MATERIALS AND METHODS

Study area: Otamiri River flows through various communities in Imo State, Nigeria and serves as a source of drinking water for these communities. In Egbu community, the river is a discharge point for wastes from an abattoir located about 300 m away. Three sampling stations namely upstream, downstream and source point which were about 300 m apart and located at N 05° 28.329t, E 07°03.113t; N 05°28.386t, E 07°02.991t and N 05°28.422t, E 07°02.803t, respectively were chosen.

Sample collection: River water samples were collected from the three sampling stations at a depth of about 10-20 cm below the water surface using three separate 5 L plastic containers. Samples were transported to the laboratory for analysis within one hour of collection. Portions of the samples were used for microbiological and physicochemical analyses while the other was used for monitoring ammonium and nitrite oxidation over a 24 h period.

Microbiological analysis: Microbial counts were estimated by plating out 0.1 mL of the 10^-1 to 10^-3 dilutions of the various samples in normal saline on appropriate agar plates. Total heterotrophic bacterial count was done in nutrient agar while ammonium and nitrite oxidizing bacterial counts were done in modified Schmidt and Belser medium (Schmidt and Belser, 1994). The medium composition for the ammonia oxidizing bacteria was (g L^-1): NH₄Cl-0.136, MgSO₄.7H₂O-0.04, KH₂PO₄-0.204, EDTA-0.005, Trace metal solution-1 mL, agar-15 g. For nitrite oxidizing bacteria the medium was composed of (g L^-1): NaNO₂-0.006, MgSO₄.7H₂O-0.0098, K₂HPO₄-0.0556, NaCl-0.1168, CaCl₂-0.0294, EDTA-0.001, Trace metal solution-1 mL, agar -15 g. The trace metal solution used was composed of (g L^-1) H₃BO₄-0.1976, ZnSO₄.7H₂O-0.1724, FeSO₄.7H₂O-3.8924, CuSO₄.5H₂O-0.250. Ketoconazole at 50 μg mL^-1 was added to the medium for the nitrifiers and the pH adjusted to 7.5. The inoculated plates were incubated at room temperature 28(±2) °C for 1-2 days for the heterotrophs and 7-9 days for the nitrifiers.

Physicochemical analysis: The physicochemical analysis of the water samples were carried out in the geological laboratory of Federal University of Technology, Owerri, by standard methods for water analysis as adopted by HACH (1992) using a portable HACH DR-EL/5 Laboratory Kit. Total carbon was determined by the wet combustion method as modified by Nelson and Sommers (1982) and total nitrogen was by the semi-micro Kjeldhal method (Bremner and Mulvany, 1982).

Estimation of ammonium and nitrite consumption: Extents and rates of ammonium and nitrite consumptions were measured using inhibitors of chemoautotrophic nitrifying bacteria during short-term 24 h incubation experiments following the procedures by Bianchi et al. (1994). Some 2.5 L portions of river water from each site were put in screw capped containers and supplemented with basic food solution consisting of (g L^-1) NaCl-0.73, CaCl₂-0.1825, MgS0₄.7H₂0-0.0625, KH₂PO₄-0.0675, FeSO₄.7H₂O-0.0000025, ZnSO₄-0.0000025 (Juliastuti et al., 2003). Ammonium was added in form of NH₄Cl to a final concentration of 50 μM while nitrite was added as NaNO₂ to a final concentration of 0.36 mM (Cebron et al., 2003). The pH of each sample was adjusted to 7.5 with NaOH. Each sample was dispensed in 250 mL amounts into three triplicate bottles labeled A, Na and W. The bottles were given different treatments which enabled the estimations of ammonium and nitrite consumptions by autotrophic and heterotrophic microorganisms as follows:

This treatment estimated ammonium consumed by heterotrophs (NH_{4 hetero}).

This treatment estimated nitrite consumed by heterotrophs (NO_{2 hetero}).

This treatment estimated:

Therefore, autotrophic ammonium (NH_{4 auto}) and nitrite consumptions (NO_{2 auto}) were estimated as follows:

The extents of ammonium and nitrite consumptions were calculated from the decrease in ammonium and nitrite concentration during the incubation. Rates of consumption were calculated by dividing the ammonium or nitrite consumed with time of estimation (h).

Incubation conditions and analysis: The samples were incubated in the dark, at room temperature 28 (±2)°C. Ten milliliter aliquots of each sample were collected at 0, 6, 12 and 24 h and held at 0-5°C prior to analysis. One milliliter aliquots of each sample collected at different time intervals were used for the determination of NH₄⁺-N and NO₂¯-N. Ammonium-nitrogen was determined by the modified Keeney and Nelson (1982) method while nitrite-nitrogen was determined by the Greiss method as modified by Promega (2005). Ammonium and nitrite consumed were estimated from reductions in ammonium and nitrite concentrations relative to the initial concentrations.

RESULTS

Physicochemical analysis of the river water (Table 1) samples showed that sample from the Source Point (SP) was the most turbid (10 FTU) and had higher conductivity (82 μmhos cm^-1), total dissolved solids (49.2 mg L^-1), nitrates, bicarbonates, total suspended solids and C:N ratio than the Upstream (UP) and Downstream (DN) stations. Microbiologically, SP sample also had the highest BOD (0.75 mg L^-1) and was richer in the various microbial populations. At the various stations, the populations of the total heterotrophic bacteria were significantly higher than those of the nitrifiers with the SP having the highest count of 16.6x10³ cfu mL^-1.

The patterns of cumulative ammonium consumptions showed active autotrophic and heterotrophic ammonium consumptions at the upstream and downstream stations throughout the incubation period (Fig. 1). After 24 h, the autotrophs have consumed 203.95 μg NH₄⁺ and 124.78 μg NH₄⁺ at the upstream and downstream stations, respectively. On the contrary, at the source point, autotrophic ammonium consumption was completely repressed ab initio.

Figure 2 showed the patterns of cumulative nitrite consumption at the three stations. In all the stations, nitrite consumptions by both groups of organisms were active throughout the incubation period. At 24 h, the autotrophs have consumed 332.09 and 278.37 μg NO₂⁺ at the upstream and downstream stations, respectively.

Table 1:	Physicochemical and microbiological properties of the river samples at the three stations
ND*: Not determined

Table 2:	Rates of ammonium and nitrite consumption by different microbial communities

Fig. 1(a-b):	Patterns of cumulative (a) autotrophic and (b) heterotrophic ammonium consumption at different sites (<0% = inhibition)

Fig. 2(a-b):	Patterns of cumulative (a) autotrophic and (b) heterotrophic nitrite consumption at different sites

At the source point, autotrophic nitrite consumption was active in the first 12 h, consuming 410.66 μg NO₂⁺. At 24 h, no additional nitrite was detected in the medium. Hence, the cumulative nitrite consumption at 24 h was same as that at 12 h.

The rates of ammonium and nitrite consumption at the different stations by the autotrophic and heterotrophic microbial communities are shown in Table 2. The highest rate of autotrophic ammonium consumption was 14.254 μg NH₄⁺-Nh^-1 observed upstream between 0-6 h, however, this steadily declined with time. The rate of autotrophic nitrite consumption upstream was constant (16.667 μg NO₂¯-Nh^-1) in the first 12 h and slightly declined thereafter. In all the stations, the rates of nitrite consumption by heterotrophs were greater than those by autotrophs. At the SP, the rate of heterotrophic nitrite consumption increased with time and the highest rate of 122.917 μg NO₂¯-Nh^-1 was observed between the 12-24 h.

Estimation of the relationships between ammonium and nitrite consumptions by autotrophs and heterotrophs at the various sampling points using the Pearson’s product moments correlation analysis showed that apart from the source point where a strong negative correlation (r = -0.96) existed between both parameters among the autotrophs, the relationships were strongly positive ( 0.96< r > 1.0) among autotrophs and heterotrophs in the other stations.

DISCUSSION

The physicochemical and microbiological conditions at the source point station which is a direct recipient of abattoir waste, compared to other stations indicate that the waste adversely affected Otamiri River. High turbidity could reduce light penetration into the water and this may inhibit primary productivity (Sandrin et al., 2009). Studies by Shah et al. (2008) indicated that water transparency is a major physical factor affecting the distribution and seasonal variation of phytoplanktons which represents the biological wealth of a water body and a vital link in the food chain (Hossain et al., 2007). In a similar study, Adesalu (2010) attributed poor phytoplankton population in River Oli partly to high turbidity. Increased organic matter can alter energy relationships in the stream and this is capable of disrupting biotic community structure and function (Wakelin et al., 2008; Spanhoff et al., 2007). This is evident in the higher populations of the various microbial groups in the source point station. Omole and Longe (2008) also reported a high population of fecal coliforms at the source point of a river impacted by abattoir effluent. Increased microbial population could lead to a disruption in microbial community structure that can create shifts in ecosystem-level carbon, energy and nutrient flows (Schimel et al., 2007). The greater population of heterotrophs relative to the nitrifiers is in line with the higher growth rate of heterotrophs relative to the nitrifiers (Rittmann and McCarty, 2001) and is consistent with the observations of Fernandes et al. (2010) in shrimp pond with elevated organic carbon availability.

Repressed autotrophic ammonium consumption at the source point indicates that the conditions imposed by the abattoir waste at the source point adversely impacted the process. Part of the reason for this could be the high organic carbon content of the waste which imposed nitrogen limitation leading to higher C:N ratio at this point compared to other points. With the high C:N ratio, the faster growing, more abundant heterotrophic bacteria would have out-competed the autotrophic nitrifying bacteria for available NH₄⁺. These observations are consistent with those of Strauss and Lamberti (2000) who reported that addition of organic carbon to aquatic environment significantly decreased nitrification rates but increased total microbial population. Another possible reason for the repressed nitrification is the microbial predilection for reduced nitrogen in form of NH₄⁺ for cellular incorporation since reduced nutrients are incorporated directly into the Krebs cycle intermediate for amino acid synthesis. Thus the NH₄⁺ must have been immobilised rather than nitrified. The impact was however relieved downstream where even though autotrophic ammonium consumption was observed, it was not as vibrant as the ammonium consumption observed at the unimpacted upstream.

Positive correlations between ammonium and nitrite consumptions among the autotrophs and heterotrophs in the upstream and downstream stations imply normal nitrification process. Thus at the source point, normal nitrification by autotrophs was impeded probably due to conditions imposed by the abattoir waste. Repressed autotrophic ammonium oxidation signifies an impairment of a critical process in the biological removal of nitrogen and has serious implications. It leads to a reduction in the formation of nitrite and nitrate which are substrates for denitrification, the second process in biological nitrogen removal and hence a reduction in the self purifying ability of the river. This can create an imbalance in the nitrogen cycle. Secondly, it could provide assimilable nitrogen for microorganisms and some phytoplanktons. Research has shown that diatoms prefer nitrates (Yool et al., 2007) while phytoplanktons such as dinoflagellates prefer ammonium as a growth nutrient (Lima et al., 2010). Increased ammonium will therefore enhance the growth of dinoflagellates relative to diatoms, thus causing a shift in the phytoplankton community composition. Furthermore it could lead to the accumulation of potentially toxic ammonium salts which could harm aquatic lives (Wee et al., 2007). Ammonium salts have also been reported to have microbicidal effects (Xiao et al., 2008) and so can affect the ammonium oxidizers themselves. This has far reaching implications because ammonium oxidizers possess the Ammonia Mono-Oxygenase (AMO) enzyme complex which has a broad substrate range that permits the co-oxidation of numerous recalcitrant aliphatic, aromatic and halogenated molecules that could cause environmental problems (Robertson and Groffman, 2007). The change in microbial community structure due to shifts in nutrient composition and reduction in substrates for denitrification could alter the aquatic nitrogen cycle and lead to eutrophication of the river. These adverse effects of the abattoir wastes are however, relieved downstream.

Although the upstream station was not impacted, a steady decline in rate of autotrophic ammonium consumption with time was observed there. This could be due to substrate limitation imposed by a progressive reduction in the concentration of the ammonium substrate with time. This however does not seem to affect nitrite whose rate of consumption by autotrophs was relatively constant in the first twelve hours. It thus appears that the overall extent of autotrophic nitrification will be affected more by the concentration of ammonium than the concentration of nitrite. This is consistent with the views expressed by Prosser and Embley (2002) that ammonium oxidation is the rate limiting step in overall nitrification. The fact that nitrite consumption rates by heterotrophs were greater than those by autotrophs suggested that the heterotrophs made more demands on nitrite than the autotrophs. This is consistent with the higher population and faster growth rate of heterotrophic bacteria relative to nitrifying bacteria (Strauss and Lamberti, 2000).

CONCLUSIONS

The abattoir waste adversely impacted nitrification in the fresh water ecosystem through its effect on autotrophic ammonium oxidation. Ammonium oxidation by heterotrophs and nitrite oxidation by both autotrophs and heterotrophs were not affected. Repression of nitrification by autotrophic microbial population acclaimed to be the most prolific nitrifiers could disrupt the nitrogen balance and have far reaching effects on the aquatic ecosystem.