The Influence of Rocket Hydrocarbon Fuel on the Activity of Soil Microbial Communities in Areas of Launch Vehicle Operation in Kazakhstan

Abstract

Hydrocarbon-based rocket fuels, particularly kerosene grades T-1 and RG-1 used during launch vehicle operations, represent a persistent source of soil contamination in areas impacted by rocket stages. This study quantitatively evaluates the response and recovery dynamics of soil microbial communities in Calcisol (Loamic) soils from the U-25 impact area near the “Baikonur” Cosmodrome (Kazakhstan) under controlled kerosene contamination. Eleven soil samples were monitored over 90 days, including one uncontaminated control and ten samples exposed to increasing concentrations of T-1 or RG-1 (100–15,000 mg/kg). Microbial indicators included total microbial count, actinomycetes, microscopic fungi, and spore-forming bacteria, expressed as CFU/g (mean ± SD, n = 3). Acute exposure caused significant reductions in total microbial abundance (28–58%) and microscopic fungi (43–75%, p ≤ 0.05), indicating pronounced short-term toxicity. By Day 90, bacterial and actinomycete populations exhibited partial to complete recovery, with some treatments exceeding control values, suggesting metabolic adaptation and hydrocarbon utilization. In contrast, fungal populations remained consistently suppressed throughout the experiment, indicating prolonged ecological stress. No strict dose–response relationship was observed, highlighting the influence of soil physicochemical properties on microbial resilience and hydrocarbon bioavailability. These findings identify microscopic fungi as the most sensitive indicators of kerosene contamination, suggesting that indigenous bacterial and actinomycete communities play a key role in natural attenuation. The results provide quantitative thresholds relevant for environmental monitoring and support the development of microbiologically informed bioremediation strategies in areas impacted by rocket launches.

1. Introduction

Kerosene-based rocket fuels such as T-1 and RG-1 are widely used in the propulsion systems of Soyuz launch vehicles due to their favorable energy characteristics and relatively low acute toxicity. These fuels consist primarily of aliphatic, cycloalkane, and aromatic hydrocarbons derived from ligroin-kerosene fractions [1]. During launch vehicle stage separation, unburned fuel may be released into the environment, where cold temperatures and limited aeration slow degradation processes. Lighter hydrocarbon components volatilize quickly, while heavier fractions remain in the soil, causing persistent contamination and altering key physicochemical properties such as aeration and water permeability. These changes adversely affect soil microbial communities, which play crucial roles in nutrient cycling, organic matter turnover, and ecosystem resilience [2,3,4,5,6].

Hydrocarbon rocket propellants (HRPs), including kerosene-based fuels RG-1 and T-1, are characterized by high persistence in soils and limited solubility in water. Oxidative processes begin immediately after their entry into the soil environment. HRPs tend to accumulate primarily in the upper soil layers, particularly within the humus horizon, which is enriched with organic matter. Following the evaporation of volatile fractions, the remaining petroleum residues undergo a series of transformation processes. These include physicochemical and partial microbial degradation of aliphatic hydrocarbons, microbial decomposition of low-molecular-weight compounds of various chemical classes leading to the formation of resinous substances, as well as the transformation of high-molecular-weight compounds such as resins, asphaltenes, and cyclic hydrocarbons.

Soils located near rocket stage impact areas in Central Kazakhstan, particularly in the U-25 zone of the Ulytau Region, periodically receive inputs of hydrocarbon-based rocket fuels (HCRFs). Although T-1 and RG-1 are classified as low-hazard substances according to the low-hazard substances according to State All-Union Standard 12.1.007 [7], their limited solubility and partial persistence can lead to long-term accumulation within the upper soil horizons. Once deposited, these fuels undergo a combination of evaporation, oxidation, adsorption, and microbial transformation, potentially forming long-lasting contamination hotspots that disrupt microbial structure and function [4,5,6].

Soil microbial communities play a crucial role in maintaining soil health, facilitating nutrient cycling, and supporting ecosystem functioning. The diversity and abundance of bacteria, fungi, actinomycetes, and spore-forming bacteria are particularly sensitive indicators of soil quality and disturbance [8]. According to Babur et al. (2025), conversion of forest to cropland driven by anthropogenic activities significantly alters soil biochemical properties and microbial indicators: forest topsoil had higher soil organic carbon, total nitrogen, microbial biomass carbon and nitrogen, as well as microbial respiration than agricultural topsoil, pointing to negative impacts of land conversion on soil biogeochemical function and microbial community status [9]. Precise quantification and qualitative assessment of microbial shifts are essential for understanding the ecological consequences of such interventions.

Previous local investigations have demonstrated that certain indigenous microorganisms are capable of metabolizing kerosene components. Several bacterial and yeast strains isolated from HCRF-impacted soils displayed substantial growth on culture media containing T-1 kerosene as the sole carbon source, even at high concentrations. Such findings support the potential for natural attenuation, although nutrient limitations, low temperatures, and the presence of recalcitrant hydrocarbon fractions may constrain the efficiency of biodegradation [6]. Hydrocarbon contamination is known to cause significant shifts in microbial community composition. Sensitive microbial groups often decline immediately after exposure, while taxa capable of utilizing hydrocarbons, such as species within Pseudomonas, Rhodococcus, Acinetobacter, and Bacillus, tend to increase in relative abundance due to their versatile catabolic enzymes [10,11]. At the same time, contamination may inhibit key soil functions, including respiration, nitrogen turnover, phosphorus depletion, and enzymatic activities, limiting microbial activity and slowing natural self-remediation, especially under nutrient-poor conditions typical of arid and semi-arid soils [10,11,12,13,14].

Although the ecological impacts of petroleum hydrocarbons are widely documented, relatively few studies have focused specifically on rocket-grade fuels under the climatic and edaphic conditions of the Ulytau Region of Central Kazakhstan. There is also a notable lack of regulatory soil quality standards for kerosene-based rocket fuels, which limits environmental risk assessment and complicates remediation planning for affected territories.

This research addresses these gaps by assessing how varying concentrations of T-1 and RG-1 kerosene influence soil microbial communities in impact zones near the “Baikonur” Cosmodrome. The study evaluates changes in microbial abundance, community structure, and recovery potential over time, with emphasis on identifying thresholds of ecological disruption and assessing the capacity of native microbial communities to contribute to natural biodegradation processes.

This study aimed to quantitatively evaluate the impacts of different soil treatments on microbial community composition, focusing on changes in total bacterial, fungal, actinomycete, and spore-forming bacterial populations over 90 days in soils treated with hydrocarbon-based rocket fuels (kerosene grades T-1 and RG-1). The data presented here provide important insights into microbial responses to tillage-induced disturbances, leading to improved soil management practices.

2. Materials and Methods

2.1. Soil Characteristics and Preparation

To evaluate the microbiological response of soils exposed to hydrocarbon rocket fuels (HRFs), a controlled laboratory experiment was conducted using 11 Calcisol (Loamic) soil samples collected from the first-stage fall zone (U-25) of the Soyuz launch vehicle in the Ulytau Region of Central Kazakhstan (47°35′24.6″ N 67°49′05.4″ E). Ten samples were artificially contaminated with kerosene grades T-1 or RG-1, while one served as an uncontaminated control.

The natural soil used in this study was collected from a depth of 0–50 cm. The soil is classified as heavy loam, characterized by 56.16% fine particles (<0.01 mm), a humus content of 0.67–0.72%, total nitrogen of 0.042–0.063%, and carbonate levels ranging from 2.29 to 7.83%. Chloride and sulfate salinity dominate the salt composition, with maximum concentrations reaching 108 mg/kg for chlorides and 583–1086 mg/kg for sulfates. The high silt fraction (25.92%) contributes to the soil’s capacity to retain petroleum hydrocarbons while limiting horizontal migration.

T-1 kerosene is produced by straight-run distillation of low-sulfur crude oil within a boiling range of 130–280 °C. Rocket kerosene T-1, like most kerosene fuels, consists primarily of saturated hydrocarbons with linear and cyclic structures, while the content of aromatic compounds does not exceed 20% (

Table 1. Average characteristics of the group composition of rocket kerosene grades T-1 and RG-1.

Kerosene Grades	Hydrocarbon Groups
	Alkanes	Cycloalkanes	Arenas	Olefins
T-1	~25–30%	~70%	<5%	absent
RG-1	~30–50%	~40–60%	15–20%	1.0–1.5%

and

Table 2. Physical characteristics of hydrocarbon rocket fuels.

Indicator	Kerosene Grades T-1	Kerosene Grades RG-1
1 Density at 20 °C, g/cm3	0.80	0.83
2. Kinematic viscosity coefficient, mm2/s:
at 20 °C, not less than	1.5	2.5
at −40 °C, not more than	16	25
3. Crystallization onset temperature, °C, not higher	−60	−60

).

RG-1 kerosene exhibits no pronounced differences from T-1 in terms of appearance, being a colorless or slightly yellowish liquid with a characteristic petroleum odor. However, despite these visual similarities, RG-1 is characterized by a substantially different group composition (Table 1 and Table 2).

All procedures for sample collection, transport, and storage followed the State All-Union Standard 17.4.4.02-2017 [15]. In the laboratory, soils were maintained at 18–20 °C and 60% of field capacity moisture to simulate natural conditions. The Fuel Samples were coded according to kerosene type and concentration (

Table 3. Fuel Treatments.

T-1 Kerosene	RG-1 Kerosene	Control
T-1-1: 108.5 mg/kg	RG-1-1: 84.75 mg/kg	1.08 mg/kg background hydrocarbons.
T-1-2: 587.5 mg/kg	RG-1-2: 600.0 mg/kg
T-1-3: 1087.5 mg/kg	RG-1-3: 895.0 mg/kg
T-1-4: 4625.0 mg/kg	RG-1-4: 4325.0 mg/kg
T-1-5: 14,925.0 mg/kg	RG-1-5: 5800.0 mg/kg

). The experiment was performed over 90 days. Microbial assessments were conducted on Days 1 and 90 to evaluate both immediate and longer-term ecological changes.

2.2. Microbiological Quantification

Microbial abundance was quantified as colony-forming units per gram of soil (CFU/g). Four functional microbial groups were analyzed:

• Total microbial count (TMC)—enumerated on general-purpose medium containing K₂HPO₄·12H₂O (1.0 g/L), MgSO₄ (0.5 g/L), FeSO₄·7H₂O (0.01 g/L), NaCl (2.0 g/L), CaCO₃ (1.0 g/L), and pH 7.5.
• Actinomycetes—cultivated on starch-casein agar.
• Microscopic fungi—quantified on Sabouraud dextrose agar supplemented with chloramphenicol (Himedia M1067, HiMedia Laboratories Private Limited, Mumbai, India).
• Spore-forming bacteria—assessed on CHROMagar (Saint-Denis, France) after heat-shocking samples at 80 °C for 20 min.

Soil suspensions were prepared through sequential 10-fold dilutions in sterile 0.9% NaCl solution. Aliquots were plated on solid media and incubated in a Binder (Tuttlingen, Germany) thermostat at 37 ± 1 °C for 72 h for bacteria; 22 ± 1 °C for 7 days for fungi and actinomycetes. Colonies were counted using an Interscience Scan^® 100 colony counter (Interscience, Puycapel, France).

To enumerate heterotrophic microorganisms utilizing organic nitrogen, samples were plated on Nutrient Agar (Himedia M001, HiMedia Laboratories Private Limited, Mumbai, India). Actinomycetes were grown on starch–casein agar prepared with soluble starch (10 g/L), casein (0.3 g/L), KNO₃ (2 g/L), MgSO₄·7H₂O (0.05 g/L), K₂HPO₄ (2 g/L), NaCl (2 g/L), CaCO₃ (0.02 g/L), FeSO₄·7H₂O (0.01 g/L), and agar (18 g/L).

2.3. Catalase Activity

Catalase activity of microbial isolates was determined qualitatively by assessing hydrogen peroxide decomposition. Briefly, 500 µL of 3% (v/v) H₂O₂ was placed on a clean glass slide, and a loopful of a 24 h culture was added. The reaction was evaluated within 5–10 s. Immediate oxygen bubble formation was recorded as a positive catalase reaction.

2.4. Protease Activity

Proteolytic activity was evaluated using a plate assay on milk agar containing casein as the protein substrate. Overnight cultures were suspended in sterile isotonic NaCl solution and adjusted to a 1.0 McFarland standard (~3.0 × 10⁸ CFU mL⁻¹). Aliquots of 1 µL were spot-inoculated onto the agar surface and incubated at 37 ± 1 °C for 18–24 h.

Protease activity was determined by the presence of clear hydrolysis zones surrounding bacterial colonies. The diameters of hydrolysis zones were measured to the nearest 1 mm.

2.5. Cellulase Activity

Cellulolytic activity was assessed using carboxymethyl cellulose (CMC) agar (Merck KGaA, Darmstadt, Germany). Isolates were spot-inoculated onto CMC agar plates and incubated at 37 ± 1 °C for 48–72 h. After incubation, the plates were stained with a 0.1% (w/v) Congo red solution for 15–20 min and then destained with 1 M NaCl for 15–20 min. Clear halos around or beneath colonies indicated cellulose hydrolysis. The diameters of the hydrolysis zones were measured with a precision of 1 mm.

2.6. Soil Dehydrogenase Activity

Total soil dehydrogenase activity was determined spectrophotometrically using the reduction assay. Soil samples (1 g) were mixed with 1 mL of 0.1 M glucose, 2 mL of 0.1 M phosphate buffer (pH 7.2), and 1 mL of 1% (w/v) Methylthiazolyl tetrazolium (MTT test (3-(4.5-dimethylthiazol or 2 (-2.5-diphenyl-tetrazolium bromide)) solution. The mixtures were incubated at 37 ± 1 °C for 18–24 h.

Formazan was extracted by adding 5 mL of dimethyl sulfoxide (DMSO), followed by centrifugation at 10,000 rpm for 2 min. Absorbance of the supernatant was measured at 540 nm. Formazan concentration was calculated using a calibration curve prepared with purified formazan (Figure 1). Dehydrogenase activity was expressed as mg formazan kg⁻¹ dry soil and calculated as:

$$DA=\frac{C\times V}{m}$$

(1)

where C is the formazan concentration (mg L⁻¹), V is the reaction volume (mL), and m is the soil mass (g).

2.7. Statistical Analysis

All microbiological measurements were performed in triplicate (n = 3) for each soil treatment and sampling time. Results are presented as mean values ± standard deviation (SD). Statistical significance was evaluated at a 95% confidence level (p ≤ 0.05).

Correlation between fuel concentration and microbial response was assessed using Spearman’s rank correlation coefficient. This non-parametric approach was selected due to the non-normal distribution of the experimental data. Statistical significance was evaluated at a threshold of p < 0.05. Data visualization was performed using scatter plots to illustrate monotonic trends.

3. Results

The experiments were conducted using soil samples contaminated with T-1 and RG-1 grades of hydrocarbon rocket kerosene. The biological activity of the soils was evaluated by measuring microbial abundance in comparison with the control samples. On both the 1st and 90th days of the study, a diverse range of microorganisms was detected in all soil samples, including TMC microscopic fungi (belonging to the genera Penicillium, Aspergillus, Mucor, Trichoderma, etc.), actinomycetes, and spore-forming bacteria (

Table 4. Comparative data on the quantitative and qualitative composition of soil microorganisms.

Soil Sample Code	Number of Bacteria, Thousand CFU/g	Percent Increase (↑)/Decrease (↓) of the Indicator Relative to the Control, %	p Value Criterion for the Reliability of the Reduction in CFU Relative to the Control	Number of Bacteria, Thousand CFU/g	Percent Increase (↑)/Decrease (↓) of the Indicator Relative to the Control, %	p Value Criterion for the Reliability of the Reduction in CFU Relative to the Control
	1st days of the experiment			90th day of the experiment
Total microbial count
Control	206.25 ± 0.057			165.33 ± 0.074
T-1.1	119.75 ± 0.210	42 ↓	0.0004	176.33 ± 0.055	6 ↑
T-1.2	147.75 ± 0.023	28 ↓	0.0007	127.00 ± 0.146	23 ↓	0.18
T-1.3	124.75 ± 0.166	40 ↓	5.3 × 10−7	246.00 ± 0.082	49 ↑
T-1.4	201.50 ± 0.044	3 ↓	0.003	158.66 ± 0.088	4 ↓	0.69
T-1.5	123.50 ± 0.199	41 ↓	5.3 × 10−7	209.66 ± 0.010	27 ↑
RG-1.1	124.25 ± 0.180	40 ↓	5.5 × 10−7	149.83 ± 0.004	9 ↓	0.26
RG-1.2	115.50 ± 0.039	44 ↓	4.0 × 10−7	122.66 ± 0.0	26 ↓	0.05
RG-1.3	87.500 ± 0.071	58 ↓	1.7 × 10−7	280.33 ± 0.267	70 ↑
RG-1.4	105.75 ± 0.028	49 ↓	2.8 × 10−7	277.00 ± 0.035	68 ↑
RG-1.5	176.75 ± 0.081	15 ↓	3.8 × 10−6	154.83 ± 0.026	6 ↓	0.44
Microscopic fungi
Control	12.360 ± 0.240			11.80 ± 0.006
T-1.1	5.620 ± 0.035	55 ↓	0.0001	3.10 ± 0.008	74 ↓	0.0003
T-1.2	5.110 ± 0.017	59 ↓	0.001	2.53 ± 0.010	79 ↓	0.0006
T-1.3	4.370 ± 0.009	75 ↓	0.001	3.70 ± 0.008	69 ↓	0.0005
T-1.4	4. 080 ± 0.011	67 ↓	6.0 × 10−5	3.97 ± 0.004	64 ↓	0.0003
T-1.5	5.250 ± 0.009	58 ↓	0.002	4.10 ± 0.008	65 ↓	0.0008
RG-1.1	7.050 ± 0.003	43 ↓	3.3 × 10−11	2.73 ± 0.011	77 ↓	0.001
RG-1.2	3.510 ± 0.008	72 ↓	4.2 × 10−7	3.10 ± 0.026	74 ↓	0.03
RG-1.3	5.850 ± 0.146	53 ↓	6.2 × 10−7	3.63 ± 0.006	69 ↓	0.0002
RG-1.4	4.580 ± 0.026	63 ↓	4.06 × 10−10	3.07 ± 0.0	74 ↓	0.002
RG-1.5	6.080 ± 0.240	51 ↓	4.8 × 10−5	5.10 ± 0.027	57 ↓	0.02
Actinomycetes
Control	87.750 ± 0.003			117.50 ± 0.03
T-1.1	75.50 ± 0.011	14 ↓	0.002	70.00 ± 0.018	40 ↓	0.002
T-1.2	78.50 ± 0.005	11 ↓	0.004	57.50 ± 0.028	51 ↓	0.0005
T-1.3	83.50 ± 0.204	5 ↓	0.004	149.7 ± 0.100	27 ↑
T-1.4	147.75 ± 0.297	41 ↑		113.17 ± 0.059	64 ↓	0.66
T-1.5	79.750 ± 0.002	10 ↓	0.002	128.50 ± 0.016	4 ↑
RG-1.1	94.00 ± 0.325	7 ↑		45.67 ± 0.052	61 ↓	0.002
RG-1.2	118.25 ± 0.081	26 ↑		30.167 ± 0.016	74 ↓	0.0003
RG-1.3	229.25 ± 0.006	62 ↑		97.17 ± 0.070	17 ↓	0.15
RG-1.4	85.50 ± 0.028	3 ↓	0.04	82.00 ± 0.010	30 ↓	0.008
RG-1.5	86.50 ± 0.005	2 ↓	0.09	49.67 ± 0.021	68 ↓	0.0003
Spore-forming bacteria
Control	119.50 ± 0.017			70.90 ± 0.020
T-1.1	39.250 ± 0.042	67 ↓	9.4 × 10−7	28.10 ± 0.625	61 ↓	0.63
T-1.2	84.00 ± 0.014	30 ↓	0.004	42.50 ± 0.339	41 ↓	0.05
T-1.3	123.50 ± 0.048	3 ↑		52.10 ± 0.054	27 ↓	0.001
T-1.4	122.25 ± 0.007	2 ↑		54.00 ± 0.124	24 ↓	0.02
T-1.5	86.00 ± 0.005	28 ↓	0.002	67.30 ± 0.195	5 ↓	0.46
RG-1.1	101.25 ± 0.019	15 ↓	0.01	42.40 ± 0.096	41 ↓	0.004
RG-1.2	89.50 ± 0.015	25 ↓	0.003	38.80 ± 0.215	46 ↓	0.02
RG-1.3	109.25 ± 0.026	9 ↓	0.07	63.40 ± 0.447	11 ↓	0.5
RG-1.4	127.00 ± 0.018	6 ↑		60.80 ± 0.158	14 ↓	0.09
RG-1.5	91.50 ± 0.024	23 ↓	0.03	46.25 ± 0.103	35 ↓	0.006
Cellulolytic bacteria
Control	23.75 ± 8.13			1.98 ± 0.17
T-1.1	3.75 ± 1.8	85 ↓	6.3 × 10−5	1.17 ± 0.38	41 ↓	0.001
T-1.2	4.25 ± 1.8	82 ↓	0.0002	7.26 ± 0.25	267 ↑
T-1.3	15.75 ± 9.5	34 ↓	6.6 × 10−5	14.61 ± 0.86	683 ↑
T-1.4	9.50 ± 2.8	60 ↓	2.1 × 10−5	8.88 ± 0.72	348 ↑
T-1.5	3.25 ± 2.8	86 ↓	2.2 × 10−5	9.27 ± 2.8	368 ↑
RG-1.1	4.25 ± 1.8	82 ↓	0.0002	6.66 ± 1.69	236 ↑
RG-1.2	4.50 ± 3.2	81 ↓	0.0001	7.63 ± 1.57	285 ↑
RG-1.3	4.75 ± 2.1	80 ↓	3.7 × 10−5	1.02 ± 0.81	48 ↓	0.001
RG-1.4	11.00 ± 7.1	54 ↓	1.1 × 10−5	1.29 ± 0.14	14 ↓	0.003
RG-1.5	5.250 ± 1.8	78 ↓	3.9 × 10−5	4.66 ± 1.5	135 ↑

Note—p-value ≤ 0.05 is considered an indicator confirming the statistical reliability and significance of the obtained results at the 95% confidence level.

, Appendix A, (Figure A1, Figure A2, Figure A3, Figure A4, Figure A5, Figure A6, Figure A7 and Figure A8)).

3.1. Initial Microbial Response Day 1

Immediately after exposure to T-1 and RG-1 kerosene, all contaminated soils exhibited reductions in the major microbial groups when compared to the control. The control sample contained:

•

TMC: 206.25 × 10³ CFU/g

•

Actinomycetes: 87.75 × 10³ CFU/g

•

Microscopic fungi: 12.36 × 10³ CFU/g

•

Spore-forming bacteria: 119.50 × 10³ CFU/g

Across contaminated samples:

•

TMC decreased by 28–58%, showing pronounced sensitivity.

•

Fungal abundance decreased by 43–75%, and the strongest suppression occurred in samples T-1-3, T-1-4, RG-1-2, and RG-1-4.

•

Actinomycetes declined in six samples (up to −14%), although some samples showed increases up to 62%, indicating heterogeneous responses.

•

Spore-forming bacteria exhibited slight increases (2–6%) in several samples, with the highest counts in T-1-3, T-1-4, and RG-1-4.

Statistical analysis confirmed that higher hydrocarbon loads were associated with significant declines across all microbial groups (p ≤ 0.05), although the absence of a clear concentration response curve suggests complex interactions between hydrocarbons and soil properties.

3.2. Microbial Dynamics After 90 Days

Microbial succession patterns differed substantially among groups:

•

Bacteria. By Day 90, bacterial populations exhibited strong recovery trends in several contaminated treatments: RG-1-3: +70% relative to control; RG-1-4: +68%; T-1-3: +49%. For example, the bacterial count in RG-1-4 reached 277.00 × 10³ CFU/g, exceeding the control (165.33 × 10³ CFU/g). This indicates metabolic adaptation and the development of populations capable of degrading hydrocarbons. However, some samples (e.g., RG-1-5) showed minimal or statistically insignificant recovery.

•

Actinomycetes. Actinomycete dynamics were mixed:

-

Day 1: increases up to 62% (e.g., RG-1-3), reflecting rapid exploitation of available hydrocarbons;

-

Day 90: notable decreases in several samples (e.g., RG-1-2: −74%, p = 0.0003), likely due to nutrient depletion or accumulation of toxic intermediates.

•

Microscopic Fungi. Fungi were the most severely affected group:

-

Day 1: reductions up to 75% (p < 0.01);

-

Day 90: Although some samples exhibited partial recovery, most remained significantly below control levels.

Persistent fungal suppression highlights the long-term ecological impact of HRFs on soil fungal communities.

•

Spore-Forming Bacteria. Day 1 showed significant declines in most samples (−58% in RG-1-3). By Day 90, most samples remained 20–60% below the control, indicating that the spores were insufficient to ensure rapid recovery under prolonged hydrocarbon stress.

3.3. Correlation Analysis

On day 1 of the experiment, Spearman’s correlation analysis revealed a weak negative relationship between fuel concentration and microbial response (r_s ≈ −0.1), which was not statistically significant (p > 0.05). In contrast, by day 90, a correlation between fuel concentration and microbial response was observed, indicating the development of a concentration-dependent microbial response over time.

Correlation analysis between kerosene concentration and microbial abundance revealed no consistent linear dose–response relationship across the tested concentration range. Pearson correlation coefficients between fuel concentration and CFU reduction varied from weak to moderate and were not statistically significant in several treatments (p > 0.05). This indicates that microbial responses were not governed solely by hydrocarbon concentration but were strongly modulated by soil physicochemical properties, fuel fraction composition, and microbial adaptive capacity (Figure 2).

The findings of this study indicate that increasing hydrocarbon concentrations lead to a statistically significant (p ≤ 0.05) decline in total microscopic fungi, actinomycetes, and spore-forming bacteria in soil. Comparative analysis of quantitative microbiological parameters in contaminated versus control soils revealed varied responses of microbial communities to carbon pollution; however, no irreversible alterations in the overall composition of microscopic organisms were observed. Analysis of individual microbial groups suggests that microscopic fungi were most sensitive to pollutant-induced stress at the initial stage of the experiment, while bacteria and actinomycetes showed signs of recovery by day 90. These patterns likely reflect morphological and physiological characteristics specific to each group, which influence the rate of their adaptation (Table 4).

4. Discussion

The data indicate that T-1 and RG-1 kerosene contamination markedly suppresses soil microbiota within 24 h, reducing TMC by 28–58% and fungal abundance by 43–75%, underscoring the high hydrocarbon sensitivity of soil microorganisms (Table 4, Appendix A, Figure A1, Figure A2, Figure A3, Figure A4, Figure A5, Figure A6, Figure A7 and Figure A8).

The experiment demonstrated that both T-1 and RG-1 kerosene exert acute toxicity on soil microbial communities. The 28–58% reduction in TMC within 24 h indicates that many soil microorganisms are highly susceptible to petroleum hydrocarbons. This finding aligns with earlier studies showing rapid suppression of saprophytic microbial groups following exposure to diesel and crude oil contaminants [16,17,18].

Microscopic fungi exhibited the strongest negative response. Their sharp decline (up to 75%) can be attributed to hydrocarbon-induced membrane damage and disruption of oxidative enzyme systems, which are critical for fungal metabolism [19,20]. The persistent suppression of fungal groups even after 90 days suggests long-lasting ecological stress that may affect soil organic matter turnover.

Actinomycetes and spore-forming bacteria showed more complex patterns. Modest increases in actinomycetes during the early stage are consistent with their known capacity to utilize hydrocarbons as carbon sources [21,22]. Spore-formers, although resilient to harsh conditions, also experienced significant initial declines, likely reflecting damage to vegetative cells and delayed germination under hydrocarbon stress.

The recovery of bacterial populations by Day 90 in many treatments highlights the adaptability of hydrocarbon-degrading genera such as Pseudomonas, Acinetobacter, Rhodococcus, and Bacillus [20,21]. These taxa rely on oxygenase enzymes capable of breaking down aliphatic and aromatic hydrocarbons, enabling them to thrive following an initial decline.

The absence of a consistent dose–response relationship indicates that microbial responses were modulated not only by hydrocarbon concentration but also by soil texture, salinity, moisture, and the physicochemical properties of individual hydrocarbon fractions [22].

Overall, the observed microbial trajectories suggest that natural attenuation processes are active in these soils. However, the pace and completeness of recovery vary among microbial groups, with fungi showing the slowest rebound.

Microbial responses to petroleum contamination differ by group: fungi exhibit the strongest short-term sensitivity, while bacteria and actinomycetes recover more quickly due to their rapid growth, metabolic flexibility, and stress tolerance. Community shifts are further influenced by interactions such as competition, cooperation, and niche partitioning. These recovery patterns emphasize the soil’s natural attenuation capacity. Using mixed microbial consortia of bacteria, actinomycetes, and fungi can enhance bioremediation by leveraging their complementary hydrocarbon-degrading abilities and supporting overall microbiome recovery.

The absence of a significant correlation at the early stage of the experiment suggests that microbial communities did not exhibit an immediate concentration-dependent response to fuel exposure. The emergence of a correlation by day 90 may reflect gradual microbial adaptation and increased involvement of microorganisms in fuel-associated metabolic processes.

It should be noted that culture-based methods capture only a fraction of the total soil microbiome. Future studies should incorporate high-throughput sequencing approaches (16S rRNA and ITS amplicon sequencing) to assess non-culturable microbial taxa potentially affected by rocket fuel contamination. In addition, measurements of soil enzyme activities (e.g., dehydrogenase, urease, and catalase) and phytotoxicity assays would provide a more comprehensive evaluation of functional soil recovery following hydrocarbon exposure.

5. Conclusions

Contamination of Calcisol soils with T-1 and RG-1 kerosene caused immediate and substantial reductions in microbial abundance, particularly among total bacteria and microscopic fungi. Fungi were the most sensitive group, while actinomycetes demonstrated partial resilience due to their ability to metabolize hydrocarbons.

Despite the initial suppression, bacterial and actinomycete populations showed noteworthy recovery within 90 days, indicating adaptive responses and highlighting the potential for natural attenuation. The lack of a clear concentration-dependent effect suggests that soil physicochemical characteristics strongly influence microbial resilience and hydrocarbon bioavailability.

The results demonstrate that contamination with T-1 and RG-1 kerosene leads to immediate and pronounced suppression of soil microbial communities, with microscopic fungi representing the most sensitive group. Persistent fungal inhibition was observed at contamination levels exceeding approximately 500–1000 mg/kg, indicating a potential ecological threshold for irreversible fungal suppression. In contrast, bacteria and actinomycetes exhibited partial recovery within 90 days, highlighting their adaptive capacity and suitability as key functional groups for bioremediation strategies in rocket stage impact zones.