Growth Rate (SGR), Aspartate Aminotranseferase (AST), Alanine Aminotransferase (ALT) and histopathological changes were studied in Oreochromis niloticus aquaculture. Water analysis results revealed that Dissolved Oxygen (DO), Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), NPP, NO3-N and O-PO4 levels were significantly (p<0.05) increased by treatment with Azotobacter bacteria while Azospirillum treatment gave lower levels. On the other hand mixed or single bacterial treatments increased fish SGR especially treated with Azotobacter (34.62% increase in growth). Although the mixed bacterial treatment improved some water parameters it showed higher ALT and AST levels and sever histopathological lesions in fish than that of single treatment. So the present study introduces the single inoculation of Azotobacter bacteria biofertilizer as a suitable probiotic which may be used in Oreochromis niloticus aquacultures.
INTRODUCTION
Organic and inorganic fertilizers are often used in fish aquacultures to increase nutrient levels of a water body for enhancing fish production; however their excessive use leads to change in water quality parameters (Yadava and Garg, 1992; Garg and Bhatnagar, 1996). The use of chemical fertilizers is not only costly and lost quickly but also has caused tremendous harm to the environment. Science the three vital natural resources, soil, water and air are liable to be affected by excessive use of chemicals through leaching, volatilization and by abrupt mineralization (Garg and Bhatnagar, 1996).
Biofertilizers can be defined as microbial inoculants which contain live or latent cells of selected strains of microorganisms. They are used as a supply of nitrogen and phosphorus to agricultural crops (Shehata and El-Khawas, 2003). Moreover, the biofertilizeres can reduce the use of inorganic fertilizers and prevents pollution (Narula et al., 1991; Lakshminarayana, 1993). The biofertilizers include cyanobacteria, nitrogen-fixing bacteria such as Rhizobium, Azotobacter, Azospirillum etc. and phosphate-solubilizing bacteria such as Bacillus etc. (Tripathy and Ayyappan, 2005; Nagananda et al., 2010).
The potential of using these biofertilizers to fix nitrogen in different aquatic systems has been demonstrated by Tripathy and Ayyappan (1998) and Tripathy et al. (2001). Moreover, Garg et al. (1998), stated that the inoculation of biofertilizers enhance plankton production, net primary productivity and fish biomass. So that biofertilizers are represented as a source of microbial inoculants which have brought hopes for many developing countries both economically and environmentally. Since they can solve problems of high inorganic fertilizers cost and ecological hazards (Gupta et al., 2003).
Azotobacter and Azospirillum are the most common nitrogen-fixing bacteria found in different types of aquatic habitats such as lakes, rivers and oceans (Terzaghi and Terzaghi, 1986). These organisms must not only serve as biofertlizer but also as detritus processors, fish food organisms and could also be supplementing as bioremediators, bioameliorators, biofilters which in a single term could be defined as probiotics (Tripathy and Ayyappan, 2005). The using of some microbial organisms as a probiotics was showed to improve the growth and the immune response of fish (Kesarcodi-Watson et al., 2008; Marzouk et al., 2008).
Although the nitrogen-fixing ability of Azotobacter and Azospirillum have been reported to occur in a wide variety of aquatic systems to optimize the productivity of freshwater fish ecosystems, the effect of such biofeltilizers on fish pathology and growth rate has not been investigated so far. Hence, the present study was designed with the objective of evaluating the efficacy of use of Azotobacter and/or Azospirillum biofertilizers as probiotics in Oreochromis niloticus aquaculture, their effect on growth rate, liver function and moreover their pathological effect on the different fish tissues.
MATERIALS AND METHODS
Experimental aquaria: The study was carried out in El-Qanater El-khayria Research Station, Egypt. Four fiberglass aquaria (1000 liter capacity) were used, cleaned and filled with freshwater. This study was conducted from 6-2010 to 10-2010.
Preparation of biofertilizers: Strains of free-living nitrogen fixer bacteria used in the present study were Azospirillum brasilense (Azos. R7) isolated from Ricinus communis and Azotobacter chroococcum (Azt.) isolated from Hordeum vulgare (Hamza et al., 1994). They were grown in N-deficient Combined Carbon Sources Medium (CCM) (Hegazi et al., 1998) at 32°C in a rotary shaker for three days for Azospirillum brasilense and five days for Azotobacter chroococcum.
Experimental treatments: A total of 120 Oreochromis niloticus fish were divided into four groups (30 of each). The first group; control group received the food without bacteria (T1), the second group was received Azospirillum brasilense treatment (T2), the third group was received Azotobacter chroococcum (T3) and the fourth one was received mixture of Azospirillum brasilense and Azotobacter chroococcum (T4). Approximately 2-5x107 bacterial cells were added daily to the fish food; the fish food consisted of 20% fish meal powder, 20% soybean, 10% corn, 45% bran, 2% vitamins and elements and 3% oils.
Water analysis: Water samples were collected every 15 days to determine the chemical parameters and bacterial count.
Chemical analysis: Dissolved Oxygen (DO) was measured using the modified Winkler method, Biochemical Oxygen Demand (BOD) with the five-day incubation method and Net Primary Productivity (NPP) with the three-hour incubation method (APHA, 1995). Chemical Oxygen Demand (COD) was carried out using the potassium permanganate method (Golterman and Clymo, 1971). Colorimetric methods were used to determine ammonia, nitrite and phosphate according to APHA (1995) and nitrate according to Mullin and Riley (1955).
Bacterial count: The pour plate technique and the plate count agar (APHA, 1995) were used for the enumeration of total bacterial counts at 32°C incubation temperatures. Total diazotrophs (associative nitrogen-fixing bacteria) were counted using the surface inoculated plate method on N-deficient Combined Carbon Sources Medium (CCM) (Hegazi et al., 1998).
Growth rate determination: At the end of experiment fish were harvested, weighed and the Weight Gain (WG) and the Specific Growth Rates (SGR) were calculated according to Priestley et al. (2006).
ALT and AST: At the end of experiment, blood samples were taken from the caudal vein of the fish by sterile syringe rinsed with heparin. Serum was separated by centrifugation of blood at 3000 rpm for 15 min and stored at -20°C until analyses. Activities of Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) were determined colorimetrically according to Reitman and Frankel (1957) using kits supplied from Biomerieux, France.
Histopathological examination: Small portions of gill, liver, spleen and intestine from control and treated Oreochromis niloticus fish were fixed in 10% formalin, dehydrated in series of alcohols, cleared in xylol, embedded in paraffin wax, sectioned by a microtome at 6 μm thicknesses and the tissue sections were stained with Haematoxylin and Eosin and then pathologically examined by light microscope (Carleton, 1967).
Statistical analysis: Data were statistically analyzed using analysis of variance, ANOVA (Freed et al., 1990) using the MSTAT and STATISTICA (6.0) computer programs. The correlation coefficients and linear regressions among the different parameters were determined as well.
RESULTS
Water analysis: Azotobacter (T3) and mixed strains (T4) treatments were significantly (p<0.05) increased DO (8.24 and 8.72 mg L-1), BOD (8.18 and 7.31 mg L-1), COD (14.64 and 15.76 mg L-1) as well as NPP (12.62 and 12.21 mg L-1), respectively. On the contrary Azospirillum treatment (T2) showed lower values of DO (6.05 mg L-1), BOD (3.98 mg L-1), COD (11.59 mg L-1) and NPP (6.53 mg L-1) compared to the control group. Regarding the soluble forms of nitrogen the treatment with Azospirillum showed the significant (p<0.05) lowest values of nitrite and nitrate (0.113 and 0.726 mg L-1), respectively. While the highest significant (p<0.05) O-phosphate concentration (0.65 mg L-1) was determined in Azotobacter treated aquaria (Table 1). Correlation coefficient indicated significant positive interactions (n = 16) among the NPP with DO (r = 0.79) and BOD (r = 0.71) also NO3-N with DO (r = 0.55), COD (r = 0.78) and O-PO4 (r = 0.59). Negative correlation between the NH4-N with NO3-N (r = -0.67) and COD (r = -0.58).
| Table 1: | Bacterial count and water analysis as affected by bacterial treatments |
| Means followed by the same letter are not significantly different (p<0.05) | |
| Table 2: | Effect of different bacterial treatments on growth rate of Oreochromis niloticus |
| Means followed by the same letter are not significantly different (p<0.05). Initial weight (g) = 25.26±7.58, n = 15, Specific Growth Rate (SGR) = {(ln Wtf - ln Wti) x100}/t, where Wtf and Wti denote final and initial weight (g) of fish, respectively, t represents duration of the experiment (days) and ln = natural log. % increase = {(treatment-control)/control}*100 | |
| Table 3: | Effect of different bacterial treatments on ALT and AST of Oreochromis niloticus |
| Means followed by the same letter are not significantly different (p<0.05). ALT, AST and final weight values were expressed in Mean±SD, n = 8. % increase = {(treatment-control)/control}*100 | |
Regarding the bacterial count, the highest bacterial count was recorded in T3, followed by T4, T2 and T1, respectively; while diazotrophic bacteria showed the highest counts in T4, followed by T3, T2 and T1, respectively (Table 1).
Growth rate: A significant increase (p<0.05) in final weight of fish and consequently in growth rate was recorded at the end of the experiment (Table 2). The highest percentage of increase was recorded in Azotobacter treatment (34.62%), followed by Azospirillum treatment (25.10%) while mixed strains treatments recorded the lowest percentage (10.27%).
ALT and AST: A significant increase in ALT and AST in both Azospirillum and Azotobacter treated fish were recorded at the end of the experiment. This increase was about 17-38% in single strain treatments (T2 and T3) and reach to 110-128% in mixed strains treatment (T4) (Table 3).
Clinical signs and histopathological changes: No abnormal clinical signs, no gross lesions or dies were observed in the four studied aquaria. Slight histopathological changes in tissues of both Azospirillum and Azotobacter treated fish were detected. Gills show hyperplasia and necrosis in fish exposed to Azospirillum sp. and lamellar fusion accompanied by congestion in fish exposed to Azotobacter sp. Intestine show few mucinus degeneration associated with congestion of submucosa of fish exposed to Azospirillum sp. and infiltiration of inflammatory cells in the lamina propria in fish exposed to Azotobacter sp. Liver show congestion in the hepatic blood vessels associated with slight degenerative changes in fish exposed to Azospirillum sp. and infiltration by melanomacrophage cells in fish exposed to Azotobacter sp. Spleen show depletion in the lymphocytic elements with hyperactivation of melanophores and melanomacrophage center numbers in fish exposed to Azospirillum sp. and slight congestion in fish exposed to Azotobacter sp. (Fig. 1-4b, c).
On the other hand, the most pathological changes were recorded in mixed bacterial treated group (T4) which represented by severe hyperplasia, hypertrophy, lamellar fusion and presence of eosinophilic granular cells in gills. Intestine showed severe hyperplasia with fusion of some mucosal layers and activation in mucous secreting cells. Liver showed necrosis, vacuolar degeneration in hepatocytes and pancreatic tissue. In addition to depletion, congestion in the spleenic blood vessels, sever hyperactivation of melanophores and melanomacrophage centers in spleen and liver tissues (Fig. 1-4d).
| Fig. 1: | Gill sections showing: (a) normal primary and secondary lamellae in fish of control group. (b) hyperplasia at the base of secondary lamellae accompanied by degenerative changes and necrosis in fish exposed to Azospirillum sp. (c) lamellar fusion in some primary lamellae with hyperplasia and congestion in fish exposed to Azotobacter sp. and (d) severe hyperplasia, hypertrophy, lamellar fusion and presence of eosinophilic granular cells (arrow) in fish exposed to mixed bacteria |
| Fig. 2: | Intestine sections showing: (a) intestine of control group (b) slight mucinus degeneration in the intestinal villar epithelium associated with congestion of submucosa of fish exposed to Azospirillum sp. (c) infiltiration of mononuclear inflammatory cells in the lamina propria in fish exposed to Azotobacter sp. and (d) severe hyperplasia of the villar epithelium lining of the mucosa with fusion of some mucosal layers and activation in mucous secreting cells (arrows) in fish exposed to mixed bacteria |
| Fig. 3: | Liver sections showing: (a) liver tissue of control group (b) congestion in the hepatic blood vessel associated with degenerative changes and chronic inflammatory cells infiltration in between the hepatocytes in fish exposed to Azospirillum sp. (c) focal necrotic area surrounding the pancreatic tissue infiltrated by melanomacrophage cells in fish exposed to Azotobacter sp. (d) necrosis and vacuolar degeneration in hepatocytes and pancreatic tissue with aggregation of melanomacrophage cells in fish exposed to mixed bacteria |
| Fig. 4: | Spleen sections showing: (a) spleen of control group (b) depletion in the lymphocytic elements (arrow) with hyperactivation of melanophores and melanomacrophage center numbers in fish exposed to Azospirillum sp. (c) congestion in between the lymphocitic tissues in fish exposed to Azotobacter sp. and (d) depletion, congestion in the spleenic blood vessels, sever hyperactivation of melanophores and melanomacrophage centers with fibrous connective tissue in between the lymphocytic tissue in fish exposed to mixed bacteria |
DISCUSSION
Now-a-days, aquaculture has become an important economic activity in many countries; using of biofertilizers is important where chemical fertilizers impact negatively on environmental pollution and human health. Azotobacter and Azospirillum were used as biofertilizers in aquaculture; they increased the phytoplankton population and consequently the yield of fish (Garg and Bhatnagar, 1996; Garg et al., 2001; Tripathy and Ayyappan, 2005). In the present study, water analysis showed that most of water parameters do not exceed the recommended water quality guidelines for the protection of cultured freshwater fish which stated by ANZECC (2000) to ensure the safety of using such biofertilizers on the fish environment.
The present study investigated that Azotobacter treatment was significantly increased BOD and COD; this indicates an increase of the oxi-disability and oxygen demand of water samples as well as increased the suitable components consumed by microorganisms or chemically. This may be explained by the fact that Azotobacter spp. in particular, are rather qualified for the bioremediation of the complex organic compounds, a mechanism for protecting their nitrogenases against high O2 concentrations (Hegazi et al., 1984) as well as the ability of Azotobacter to form capsular polysaccharides qualifies these particular organisms to entrap heavy metals, such as cadmium and lead ions (Pasetti et al., 1996), these also illustrated increase total bacterial counts and net primary productivity. In addition, NO3-N and O-PO4 concentration were also significantly high in Azotobacter treatment as compared to the other treatments, this agreement with (Garg et al., 2001) studies showed that Azotobacter can be used as a biofertilizer for enhancing nitrogen input and phosphate solubilization in fish ponds.
Although Azotobacter is a highly aerobic organism (Lakshminarayana, 1993) and its inoculation reduces DO concentration in water, our results showed that there was no decrease in DO concentration; this may be attributed to mix the bacteria strain with the fish food where Garg et al. (2001) observed a slight reduction in DO when Azotobacter were mixed with organic substrate.
The highest weight gain and specific growth rate recorded in Azotobacter treated fish may be attributed to the highest NPP, NO3 and PO4 recorded in that treatment. This indicates a high trophic status in that treatment over the other ones. And this in turn make Azotobacter is better used as a biofertilizer than Azospirillum. This was in agreement with Tripathy and Ayyappan (2005) who stated that Azotobacter seems to be a relatively abundant species in aquatic ecosystems due to its aerobic nature compared to the micro-aerophilic nature of Azospirillum. Also the inoculation of freshwater fish aquaculture ponds in India with Azospirillum sp. and Azotobacter sp. were significantly increased the phytoplankton population and consequently the yield of fish (Garg and Bhatnagar, 1996).
ALT and AST are the most sensitive tests for diagnosis of infectious diseases. Although the present study showed an increase of ALT and AST in Azotobacter and Azospirilum treated fish, there is no dangerous effect on fish health or growth. This increase may be attributed to the fact that the liver is the primary organ of detoxification; also, exposure of fish to any external stressful stimuli may cause adverse physiological reactions affecting its essential life functions as stated by Abdel-Moneim et al. (2008) and Dobsikova et al. (2009). On the other hand the marked elevation in ALT and AST in the mixed bacteria treatment revealed a serious problem in that case.
Unfortunately, there is no available data about the histopathological effects of bacterial biofertilizers treatment on fish tissue. While in present study, the treatment with Azotobacter sp., revealed the slight histopathological alterations in fish tissues than Azospirilum sp. and mixed bacterial treatment.
The prominent improve in growth in addition the hyperactivation of melanophores and melanomacrophage centers in spleen and liver of fish treated by Azotobacter sp., point out to the use of this bacteria as a probiotic in fish aquaculture, this agreed with several studies which demonstrated certain modes of probiotic action in the aquatic environment, they improved feed conversion ratio and feed utilization consequently increased weight gain and specific growth rate (El-Haroun, 2007; Soundarapandian and Sankar, 2008). Also, many researches showed improvement in the immune response of fishes treated with probiotices (Marzouk et al., 2008; Kesarcodi-Watson et al., 2008).
CONCLUSION
Based on the present study results, the usage of Azotobacter or Azospirillum biofertilizer, singly-particularly Azotobacter-in fish aquacultures appears to be safer on fish than using mixed bacteria treatment. However, more work need to be done to decide the extent to which it can be incorporate in fish cultures. Also, it needs more work to evaluate its use as a probiotics to promote the growth and enhance the immune response of fish.