Bioremediation of Petroleum Pollutants: Comparison
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Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Vishnu D. Rajput.

The contamination of the soil, agricultural lands, and water bodies with petroleum wastes and other hydrocarbon pollutants has become a serious environmental concern as perceived by the impacts on the aquatic and marine ecosystem. Various investigations have provided novel insights into the significant roles of microbial activities in the cleanup of hydrocarbon contaminants. 

  • bioremediation
  • eco-sustainable biotechnology
  • environmental cleanup

1. Introduction

Petroleum contaminants are the most important pollutants worldwide, and they should be handled effectively to preserve marine lives and the ecosystem. The primary anticipation has been for evaluating the degradability of the toxic chemicals in the presence of the native microbial environment [15,16,17,18][1][2][3][4]. The hydrocarbon-contaminated drill mud waste from different tanks and petroleum waste sludge from refineries depicts the seriousness of the problem [19,20][5][6]. The bioremediation trials were made for the OMW sludge collected from seven long-term evaporation ponds polluted by abundant complex organic compounds [11][7]. The understanding of the associated mechanisms and the courses of action using microbes can guide better approaches for the bioremediation of contaminants. The treatment method proved to be versatile for the degradation of various organic hydrocarbon pollutants, including explosives, pesticides, chlorophenols, and PAHs (Figure 1). Recent works based on the PAH-contaminated aged field soil samples collected from a producer gas manufacturing plant and soil samples from an old diamond mining field proved the feasibility of bioremediation [11,21,22,23,24,25,26,27][7][8][9][10][11][12][13][14]. Many researchers made several trials to remediate generic hydrocarbon-contaminated soil and performed experiments on oily sludge collected from refineries [26][13], using amendment techniques for the pollutant sulfamethoxazole during wetland remediation [28][15]. These efforts are important to understand the impact of bioremediation in the treatment of hydrocarbon pollutants.
Figure 1.
Schematic representation of the bioremediation used to treat hydrocarbon pollutants.

2. Role of Microorganisms in Hydrocarbon Biodegradation

Hydrocarbon degradation can occur by complex mechanisms involving microbial activities associated with the conversion of the complex hydrocarbons to simpler forms (Figure 2). The major pathways by aerobic and anaerobic microorganisms follow enzyme activation and then catalysis to simpler forms in optimized experimental conditions. The Acinetobacter radioresistens strain KA2 was isolated from oily waste sludge and performed two-stage methods. The experiment resulted in removing total petroleum hydrocarbon (TPH) up to 80% in 16 weeks. The technique successfully remediated the crude oil [8,9][16][17]. In another study, A. radioresistens strain KA5 and Enterobacter hormaechei strain KA6 were isolated from petroleum waste sludge (PWS) and two-stage bioremediations conducted for three months have been reported to remove the TPHs by 84% in 16 weeks. Oily sludge (OS) contaminant degraded using a culture-based medium consisting of E. hormaechei strain KA6. The in vessel experiment was conducted for a period of four months, and the rate of TPH removal was found to be up to 80% [26][13].
Figure 2.
The schematic representation of aerobic and anaerobic biodegradation mechanisms.
The rapidly growing bacteria were isolated from heavy oil sludge, including Staphylococcus equorum strain KA4 and E. hormaechei strain KA3. The experiment was performed in a bioreactor for eight + eight weeks to degrade the mineral-based medium, and the TPH removal efficiency was up to 89% [7][18]. The fungal species Fomitopsispinicola, Daedalea dickinsii, and Gloeophyllum trabeum reduced the DDT contamination in the soil through bioremediation significantly. A. radioresistens strain KA5 and E. hormaechei strain KA6 were isolated from petroleum waste sludge (PWS) using 1% crude oil and mineral Bushnell-Haas (BH) medium. The rate of growth of the cells at various intervals was evaluated by measuring the optical density using a spectrophotometer. The strains were identified using the tests, such as catalase, citrate, oxidase, urease, triple sugar iron, nitrate reduction, H2S production, indole production, and gram staining [20][6]. The fungal species Aspergillus ochraceus H2 and Scedosporium apiospermum H16 were isolated from OMW for the in situ method analysis, and microorganisms, such as Proteobacteria (α, β, γ), Actinobacter, Thermobifida, and Streptomyces for effective biodegradation of pyrene, anthracene, phenanthrene, fluorene, naphthalene, acenaphthalene, and PAH contamination [11,13][7][19].
An experimental study using a hydrocarbon-contaminated drill mud waste along with cow bile and bacterial species Brevibacterium casei and Bacillus zhangzhouensi (as indigenous and combined experiments) resulted in TPH removal of up to 90% [19][5]. Similar observations have been summarized in Table 1 and Table 2. These experimental observations and results are important for planning and designing large-scale studies for the bioremediation of hydrocarbons. However, there is a need for physical parameter optimization as well as scale-up analysis.
Table 1.
Petroleum degrading microorganisms isolated from various contaminated sites.

Microorganisms Degrading

Xenobiotics

Isolation Sites
Table 2.
Summary of the efficiency of the removal of hydrocarbons according to potential microbes, substrate (s), and duration details.

Substrate

Microbes

References

Duration

Removal

Efficiency (%)

References

Micrococcus and Pseudomonas

Soil samples contaminated with spent engine oil; from a workshop in Ado-Ekiti

[29][20]

Oily sludge

Acinetobacter radioresistens KA2

Two stage (8 + 8 weeks)

90

[9][17]

Proteus vulgaris SR1

Freshly killed fish samples close to the point of oil spill in the Niger Delta, Nigeria

[

Petroleum waste sludge

30

][21]

Acinetobacter radioresistens KA5, Enterobacter hormaechei KA6

Two stage composting, 12 weeks

84

[20][6]

Pseudomonas sp., Achromobacter sp., Bacillus sp. and Flavobacterium sp.

Soil sample; obtained from a diesel spill region in north-central Alberta, British Columbia

[31][22]

Olive mill wastewater

-

Two stage composting

84

[29][20]

Flavobacterium sp., and Acinetobacterium calcoaceticum

Soil sample; collected from Amanzimtoti, South Africa

Petroleum sludge

Acinetobacter radioresistens KA2

[32][23]

In vessel reactor, two phase composting (8 + 8 weeks)

88

[9][17]

Bacillus coagulans CR31, Klebsiella pneumonia CR23, Klebsiella aerogenes CR21 and Pseudomonas putrefacience CR33

Rhizosphere soil contaminated with spent engine oil in Sokoto, Nigeria

Oily sludge

Enterobacter hormaechei KA6

[33][24]

In vessel experiment, 16 weeks

81

[26][13]

Pseudomonas sp., Acinetobacter sp., Bacillus sp., Corynebacterium sp. and Flavobacterium sp.

Soil samples from auto-mechanic workshops at Mgbukankpor, Nigeria

[34

Contaminated soil

-

][25]

Soil inoculated sewage sludge, wood chips and incubated for 19 months

99

[23][10]

Pseudomonas putida, (Strain G1) and Pseudomonas aeruginosa (Strain K1)

Soil samples from abandoned coal power plant (PHC) at Ijora-Olapa, Lagos

Heavy oily sludge

Staphylococcus equorum KA4, Enterobacter hormaechei KA3

[25][12]

Composting bioreactor (2 phase composting process 8 + 8 weeks)

89

[19][5]

Bacillus sp. S6 and S35

Soil samples from storage centre of oil products in Tehran refinery and Siri Island

Hydrocarbon contaminated drill mud waste

Brevibacterium casei, Bacillus sp.

[35][26]

Composting bioreactor, 6 weeks process

99

[

7

]

[18]

3. Optimization of Bioremediation Conditions

The performance criteria depend on various biotic and abiotic factors, such as microbial populations, aeration status, moisture content, temperature, etc. [36][27]. Further, the selection of a suitable method is significant for efficient bioremediation. There are various sequencing approaches now available to easily identify novel microbes from unique extreme environments [30,37][21][28]. The advancements in genome sequencing have paved the way for rapid microbial identifications and characterization of microbial strains [38,39][29][30].
The right microbial population determines the efficiency of the process. The optimum moisture conditions to be maintained are in the range of 50–55%. The pH value should not be too acidic or too basic. The microbial population is sensitive to these changes. The pH near neutrality is preferable, and a minimum of 40% organic content must be present, while the C/N ratio is also important and should exist below 50 for rapid biodegradation. The temperature should be in the range of 65–70 °C [40][31]. It is to be noted that the use of chemometrics methods can help optimize the conditions for bioremediation and improve the efficiency of the degradation process [41,42][32][33]. By analysing and modelling the relationship between the input variables and the output variables, chemometrics methods can help identify the key factors, such as temperature, pH, and nutrient concentration, that affect the efficiency of bioremediation and optimize the conditions accordingly. This is conducted by monitoring the progress of the biodegradation process by analysing the complex data sets generated, such as the changes in microbial populations and production of the metabolites [43,44][34][35]. Some of the chemometric methods commonly used in the optimization of bioremediation conditions include the design of experiments (DoE), response surface methodology (RSM), artificial neural networks (ANN), principal component analysis (PCA), and genetic algorithms (GA) [45,46][36][37]. Based on the current trends in bioinformatics and data analytics, the applications of chemometrics in bioremediation may give more efficient and cost-effective solutions for the sustainable implementation of bioremediation plans.
The biodegradation process is said to be of two stages, the maturation stage {including the mesophilic phase (25–45 °C) and the thermophilic phase (>45 °C)} and the curing stage (second mesophilic phase). The process also mainly depends upon the mixing ratio because inappropriate mixing leads to the inhibition of target microorganisms [13][19]. These two-stage methods are widely used for petroleum contaminants. For post-diamond mining soil, open-state biodegradation was preferred to remediate the contaminated soil [22][9]. In another approach, in vessel reactors for the bioremediation of petroleum sludge were widely preferred for laboratory experiments [7,9,26][13][17][18]. A lab-scale bioreactor was used for treating the PWS obtained from a petroleum refinery with finished compost of around three kilograms and pre-inoculum as the bulking material [20,26][6][13]. The findings revealed that maximum degradation can be achieved by near neutral pH and the maximum degrading ability possessed by isolated species from PWS compared to indigenous microbes. It was reported that the optimum moisture range is 12–25%, and the biodegradation rate is directly proportional to temperature and pH [12][38]. Another in situ bioremediation process was carried out to degrade the contaminated olive mill waste (OMW) using biowaste and animal waste, along with vermicomposting techniques [29][20]. Their finding reveals that trapezoidal pile methods of vermicomposting are versatile enough to degrade phenol compounds. Similar observations were found from a bioremediation experiment in an evaporation pond using a novel microbial-fungal consortium isolated from OMW [11][7]. For the pyrene-contaminated soil, an additional 14 days in vessel method remediated was required apart from 60 days under the mesophilic and thermophilic conditions. The process degraded various emerging petroleum contaminations, including PAHs, anthracene, phenanthrene, fluorene, naphthalene, and acenaphthalene [13][19]. For a 30-day study, an open vessel method was employed by using cow manure and diamond mining soil and was found to remove up to 78% of contaminants [22][9]. Similarly, a static pile method for the substrate petroleum hydrocarbon and sewage sludge was also performed, and efficient results were obtained [23][10].
An in vessel method using matured compost as bulking material along with oily sludge in a bioreactor was found to degrade the TPHs successfully [26][13]. A bioremediation experiment using a cylindrical bioreactor with heavy oil sludge was reported where finished compost was made of food waste and green waste for four months [7][18]. Since the isolated micro-organisms or microbial consortiums must grow properly to inoculate in the bioreactors or piles or windrows, the method of inoculation depends upon the substrates, contaminants, and prevailing biogeochemical conditions [9][17]. Researchers also inoculated 0.5 Mcfarland isolate solution to the cylindrical bioreactor initially and continued the same bacterial inoculation after eight weeks [7][18]. Abtahi et al. (2020) [20][6] selected two bioreactors for petroleum biodegradation using 1.5 × 108 CFU/g dry mixture inoculum in it. Another study reported the usage of 40 L of produced inoculums (7 × 107 CFU/vol. of material) for the olive mill waste sludge biodegradation [11][7]. Petroleum hydrocarbon-contaminated soil, when inoculated with a mix ratio of microbial consortium, has four species: Pseudomonas poae, Actinobacter bouvetii, Stenotrophomonas rhizophila, and P. rhizosphaerae has resulted in significant biodegradation of hydrocarbons, indicating the significance of microbial consortia in place of single population type [31][22]. The inoculation medium details and culture conditions have been summarized in Table 3.
Table 3.
This table summarizes medium and conditions for bioremediation assays.

Substrate (s)

Medium

Conditions

References

Oil sludge

Bushnell-Haas, 1% Kerosene

150 rpm shaking, 1 week at 35 °C

[26][13]

Heavy oil sludge

Bushnell-Haas, 1% Crude Oil

160 rpm shaking, 1 week at 30 °C

[7][18]

Oily waste sludge

Bushnell-Haas, 1% Crude Oil

160 rpm shaking, 1 week at 30 °C

[7][18]

Petroleum sludge

Bushnell-Haas, 1% Crude Oil

120 rpm shaking, 12 days at 30 °C

[9][17]

Petroleum sludge

Bushnell-Haas, 1% Crude Oil

120 rpm shaking, 12 days at 30 °C

[20][6]

Olive mill sludge

Remazol brilliant blue R (RBBR) plate count agar-tannic acid or potato dextrose agar-tannic acid

Incubation at 30 °C for 48 h (bacteria) and 96 h fungi

[11][7]

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