Study on Heterotrophic and Chemo-autotrophic Bacteria (nitrifying) as Bioremediator of Ammonia and Nitrate in the Simulated Aquaculture System

Study on Heterotrophic and Chemo-autotrophic Bacteria (nitrifying) as Bioremediator of Ammonia and Nitrate in the Simulated Aquaculture System

Anjulata Suman Patre^1* , S. B. Gupta¹

¹Department of Agricultural Microbiology, College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur, Chhattisgarh, India

Corresponding Author Email: anjulatasuman90@gmail.com

DOI : http://dx.doi.org/10.53709/CHE.2021.v02i01.003

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INTRODUCTION

Chhattisgarh state of our country is blessed with various water bodies in the form of reservoirs (83,873 ha), ponds (70,000 ha), and rivers (3,573km). However, natural and manufactured ponds constitute a significant source to fish culture. Due to tropical climate location, there is a considerable variation in environmental factors like temperature, rainfall, photoperiod, etc., which also physically affect the water bodies. Fish production is influenced by physical characteristics like temperature, pH, sunlight and chemical factors like dissolved O₂ and CO₂ levels, and levels of inorganic nutrients [1]. For location-specific optimum fish production, it is necessary to know about the variations in environmental factors under local conditions, which can be matched with farmer managerial practices for maximizing fish production.

Heterotrophic bacteria use ammonia from consumed food, fecal waste, etc. as nitrogen sources that are decomposed anaerobically. However, for bacterial growth and energy generation, supplemental carbon sources are needed. Their decomposition depends on environmental factors such as temperature and oxygen. Thus, the use of labile sources such as molasses and dextrose, as well as the use of sources with lower dissolution, as in the case of plant meals, are necessary as carbon sources to heterotrophic bacteria [2-3]. Commercially available nutrient amendments with complex compositions that are known to stimulate microbial growth include molasses. Molasses contains about 50% sugar in the form of sucrose, glucose, and fructose, and is rich in mineral elements [4]. In recent years, manipulation of C:N ratio infeed has shown promising results in aquaculture [2]. The C:N ratio can be manipulated by the application of various carbohydra te sources such as molasses, rice flour, tapioca powder, etc. Plant meals are alternative carbon sources that contrast with molasses in their cost and dissolution in culture water. BFT farming system is viable with low-cost carbon-rich sources [3]. Recommend using low-cost carbon sources because it is an alternative that enables economics sustainability, provides an additional source of protein, and improves the nutritional efficiency of the culture system [5].

The recent approach to improve the quality of polluted water bodies in aquaculture was the application of beneficial eco-friendly microbes, enzymes, and other bio-chemicals to the ponds, known as ‘bioremediation, which involves the manipulation of microbes in ponds to enhance mineralization, nitrification, and denitrification processes to minimize toxic effects in order to increase sustainable, eco-friendly fish production. Bacteriological nitrification was the most practical method for the removal of excess ammonia from polluted urban water bodies and it can be achieved by setting of sand and gravel bio-filter through which water was allowed to circulate. The ammonia oxidizers for conversion of ammonia to nitrite are placed under five genera, Nitrosomonas, Nitrosovibrio, Nitrosococcus, Nitrolobus, and Nitrospira, and nitrite oxidizers for conversion of nitrite to nitrate, under three genera, Nitrobacter, Nitrococcus and Nitrospira. Mixed cultures of nitrifiers have been demonstrated to nitrify more efficiently. Nitrification produces nitrate and alters the pH slightly towards the acidic range, facilitating the availability of soluble materials. The vast majority of urban water bodies accumulate nitrate, as they do not contain a suitable effective eco-friendly denitrifying filters. Denitrifying filters also helps to convert nitrate to nitrogen. It creates an anaerobic region where anaerobic bacteria can grow and reduce nitrate to nitrogen gas for increasing fish production in heavily polluted water bodies. Therefore, the study aims to develop microbial biofloc for culture using carbohydrate materials (sugarcane molasses) as a carbon source to boost production by improving the conversion of nutrients into harvestable products while maintaining good water quality.

MATERIALS AND METHODS

A sampling of soil and water for bacterial isolation

Twenty-five soil samples (0-15 cm) were collected from the corner bottom of 25 fish ponds belonging to the Raipur district (Chhattisgarh). The samples were taken from five places of each pond bed. Just after the collection of samples, they were kept in air-tight polythene bags and used for isolation of heterotrophic bacteria, then, after muddy soil samples were kept for air drying for isolation of the autotrophic bacteria (nitrifying). Further, these bacterial isolates were tested as bio-remediators in vitro and in the set of fish aquariums containing polluted urea water to observe their efficiency, especially their effect on the rate of fish growth.

Isolation of autotrophic (nitrifying) bacteria

Autotrophic (nitrifying) bacteria were isolated from moist fish pond soil utilizing serial dilution and plating [6] using nitrifying agar media containing red phenol [7]. The agar plates were incubated for 4 weeks at 30 °C. For their multiplication and testing in nitrifying broth media, desirable colonies were picked up.

Culture media composition

Nitrosomonas culture medium

The chemical composition of ammonia broth medium for Nitrosomonas as follows: ammonium sulfate (NH₄)2 SO₄– 2.00gm, magnesium sulfate (MgSO₄ 7H₂O) – 0.50gm, Ferrous sulfate (FeSO₄ 7H₂O)- 0.03gm, sodium chloride (NaCl)- 0.30gm, magnesium carbonate (MgCO₃) – 10.00gm, di-potassium hydrogen phosphate(K₂HPO₄) – 1.00gm, double distilled water- 1000ml

Nitrobacter culture media

The chemical composition of nitrite broth medium for Nitrobacter was as follows:

Sodium nitrite (NaNO₂) – 1.00g,magnesium sulfate (MgSO₄.7H₂O) – 0.50 gm, ferrous sulfate (FeSO₄. 7H₂O) – 0.03gm, sodium chloride (NaCl) – 0.30gm, sodium carbonate (Na₂CO₃)- 1.00gm, di-potassium hydrogen phosphate (K₂HPO₄)- 1.00gm, double distilled water – 1000ml.

Isolation of heterotrophic (denitrifying) bacteria

After collecting samples, heterotrophic bacteria were isolated from the muddy moist fish pond soil using the serial dilution and plating process [6]. In this relation, for the isolation of the heterotrophic bacterium, PS5 Micrococcus luteus CP001628 and PS16 Ochrobactrum pituitosum AM490609, its medium was used for its multiplication and testing as bio-remediators for contaminated water to increase fish growth rate, as defined by the writer/edit or handbook.

Using a sterilized dropper, roughly 1gram of the muddy soil was aseptically transferred to a sterile test tube containing 9ml of the diluents. This yielded a 10^-1 dilution. Up to six-fold (10^-6), serial dilutions were subsequently prepared from the 10^-1 dilution [8]. In addition, sufficient dilution of the aliquot was used for inoculation on its agar medium. The inoculated agar plates were incubated for up to 72 hrs at 28 ± 2oC. They mentioned, the identified selected colonies were counted at an interval of 24 hours.

Culture media composition:

The chemical composition of the heterotrophic medium for heterotrophic bacteria was as follows (composition for 1 liter): Agar -20.0gm, Na₂HPO₄-7.9gm, KH₂PO₄-1.5gm, NH₄Cl -0.3gm, MgSO₄.7H₂O -0.1gm, Yeast extract solution -10.0ml, Trace elements solution SL10 -1.0ml, pH- 7.5.

Determination of ammonium and nitrate

The concentration of ammonia and nitrate was determined by Kjeldhal method by using Devardaalloy. The Devardaalloy  was used as a reducing catalyst that once the ammonium was driven off during the first part of the N-distillation, then after, it was added to the solution to help for conversion (reduce) nitrate to ammonium. The solution still has a high pH so that the NO₃ is converted to NH₄ easily, which can be again distilled off for analysis [9].

RESULTS

Location of soil and water samples collection for isolation of nitrifying autotrophic bacteria (Tehsil and District: Raipur; Chhattisgarh)

            Data presented in (Table 1) related to information about Nitrosomonas and Nitrobacter isolates which were isolated from 25 soil and water samples of polluted ponds belonging to area of Raipur, Chhattisgarh. Further, these isolates were tested for their ability to convert ammonia into nitrite by using an ammonia broth medium. Results of weekly qualitative analysis indicate that ammonia started to transform after two weeks of inoculation of nitrifying bacterial isolates under controlled conditions. The selection of effective nitrifying bacterial isolates was made based on the rate of conversion of ammonia into nitrate [10].

            Isolation of nitrifying bacteria was done by using the serial dilution-plating method as also practiced by several researches [6, 7 and 11]. They indicate that isolates of nitrifying bacteria can be obtained by making serial dilution of soil samples. [12] reported that nitrifying bacteria could be isolated by making appropriate serial dilution of drained fish pond soil. In this connection, [11] said that the presence of nitrifying bacteria could be observed up to 10^-7diluation. However, in the present studies, isolation was made from 10^-3 dilution, which bears a low population density of nitrifying bacteria in soils of Chhattisgarh.

Table 1. Location of soil and water samples collection for isolation of nitrifying autotrophic bacteria (Tehsil and District: Raipur; Chhattisgarh

NS =Nitrosomonas bacterial isolate, NB = Nitrobacter bacterial isolate

Location of soil and water samples collection for isolation of heterotrophic bacteria (Tehsil and District: Raipur; Chhattisgarh

            Heterotrophic bacteria were isolated from 25 soil and water samples of polluted ponds belonging to the area of Raipur, Chhattisgarh (Table 2a). Further, these isolates were tested for their ability to convert ammonia into nitrate by using the proper medium.

            During isolation of denitrifying bacteria Ochrobactrum pituitosumAM 490609(PS16) and Micrococcus luteus CP001628 (PS5), media become more turbid within a week. This further confirms that they belong to the heterotrophic group of bacteria. Gram’s staining and microscopic observation also confirmed the presence of these bacteria.

Table 2a.Location of soil and water samples collection for isolation of heterotrophic bacteria (Tehsil and District: Raipur; Chhattisgarh)

PS= Heterotrophic bacteria isolate, PS5=Micrococcus luteusCP001628 bacterial isolate, PS16 =Ochrobactrum pituitosum AM490609 bacterial isolate

Ammonia and nitrate dynamics in polluted pond water as influenced by selected pond soil heterotrophic bacterial isolates under controlled conditions.

            Heterotrophic bacteria isolates were isolated from 25 pond soils belonging to the area of Raipur district, Chhattisgarh. Among these, 7 isolates (PS1, PS3, PS5, PS6, PS10, PS16, and PS25) were selected based on their growth performance during isolations from pond soils. Data presented in Table 2b revealed that out of 7 isolates, PS-5 and PS-16 isolates were found more effective concerning conversion of ammonia. The ammonia concentration due to PS-5 was decreased from 0.20 mg/l (15 DAI) to 0.09 mg/l (30 DAI). Similarly, the PS16 was also showed a decreasing result from 0.15 mg/l (15 DAI) to 0.05 mg/l (30 DAI). These findings are close to the findings of [13].

            In table 4.2(b), the nitrate concentration due to PS-5 was increased from 135 mg/l (15 DAI) to 170 mg/l (30 DAI) because of the effective transformation of ammonia. Similarly, the PS16also gave the similar type of promising result to a reduced harmful concentration of ammonia by nitrification process. The nitrate concentration increased from139 mg/l (15 DAI) to 171 mg/l (30 DAI). These findings are also close to results of [13 and 14].

Table 2b. Ammonia and nitrate dynamics in polluted pond water as influenced by selected pond soil heterotrophic bacterial isolates under controlled conditions.

PS 5 = Micrococcus luteus CP001628, PS16 = Ochrobactrum pituitosumAM490609

Note =Water were collected from the polluted pond which was contaminated by open drain water

Ammonia and nitrate dynamics in molasses amended water of polluted pond as influenced by autotrophic and heterotrophic bacteria under controlled conditions.

            The data presented in Table 3 revealed that at 2-ppm organic N level, T₄ gave best results with reference to decrease in the ammonium concentration from 15 DAI (1.41 ppm) to 30 DAI (1.27 ppm) and increase in nitrate concentration from 30.0 to 44.2 ppm when comparing the treatments of T₃ and T₄ with the treatment T₂ for the analysis of the ammonia concentration. Similarly, at 8-ppm concentration of organic N when comparing the T₅ with T₆ and T₇ treatment, we got the best result with treatment T_7. The decrease in the ammonia concentration from 15 DAI(4.77) to 30 DAI(2.73). While the next organic N level of 12ppm, it had the most negligible effect upon lowering the ammonia concentration from 8.33 ppm at 15 DAI to 6.52 ppm at 30 DAI.

A similar finding has been obtained that molasses pretreatment can significantly stimulate the process of bacterial attachment on the ceramic bottom pieces for faster degradation of ammonia in the submerged condition. These types of the finding of the present investigation are close to results of [15]. The ammonia-nitrogen range was 0.02 – 0.07 mg L^-1, the nitrite-nitrogen field was between 0.20 – 0.43 mg L^-1, whereas in the nitrate-nitrogen 0.90 – 3.20 mg L^-1 and the carbon source from the molasses was effective in reducing the concentration of ammonia, when cultured with the biofloc technique [16]. The ammonia concentration was decreasing as per the results given by [17].

Table 3.Ammonia and nitrate dynamics in molasses amended* water of polluted pond as influenced by autotrophic and heterotrophic bacteria under controlled conditions.

* = 5% Molasses water solution,

I₁ = Mixed culture of heterotrophic bacteria (PS5, PS16), I₂ = It was a mixture of culture of NS8, NB8 and PS5, PS16

Ammonia and nitrate dynamics in skimmed milk amended polluted pond water as influenced by autotrophic and heterotrophic bacteria with different levels of organic under controlled conditions.

Data presented in Table 4related to ammonia and nitrate dynamics in skimmed milk amended polluted pond water as influenced by autotrophic and heterotrophic bacteria with different organic N levels under controlled conditions at 15 and 30 DAI. The results were recorded at 15 DAI (0.28 to 8.41 ppm) and 30 DAI (0.275 to 6.44 ppm), respectively. However, the ammonia concentration showed a decreasing trend from 15 DAI to 30 DAI, in both intervals (15 DAI & 30 DAI).The minimum ammonia concentration obtained in the T₄ treatment (1.46 to 1.00 ppm) at 2 ppm organic N source and T₇ also gave minimum ammonia concentration at 8 ppm organic N source application at 30DAI (4.80 to 2.77 ppm) i.e., reduced by 2.03 ppm, thus in both the treatments significant reduction in the ammonia concentration was observed. When we used the area at 12 ppm, the maximum concentration of ammonia was recorded in T₈ (8.41to 6.44 ppm). A similar finding has been obtained by [18].

Table 4. Ammonia and nitrate dynamics in skimmed milk* amended polluted pond water as influenced by autotrophic and heterotrophic bacteria with different levels of organic N under controlled condition.

* = 1% Skimmed milk amended polluted pond water solution,

I₁ = Mixed culture of heterotrophic bacteria (PS5, PS16), I₂ = It was a mixture of culture of NS8, NB8 and PS5, PS16

DISCUSSION

During isolation of nitrifying bacteria of Nitrosomonas and Nitrobacter, both media become turbid, and this further confirms the presence of nitrifying bacteria. Gram’s staining and microscopic observation also confirmed the presence of nitrifying bacteria. Additionally selected isolates of Nitrosomonas and Nitrobacter were multiplied for their exploitation as bio-remediator in simulated aquaculture systems. Nitrosomonas and Nitrobacter strains were also isolated from drained fish ponds by [13]. They expressed similar views and reported that the soil of Chhattisgarh plains was exposed to an arid and hot climate during summer resulting in drastic loss in the population of mesophilic microbes, including nitrifying bacteria:

Further, a similar view was also given by [6]. They suggested that 10^-3 dilution can be considered as appropriate diluation for isolation of nitrifying bacteria from drained fish pond soil. Nitrification has been reported to get inhibited in polluted waters [11]. Nitrosomonas and Nitrobacter genera were also isolated from polluted pond water by [13 and 19] and from activated sludge by [11], (2001) and [20], biofilm in the aquarium by [21], garden soil by [10], and from pond mud by [12]. Selection of effective nitrifying bacterial isolates was made on the basis of the rate of colour change due to thenitrifying process [10]. Selected isolates of Nitrosomonas and Nitrobacter were multiplied for their exploitation as bioremediator in aquaculture systems. For fast multiplication purposes, temperature and pH were maintained at 30^oC and 8, respectively, throughout the 4 weeks of incubation period as described by [11].

Further selected heterotrophic bacterial isolates were multiplied for their testing as bioremediators in a simulated aquaculture system. Ochrobactrum pituitosum and Micrococcus luteus strains were also isolated polluted from fish ponds water by [22]. Ochrobactrum pituitosum genera were also isolated from the industrial environment by [23]. The selection of effective denitrifying bacterial isolates was made on the basis of the rate of colour change due to denitrifying process [24].

The best result associated with the T₄ treatment as it showed a higher rate of nitrification, the increase in nitrate from 15 DAI (30.00ppm) to 30 DAI (44.2 ppm). Similarly, at 8-ppm concentration of organic N, when comparing between the T₅ with T₆ and T₇ treatment, got the treatment T₇ was also having the best result among them as it was showing the increase in the nitrate concentration from 15 DAI (59.17 ppm) to 30 DAI (72.90 ppm). In the next urea concentration level of 12-ppm, it had the most negligible effect upon increasing the nitrate concentration from 15 DAI (70.00 ppm) to 30 DAI (77.50 ppm). A similar finding was obtained by [15], who emphasized molasses pretreatment, which can significantly stimulate the process of bacterial attachment on the ceramic carrier and the degradation of ammonia in the submerged condition. The ammonia-nitrogen range was 0.02 – 0.07 mg L^-1, Nitrite-nitrogen range was between 0.20 – 0.43 mg L^-1 whereas in the Nitrate-Nitrogen 0.90 – 3.20 mg L^-1 and carbon source from the molasses was effective in reducing the concentration of ammonia when cultured with the biofloc technique [16] and also another finding was that the nitrate concentration was increasing under the similar in-vitro condition as reported by [17].

In the present investigation, the nitrate concentration was observed at 15 DAI and 30 DAI, we have used the 3 levels of the urea concentration as (2 ppm, 8 ppm, 12 ppm) in which under the 2-ppm urea concentration when comparing the treatments T₃ and T₄  with the treatment T₂ with respect to nitrate concentration we obtained the best result in T₄ treatment as it helps in increasing the level of nitrate concentration in the treatments from 15 DAI (28.8) to 30 DAI (42.9 ppm) and under the 8-ppm concentration when comparing T₅ with T₆ and T₇ treatment we get that the treatment T₇ was also having the best result among them as it was shown an increase in the nitrate concentration from 15 DAI (57 ppm) to 30 DAI (70.07 ppm). In the next urea level of 12-ppm, it had the most negligible effect upon increasing the nitrate concentration from 15 DAI (70.0 ppm) to 30 DAI (71.1 ppm). A similar finding has been reported as the acceptable range of nitrate was (0 – 100 ppm), and the desirable range was (0.1 – 4.5 ppm) and under stress conditions, the concentration ranges >100, <0.01 as reported by [25]. The result was also similar to the finding of [14], were observed maximum nitrate concentration in the sample (Moti talab, Rajnandgaon).

CONCLUSION

Upon collecting the water and soil samples from different 25 different ponds of different village/n. nigam/ward the best performance of Nitrosomonas (soil-NS8S and water-NS8W) and Nitrobacter (soil-NB8S and water-NB8W) were the autotrophic bacteria has been seen which was taken from the 1^st Dudhadhari pond.

The heterotrophic bacteria (Micrococcus luteus and Ochrobactrum pituitosum) of isolate number PS5 and PS16 have the best result among the 7 best performing samples of different village/n. nigam/ward, which was taken from the Katora talab pond and Budha talab pond respectively.

In the polluted pond water, the heterotrophic bacteria (Micrococcus luteus and Ochrobactrum pituitosum) PS5 and PS16isolateare performing best results in decreasing the ammonia concentration and increasing the nitrate concentration, respectively.

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