Monitoring of soil pollution with heavy metals using some microbiological parameters

ABSTRACT

Although heavy metal pollution significantly impacts living organisms, the monitoring biomarkers can guide remediation. This study examined the response of Pb²⁺, Cd²⁺, Cu²⁺, Zn²⁺, Cr⁶⁺, Ni²⁺, and Hg²⁺ to soil microorganisms. Bacterial counts were measured at three metal concentrations (T₁, T₂, and T₃). Results revealed that Hg²⁺-T₁ caused a 91.4% reduction in bacterial counts within 10 days, while the other metals reduced counts by 32.9% to 71.8%. Higher concentrations (T₂ and T₃) of all metals, except Ni²⁺ (48.8%) at T₂, reduced counts by 92.1% to 99.6%. After 40 days, reductions were 74.5% for Hg²⁺-T₁, 97.0% for Cr⁶⁺-T₂, and 95.2 to 98.0 % for Hg²⁺, Cr⁶⁺, Cu²⁺ and Zn²⁺-T₃. Intrinsically resistant bacteria were observed after 40 days, constituting 34.2%–71.8% in T₁ of most metals, except Hg²⁺ (25.5%). These percentages decreased in T₂ and T₃. 55.3% of the initial organic-N was oxidized after 20 days in untreated soil, while Pb²⁺ was the least toxic for ammonification, and Hg²⁺ was the highest. In untreated soil, 67.4% of ammoniacal-N was oxidized after 20 days. Nitrification was unaffected by T₁, except for Hg²⁺. At T₂, Hg²⁺, Ni²⁺, and Cd²⁺ were the most suppressive, while at T₃, Hg²⁺, Zn²⁺, and Cd²⁺ showed the most harmful effects.

KEYWORDS:

• Pollution
• heavy metals
• soil microorganisms
• ammonification
• nitrification
• monitoring of soil pollution

Introduction

Rapid technological progress, the growth of the world’s population, and the consumption of natural resources to meet food, energy, and other needs have upset the balance of nature. Pollution has become one of the world’s most important problems since the industrial revolution. Water, soil, and air pollution are the main types of pollution caused by various emerging pollutants that threaten the health and well-being of millions of people and global ecosystems [1]. From an ecological perspective, soil is a fundamental resource for human life. It is the most basic element of human production and the carrier that can connect various human economic relations [2]. Thus, soil pollution has become a global risk that directly threatens all soil functional parameters [3] as soil is the link for organic pollutants and heavy metals from crops to humans and their bioaccumulation along the food chain increases the potential risks to human health.

Anthropogenic activities such as mining and metal smelting have increased heavy metals in soils and food crops cultivated within industrial areas [4]. They added that well-developed transportation and agricultural activities are also important sources of heavy metals. The soil quality of arable and cultivated lands near industrial and abandoned multiple metal mines is concerning. As reported by the Ministry of Environment Protection [5] about 16.1% of soils, including 19.4% of the arable lands and 36.3% of enterprise surrounding soils were over the Grade II of the environment quality standard.

Heavy metals are one of the largest sources of pollution in the environment and have increased dramatically in recent years due to various human activities such as agriculture, mining, and other industrial processes [6]. They generally refer to a metal with a density greater than 5.0 g/cm3, including 45 elements such as Pb²⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Hg²⁺, which were common and important refractory pollutants. Moreover, they are usually present in relatively low concentrations in soil, whereas they are known to be toxic to most organisms when present in too high concentrations. Consequently, the higher concentrations of these elements above threshold levels have a very detrimental effect on microbial communities and their vital activities. Moreover, once they enter the soil, they become irreversibly immobilized soil components. From a biochemical point of view, heavy metals in soil can be divided into two categories. One is harmful to plants, humans, microorganisms, and animals, such as Pb²⁺, Cd²⁺, and Hg²⁺; the other is beneficial when present in constant amounts, but when present in excess it damages biological activity, such as Cu²⁺ and Zn²⁺.

The toxicity of heavy metals to microorganisms, as mentioned by [7,8] is primarily about the bioavailability of metals, i.e. the amount of the metal eventually absorbed by the microorganism. In addition, heavy metals and microorganisms have a strong affinity to bind easily with some biological macromolecules such as the activity center of enzymes and electron-donating groups, nucleic acid bases, and phosphate combinations, leading to their inactivation. Recently [9] added that once heavy metals enter the soil, they primarily affect the number of soil bacteria, fungi, actinomycetes, and other microbial populations as they affect microbial quality and quantity. Their contamination can lead to different microbial community patterns. Long-term soil contamination with heavy metals will select microorganisms that can adapt specifically to the contaminated soil. The higher the organic carbon content in heavily polluted soils, the lower the efficiency of microbial populations in organic mineralization. This may be a simple indication of the impact of heavy metal pollution on soil microbial communities and biological activities.

In addition, as a key limiting nutrient in terrestrial ecosystems, nitrogen plays an important role in determining soil quality [10]. It is an essential nutrient for plant growth and ecosystem functioning, and its cycling is closely related to soil microbial activity and plant productivity [11,12]. However, heavy metals in soil can affect the nitrogen cycle by altering soil microbial communities, absorbing nitrogen, and changing nitrogen transformation pathways. As soil heavy metal pollution can inhibit the activity of nitrogen-fixing bacteria, which play an important role in the nitrogen cycle, this can lead to lower nitrogen availability in the soil and negatively affect plant growth and soil productivity [13].

[14,15] found that microbial parameters appear as very useful in monitoring soil pollution by heavy metals. These parameters can be divided into three main groups: The first measures the size of the microbial population at the individual organism, functional group, or whole population level, the second measures the activity of the whole microbial population, e.g. C and N mineralization, and the third option is a combination of activity and biomass data that provides specific activities of the microbial population.

Microbial parameters serve as sensitive indicators of soil pollution with heavy metals, providing early detection of the impact of heavy metals on soil health by evaluating changes in microbial populations and activities. Moreover, soil monitoring reveals how heavy metals shape the structure and function of microbial communities, highlighting shifts toward metal-tolerant species. One of the most sensitive indicators for heavy metal soil pollution is nitrogen transformations, such as ammonification and nitrification, which are vital for soil fertility and ecosystem function. This provides insights into the dynamics of soil ecosystems under heavy metal stress, aiding in understanding long-term contamination effects and informing the development of strategies for sustainable soil management and effective remediation techniques. Finally, all of these parameters contribute to broader environmental health assessments by linking soil microbial health with ecosystem services.

From this point of view, the present work was carried out to evaluate the effects of soil pollution with elevated concentrations of seven heavy metals (Pb²⁺, Cd²⁺, Cu²⁺, Zn²⁺, Cr⁶⁺, Ni²⁺, and Hg²⁺) on the microbial population density and some microbial activities, as sensitive indicators of heavy metal pollution in soil. The changes in the total microbial population were estimated at regular intervals and the nitrogen transformation processes (ammonification and nitrification), affected by heavy metals stress, were determined as well.

Materials and methods

Heavy metals

Seven heavy metals were used: Pb²⁺ as Pb(CH,COO)₂.3 H₂O, Cd²⁺ as CdCl₂ H₂O, Cu²⁺ as CuSO₄.5 H₂O, Zn²⁺ as ZnSO₄.7 H₂O, Cr⁶⁺ as K₂Cr₂O₇, Ni²⁺ as NiCl₂.6 H₂O and Hg²⁺ as HgCl₂. Standard stock solutions of each metal salt were prepared and used to supplement either soil or culture media to achieve the desired metal-ion concentrations.

Soil sampling

Soil samples were collected from an experimental farm in the Faculty of Agriculture, Cairo University at Giza, Egypt. This site is specialized for field experiments and research, so it doesn’t provide any treatments e.g. pesticides or fertilizers. Moreover, the experimental field is away from any source of pollution which makes it considered uncontaminated soil. The fertile clay-loam soil (top 10 cm layer) was obtained, air-dried and 2 mm sieved in preparation for the experimental part.

Chemical determinations

Ammoniacal-, nitrate- and total-inorganic nitrogen were determined according to [16].

Determination of NH₄⁺-N

40 g of a representative soil sample was mixed with 1 M KCl, shaken for 1 hour and the suspension filtered. 50 ml of the extract was distilled in the presence of 0.5 g MgO and 10 ml boric acid and titrated against 0.01 M HCl with methyl red-bromocresol green indicator. The blank test was carried out with 50 ml 1 M KCl solution. The determined titration should be subtracted from the sample titration to obtain a corrected titration volume.

Determination of NO₃⁻-N

After NH₄⁺-N distillation, the solution was cooled, provided with 0.5 g Devara’s alloy, and the distillation was continued. The distillate was then received in 5 ml boric acid and titrated against 0.01 M HCL using methyl red-bromocresol green indicator. Blank was carried out using 50 ml of 1 M KCl solution. The determined titration should be subtracted from the sample titration to obtain a corrected titration volume.

Determination of total mineral-N

Total mineral-N was determined by adding 1 drop of Octan-2-ol, 0.5 g of Devarda’s alloy followed by 0.5 g MgO and the procedure was carried out as described above.

Calculations:

NH₄⁺-N, NO₃⁻-N, and total mineral-N were each calculated using the following equation:

$\mathit{\mu }\text{g\hspace{0.17em}N}.{\text{g}}^{\text{-1}}\text{soil=}\frac{\text{2}.\text{8\hspace{0.17em}x\hspace{0.17em}corrected\hspace{0.17em}titration\hspace{0.17em}volume\hspace{0.17em}x\hspace{0.17em}total\hspace{0.17em}liquid\hspace{0.17em}volume}}{\text{oven-dried\hspace{0.17em}soil\hspace{0.17em}mass}}$

Data analysis

In the present study, the results were presented as means using Microsoft Excel from the Microsoft Office 365 suite.

Experiments

Effect of soil pollution with heavy metals on the total microbial population

A laboratory experiment was conducted to evaluate the effects of seven heavy metals on the total microbial population by determining the periodic changes in the microbial counts and the metal(s) resistant ones, as well.

Soil was distributed in glass jars at the rate of 150 g and supplemented with different concentrations of each of the test heavy metals. Three jars were prepared for each treatment as replicates. After that, soil moisture was adjusted at 75% water holding capacity (WHC) using distilled water with thorough mixing and incubated at 28°C for 40 days.

In addition to the control (untreated) soil (C), three other treatments were prepared using the standard stock salt solutions. The first treatment was T₁ (low-metal concentration); in which the soil was supplied with 25 µg.g⁻¹soil of each of the seven metals, the second one was T₂ (moderate-metal concentration); it included the addition of 250 µg.g⁻¹ soil of Pb²⁺, Cd²⁺, Cu²⁺ and Zn²⁺, 1000 µg.g⁻¹ soil of Cr⁶⁺ and Ni²⁺ and 500 µg.g⁻¹ soil of Hg²⁺, and the third one was T₃ (high-metal concentration): comprised the addition of 500 µg.g⁻¹ soil of Pb²⁺, Cd²⁺, Cu²⁺, and Zn²⁺; 200 µg.g⁻¹soil of Cr⁶⁺ and Ni²⁺ and 75 µg.g⁻¹ soil of Hg²⁺. These concentrations which caused a reduction in total microbial counts by < 25, ca. 50, and > 90% of the initial population were chosen based on results obtained from preliminary experiments.

A representative soil sample of 20 g was taken at 10-day intervals by mixing equal parts of the three replicates representing a single treatment. Total plate counts were determined on metal-free nutrient agar medium according to [17]. The same medium provided with known concentrations of heavy metal ions was used to determine the periodical changes in the intrinsically resistant microorganisms during the 40-day incubation period.

Impact of heavy metals on some nitrogen transformations processes in soil

Ammonification

The same clay-loam soil used in the previous experiment was used to elucidate the effect of Pb²⁺, Cd²⁺, Cu²⁺, Zn²⁺, Cr⁶⁺, Ni^2+, and Hg²⁺ on the ammonification process. Two-hundred-gram portions of the air-dried soil were distributed in glass jars and mixed with 1% (w/w) peptone (12.6% N). In addition to the control (untreated) soil, the experiment comprised three other treatments prepared by adding 200 µg.g⁻¹ soil (low-metal concentration, T₁), 1000 µg.g⁻¹ soil (moderate-metal concentration, T₂) and 2000 µg.g⁻¹ soil (high metal concentration, T₃) of the seven heavy metals under investigation. These metal-ions concentrations were achieved by adding certain volumes of the standard stock solutions and sufficient amounts of distilled water to adjust WHC at 75%. Three jars were prepared as triplicates for each treatment, the soil was then thoroughly mixed and incubated at 28°C for 20 days. A representative soil sample of 40 g was taken from each of the three replicates at intervals of 5 days and subjected to the determination of total mineral nitrogen (NH₄-N and NO₃-N) according to [16].

Nitrification

Using the same soil, a further experiment was carried out to investigate the effect of the seven heavy metals on the nitrification process. The soil was supplemented with 400 µg NH₄-N.g⁻¹ soil in the form of (NH₄)₂SO₄ and the experiment proceeded under the same prevailing experimental conditions as described above. At 5-day intervals, a representative soil sample of 40 g was obtained from each three triplicates and the NH₄-N and NO₃-N were determined individually according to [16].

Results

Effect of soil pollution with heavy metals on the total microbial population

The periodic changes in microbial population given in Table 1 and illustrated in Figure 1 showed that counts determined on the metal-free agar medium increased in the control soil (C) by about 10-folds compared with the other three soil metal-treatments of the seven heavy metals. After 10 days of incubation, the low-metal treated soil (T₁) with Hg²⁺ recorded the highest reduction percentage in total bacterial counts by 91.4% corresponding to the control soil, while the moderate inhibition in the bacterial counts was 58.1% in the soil treated with Pb²⁺- and the lowest percentage (32.9%) was observed in soil supplemented with Ni²⁺. On the other hand, a reduction range in bacterial counts between 48.8 to 71.8% was recorded for the other four heavy metals. A more suppression effect was observed with the other two metal-soil treatments, T₂ and T₃. Except for Ni^{2 +} where a 48.8% reduction in total bacterial counts in soil was observed, the T₂-treatment caused a drastic 92.1 to 99.6% reduction for the other six metals after 10 days. This percentage range increased slightly to 94.6–99.6 in T₃-treatment after the same period for the seven tested heavy metals.

Figure 1. Periodical changes in the total bacterial counts (CFU.g⁻¹ air-dried soil) in soil treated with different concentrations of seven heavy metals.

Table 1. Periodical changes in the total bacterial counts (CFU.g⁻¹ air-dried soil) in soil treated with different concentrations of seven heavy metals.

Time (days)	10				20			30			40
Metal concentrations in soil (µg. g^−1 soil)	Total bacterial counts (FCU x 10^6 .g^−1 soil)
	Count	%^a		%^b	Count	%^a	%^b	Count	%^a	%^b	Count	%^a	%^b
1- Untreated (control soil)
C (untreated)	31.30	100	100		29.40	100	100	26.40	100	100	27.60	100	100
2- Soil + Pb^2+
T1 (25)	13.10	58.1		41.9	19.00	35.4	64.6	17.00	35.6	64.4	18.20	34.0	66.0
T2 (250)	2.15	93.1		6.9	7.60	74.2	25.8	7.03	73.4	26.6	10.41	62.3	37.7
T3 (500)	0.25	99.2		0.8	0.23	99.2	0.8	5.20	80.3	19.7	7.82	71.7	28.3
3- Soil + Cd^2+
T1 (25)	16.03	48.8		51.2	22.00	24.8	75.2	18.40	30.0	70.0	19.83	28.2	71.8
T2 (250)	2.00	93.6		6.4	2.44	91.7	8.3	5.10	80.7	19.3	8.03	70.9	29.1
T3 (500)	0.13	99.6		0.4	1.40	95.3	4.7	3.00	88.6	11.4	5.56	79.9	20.1
4- Soil + Cu^2+
T1 (25)	9.40	69.9		30.1	11.40	61.2	38.8	10.00	62.2	37.8	9.43	65.8	34.2
T2 (250)	2.02	93.5		6.5	8.50	71.1	28.9	8.10	69.3	30.7	7.43	73.1	26.9
T3 (500)	0.20	99.4		0.6	0.20	99.3	0.7	0.34	98.7	1.3	0.58	97.9	0.1
5- Soil + Zn^2+
T1 (25)	8.83	71.8		28.2	16.00	45.6	54.4	14.00	47.0	53.0	14.33	48.0	52.0
T2 (250)	2.50	92.1		7.9	2.10	92.8	7.2	3.03	88.5	11.5	3.75	86.4	13.6
T3 (500)	0.70	97.7		2.3	0.70	97.6	2.4	0.52	98.0	2.0	0.50	98.0	2.0
6- Soil + Cr^5+
T1 (25)	12.10	61.4		38.6	21.50	26.9	73.1	17.30	34.5	65.5	19.20	30.5	69.5
T2 (100)	0.70	97.8		2.2	0.85	97.1	2.9	0.71	97.3	2.7	0.83	97.0	3.0
T3 (200)	0.13	99.6		0.4	0.62	97.9	2.1	0.60	97.8	2.2	0.76	97.3	2.7
7- Soil + Ni^2+
T1 (25)	21.00	32.9		67.1	16.10	45.3	54.7	16.03	39.3	60.7	18.50	33.0	67.0
T2 (100)	16.03	48.8		51.2	12.20	58.5	41.5	11.03	58.2	41.8	9.00	67.4	32.6
T3 (200)	1.50	95.2		4.8	2.50	91.5	8.5	3.03	88.5	11.5	3.45	87.5	12.5
8- Soil + Hg^2+
T1 (25)	2.71	91.4		8.6	7.23	75.4	24.6	6.10	76.9	23.1	7.00	74.5	25.5
T2 (50)	2.30	92.7		7.3	2.42	91.8	8.2	3.03	88.5	11.5	2.81	89.8	10.2
T3 (75)	1.85	94.6		5.4	1.96	93.4	6.6	1.10	95.8	4.2	1.33	95.2	4.8

N. B. 6.7 x 106 CFU.g-1 soil was recorded at 0-time.

(%^a): Decreasing percentage in the bacterial counts relative to the corresponding one in untreated (control) soil.

(%^b): Percentage of the metal-tolerant bacterial counts relative to the corresponding one in untreated (control) soil.

C: Control (untreated) soil.

T1: Soil was initially provided with 25 µg.g-1 soil of all metals at zero time.

T2: Soil was initially provided with 250 µg.g-1 soil of Pb2+, Cd2+, Cu2+ and Zn2+, 100 µg.g-1 soil of Cr6+ and Ni2+ and 50 µg.g-1 soil of Hg2+ at zero-time.

T3: Soil was initially provided with 500 µg.g-1 soil of Pb2+, Cd2+, Cu2+ and Zn2+, 200 µg.g-1 soil of Cr6+ and Ni2+ and 75 µg.g-1 soil of Hg2+ at zero-time.

The results indicated that within the three soil metals treatments, the total bacterial counts either remained stable or gradually increased over the 10 days of the experiment. But remained markedly lower than the corresponding counts in the control one. After 40 days of incubation with the three metals treatments, 28.2% in T₁- Cd²⁺, 48.0% in T₁- Zn^2+, and 74.5% T₁- Hg²⁺ were found to be the lowest, moderate, and highest inhibition percentages, respectively, in reducing the bacterial counts. With the except Cu²⁺, which caused a percentage reduction in total bacteria counts of 65.8, treatment of the soil with the other three heavy metals resulted in a percentage reduction of between 30.5 and 34.0% compared to the untreated soil.

Providing the soil with different concentrations of heavy metals at the higher level, T_2, leads to a greater reduction in the total bacterial count. The highest percentage reduction was observed with soil treated with 100 µgCr⁶⁺.g⁻¹ soil with 97.0%, while the least effect was recorded at 250 µgPb²⁺.g⁻¹ soil with 62.3% compared to the control soil. On the other hand, a percentage reduction ranging from 67.4 to 89.8 was observed in the soils treated with the other five heavy metals at the same concentrations of T₂. The strongest suppression effect in the total bacterial counts was recorded after the addition of the metals concentrations at the highest level, T₃, where 95.2 to 98.0% of the total bacterial counts were reduced by treating the soils with Hg²⁺, Cr^6+, Cu²⁺ and Zn²⁺, while less effect was observed in soil treated with Pb²⁺, Cd²⁺, and Ni²⁺ at the same level of T₃ where the reduction in total bacterial counts was 71.7, 79.9 and 87.5% compared to the control soil, respectively.

It is of interest to note that the percentages of inhibition of the total bacterial counts were slightly lower after 40 days than after 10 days but still showed the same pattern. This difference in percentages was translated into the number of bacteria able to tolerate specific concentrations of the respective metal. Concerning this point, it was of importance to test the possible development of metal-tolerant bacteria.

A clear increase in the number of intrinsically resistant bacteria was observed throughout the experiment (Table 1). After 40 days in the T₁ treatment, they comprised 34.2–71.8% of the initial bacterial counts in the different six metals, except for Hg²⁺, where they amounted to 25.5%. This range of intrinsically resistant bacterial counts decreased with increasing metal concentration, reaching 26.9–37.7% in T₂ treatment, except for Zn²⁺, Cr^6+, and Hg²⁺ where 13.6, 3.1, and 10.2% of the initial bacterial counts were recorded, respectively. The T₃ treatment of the seven tested metals was tolerated by a lower number of bacteria, with 28.3, 20.1 and 12.5% tolerable for Pb²⁺-, Cd²⁺- and Ni²⁺-soil treatments, respectively, while only a few thousands could tolerate the concentrations of this treatment with the other four heavy metals.

Impact of heavy metals on some nitrogen transformation processes in soil

The results of the previous part of this study indicated the negative effects of seven heavy metal salts on soil microorganisms, with the possible development of resistant species that can withstand the toxic effects of these metals. It was of interest to investigate to what extent the toxicity of these metals can influence the biological activity in soil. In this part of the study, the mineralization of nitrogen-rich organic compounds (ammonification) and the subsequent oxidation of the resulting NH₄-N (nitrification), were investigated, which could be influenced by the increasing load of heavy metal salts in fertile soils.

Ammonification

Ammonification is the process by which organic N is mineralized to NH₄⁺. It is the first step in organic N decomposition and is often referred to as N mineralization. This process is carried out by microorganisms in the soil that mineralize dissolved low molecular weight, dissolved, organic molecules containing amine or amide groups (with the general formula R-NH₂) and produce ammonium (NH₄⁺).

In the present experiment, peptone, as a nitrogen-rich organic matter was added to the same fertile soil under investigation at the rate of 1% (W/W) (12.6% N). Then, in addition to the untreated one, the soil was supplemented with heavy metal salts at the aforementioned three levels of 200, 1000, and 2000 µg.g⁻¹ soil. The changes in total inorganic-N in four consecutive periods successive periods were followed as a reliable indicator of organic nitrogen mineralization in the soil under optimal conditions.

The periodic increase in total inorganic nitrogen was shown in (Figure 2) and recorded in (Table 2). It is quite clear that, apart from the type of heavy metal and its concentration applied in the soil, the periodical changes in inorganic nitrogen production followed a more or less constant pattern in all soil treatments with few exceptions.

Figure 2. Periodical changes in the total inorganic-N (µg. g⁻¹ soil) in soil treated with three concentrations of each of the tested seven heavy metals.

Table (2): Periodical changes in the total inorganic-N (µg.g⁻¹ soil) in soil treated with three concentrations of each of the tested seven heavy metals.

Soil treatments^1	Total inorganic-N (µg.g^−1 soil)				Increase in mineralized-N (µg.g^−1 soil)^2 (%)^3				Total inorganic-N (µg.g^−1 soil) produced within one time interval (5 days)
	Days
	5	10	15	20	5	10	15	20	0–5	5–10	10–15	15–20
Untreated (control soil)
C (untreated)	893	1117	1264	1296	709 (35.3)	933 (46.4)	1080 (53.7)	1112 (55.3)	709	224	147	32
1- Soil + Pb^2+
T1 (200)	797	1064	1147	1184	613 (30.5)	880 (43.8)	963 (47.9)	1000 (49.8)	613	267	83	37
T2 (1000)	757	973	1008	1011	573 (28.5)	789 (39.3)	824 (41.0)	827 (41.1)	573	216	35	3
T3 (2000)	597	832	872	896	413 (20.1)	648 (32.2)	688 (34.2)	712 (35.4)	413	235	40	24
2- Soil + Cd^2+
T1 (200)	616	661	832	923	432 (21.5)	477 (23.7)	648 (32.2)	739 (36.8)	432	45	171	91
T2 (1000)	475	536	728	757	291 (14.5)	532 (17.5)	544 (27.1)	573 (28.5)	291	61	192	29
T3 (2000)	320	397	605	667	136 (6.8)	213 (10.6)	421 (21.0)	483 (24.0)	136	77	208	62
3- Soil + Cu^2+
T1 (200)	779	805	981	981	595 (29.6)	621 (30.9)	797 (39.7)	797 (39.7)	595	26	176	0
T2 (1000)	755	760	856	891	571 (28.4)	576 (28.7)	672 (33.4)	707 (35.2)	571	5	96	35
T3 (2000)	541	555	659	696	357 (17.8)	371 (18.5)	475 (23.6)	512 (25.5)	357	14	104	37
4- Soil + Zn^2+
T1 (200)	371	797	855	1035	547 (27.2)	613 (30.5)	671 (33.4)	851 (42.3)	547	66	58	180
T2 (1000)	715	744	840	869	531 (26.4)	560 (27.9)	656 (32.6)	685 (34.1)	531	29	96	29
T3 (2000)	517	571	765	819	333 (16.6)	387 (19.3)	581 (28.9)	635 (31.6)	333	54	194	54
5- Soil + Cr^2+
T1 (200)	760	899	957	1061	576 (28.7)	715 (35.6)	773 (38.5)	877 (43.6)	576	139	58	104
T2 (1000)	523	672	795	840	339 (16.9)	488 (24.3)	611 (30.4)	656 (32.6)	339	149	123	45
T3 (2000)	475	549	771	805	291 (14.5)	365 (18.2)	587 (29.2)	621 (30.9)	291	74	222	34
6- Soil + Ni^2+
T1 (200)	680	904	965	1112	496 (24.7)	720 (35.8)	781 (38.9)	928 (46.2)	946	224	61	147
T2 (1000)	677	848	901	923	493 (24.5)	664 (32.0)	717 (35.7)	739 (36.8)	493	171	33	22
T3 (2000)	469	661	784	781	285 (14.2)	477 (23.7)	600 (29.9)	597 (29.7)	285	192	123	3
7- Soil + Hg^2+
T1 (200)	656	693	731	747	472 (23.5)	509 (25.3)	547 (27.2)	563 (28.0)	472	37	38	16
T2 (1000)	336	611	624	635	152 (7.6)	427 (21.2)	440 (21.9)	451 (22.4)	152	275	13	11
T3 (2000)	280	504	520	536	96 (4.8)	320 (15.9)	336 (16.7)	352 (17.5)	96	224	16	16

1- Soil treatments.

C: Control (untreated) soil.

T1: Soil was initially provided with 200 µg.g-1 soil of all metals at zero time.

T2: Soil was initially provided with 1000 µg.g-1 soil of all metals at zero time.

T3: Soil was initially provided with 2000 µg.g-1 soil of all metals at zero time.

2- A net increase of inorganic-N related to total inorganic-N content in the soil at zero time.

3- Percentage of the soil organic-N mineralized related to total organic -N content in soil at zero time.

N.B.

*Total organic -N content in soil at the beginning of the experiment was 2010 µg.g-1 soil; (750 µg.g-1 soil organic -N content originally present in the soil plus 1260 µg organic-N.g-1 added in the form of peptone (1% w/w) at zero time.

*Total inorganic-N content in the soil at the beginning of the experiment was 184 µg.g-1 soil.

The amounts of mineralized nitrogen reached their maximum in the control soil within the first period (5 days), including all metal treatments, followed by a rather slow increase until the end of the period of 20 days. The total inorganic nitrogen in the control soil (C) increased from 184.0 µg.g⁻¹ soil at 0-time to 893 µg.g⁻¹ soil after 5 days, which means about 35.3% of the organic nitrogen in the soil was transformed to the inorganic form. The application of 200, 1000, or 2000 µg.g⁻¹ of the seven metals (T₁, T_2, and T₃) resulted in a marked reduction in the mineralization rate where the increase in the amount of inorganic nitrogen within this period (5 days) ranging from 432 to 613, 152 to 573 and 96 to 413 µg.g⁻¹ soil for treatments T₁, T₂ and T₃, respectively. The corresponding amounts of mineralized nitrogen represented 21.5–30.5, 7.6–28.5, and 4.8–20.1% of the initial organic nitrogen content. On the other hand, it was noticeable that the greatest reduction in nitrogen mineralization was observed after 5 days with Hg²⁺ at the three levels (T₁, T₂, and T₃), which recorded 23.5, 7.6, and 4.8% of the initial organic-N. Hg²⁺ was followed, in its inhibitory effect, by Cd²⁺ except at T₁, as the percentages of mineralized nitrogen in the three soil treatments were 21.5, 14.5, and 6.8 of the initial organic-N, respectively.

As recorded in (Table 2), mineralization increased in all treatments with metals at the beginning of the experiment, then decreased significantly after 5 days, and then increased slightly until the end of the experiment. After 20 days, 28.0–49.8, 22.4–41.1, and 17.5–35.4% of the initial organic nitrogen were mineralized in T₁, T₂, and T₃, respectively in the different metal-treated soils against 55.3% in the untreated soil. Moreover, Hg²⁺ recorded the lowest percentages of mineralized nitrogen after 20 days, namely 28.0, 22.4, and 17.5 in the three levels (T1, T₂, and T3) of initial organic nitrogen.

Moreover, the maximum values of inorganic nitrogen production were recorded in 0–5-day intervals of 709 µg.g⁻¹ soil in control soil and in the other three metals treatments. In contrast with the untreated soil, the amounts of inorganic-N decreased and ranged from 613.0 to 432.0 µg.g⁻¹ soil in T₁, 573.0 to 152.0 µg.g⁻¹ soil in T₂ and 413.0 to 96.0 µg.g⁻¹ soil in T₃. On the other hand, and because the mineralization rate decreased until the end of the experiment, the lowest values of inorganic-N produced were observed in the 15–20 day interval.

Nitrification

The present experiment was conducted to study the effect of the investigated heavy metals on the biological oxidation of ammonium salts in the same fertile soil under optimal conditions of aeration (75% WHC) and temperature of (28°C). The end product of NH₄-N oxidation yields NO₃-N, the most appropriate form of inorganic nitrogen for plants. The fertile clay-loam soil was amended with 400 mg NH₄-N.g⁻¹ soil in the form of (NH₄)₂SO₄ as the sole energy source for the nitrifying bacteria as a group, in addition to the elevated levels of the seven heavy metals studied.

It is worth mentioning that the assessment of nitrification can be achieved either by the determination of NH₄-N or by the increase in NO₃-N on condition that no other biological processes that cause any quantitative changes in soil inorganic nitrogen may occur. Actually, under the prevailing experimental conditions, immobilization of inorganic nitrogen and denitrification are not expected to take place where suitable oxidation is provided and at the same time, no poor-N organic materials are present in soil. At any rate, and apart from an experimental error that may take place equally in the different soil treatments, the comparison between treatments in terms of the amounts of NO₃-N produced was considered a reliable parameter for nitrification which could be influenced by the addition of heavy metals in soil.

(Table 3) and Figure 3 show the periodical increase in NO₃-N throughout the successive periods. Nitrification started in all soil treatments from the early beginning and continued, albeit at relatively lower rates, until the end of the experiment. The results recorded a progressive increase in NO₃-N production, with the highest net nitrified nitrogen occurred within the first 5 days (0–5 days) in some treatments, while in other treatments, it occurred at the 5–10, 10–15 and 15–20 day intervals. It was found that, in untreated soil (C) nitrification led to the production of 145 µg NO₃-N.g⁻¹ soil within the initial five days, indicating that approximately 29% of the soil’s Ammoniacal nitrogen was oxidized during this short period. Subsequently, the oxidation process continued at a comparatively slower pace, resulting in the oxidation of about two-thirds of NH⁺₄-N in soil by the end of the 20-day period, corresponding to the production of 337 µg NO₃-N.g⁻¹ soil.

Figure 3. Periodical changes in the total nitrate-N (µg.g⁻¹ soil) in soil treated with three concentrations of each of the tested seven heavy metals.

Table (3): Periodical changes in the total nitrate-N (µg. g⁻¹ soil) in soil treated with three concentrations of each of the tested seven heavy metals.

Soil treatments^1	Total NO3-N (µg.g^−1 soil)									Total nitrate-N (µg.g^−1 soil) produced within one time interval (5 days)
	Determined^2					Nitrified^3 (%)^4
	Days
	5	10	15	20	5		10	15	20	0–5	5–10	10–15	15–20
Untreated (control soil)
C (untreated)	207	243	371	399	145 (29.0)		181 (36.2)	309 (61.8)	337 (67.4)	145	36	128	28
1- Soil + Pb^2+
T1 (200)	196	228	361	367	134 (26.8)		166 (33.2)	299 (59.8)	305 (61.0)	134	32	133	6
T2 (1000)	179	215	341	346	117 (23.4)		153 (30.6)	279 (55.8)	284 (56.8)	117	36	126	5
T3 (2000)	137	188	297	298	75 (15.0)		126 (25.2)	235 (47.0)	236 (47.2)	75	51	109	1
2- Soil + Cd^2+
T1 (200)	90	171	205	314	28 (5.6)		109 (21.8)	143 (28.6)	252 (50.4)	28	81	34	109
T2 (1000)	70	125	192	278	8 (1.6)		63 (12.6)	130 (26.0)	216 (43.2)	8	55	67	86
T3 (2000)	64	75	118	211	2 (0.4)		13 (2.6)	56 (11.2)	149 (29.8)	2	11	43	93
3- Soil + Cu^2+
T1 (200)	207	222	323	339	145 (29.0)		160 (32.0)	261 (52.2)	277 (55.4)	145	15	101	16
T2 (1000)	117	220	292	324	55 (11.0)		158 (31.6)	230 (46.0)	262 (52.1)	55	103	72	32
T3 (2000)	92	117	237	314	30 (6.0)		55 (11.0)	175 (35.0)	252 (50.4)	30	25	120	77
4- Soil + Zn^2+
T1 (200)	113	226	322	322	51 (10.2)		164 (32.8)	260 (52.0)	260 (52.0)	51	113	96	0
T2 (1000)	80	215	250	294	18 (3.0)		153 (30.6)	188 (37.6)	232 (46.4)	18	135	35	44
T3 (2000)	77	145	205	208	15 (3.0)		83 (16.6)	143 (28.6)	146 (29.2)	15	68	60	3
5- Soil + Cr^2+
T1 (200)	90	122	256	331	28 (5.6)		60 (12.0)	194 (38.8)	269 (53.8)	28	32	134	75
T2 (1000)	70	86	139	290	8 (1.6)		24 (4.8)	77 (15.4)	228 (45.6)	8	16	53	151
T3 (2000)	64	70	126	256	2 (0.4)		8 (1.6)	64 (12.8)	194 (38.8)	2	6	56	130
6- Soil + Ni^2+
T1 (200)	181	233	262	341	119 (23.8)		171 (34.2)	200 (40.1)	279 (55.8)	119	52	29	79
T2 (1000)	70	213	230	241	8 (1.6)		151 (30.2)	168 (33.6)	179 (35.8)	8	143	17	11
T3 (2000)	64	160	160	222	2 (0.4)		98 (19.6)	98 (19.6)	160 (32.0)	2	96	0	62
7- Soil + Hg^2+
T1 (200)	77	211	215	226	15 (3.0)		149 (29.8)	153 (30.6)	164 (32.8)	15	134	4	11
T2 (1000)	75	173	173	192	13 (2.6)		111 (22.2)	111 (22.2)	130 (26.0)	13	98	0	19
T3 (2000)	64	100	113	188	2 (0.4)		38 (7.6)	51 (10.2)	126 (25.2)	2	36	13	75

1- Soil treatments.

C: Control (untreated) soil.

T1: Soil was initially provided with 200 µg.g-1 soil of all metals at zero-time.

T2: Soil was initially provided with 1000 µg.g-1 soil of all metals at zero-time.

T3: Soil was initially provided with 2000 µg.g-1 soil of all metals at zero-time.

2- Total NO3-N at a certain time.

3- The amount of NO3-N increased over the initial amount.

4- Percentage of the amount of NO3-N increased related to total NO3-N content in the soil at zero time.

N.B.

*Total NH4 -N content in soil at the beginning of the experiment was 500 µg.g-1 soil; (100 µg.g-1 soil originally present in the soil plus 400 µg added in the form of NH4-N at zero time.

*Total NO3-N content in the soil at the beginning of the experiment was 62 µg.g-1 soil.

Providing the soil with any of the test heavy metals resulted in a slowdown in the biological oxidation of NH₄-N. Within the 5-day period, the T₁ soil treatment showed a reduction in the biological oxidation of initial NH⁺₄-N content, ranging from 10.2 to 29.0% in Pb²⁺, Cu²⁺, Zn^2+, and Ni²⁺, except for Cd²⁺, Cr^6+, and Hg²⁺ which exhibited a more inhibitory effect (5.6, 5.6 and 3.0% of the initial NH₄-N oxidation, respectively). The low concentration (200 µg.g⁻¹ soil, T₁) of any test metal had a relatively minor negative effect on nitrification, with NO₃-N production after 20 days ranging from 252 to 305 µg.g⁻¹ soil, representing 50.4% to 61.0% oxidation of the initial NH₄-N content. Comparatively, it was clear that mercury proved to be more toxic than other metals under investigation, as the addition of 200 µg Hg²⁺.g⁻¹ soil reduced nitrification output to 164 µg NO₃-N.g⁻¹ soil, equivalent to 33% oxidation of the initially found NH₄-N in soil.

As might be expected, an increase in the concentration of any test heavy metal in the soil led to a corresponding decrease in the rate of ammonium oxidation. Results showed that the presence of 1000 µg.g⁻¹ soil (T₂) was more toxic to the nitrification process than the lower concentration (T₁). Among the metals, Hg²⁺ followed by Ni²⁺ and Cd²⁺ exhibited the most suppressive effects resulting in the production of 130, 179, and 216 µg NO₃-N.g⁻¹ soil, respectively throughout the 20-day experimental period, representing 26.0, 35.8 and 43.2% of the initial NH₄-N. On the other hand, the other four metals, Pb²⁺, Cu²⁺, Zn^2+, and Cr⁶⁺ were comparatively less toxic, with a range of 45.6 to 56.7% of the original NH₄-N transformed to NO₃-N.

More retardation in the biological oxidation of NH₄-N was observed when the soil was supplemented with 2000 µg.g⁻¹ soil (T₃). Results showed that Pb²⁺ and Cu²⁺ at this concentration were less toxic where 47.2 and 50.4% of the initial NH₄-N were oxidized after 20 days, respectively, compared to 67.4% in the control soil (C). The more toxic effect was given by Ni²⁺ and Cr⁶⁺ at the same concentration (T₃), with a total NO₃-N found at the end of the experiment reaching 160 and 194 µg.g-1 soil, respectively, equivalent to 32.0 and 38.8% of the NH₄-N oxidized. Meanwhile, the highest suppressive effect on nitrification, at this concentration (T₃) was observed in soil treated with Hg²⁺, Zn^2+, and Cd²⁺ where 125, 146, and 149 µg NO₃-N. g⁻¹soil were produced, representing 25.1, 29.2, and 29.8% oxidation of the initial soil NH₄-N, respectively.

Discussion

Effect of soil pollution with heavy metals on the total microbial population

Soil contamination by heavy metals is a prevalent environmental concern, primarily stemming from human activities like mining, smelting, and agriculture [10]. As a result, the buildup and harmful impacts of heavy metals at elevated levels can lead to a deterioration in soil quality, soil microorganisms, and a decrease in soil fertility.

The findings of the current research indicate a substantial decline in the overall bacterial populations across the three soil treatments (T₁, T_2, and T₃). As the concentrations of metals increased, the reduction in bacterial populations also escalated, with a reduction of 71.8% in the initial bacterial counts in the T₁-treatment, and a range of 92.1% to 99.6% in the 92.1–99.6% in T₂- and T₃-treatments after 10 days. A minor decrease in the total bacterial count was noted after the experiment. This decline underscores the detrimental influence of heavy metals on the microbial community [2] attributed the harmful effect of heavy metals on microorganisms to their bioavailability highlighting a strong binding affinity between heavy metals and microorganisms. Furthermore, he suggested that as the concentration of heavy metals rises, the activity of numerous enzymes experiences a significant decrease, with this decline potentially being a direct result of the interaction between the enzyme and the heavy metals, leading to a direct impact on the spatial structure of the enzyme’s active groups and ultimately causing its degradation. Consequently, the growth and reproduction of microorganisms are inhibited. Another explanation was introduced by [7,8,14]who suggested that the toxicity of heavy metals, such as lead, occurs due to its interaction with sulfhydryl groups, leading to the destruction of nucleic acids and proteins. This interaction also inhibits microbial respiration, enzyme activities, transcription processes, and disrupts cell membranes. Heavy metals like lead enter bacterial cells through pathways used for essential divalent metals like Mn²⁺ and Zn²⁺, causing toxic effects by altering the osmotic balance within the bacterial cells [18].

Considering the concentrations of metals introduced into the soil in the study, the toxicity toward soil microorganisms can be ranked as follows: Hg²⁺ ≥Cr²⁺> Ni²⁺ > Cu²⁺ ≥Zn²⁺ > Cd²⁺> Pb²⁺. The present study reported that mercury and, to some extent, chromium showed the highest reduction percentage in total bacterial counts compared with the other five metals. Treatments with Hg⁺² recorded reductions of 91.4, 92.7, and 94.6% reduction in total bacterial counts in the T₁-,T₂-, and T₃- treatment, respectively, after 10 days. On the other hand, the Cr⁶⁺ treatment showed a reduction of 61.4, 97.8, and 97.7% in the same corresponding treatments. Concerning this point [19] mentioned that mercury ions induce toxic effects through protein precipitation, enzyme inhibition, and corrosive actions. Mercury binds not only to sulfhydryl groups but also to phosphoryl, carboxyl, amide, and amine groups. Proteins, including enzymes, containing such groups are highly susceptible to reactions with mercury, leading to their inactivation upon binding.

The study was extended to investigate the possible development of metal-tolerant bacteria throughout the relatively short period of metal-soil contact (40 days). The results obtained from the experiment after 40 days claimed that the presence of heavy metal salts in soil may encourage the development of metal-tolerant microorganisms which are adapted to the toxic effect of heavy metal pollution. The highest percentage of metal-tolerant bacteria was recorded in the T₁-treatment of all tested metals and ranged from 25.5 to 71.8% of the total initial bacterial count. However, as the concentration of metals increased (T₂ and T₃), the metal-tolerant bacteria percentages decreased. This finding aligns with the study of [20,21] who suggested that the development of heavy metal-resistant microorganisms can take place in a relatively short time. On the other hand [2] reported that heavy metal contamination can produce different microbial community patterns. Despite significant changes in the chemical and biological properties of the soil, the original microorganisms in the soil’s microbial community persist. In the case of long-term heavy metal-contaminated soil, the selection process favors microorganisms that possess specific adaptations to thrive in polluted soil.

Impact of heavy metals on some nitrogen transformation processes in soil

Ammonification

According to the saying of [10] soil nitrogen and heavy metals are two distinct components that can interact and influence each other in various ways. The relationship between soil nitrogen content, form, and heavy metals is indeed complex and influenced by various factors, including soil characteristics, environmental conditions, and management practices.

The results obtained in this experiment demonstrated the suppressive effects of the tested seven heavy metals on the biological activity of ammonification. A quite marked differences between treatments were observed which could be attributed to the toxic nature of the heavy metal salt and its concentration. [14] explained the reduction in the ammonification process by stating that the bacterial community is highly sensitive to heavy metals, leading to a decrease in N transformation in soil. Ammonifying bacteria, which are crucial for nitrogen cycling, were found to be more abundant in uncontaminated soil compared to soil treated with Zn²⁺, Cd^2+, and Cu²⁺, treatments. The presence of heavy metals in the soil also inhibits the growth of Azotobacter, which plays a key role in the nitrogen cycle and subsequently hinders nitrogen mineralization.

The comparison of the seven test heavy metals in terms of their impact on organic nitrogen mineralization revealed that Pb²⁺ exhibited the lowest suppressive effect. After 5 days at the three different levels (T₁, T₂, and T₃) of the initial organic-N, nitrogen mineralization percentages were recorded as 30.5, 28.5, and 20.1, respectively. However, these percentages increased after 20 days to reach 49.8, 41.1, and 35.4 of the initial organic-N for the same treatments. This result appears to be similar to the findings of a previous investigation of [21] who suggested that Pb²⁺ has a limited toxic effect due to its rapid binding to the soil matrix, resulting in minimal impact on microbial activity. In contrast, Hg²⁺ proved to be the highest toxicity among the metals in the mineralization process. The remaining five metals exhibited a moderate effect on nitrogen mineralization, with Pb⁺² as the least suppressive ion and Hg²⁺ as the most suppressive one.

Furthermore, Hg²⁺ displayed the lowest percentages of mineralized nitrogen after 20 days, measuring 28.0, 22.4, and 17.5 at the three levels (T₁, T_2, and T₃) of the initial organic-N. This observation can be attributed to the process of ammonification, which is carried out by a group of bacteria known as ammonifiers, that is considered very sensitive to the toxicity of heavy metals. This result is consistent with the findings of [19] who proposed that the toxicity of mercury stems from its impact on protein precipitation, enzyme inhibition, and binding to various functional groups such as sulfhydryl, phosphoryl, carboxyl, amide, and amine, rendering the proteins inactive.

Nitrification

As reported by [22] nitrification is the process in which NH₄⁺ is oxidized to nitrite (NO₂⁻) by Nitrosomonas and further to nitrate (NO₃⁻) by Nitrobacter bacteria. Other organisms involved are heterotrophic bacteria e.g. Arthrobacter globiformis, and various Pseudomonas spp., fungi (Aspergillus flavus), and autotrophic bacteria e.g. Nitrosococcus, Nitrosospira, and Nitrosovibrio. They added that soil nitrification was often used as an index to evaluate the toxicity of pollutants to microorganisms in virtue of its sensibility to the change of environmental factors, especially to heavy metals.

The results of the study indicated that an increase in the concentration of any tested heavy metal in the soil led to a corresponding decrease in the rate of ammonium oxidation. The most suppressive effect on nitrification was observed in soil treated with the highest concentration of Hg²⁺, Zn^2+, and Cd²⁺, where after 20 days of incubation, only 25.2, 29.2 and 29.8% of the ammonium-N were oxidized, respectively. This finding was consistent with the findings of [15,23,24] who also reported a negative effect of Cd²⁺ on nitrogen transformation. Regarding Zinc, it was found that 100 ppm of Zn²⁺ in soils can hinder nitrification processes, while approximately 1000 ppm inhibits the majority of microbiological processes in soils. However, the concentration of < 10 ppm Hg²⁺ was found to have the highest toxic effects on nitrifiers in soils. On the other hand, the results showed that Cu²⁺, Pb²⁺, Cr^6+, and Ni²⁺ had the least toxicity, with 50.4, 47.2, 38.3, and 32.0% of the ammonium-N being oxidized, respectively. The trend of metal toxicity obtained in this experiment agrees or may slightly differ from results reported by [24] and any slight difference here could be attributed to variation in soil characteristics in addition to possible variation in the environmental condition prevailing.

In another study by [25,26] they analyzed the response difference of soil potential nitrification rate to Cu²⁺ stress. The findings revealed that the addition of 1000 µg. g⁻¹ soil of Cu²⁺ caused a significant reduction in soil nitrification by 40.0%. These results agreed with the obtained results in the present experiment, where the addition of 1000 µg Cu²⁺.g⁻¹ soil led to a 47.9% reduction in the nitrification biological process after 20 days. This reduction was attributed to the inhibition of growth in ammonia-oxidizing microorganisms, as the heavy metals were found to hinder the amoA functional genes of transcription, replication, and growth of these groups of microorganisms. This gene (amoA) was often chosen as a molecular marker of ammonia-oxidizing microbes to investigate ammonia-oxidizing communities shift in polluted soils. This explanation also was confirmed by [27]; who reported an obvious decrease in soil potential nitrification rate due to the inhibition of amoA functional genes in the ammonia-oxidizing microbes.

As the present study showed that both Cu²⁺ and Cr⁶⁺ soil treatments at the highest level (T₃) caused an inhibition in the nitrification by 70.2 and 61.2%, respectively [14], stated that the decrease of mineralization rates as a consequence of Cu²⁺ pollution might reduce the turnover of organic matter and availability of nutrients in the ecosystem due to the inhibition of a specific group of microorganisms as nitrifying bacteria. Consequently, the nitrification rate appears to serve as a valuable biomarker for soil monitoring of both metals Cu²⁺ and Cr⁶⁺ and other heavy metals.

Conclusion

Summing up the results obtained in the present study, it can be concluded that:

1. Concerning the effect of different concentrations of heavy metals on the total bacterial counts, after 10 days, Hg²⁺ caused the highest reduction in T₁-treated soil (91.4%), followed by Pb²⁺ (58.1%) and Ni²⁺ (32.9%), meanwhile, in T₂ treatments, was ranged from 92.1% to 99.6% and increasing to 94.6% to 99.6% in T₃. After 40 days, inhibition was at maximum by 74.5% of Hg²⁺ in T₁ and reached 97.0% by Cr⁶⁺ in T₂ and from 95.2% to 98.0% by Hg²⁺, Cr⁶⁺, Cu^2+, and Zn²⁺ in T₃.

2. The intrinsically resistant bacteria comprised only 25.5% for Hg²⁺ and 34.2-71.8% in the different six metals after 40 days in the T₁ treatment. In T₂ treatment, their percentage reached 13.6, 3.1, and 10.2%, for Zn²⁺, Cr^6+, and Hg²⁺, respectively. On the other hand, 28.3, 20.1 and 12.5% were tolerable for Pb²⁺-, Cd²⁺- and Ni²⁺-soil in T₃ treatments, respectively.

3. About 35.3% of the organic nitrogen in the soil was transformed to the inorganic form after 5 days in the untreated soil. The application of the tree treatments, T₁, T_2, and T₃, resulted in a marked reduction in the mineralization rate of 21.5-30.5, 7.6-28.5, and 4.8-20.1%, respectively. The most nitrogen mineralization reduction percentages were observed after 5 days by Hg²⁺ at T₁, T₂, and T₃ of 23.5, 7.6, and 4.8%, respectively.

4. After 20 days, also, Hg²⁺ recorded the least percentages in the amount of mineralized nitrogen of 28.0, 22.4, and 17.5 at the three levels (T₁, T₂, and T₃) of the initial organic-N.

5. At the period of 5 days, the T₁ soil treatment with Cd²⁺, Cr⁶⁺ and Hg²⁺ resulted in retardation in the biological oxidation of initial NH⁺₄-N content by 5.6, 5.6 and 3.0%, respectively. After 20 days, also, mercury proved to be more toxic than other metals under investigation, where 200 µg Hg²⁺.g⁻¹ soil resulted in a 33% reduction in the output of nitrification. Presence of 1000 µg. g⁻¹ soil (T₂) was more toxic to the nitrification process where, Hg²⁺ followed by Ni²⁺ and Cd²⁺ caused suppressive reduction by 26.0, 35.8 and 43.2%, respectively. On the other hand, the highest suppressive effect on nitrification, at (T₃) was observed in soil treated with Hg²⁺, Zn^2+, and Cd²⁺ of 25.1, 29.2, and 29.8% of initial soil NH₄-N were oxidized, respectively.

6. The results obtained in this study clearly showed that the microbial community was affected, at different levels, by the heavy metals contamination. This led to a change in their number, activity, structure, and function which gives a chance to use these parameters as indicators of soil pollution with heavy metals by monitoring these microbial communities and their biochemical functions in polluted soils to provide valuable insights for sustainable soil management and remediation. More studies in this research field are needed in the future.

Disclosure statement

No potential conflict of interest was reported by the author(s).