OD, pH and ammonium accumulation by
1.Introduction
Nitrogen is an essential element for many biological processes, including those occurring in plants (Ogura et al., 2006). Despite the abundance of atmospheric nitrogen, production of nitrogen fertilisers by the Harber–Bosch process is increasing annually due to the deficiency of ammonia produced by biological nitrogen fixation—the enzyme-catalyzed reduction of nitrogen gas (N2). Concern over ‘greenhouse’ gasses emitted by the Harber–Bosch process has resulted in a research focus on nitrogen-fixing bacteria, and in particular, their genetic modification to excrete excess ammonia for agricultural purposes (Terzaghi, 1980;Saikia& Jain, 2007).
There are three main biological processes in the natural cycle of nitrogen (Fig. 1): fixation, nitrification and denitrification, which involve nitrogen-fixing, nitrifying and denitrifying bacteria, respectively.
Blue arrows indicate nitrogen fixation, including biological and industrial processes. Green arrows indicate microbial nitrification processes involving nitrifying bacteria, and pink arrows indicate microbial denitrification processes involving denitrifying bacteria. Black arrows indicate the flow of each compound in soils. The NH3 produced by nitrogen fixation may be assimilated into amino acids and thence to protein and other N compounds, or it may be converted by nitrifying bacteria to NO2 - and NO3 -. In turn, NO3 - may enter metabolism through reduction to NH4 + and subsequent assimilation to amino acids by bacteria, fungi and plants or can serve as an electron acceptor in denitrifying bacteria when oxygen is limiting. Losses from the nitrogen pool occur physically, when nitrogen (especially nitrate) is leached into inaccessible domains in the soils, and chemically, when denitrification releases N2.
2. Biological nitrogen fixation
Decomposers use several enzymes to break down proteins in dead organisms and their waste, releasing nitrogen in much the same way as they release carbon. Proteinases break large proteins into smaller molecules. Peptidases break peptide bonds to release amino acids. Deaminases remove amino groups from amino acids and release ammonia.
According to Kneip et al. (2007), during biological nitrogen fixation (BNF), molecular nitrogen is reduced (Formula 1) in multiple electron-transfer reactions, resulting in the synthesis of ammonia and release of hydrogen. Ammonium is then used for the subsequent synthesis of biomolecules. This reduction of molecular nitrogen to ammonium is catalysed in all nitrogen-fixing organisms via the nitrogenase enzyme complex in an ATP-dependent, highly energy-consuming reaction (Fig. 2). The nitrogenase complex is composed of two main functional subunits, dinitrogenase reductase (azoferredoxin) and dinitrogenase (molybdoferredoxin). The structural components of these subunits are the Nif (nitrogen fixation) proteins: NifH (γ2 homodimeric azoferredoxin) and NifD/K (α2β2 heterotetrameric molybdoferredoxin). Three basic types of nitrogenases are known based on the composition of their metal centres: iron and molybdenum (Fe/Mo), iron and vanadium (Fe/V) or iron only (Fe). The most common form is the Fe/Mo-type found incyanobacteria and rhizobia.Electrons are transferred from reduced ferredoxin (or flavodoxin) via azoferredoxin to molybdoferredoxin. Each mole of fixed nitrogen requires 16 moles ATP to be hydrolysed by the NifH protein. The NH3 produced is utilised in the synthesis of glutamine or glutamate for N-metabolism. NifJ: pyruvate flavodoxin/ferrodoxin oxidoreductase, NifF: flavodoxin/ferredoxin). An important feature of the nitrogenase enzyme complex is its extreme sensitivity to even minor concentrations of oxygen. In aerobic environments and in photoautotrophic cyanobacteria, in which oxygen is produced in the light reaction of photosynthesis, nitrogenase activity must be protected. This protection is mediated by different mechanisms in nitrogen-fixing bacteria, depending on their cellular and physiologic constitutions. Aerobic bacteria (like
General reaction of molecular nitrogen fixation. Schematic of the structure and operation of the nitrogenase enzyme complex and subsequent metabolism of nitrogen.
Ultraviolet mutagenesis, the most easily controllable method of mutation, was thus often the first choice. Ultraviolet irradiation was used to modify
The same results arose from mutation of
With regard to the carbon sources used to culture these two species, most of the studies described above used Burk’s medium, which contains 2% sucrose, or modified Burk’s medium (0.5% or 2% glucose) as carbon sources. The latest researches on
3. Screening and identification of nitrogen-fixing bacteria
3.1. Screening of nitrogen-fixing bacteria
To screen for nitrogen-fixing bacteria, 1 g of soil was suspended in 10 mL of sterilized dH2O in a 15-mL Eppendorff tube that was left to stand until the soil solution settled. A 1-mL aliquot of supernatant was then added to 200 mL of fresh NFMM or NFMM liquid medium and incubated for 1 week on a rotary shaker at 120 rpm and 30 C. Subculture was carried out twice by adding 2 mL of liquid culture to 200 mL of new C–NFMM medium and incubated as before. Single-colony isolation was performed on NFMM plates. Nitrogen-fixing activity was tested by growing the strains on glucose–NFMM plates substituted with BTB.From the 20 soil samples collected, we obtained four strains that showed a colour change in BTB-containing medium, suggesting excretion of ammonia. These strains were named C4, E4, G6 and G7.
3.2. Identification of nitrogen-fixing bacteria
DNA extraction was performed using a Miniprep DNA Purification Kit (TaKaRa). Bacterial 16S rDNA was amplified over 35 PCR cycles. Each cycle consisted of denaturation for 1 min at 94 C, annealing for 30 s at 60 C and extension for 4 min at 72 C. DNA purification was performed using the Agarose Gel DNA Extraction Kit (Roche Diagnostics GmbH). Ligation was conducted using the DNA Ligation Kit (TaKaRa) and the pT7 Blue T-vector (Novagen) as the plasmid. Transformation used
The nucleotide sequences of C4 and G7 showed high similarity (99%) to
3.3. Classification of isolated strains
RFLP of the amplified
4. Mutation of Azotobacternif genes for ammonia accumulation
5. Accumulation of ammonia by wild-type strains
When wild-type
6. Ammonia detection and estimation
Ammonia concentration was estimated using the Visocolor Alpha Ammonia Detection Kit (Macherey-Nagel). After centrifugation at 13,000 rpm for 10 min at room temperature (RT), supernatant (1 mL) was transferred into a test tube. Two drops of NH4-1 were added to the sample and mixed well, after which one-fifth of a spoonful of NH4-2 was added. After mixing, the sample was left at RT for 5 min. One drop of NH4-3 was then added, mixed well and left at RT for 5 min.
Ammonia concentration was also estimated using ion chromatography. After centrifugation at 13,000 rpm for 10 min at RT, the supernatant was passed through a 0.2-μm filter and the ammonium concentration determined using an 861 Advanced Compact Ion Chromatography (Metrohm). The cation eluent used was 4 mM H3PO4 with 5 mM 18-crown 6-ether. The separation column was an IC YK-421 (Shodex) and the guard column was an IC-YK-G (Shodex). Standard ammonium solution was prepared from (NH4)2SO4; the concentration was adjusted to 1000 parts per million (ppm) and diluted appropriately to obtain a standard curve. All experiments were performed in triplicate.
7. Cultivation of nitrogen-fixing Lysobacter sp.
8. Effect of carbon concentration
The optimum carbon source concentration was used to determine the correlations among incubation time, ammonia accumulation and carbon uptake.
Glucose concentration | 0.10% | 0.25% | 0.50% | 0.70% | 1.00% | 2.00% | |
A. beijerinckii | OD pH NH4 + |
0.145 7.0 (7.0)* 0.062 |
0.486 7.0 (7.0)* 0.117 |
1.109 6.8 (7.1)* 0.202 |
1.406 6.6 (7.1)* 0.080 |
1.698 6.4 (7.1)* 0.026 |
1.522 6.3 (7.1)* 0.001 |
A. vinelandii | OD pH NH4 + |
0.189 7.1 (7.1)* 0.010 |
0.478 6.8 (7.1)* 0.024 |
0.950 6.1 (7.1)* 0.020 |
1.391 4.9 (7.1)* 0 |
1.710 4.7 (7.1)* 0 |
1.948 4.7 (7.0)* 0 |
OD: optical density (600 nm). *Figures in parentheses show the value before incubation. | |||||||
Note: ammonium ion concentration is in mM. Presence of ammonium was primarily tested using Nesler’s reagent before the concentration was determined by ion chromatography. |
Glucose | Fructose | Galactose | Mannose | Sucrose | Citrate | Succinate | ||
A. beijerinckii | OD | 0.518 | 0.739 | 0.564 | 0.029 | 0.656 | 0.005 | 0.212 |
pH | 7.3 (7.0)* | 7.2 (7.0)* | 7.1 (7.1)* | 7.1 (7.1)* | 7.1(7.1)* | 7.4 | 8.6 (7.2)* | |
NH4+ | 0.296 | 0.315 | 0.201 | 0.041 | 0.192 | (7.0)* | N. D. | |
N. D. | ||||||||
A. vinelandii | OD | 0.442 | 0.704 | 0.573 | 0.122 | 0.655 | 0.361 | 0.361 |
pH | 7.0 (7.0)* | 7.2 (6.9)* | 7.1 (7.0)* | 7.1 (7.1)* | 7.2(7.0)* | 8.4 | 8.8 (7.2)* | |
NH4+ | 0.026 | 0.179 | 0.025 | 0.017 | 0.63 | (7.0)* | N. D. | |
N. D. | ||||||||
N.D.: not determined, OD: optical density (600 nm). *Figures in parentheses show the value before incubation. | ||||||||
Note: ammonium ion concentration is in mM. Presence of ammonium was primarily tested using Nesler’s reagent before the concentration was determined by ion chromatography. |
9. Time course of ammonia accumulation
As the
10. Time course of ammonia accumulation byLysobacter sp.
Time-course experiments suggested that ammonia accumulation began upon glucose depletion. In the 0.30% medium, no glucose remained after incubation for 3 days, resulting in ammonia accumulation. In media with higher glucose concentrations, residual glucose was present after 3 days. As a result, no ammonia accumulation occurred; longer incubation times may have resulted in production of detectable levels of ammonia (Fig. 5A).
11. Effect of remaining sugar on ammonia accumulation
Residual sugar levels were determined using a glucose detection kit, according to the manufacturer’s protocol (Miwa et. al., 1972). For
These data suggest that ammonia accumulation by strain E4 is dependent on sugar concentration. Glucose is required for bacterial growth until the middle of the logarithmic phase, and fixation of nitrogen during this period likely supports bacterial growth. Ammonia starts to accumulate when no more glucose remains in the culture, as shown by glucose and ammonia determinations after 14 h incubation (Fig. 5B).
For
Thus, in both strains, ammonia began to accumulate at the end of log phase or in early stationary phase; no carbon source could be detected in the medium at this time. Higher ammonia levels in the medium will likely be detected after moreover 30 hours, longer incubation times, suggesting that the mechanism of nitrogen fixation might be influenced by sugar levels in the medium.
E4 strain grew well at pH 7.0 and produced the highest concentration of ammonia (~0.4 mM). Although media at pH 8.0 resulted in the greatest growth, ammonia accumulation was lower than at pH 7.0, suggesting that accumulated ammonia at the higher pH value may have been used for bacterial growth (Fig. 5B).
Ammonia was detected in E4 cultures incubated at 30 C, but not at 20 C. Ammonia may accumulate at 20 C after longer incubation times, since some glucose remained after 3 days incubation.
12. Conclusions
From the above, the following conclusions could be drawn. Firstly, the ammonium accumulation is clearly dependent on the carbon source concentration. Higher ammonium accumulation occurred in media with lower concentrations of the carbon source. Glucose was required for growth of
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