Performance Evaluation Of Bacterial Consortium For Biodegradation Of Total Petroleum Hydrocarbon: A Comparative Strategic Biostimulation Study

This research work demonstrated the comparative study of the efficacy of poultry litter extract (PLE) and 23 inorganic fertilizer (NPK) as biostimulating agents for enhancing total petroleum hydrocarbon (TPH) 24 degradation of petroleum refinery sludge (PRS) with bioaugmentation of an indigenously developed bacterial 25 consortium. In this study, six sets of treatments such as natural attenuation, bioaugmentation with the 26 indigenously developed bacterial consortium, and various biostimulation strategies with (MSM100, PLE100, 27 NPK100, MSM50+PLE50, and MSM50+NPK50) were performed to meet 100% nutrient source for bacterial 28 growth to enhance TPH degradation. Among all, the combined sources of MSM50+PLE50 showed the best 29 performance by degrading the TPH up to 91.3 ± 4.1% within 28 d of the incubation period. The GC-FID 30 analysis confirmed the efficacy of TPH degradation of PRS when PLE amendment with MSM. Further, the 31 removal of maltene and asphaltene was also achieved 92 ± 3.7% and 52 ± 2.2% during this treatment. TPH 32 degradation fitted to first-order kinetics with a rate constant 0.09 d -1 and half-life period of 7.7 d for 33 MSM50+PLE50 amendment treatment along with bacterial consortium as bioaugmentation. This study revealed 34 the implementation of the PLE amendment not only preferred as a nutrient source for bacterial growth but also 35 enhanced TPH biodegradation in an eco-friendly strategic way by dropping the practice of inorganic salts.


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This research work demonstrated the comparative study of the efficacy of poultry litter extract (PLE) and 23 inorganic fertilizer (NPK) as biostimulating agents for enhancing total petroleum hydrocarbon (TPH) 24 degradation of petroleum refinery sludge (PRS) with bioaugmentation of an indigenously developed bacterial 25 consortium. In this study, six sets of treatments such as natural attenuation, bioaugmentation with the 26 indigenously developed bacterial consortium, and various biostimulation strategies with (MSM100, PLE100, 27 NPK100, MSM50+PLE50, and MSM50+NPK50) were performed to meet 100% nutrient source for bacterial 28 growth to enhance TPH degradation. Among all, the combined sources of MSM50+PLE50 showed the best 29 performance by degrading the TPH up to 91.3 ± 4.1% within 28 d of the incubation period. The GC-FID 30 analysis confirmed the efficacy of TPH degradation of PRS when PLE amendment with MSM. Further, the 31 removal of maltene and asphaltene was also achieved 92 ± 3.7% and 52 ± 2.2% during this treatment. TPH 32 degradation fitted to first-order kinetics with a rate constant 0.09 d -1 and half-life period of 7.7 d for

Introduction
The rapid evolution in industrial sector has instigated in generation of a huge quantity of pollutants to the 48 environment (Gaur et al. 2021). Amongst these, petroleum hydrocarbon pollutants from the petroleum industries 49 need a special attention due to its carcinogenic and toxic effects towards health and environment. The release of 50 hydrocarbon contaminants during the crude oil processing, extraction and transportation causes a serious 51 environmental concern due to its hazardous nature. A significance amount of oily sludge generates from the 52 petroleum industries in which total petroleum hydrocarbon (TPH) is a major concern. TPH refers to petroleum-    In this study, the petroleum refinery sludge (PRS) was collected from Indian oil corporation limited Haldia,

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West Bengal, India. The PRS sample was stored in a closed container at 4 ℃ to avoid volatilization and to avoid  (Table S1).

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The MSM used for degradation study composed (g L -1 ) KH 2 PO 4 (0.17), K 2 HPO 4 (0.435), Na 2 HPO 4 . (25 g L -1 ) was prepared and sterilized for required media for bacterial growth. Different strategic media were 112 prepared by adding PLE and NPK with MSM for biodegradation study of TPH.

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The hydrocarbon degrading bacterial cultures used in this study were previously isolated from the same PRS    136 Table 1 137 Total hydrocarbon degrading bacteria

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To enumerate the total bacterial population (CFU mL -1 ), in each three days intervals 1 mL of media  Kinetic study is essential to (a) measure the biodegradation speed, (b) to understand biodegradation process.

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Further, kinetic study exemplifies the existence of residual TPH at any time and future prediction of its where (g kg -1 ) and (g kg -1 ) represents the initial ( = 0) and final ( = ) TPH concentration respectively,

Results and discussions
174 Total bacterial population

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The sample from each treatment were individually analyzed for total bacterial papulation by plate count method 176 at different time (0 th , 3 rd , 6 th , … and 28 th day) and the result were depicted in Fig. 1. The increased biomass with 177 the period of time was represented by CFU mL -1 (Fig. 1). The result of this current study displayed maximum populations in the treatments T5. In treatment T5, the bacterial population was increased from 0.97 ± 0.03 × 10 8 at 15 th day of incubation. It was observed that the bacterial population rapidly increased during the first 15 days 181 of incubation then reduced to 2.2 ± 0.24 ×10 9 CFU mL -1 on 28 th day. The rapid increased in biomass is due to 182 the combined effort of presence of hydrocarbon of the PRS, MSM, and additional source of nutrients in PLE.

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Similar type of trends was also observed in different treatments, but the increased biomass varies in different 184 strategies. Next to the PLE and MSM amendment, the PLE100 (T3) showed the total bacterial population 185 reached to maximum 9.5 ± 0.23 ×10 8 CFU mL -1 followed by MSM100 (T2) strategy (Fig. 1). In case of   was noticed for the treatment T5 and the TPH degradation was achieved 91.3 ± 4.1% after 28 days of incubation 199 (Fig. 2). However, a substantial amount (82.55 ± 3.4%) of TPH degradation was observed in treatment T2.

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Further the percentage of TPH degradation was 78.5 ± 2.5 and 62.7 ± 3.1% for the treatment T3 and T4 201 respectively. In this study the NPK amendments showed comparatively lower degradation than PLE 202 amendments but higher than the control. This may be due the bioavailability of the nutrients in PLE amendment 203 is more than the NPK amendment for bacterial activities. However, treatment T5 showed the maximum TPH 204 degradation, where both MSM and PLE were supplied (1:1) (Fig. 2). The presence of humic and fulvic  Table 2.
214 Table 2 215 The extracted maltene fractions comprised of aliphatic, aromatics and NSO in the range 52 ± 4%, 39 ± 216 2%, and 9 ± 1%, respectively (Behera et al. 2020). The heavier asphaltene was found to be 90 ± 3 g kg -1 , which 217 is 50% of TPH in this PRS sample. The degradation of maltene and asphaltene fractions for different 218 amendment strategies has been described in Fig 3. It was noticed that both the maltene and asphaltene fractions 219 reduced in each strategy as compared to control (T1) (Fig. 3). At the end of 28 th day, the degradation of maltene 220 was highest (92 ± 3.7%) and asphaltene was found to be (52 ± 2.2%) for T5. Presence of particulate matter  (Fig. 4).

Figure 4
Degradation rate of TPH

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Kinetic modelling was practiced to evaluate the rate of TPH degradation in the studied systems. The regression 236 analysis data of TPH degradation fitted to both first and second order kinetics. The corresponding R 2 value, k 1 , 237 k 2 , and half-life period (t 1/2 ) for individual treatment were summarized in Table 3. For the treatment T5 the TPH second order kinetics model were higher than those of first order model (Table 3). Therefore, the second order- 248 Table 3 249

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In this current study, the supply of PLE as nutrient amendments minimized the dose of MSM for TPH