The crude oil contaminated soil was obtained from Wonocolo public crude oilfields. Thirty-eight samples were taken from 0-30 depth from old wells, transportation line, and refinery area with different patterns of diagonal, random, and line, respectively. The samples were mixed and filtered using 2 mm sieves to obtain a homogenous composite sample, then stored at 4 °C [21]. Soil texture was identified as silty-clay loam (25% clay, 56% silt, and 20% sand) according to USDA classification using hydrometer method [22].

The organic waste was prepared by mixing yard waste and rumen residue in a ratio of 3:1 (Y/R) to fulfill the composting nutrient requirements [1], [23]. Both of organic wastes were shredded and sieved into 5 mm size before mixing. The C/N ratio, moisture, and pH of the organic waste were 29.96, 62.50% and 5.00, respectively. The initial concentration of TPH was 2,153.33 mg/kg, and bacterial population was 1.76 × 10¹⁰ CFU/g.

The composting process was conducted in laboratory scale experiment for 150 days. About 1.00 kg Contaminated Soil (CS) and soil-YR mixture, S/YR (1:1 ratio), were placed in glass reactor of 3.50 L volume capacity. The composting was performed at existing temperature and pH which were measured daily (thermometer) and every 3 days (Takemura soil tester), respectively. Furthermore, the composting process was conducted under aerobic condition through manual agitation every 3 days to allow good aeration and homogenization. Moisture was maintained in optimal value of 50-60% [24], [25] during the composting period. This experiment was implemented with two replications. Compost samples were collected on day 0, 20, 40, 60, 80, 100, 120, and 150 to be analysed.

The carbon and nitrogen (C/N) ratio were calculated based on organic carbon with total nitrogen content. Organic carbon and total nitrogen content were analyzed using Walkley and Black [26] and Kjehdahl [27] method, respectively.

The determination of heterotrophic bacteria population was performed by serial dilution of compost samples using 0.90% NaCl, then plated on nutrient agar medium. The bacteria population was counted after incubation at room temperature for 24 hours, and the results were expressed as Colony-Forming Units (CFU) per gram. Furthermore, dominant colonies were isolated and streaked into nutrient agar medium for bacterial strain characterization and identification [28]. Morphological and physiological characteristics were observed using microscope, Gram staining [29], and Microbact Identification Kits (Microbact^TM GNB12A and 12B). Identification of bacterial strain was conducted following Bergey’s Manual of Determinative Bacteriology [30].

The changes of TPH in crude oil contaminated soil were observed on 0^th and 150^th day of composting. The samples were extracted using conventional Soxhlet extraction and Gravimetric method following APHA-AWWA and WEF standard No. 5520D and 5520F [31], respectively. Quantification of TPH in the extract was done by Fourier Transform Infrared spectroscopy (FT-IR). The FT-IR was performed using Thermo Scientific^TMNicolet iS5 at 2,930 cm⁻¹ absorbance according to ASTM D7066-4 with 0.1 µL injection volume of TPH extract [32]. The fractions of TPH were measured by Gas Chromatography-Mass Spectrometry (GC-MS), using Agilent HP 1 MS column. Chromatographic resolution was achieved by a 30 m × 0.25 mm capillary column with a 0.25 µm film thickness. The carrier gas was helium at a flow rate of 3 mL/min. 0.1 µL TPH extract was injected to GC-MS with analytical conditions, such as initial temperature 60 °C with isothermal operation for 5 min, heated to final temperature of 280 °C at a constant rate of 5 °C min⁻¹ with a 71 min isothermal. The measurement was conducted by internal calibration method, and TPH analysis was performed by comparing their peaks standards. The TPH concentration was expressed in mg/kg.

The initial concentration of TPH, moisture, pH, and C/N ratio for CS were 51,083.04 mg/kg, 50.00, 7.00 and 23.37, respectively. The C/N ratio of CS indicated that crude oil contaminated soil was reasonable for composting process, which the criteria was ranged from 15-30 [33]. There was also 4.26 × 10⁷ CFU/g of indigenous bacterial population in soil. Meanwhile, the S/YR values were 27,743.57 mg/kg, 58.00%, 5.20, 34.40, 3.03 × 10¹⁰ CFU/g for initial concentration of TPH, moisture, pH, C/N ratio, and bacterial population, respectively. The pH value of S/YR was lower than CS, it might influence by the pH of organic waste [33], [34]. Moreover, organic waste amendments led to C/N ratio and bacterial population increasing which indicated that it could act as nutrient and exogenous bacteria sources [33], [35]. The C/N ratio of S/YR was close to Zhang et al. [18] criteria value for contaminated soil composting of 25-40.

Moisture content, temperature, pH, C/N ratio, and bacterial population showed fluctuating values during 150 days of crude oil contaminated soil composting process. The values of these parameters in this experiment was expressed in the mean of two replicates. Figure 1a showed that the composting of CS and S/YR were carried out in maintained condition of moisture content (50.00 to 60.00%) to ensure the process worked well [36].

Figure 1.

The changes of moisture content, temperature, and pH

During composting process, temperature in CS showed almost similar values to those of the ambient temperature values (31.28-33.50 °C) which was lower than S/YR (Figure 1b). The temperature of S/YR was increased in the 10^th first day of composting process from 34.50 to 35.05 °C. Then, it showed similar trend of changes with CS which was decreased up to day 30^th (31.70 °C) of composting process. On day 40^th, the temperature was increased (33.03 °C), then decreased at50^th (32.38 °C), and slightly increased following ambienttemperature from 60^th up to the end of composting process(33.13-33.25 °C).

The increased temperature indicated that organic substrate decomposition by microbial microorganism which produced heat energy, was high [25], [37]. It might lead to TPH degradation in contaminated soil, to occur at the same time. t is related to S/YR pH value decreasing from day 30^th to 45^th of composting process (Figure 1c), which due to the accumulation of organic acids fromorganic substrate hydrolysis [25].Then, the organic acids were decomposed into CO₂ and H₂O,which made its availability low. This caused the pH to increase to 6.66 and itremained stable in the range of 6.82-6.94 until the end of the process. It alsocaused the pH value of CS to range in neutral values (6.85-7.00) from day0^th to 150^th composting process.

The C/N ratio values of CS and S/YR declined from 23.37 and 34.40 to 3.76 and 13.29 at the end of composting process, respectively (Figure 2). Prior to declining, the C/N ratio values of CS reactor were slightly increased from day 0^th (23.37) to 30^th (26.02) of composting period. It might be caused by high release of nitrogen under anoxic-anaerobic conditions, which might occur due to inadequate mixing of air supply during the process.

Figure 2.

The changes of C/N ratio and bacterial population

The decrease of C/N ratio was caused by the occurrence of microbial mineralization. Declined C/N ratio value of S/YR was three times higher than CS, which showed carbon utilization of organic waste as energy source was high [34]. It was expressed by a slightly increased of S/YR bacterial population from initial (3.03 × 10¹⁰ CFU/g) to 120^th day (1.02 × 10²¹ CFU/g) on S/YR. This may be related to the adapted (lag phase) and growth (log phase) ability of heterotrophic bacterial from organic waste with the TPH presence in the mixture. At the end of S/YR composting process, the bacterial population declined to 7.28 × 10¹⁹ CFU/g reflecting the occurrence of maturation phase. Meanwhile, CS bacterial population was in the log phase from day 0^th to 150^th which increased from 4.26 × 10⁷ to 6.89 × 10¹⁹ CFU/g, respectively. It indicates that substrate availability was still sufficient for bacterial growth, shown by high C/N ratio at the end of the composting process.

The concentration of aliphatic and aromatic petroleum hydrocarbon (TPH) before and after 150^th days composting is listed in Table 1. The addition of YR mixture into polluted soil could reduce the initial TPH level from 51,083.04 to 27,743.57 mg/kg, which eases bacteria load to reduce TPH.

After the treatment, TPH concentration on S/YR was 6,974.58 mg/kg and fulfilled soil quality standard of 10,000 mg/kg. The S/YR treatment showed higher TPH removal than CS in average of 20,768.99 mg/kg (74.86%) and 1,205.67 mg/kg (2.36%), respectively. This is due to the intense microbial activity in S/YR composting process, which was shown by higher population than CS. It was also related to high temperature achievement and pH values fluctuation.

The biodegradable substrate in organic waste provided an opportunity for bacteria to adapt and grow simultaneously, while also producing required biosurfactant to improve TPH bioavailability for them [38]. The increasing of TPH bioavailability was also assisted by the presence of compost Humic Acid Like (cHAL) which was formed in humification process and had surfactant like-ability [39], [40]. Furthermore, that process could lead to greater TPH catabolic activity of indigenous bacteria in crude oil contaminated soil [41]. This was the reason for low TPH degradation in CS composting, although the bacterial population was similar to S/YR.

The occurrence of high TPH degradation was also caused by higher solubility of aliphatic (C_5-28) and aromatic (C_5-17) hydrocarbon in samples than other complex TPH fractions of 0.65-5.80 and 0.03 mg/L [6], respectively. During S/YR composting process, the dominance of TPH fraction degradation was found at C_17-20 aliphatic and C_9-12 aromatic hydrocarbons of 18,409.96 mg/kg (96.13%) and 1,025.26 mg/kg (100.00%), respectively. For CS, the dominant degraded fractions were C_13-16 aliphatic (10,245.52 mg/kg, 92.03%) and C_9-12 aromatic (2,394.80 mg/kg, 100%). In contrast, C_5-8 and C_21-28 concentrations of aliphatic fraction were increases. This may be due to the accumulation of decomposed aromatic fractions, because it was completely removed at the end of the process.

Compared to the previous research, highest TPH removal in this study (20,768.99 mg/kg, 74.86%) was lower than the research conducted by Handrianto et al. [1], who composted crude oil contaminated soil from Wonocolo oilfields using peanut (Arachishypogaea L.) compost for 30 days (21,090-27,532 mg/kg, 51.20-66.80%). Peanut composting was conducted by adding livestock dung, husks, molasses, and EM4 as stimulant. This suggested that rumen residue and yard waste were favorable bulking agent to be added in the treatment of crude oil contaminated soil in Wonocolo oilfields, though it took a longer time.

The difference in bioremediation efficiency of each study was significantly influenced by environmental factors such as temperature, pH, moisture content, C/N ratio, air supply, particle size, and incubation time [12], [21]. In addition, the TPH fraction in crude oil contaminated soil also affected the degradation efficiency which referred to its hydrophobicity [42].

At the end of composting process, three and two dominant bacteria colonies from S/YR and CS, respectively, were isolated. Morphological (Table 2) and physiological (Table 3) characteristics of isolated colonies belonged to Bacillus genera [43]. The characteristics and the prediction following Bergey’s Manual of Determinative Bacteriology of S/YR confirmed that the colonies belong to Bacillus sp. (73.68%), Bacillus pantothenticus (75.00%), and Bacillus cereus (82.00%). For CS, the colonies were Bacillus sp. (72.00%) and Bacillus cereus (77.00%).

Bacillus sp., and Bacillus cereus were confirmed as hydrocarbon-degrading bacteria by Prakash et al. [44] and Cerqueira et al. [45]. Both bacteria might be responsible for TPH removal in composting process [26]. Bacillus sp., was capable to remove naphthalene and pyrene [44], [45]. Similar capabilities were shown by Bacillus cereus, which was able to degrade TPH [46], naphthalene [47], and benzene [48]. Meanwhile, Bacillus pantothenticus was a protease producer [49] that might act as organic substrate decomposer. However, its ability to degrade hydrocarbon was unknown until now.