Heliyon 8 (2022) e08945

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Unravelling the genetic and functional diversity of dominant bacterial

communities involved in manure co-composting bioremediation of complex
crude oil waste sludge
Onyedikachi Ubani a, *, Harrison I. Atagana b, Ramganesh Selvarajan a, c, e, Henry JO. Ogola a, d
a
Department of Environmental Sciences, College of Agricultural and Environmental Sciences, University of South Africa, Florida Campus, Roodepoort, 1709, South Africa
b
Institute of Nanotechnology & Water Sustainability, College of Science, Engineering and Technology, University of South Africa, Florida Campus, Roodepoort, 1709, South
Africa
c
Laboratory of Extraterrestrial Ocean Systems (LEOS), Institute of Deep-Sea Science and Engineering, Chinese Academy of Sciences, No. 28, Luhuitou Road, Sanya,
572000, Hainan Province, PR China
d
School of Agricultural and Food Sciences, Jaramogi Oginga Odinga University of Science and Technology, Bondo, P.O Box 210-40601, Kenya
e
PG Research Department of Microbiology, J.J College of Arts and Science (Autonomous), Sivapuram, Pudukkottai, 622 422, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: The present study aimed to characterize the bacterial community and functional diversity in co-composting mi-
Animal manure crocosms of crude oil waste sludge amended with different animal manures, and to evaluate the scope for bio-
Bioremediation stimulation based in situ bioremediation. Gas chromatography–mass spectrometry (GC–MS) analyses revealed
Bacterial diversity
enhanced attenuation (>90%) of the total polyaromatic hydrocarbons (PAHs); the manure amendments signifi-
Co-compost
Catechol 2,3-dioxygenase
cantly enhancing (up to 30%) the degradation of high molecular weight (HMW) PAHs. Microbial community
analysis showed the dominance (>99% of total sequences) of sequences affiliated to phyla Proteobacteria, Fir-
micutes, Actinobacteria and Bacteroidetes. The core genera enriched were related to hydrocarbon metabolism
(Pseudomonas, Delftia, Methylobacterium, Dietzia, Bacillus, Propionibacterium, Bradyrhizobium, Streptomyces, Achro-
mobacter, Microbacterium and Sphingomonas). However, manure-treated samples exhibited high number and
heterogeneity of unique operational taxonomic units (OTUs) with enrichment of additional hydrocarbon-
degrading bacterial taxa (Proteiniphilum, unclassified Micrococcales, unclassified Lachnospiraceae, Sphingobium
and Stenotrophomonas). Thirty-three culturable hydrocarbon-degrading microbes were isolated from the co-
composting microcosms and mainly classified into Burkholderia, Sanguibacter, Pseudomonas, Bacillus, Rhodo-
coccus, Lysinibacillus, Microbacterium, Brevibacterium, Geobacillus, Micrococcus, Arthrobacter, Cellulimicrobacterium,
Streptomyces Dietzia,etc,. that was additionally affirmed with the presence of catechol 2,3-dioxygenase gene.
Finally, enhanced in situ degradation of total (49%), LMW (>75%) and HMW PAHs (>35%) was achieved with an
enriched bacterial consortium of these microbes. Overall, these findings suggests that co-composting treatment of
crude oil sludge with animal manures selects for intrinsically diverse bacterial community, that could be a driving
force behind accelerated bioremediation, and can be exploited for engineered remediation processes.

1. Introduction biodegradation and are thus recalcitrant in the environment under

normal conditions. Additionally, COWS and its components are sub-
Globally, crude oil is an essential and important strategic natural stantially cytotoxic, mutagenic and carcinogenic, especially polycyclic
energy resource for many anthropogenic activities. However, it is asso- aromatic hydrocarbons (PAHs) [1, 2]. Environmental pollution with
ciated with generation of large amounts of waste from its extraction and COWS has also been linked to physical and chemical changes of natural
processing such as crude oil waste sludge (COWS). Chemically, COWS is habitats, with potential lethal and sub-lethal toxic effects on aquatics and
composed of low and high molecular weights polycyclic aromatic hy- terrestrial ecosystem [3]. As consequence, COWS has been classified as a
drocarbons (LMW/HMW-PAHs) with characteristic strong molecular hazardous organic contaminant that must be treated before discharge
bonds and hydrophobicity. These properties make them less amenable to into environment [4, 5].

* Corresponding author.
E-mail address: onyedika.ubani@gmail.com (O. Ubani).

https://doi.org/10.1016/j.heliyon.2022.e08945
Received 9 June 2021; Received in revised form 7 January 2022; Accepted 9 February 2022
2405-8440/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
O. Ubani et al. Heliyon 8 (2022) e08945

The challenges confronting oil refineries and petrochemical in- individually or in consortia, of adapted endogenous bacterial populations
dustries to meet the regulatory requirements has given rise to research (mesophilic, thermophilic and maturation bacteria) isolated from various
interest on safe disposal and treatment technologies for crude oil wastes manure co-compost piles with COWS was evaluated using cultural, mo-
sludge [3]. Amongst the currently available methods, bioremediation lecular and high throughput deep sequencing of 16S rRNA genes. Thus, the
technologies, involving the use of microorganisms to degrade crude oil study elucidated a detailed composition of bacterial community residing in
waste sludge stands out as a greener approach. This is attributed to their co-composting pile mixtures of crude oil sludge and animal manures and
cost-effective and less disruptive nature to the environment. These explored the scope for co-composting bioremediation of COWS.
technologies focus on improving microbial growth and metabolic activity
that subsequently activates the oxidation-reduction of the contaminants 2. Materials and methods
into simple harmless products such as water and CO2 [6, 7, 8, 9]. How-
ever, there is emerging paradigm that bioremediation techniques are 2.1. Composting experiments
scientifically intense procedures, owing to the inherent nutrient defi-
ciency and recalcitrance of HMW- PAHs in crude oil sludge. This may Crude oil waste sludge was collected from an oil refinery company in
seriously impede the catabolic activities of indigenous microorganisms Durban, KwaZulu-Natal, South Africa, and its composition characterized
and limit the rate of intrinsic bioremediation [10]. Consequently, these using automated Soxhlet extraction with dichloromethane and gas
techniques must optimize both intrinsic and environmental conditions to chromatography/mass spectrometry (GC/MS) as described Haleyur et al.
promote both microbial growth and bioremediation efficiency to be [20]. The typical PAH composition of the COWS used in this study
tailored for specific applications and sites. included: 98.2 mg/kg naphthalene; 6.0 mg/kg acenaphtylene; 9.2 mg/kg
Engineered bioremediation strategies, involving amendment of oil acenaphthene; 27.5 mg/kg fluorene; 14.9 mg/kg phenanthrene; 41.6
sludge-contaminated sites with suitable nutrients (N and/or P) to improve mg/kg anthracene; 2.4 mg/kg fluoranthene; 14.1 mg/kg pyrene; 4.1
in-situ microbial growth and activity to expedite PAHs bioremediation, has mg/kg benzo[a]anthracene; 54.8 mg/kg chrysene; 23.7 mg/kg benzo[b]
been demonstrated [11]. In addition, we have also previously reported fluoranthene; 2.6 mg/kg benzo[k]fluoranthene; 10.0 mg/kg benzo[a]
that co-composting with animal manure have great potential application pyrene; 5.1 mg/kg perylene; 10.1 mg/kg indenol(1,2,3-cd)pyrene; 11.6
to decontaminate sites heavily contaminated with PAHs and crude oil [11, mg/kg dibenzo[a,h]anthracene; 9.4 mg/kg benzo[ghi]perylene; and 3.9
12, 13]. In this technique, the introduction of organic matter improves the mg/kg benzo[e]acephenathrylene.
nutrient availability and aeration, in addition to introducing ex situ mi- Cow (CM), pig/swine (SM), horse (HM), and poultry (PM) manures
crobes to improve bioremediation of contaminants [14, 15, 16]. The were collected from the University of Pretoria farm, Onderstepoort,
resultant microbial activities induce elevated temperatures, which im- Pretoria, South Africa. These manures were characterized for total
proves the solubility of contaminants and the microbial co-metabolism organic carbon (TOC), nitrogen (TN), phosphorus (TP) content using
that degrades and transforms pollutants into humus and inert products standard methods as described previously [21]. Garden soil was also
as the compost mature. These features have made co-composting poten- collected, homogenized, air-dried and analysed to determine the soil
tially popular bioremediation method, coupled with its ability to degrade type, TOC, TN, TP, pH and metal content [22]. Metals in the COWS and
of large quantities of organic pollutant at low cost with minimal envi- soil samples were quantified using PerkinElmer Optima 5300 DV
ronmental disruption. The potential to integrate of co-composting with inductively coupled plasma optical emission spectroscopy, ICP-OES
other physical or chemical techniques to achieve a better and efficient (PerkinElmer Inc., Massachusetts, USA) after aqua-regia (1/3
biodegradation outcome has further increased interest in the technique. HNO3–HCl, v/v) digestion as described by Sibanda et al. [23]. The
Several research studies have reported the involvement of various average values for the physicochemical characteristics of manures and
and complex groups of in-situ aerobic and anaerobic bacteria and archaea soil used for the microcosms experiments is summarized in Table 1.
for hydrocarbons biodegradation and associated nutrient recycling pro-
cesses in crude oil sludge contaminated environments [10, 17, 18]. The
key roles played by niche-specific guilds of known hydrocarbon utilizing Table 1. Characteristics of animal manures and soil used for the microcosm
experiments.
aerobic/facultative anaerobic (Mycobacterium, Pseudomonas, Longilinea,
Geobacter, etc.), nitrate reducing (Gordonia, Novosphigobium, etc.) and Parameter Manures Garden
soil
nitrogen fixing (Azovibrio, Rhodobacter, etc.) bacteria with strictly PM CM HM SM
anaerobic, fermentative, thermophilic, sulfate-reducing bacteria (Cop- Total organic 49.2
14.2 54.9
5.9 52.7
2.7 50.6
5.9 13.01
rothermobacter, Fervidobacterium, Treponema, Syntrophus, Thermodesulfo- C (%)
vibrio, Anaerolinea Syntrophobacter, Anaerostipes Anaerobaculum) and Total N [mg/L] 277
63 109
8 81
3 104
84 3.94
methanogenic archaea (such as Methanobacterium, Methanosaeta, Ther- Total P [mg/L] 254
14 46
8 50
2 252
29 4.4
moplasmatales, etc.) in situ biodegradation technologies have been re- pH 5.56
ported [17, 19]. In contrast, the in-situ and ex-situ microbial communities’ Moisture (%) 9.52
diversity, and their metabolic capability and perturbations in community Dry matter (%) 90.48
composition under the co-composting environmental conditions (such as Texture Sandy
nutrient availability, temperature, pollutant surface area, oxygen con- loam
tent, pH, salinity, oil composition, and many more), are not yet well Sand (% wt) 61.3
described. Therefore, to develop a co-composting as a tailored biore- Silt (% wt) 21.3
mediation technology, elucidation of a detailed composition of microbial Clay (% wt) 9.3
community diversity and dynamics is essential. Cr (mg/kg) 121.7
In this study, a microcosm-based culture dependent and culture- Pb (mg/kg) 31.9
independent metagenomic method was adopted to elucidate on the na- Ni (mg/kg) 10.13
ture of autochthonous microbial community structure and dynamics
Cu (mg/kg) 38.08
within co-composting of COWS with different animal manures. Specif-
Zn (mg/kg) 9.65
ically, the effect of co-composting with pig/swine manure (SM), cow
Mn (mg/kg) 92.38
manure (CM), horse manure (HM) or poultry manure (PM) on biodegra-
Fe (mg/kg) 67.04
dation potential as well as change in native prokaryotic diversity compo-
Co (mg/kg) 2.45
sition of crude oil sludge was analysed using high throughput sequencing
Mg (mg/kg) 22.37
of 16S rRNA genes. Further, the identity and degradation potential, either

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O. Ubani et al. Heliyon 8 (2022) e08945

For each co-composting treatment, 300 g COWS was initially dis- chemistry along with its multiplex sample identifiers on the Illumina
solved in 400 ml of tetrachloromethane (CCl4, 99.55%, molar mass MiSeq Platform (Illumina Inc., San Diego, CA, USA) at the University of
153.81 g/mol, purchased from Merck Pty, South Africa), then added to 1 South Africa according to standard protocol.
kg of garden soil. The resultant soil-oil sludge mixture (SSM) was mixed
to a homogenous slurry, before being air-dried at room temperature to 2.3.2. Bioinformatic analyses
evaporate excess CCl4. The amended soil was mixed with wood chips in a Raw sequences were initially screened for PCR artefacts and low-
ratio of 1:2 (w: v). For composting experiments, SSM þ wood chips quality reads using ngsShoRT (next generation sequencing Short
mixture was separately mixed with each SM, CM, HM and PM manures in Reads) trimmer [28], before being analyzed using Mothur v1.25 pipeline
a ratio of 2:1 (w:w). A portion of SSM þ wood mixture with no manure [29]. Chimeric sequences were removed using UCHIME algorithm [30].
supplementation was used as the control (CT). All treatments were Quality filtered non-chimeric reads were used for closed-reference
incubated under laboratory conditions (25
5  C, 65
12% relative picking and taxonomy assignation of Operational Taxonomic Units
humidity) for a period of 10 months in triangular PVC troughs (OTUs) based on the SILVA SSU database release 132 (https://www.arb
(measuring 22 cm (length) x 9.2 cm (Depth) x 20 cm (width)) with -silva.de/download/arb-files/), with the similarity threshold set at 0.97.
openings on the lids and sides for aeration. All treatments were replicated The dominant OTUs at different taxonomic levels were used to
three times. generate stacked bar charts and heatmap using ggplot2 [31] and heat-
During the composting experiments, temperature changes and mois- map.2 packages [32] in R version 3.6.1 [33], respectively, to visualize the
ture content were monitored periodically. Water was added to the variations and distributions of bacterial communities. Alpha diversity
compost mixture when necessary to maintain moisture level between 60- indices were calculated at the genetic distance of 0.03 using the plo-
80%. pH changes and carbon dioxide evolution, used to monitor mi- t_richness function of phyloseq [34]. β-diversity based Bray-Curtis
crobial activities, was also measured monthly using the closed jar method dissimilarity distance and canonical correspondence analysis (CCA) to
as described in previous studies [24, 25]. At the end of composting, visualize the community relationships between and within each com-
samples were collected from composts mixture for residual PAH analysis, posting treatment with explanatory environmental variables was also
in-situ PAH-degrading bacteria isolation and characterization, and met- performed using vegan package [35].
agenomic analysis. All samples for metagenomic analysis were stored at
-80  C until analysed. 2.4. Culture dependent microbiological analyses

2.2. Residual PAH analysis 2.4.1. Isolation of crude oil degrading bacteria
Samples consisting of 15 g of compost piles with COWS and different
Residual PAH-concentration from compost samples was recovered by manures and control were suspended in 100 ml sterile mineral salt media
extraction using automated Soxhlet technique based on EPA method (MSM) supplemented with 10 ml of crude oil sludge as sole source of
3541 [26] and dichloromethane (99.5%, Sigma-Aldrich) as solvent carbon in 250 mL conical flask. The MSM stock contained in 1 L solution:
[20]. Briefly, 10 g of compost samples was transferred into a cellulose 500 mg KH2PO4, 500 mg MgSO4. 7H2O, 500 mg NaH2PO4.H2O, 500 mg
thimble and subjected to Soxhlet extraction. For each sample, a triplicate NH4Cl, 4000 mg NaCl, 500 mg NaHCO3, 500 mg Na2CO3 and 1 mL trace
was prepared and extracted. The PAHs present in the extracts was element mix. Trace element mix contained in mg L1: 1500 mg
quantified on GC/MS Agilent 7860GC system and 5975C MSD, equipped FeCl2.H2O, 9000 mg NaCl, 197 mg MnCl2. 4H2O, 900 mg CaCl2, 238 mg
with a 7683B autosampler (Agilent Technologies Inc., California, USA). CoCl2.H2O, 17 mg CuCl2.H2O, 287 mg ZnSO4, 50 mg AlCl3, 62 mg
The column used was Agilent HP-5 MS ultra-inert 30 m  0.25 mm x 0.25 H3BO3, 24 mg NiCl2.6H2O, filter-sterilised through 0.2 μm Millipore
μm film thickness (Agilent Technologies Inc., California, USA) and the filter membrane. The flasks were incubated in the dark at 28  C on a
GC-MSD conditions used for quantification were based on the optimised rotary shaker at 150 rpm for 21 days. At the end incubation, 1 mL ali-
method described by Agilent Application Note (https://www.agilent.co quots of the enrichment cultures were aseptically transferred into new
m/cs/library/applications/application-optimized-gc-ms-analysis-for- 250 mL flasks containing 100 ml sterile MSM spiked with 10 mL crude oil
PAHs-in-challenging-matrices-8890-5977b-single-quadrupole-gc-ms- sludge, and again incubated for another 21 days at 28  C on a rotary
5994-0499en-agilent.pdf). Prior samples analysis, the GC-MSD was shaker in the dark. Crude oil-degrading bacteria were isolated from the
calibrated with 100 μg/mL PAH standards (Sigma Aldrich Ltd). enrichment cultures by serial dilutions (103-108) and spread on min-
The percent PAH degradation was calculated as follows: eral salts agar (MSA) plates supplemented with 1% crude oil sludge. The
plates were incubated for 21–28 days at 28  C in the dark.
ð½Initial PAH  ½Final PAHÞ Distinct colonies were purified by streaking several times on nutrient
Percentage PAH degradation ¼  100
½Intial PAH agar plates to obtain pure single colonies. The pure colonies were again
screened for their ability to grow and utilise crude oil by streaking on
2.3. Targeted 16S rDNA amplicon sequencing MSA plates overlaid with 1.5% oil sludge, and the plates incubated at 37

C for 3–7 days. All the positive isolates were sub-cultured on nutrient
2.3.1. DNA extraction, library preparation and sequencing broth at 28  C for three days and the culture used for DNA extraction for
Fifteen grams (15 g) of compost piles were suspended in 50 mL identification, catechol 2,3-dioxygenase gene screening, PAH biodegra-
phosphate buffered saline (PBS) overnight, homogenised and centrifuged dation screening with 2,6-dichlorophenol indophenol (2,6-DCPIP) test
at 12,000 rpm for 5 min at 4  C. The supernatants were subjected to total and bacterial consortia development for COWS degradation.
DNA extraction using the Faecal/Soil Total DNA™ extraction kit (Zymo
Research Corporation, CA, USA), according to the manufacturer's pro- 2.4.2. PAH biodegradation screening test
tocol. The extracted DNA having A260:A280 ratios between 1.8–2.0 and Cell growth and PAH degradation ability of isolates that exhibited
concentrations of 20–150 ng/μL. The extracted DNA were amplified hydrocarbonoclastic activity (utilising oil sludge as sole carbon source)
following a two-step PCR method; firstly using 16S rDNA 27F (50 - were further checked by rapid colorimetric test based on 2,6-dichloro-
AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -TACGGY- phenol indophenol (2,6-DCPIP) reduction [36]. Each isolate was
TACCTTGTTACGACTT-30 ) primers to cover the whole variable region; cultured in Bushnell Hass (BH) broth for 24 h at 37  C while being shaken
and secondly to cover the V1–V3 region using 27F and 518R primer pairs at 180 rpm. After 24 h, the culture was supplemented with a sterile
with adapter sequences that are compatible with Illumina index as mixture of 0.5% (w/v) 2,6-dichlorophenol indophenol (2,6-DCPIP),
described by Selvarajan et al. [27]. The resultant PCR were subsequently 0.1% Tween 80 and 3% (v/v) of crude oil sludge and further subcultured
purified then sequenced by paired end (300 bp reads) sequencing v.3 for 7 days at 28  C. The degradation of crude oil sludge was monitored

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O. Ubani et al. Heliyon 8 (2022) e08945

daily by colour change from blue to colourless and finally spectropho- on nutrient broth overnight at 37  C. After 24 h, 200 μL of each enriched
tometrically at 600 nm at the end of the culturing [37]. were added simultaneously to 100 mL MSM broth supplemented with 5%
Percentage biodegradation was calculated by: crude oil waste sludge and the media incubated at 28  C for 24 h at 120
  rpm for 30 days. The experiment was performed in duplicate, and un-
A600nmðTreatmentÞ inoculated flasks considered as controls. After 30 days, 5 mL of the
%degradation ¼ 1   100
A600nmðControlÞ bacterial consortia was resubcultured on fresh 100 mL MSM containing
5% crude oil waste sludge for 24 h at 28  C while shaking at 120 rpm for
2.4.3. 16S rRNA and catechol 2,3-dioxygenase (C23O) gene profiling 30 days. Similar experiments were performed using pyrene and anthra-
DNA was extracted from pure bacterial isolates using the Quick g- cene as the sole carbon source. Using GC-MSD, the residual concentra-
DNA Extraction Kit™ (Zymo Research Corporation, CA, USA) according tions of the crude oil sludge and its constituent PAHs was determined.
to the manufacturers' instruction and stored at 20  C prior to further The total DNA of the enriched bacterial consortium at the end of the
analysis. PCR amplification of the whole variable region of bacterial 16S experiments was also extracted and subjected to Illumina Miseq
rRNA was done using the forward primer 27F (50 -AGAGTTT- sequencing to establish the bacterial community diversity and composi-
GATCCTGGCTCAG-30 ) and the reverse primer 1492R (50 -TACGGY- tion as described in subsection 2.3.1 and 2.3.2.
TACCTTGTTACGACTT-30 ). Each 25 μL reaction volume contained 0.5
μM of each primer, 1X OneTaq® Hot Start Master Mix (New England 2.6. Data availability
Biolabs, Ipswich, MA, USA) and 20 ng DNA. PCR was performed under
following cycling conditions (95  C, 5 min; 32 x [95  C, 1 min; 55  C, 1 All the raw datasets from Illumina sequencing have been deposited at
min; 72  C, 1 min]; 72  C, 7 min; 4  C, ∞), and resultant amplicons the NCBI database (https://www.ncbi.nlm.nih.gov/) sequence archive
checked on a 1.5% agarose gel. The resultant PCR products were purified (SRA) as BioProject ID PRJNA794053. The 16S rDNA sequences were
with ZR DNA Clean and Concentrator Kit (Zymo Research Corporation, also deposited at the NCBI GenBank database under accession numbers
CA, USA) according to the manufacturer's instructions and sequenced an MK854826 - MK854993. The data analysis results obtained during this
ABI-3730 DNA Analyzer (Inqaba Biotech, Pretoria, South Africa). All the study are included in the manuscript.
16S rRNA sequences were checked and edited with BioEdit software to
manually correct the chromatograms obtained from the Sanger 3. Results
sequencing. Prior to constructing phylogenetic tree, sequences and their
top BLAST hit in NCBI database were aligned using CLUSTAL-W. 3.1. Changes in physicochemical conditions during co-composting
Phylogenetic tree was then constructed using the maximum likelihood
(ML) algorithm in MEGA7 as described previously [38]. Changes in temperature, CO2 evolution (respiration rate) and pH are
To further characterize the crude oil sludge degrading bacteria, PCR presented in Figure 1. Overall, higher temperature and respiration rates
amplification of the extradiol ring-cleavage catechol 2,3-dioxygenase were recorded in all the composts piles amended with manure than the
(C23O) was performed using specific primers C23OF (50 -AAG AGG control treatment (CT). However, PM showed a higher temperature than
CAT GGG GGC GCA CCG GTT CGA-30 ) and C23OR (50 -TCA CCA GCA other manure-amended treatments, recording peak mean temperature of
AAC ACC TCG TTG CGG TTG CC-30 ) in a 25 μl reaction mixture [0.5 μM (27.3
0.6  C) after 1 month, before fluctuating to 25.2
0.3 and 23.0

of each primer, 1X OneTaq® Hot Start Master Mix (New England Biolabs, 0.1  C after 5 and 10 months of co-composting, respectively (Figure 1a).
Ipswich, MA, USA) and 20 ng DNA]. The PCR conditions included: 1 In contrast, CM and CT recorded the least temperature changes during
cycle at 98  C for 10s, then 34 cycles at 98  C for 1s, 55  C for 1 min and composting. Similarly, PM exhibited significantly higher CO2 evolution
72  C for 15s. Then, a final elongation stage at 72  C for 1 min. The (~18 μg/dry weight/day) after 5 months of composting, with other
resultant PCR fragment size (912 bp) spanning the open reading frame manure compost piles (HM, CM and SM) recording moderate CO2 evo-
(ORF) of the C23O gene [39], was visualized by agarose gel electro- lution (~10 μg/dry weight/day) during the same period (Figure 1b). The
phoresis. E coli DHα lacking the ability to utilise PAHs in crude oil sludge control treatment (CT), exhibited relatively lower CO2 evolution values
was used as a negative control. during the whole co-composting period. These results showed that
manure treatments accelerated the rate of temperature and respiration
2.5. Crude oil sludge and PAHs degradation by bacterial consortia rate increase.
During the composting, the pH in PM, CM, SM, HM and CT micro-
A consortia consisting of 34 bacterial isolates exhibiting crude oil cosm increased from 5.9-7.9, 5.8–7.6, 5.6–7.8, 5.6–7.7 and 5.6–6.8,
waste degrading ability was prepared by initially culturing each colony respectively (Figure 1c). There was an overall trend of slight increase in

Figure 1. The changes in physicochemical properties during 300 days co-composting treatments of crude oil waste sludge with different manures. (a) Temperature,
(b) respiration rate and (c) pH. Soil-crude oil sludge mixture (SSM) þ wood chips amended with poultry manure (PM); horse manure (HM); cow manure (CM); pig
manure (SM), and no manure amendment (CT).

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pH for all treatments during the first five months, before fluctuating to Overall, 36.5–99.9% PAH reduction levels were achieved under the
pre-composting values after 8 months. The only exception was PM composting conditions with reduction efficiency depending on the
treatment that exhibited a sharp increase in pH to 7.9 in the eighth compost treatment and the molecular weight of the PAHs. Compost
month, before finally fluctuating to pH 6.1 at the end of composting (10 treatments, irrespective of the manure type, resulted in comparatively
months). higher reduction of total PAHs (ΣPAHs), LMW- and HMW-PAHs than in
control samples. Manure microcosms (PM, HM, SM and CM) had >300
3.2. Crude oil waste sludge PAHs reduction during composting mg/kg soil ΣPAHs reduction compared to control (CT) 175.8 mg/kg soil.
HMW-PAHs such as perylene, dibenzo[a,h]anthacene, benzo[ghi]per-
GC/MS analysis identified 18 PAHs in crude oil waste sludge ranging ylene and benzo[e]acephenathrylene exhibited lower reduction
from low- (LMW) to high molecular weight (HMW) compounds. The (39.7–81.7%) (Figure 2b), whereas reduction levels up to 99.9% was
average reduction levels of each PAH in the samples after composting achieved for LMW-PAHs such as naphthalene, acenaphthene, anthra-
with different manure amendment is presented in Figure 2. cene, fluorene and phenanthrene (Figure 2a).

Figure 2. The average reduction of selected crude oil sludge PAHs in poultry (PM), horse (HM), cow (CM), pig (SM), and Control (CT) compost mixtures after 300
days. Degradation of low molecular weight (LMW-PAHs; 2–3 rings) (a) and high molecular weight (HMW-PAHs; 4–6 rings) (b). Error bars represent the standard error
of the mean of triplicate microcosms.

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3.3. Bacterial community diversity during co-composting of crude oil waste

sludge

Summary of the sequencing outputs and diversity indices for bacterial

communities in co-composting experiments is presented in Table 2.
Overall, a total of 125,972 high quality reads (ranging from 15,377 to
40,129) with an average read length of 527 bp were obtained based on
16S rDNA amplicon sequencing analysis. Good's coverage across the
samples was >98.5%. This indicated that the sampling depth was suffi-
cient to estimate the microbial diversity enclosing all major bacterial
groups involved in co-composting of crude oil waste sludge. This was
further supported by the rarefaction curves (Figure 3) that asymptotically
approached a plateau, suggesting that the sampling depth accurately
reflected the bacterial communities. Comparatively, higher species
richness estimates (OTUs and Chao-1) were observed for PM and CM
samples than HM and SM treatments (Table 2). Additionally, Chao-1
index revealed lower species richness in CT samples, whereas higher Figure 3. Rarefaction curves indicating the observed number of OTUs within
bacterial diversity (Shannon_H) was observed for HM followed by PM, the 16S rRNA gene sequences of crude oil sludge samples composted using
with SM and CM exhibiting comparable values to CT samples. different manures. OTUs are shown at the 3% genetic distance levels.

3.4. Distribution of taxa and phylotypes in co-composted samples and 4.0% relative abundance, respectively (Figure 4a). Another notable
observation was detection of members of order Streptomycetales (11.7%
In total, 17 phyla, 42 classes, 83 orders, 162 families and 359 genera relative abundance) in PM treatment only. On the other hand, members
were detected in the study. OTUs assigned to the phylum Proteobacteria, of the order Acidithiobacillales were highly enriched in CM (80%). In
Firmicutes, Actinobacteria and Bacteroidetes were dominant taxa, ac- contrast, order Pseudomonadales was relatively more abundant in most
counting for >99% of sequences across the co-composting treatment co-composting treatments including control samples, but very low (3%
(Figure 4a). However, subtle variation in the relative distribution of relative abundance) in CM. Other important orders detected included
bacterial phyla in each co-composting treatment was discernible. Rhizobiales, Burkholderiales, Xanthomonadales, Caulobacterales, and
Comparatively, members of Proteobacteria were highly enriched in HM Enterobacteriales (only detected in SM and CT) (Figure 4c). Figure 4d also
(99%), PM (90%), CT (85%), SM (50%). CM was dominated by Actino- shows that members of order Clostridiales accounted for 90% of Firmicutes
bacteria (35%) and Firmicutes (45%) with members of phyla Proteobac- taxa in SM, whereas Bacillales was dominant in all samples with excep-
teria accounting for 20% of the detected sequences (Figure 4a). In tion of SM. However, only members of Bacillales, Lactobacillales and
contrast, Bacteroidetes were only detected in SM (20%) and PM (5%). Selenomonadales (10% only detected in CT) were detected in CT sample.
Other minor phyla detected included Verrucomicrobia, TM6_(Depende- At genus level, Pseudomonas, Rhodanobacter, Proteiniphilum, Entero-
tiae), Acidobacteria, Saccharibacteria, Tenericutes, Spirochaetae, Cyano- bacter, Achromabacter, Delftia, Bacillus, and Methylobacterium were
bacteria, BRC1 and Chloroflexi. At class level, γ-proteobacteria, dominant groups (Figure 5a). Overall, the bacterial community profiles
α-proteobacteria, Actinobacteria, β-proteobacteria, Clostridia, Bacilli, of the main genera were clustered into three groups, whereby the bac-
Bacteroidiia and Sphingobacteriia were the dominant groups in all terial community structures in PM and HM, SM and CT differed
samples. remarkably from CM. Comparatively, genera Pseudomonas (49.0%),
The relative distribution of the bacterial orders belonging to the top Rhodanobacter (22.6%), Achromobacter (10.7%), unclassified Rhizobiales
three phyla is illustrated in Figure 4b, c and d. Generally, the relative (4.6%), Marinilabiaceae_ge (2.2%) and Propionbacterium (1.4%) were
abundance of each bacterial taxonomic group varied among the co- highly enriched in PM, whereas Pseudomonas (70.2%), Achromobacter
composting treatments. Order Propionibacteriales and Micrococcales was (12.0%), Stenotrophomonas (4.5%), Sphingobium (3.2%), Pseudox-
highly enriched in most co-composting treatments (SM, CT, PM and HM) anthomonas (2.3%), Delftia (1.2%) Massilia (1.2%) and Methylobacterium
accounting for >45 and >19% relative abundance, respectively, of (1.1%) were the main genera in HM. By contrast, genera Bacillus (17.4%),
Actinobacteria detected. Interestingly, CM was enriched with Cor- KCM-B-112_ge (11.3%), Nocardiopsis (7.6%), Gordonia (4.0%), Clos-
ynebacteriales (67.4%) and Streptosporangiales (23.1%), whereas members tridium sensu stricto_1 (3.0%), Mycobacterium (2.4%), Corynebacterium
of the orders Propionibacteriales and Micrococcales were detected at 0.7 (2.2%) and Rhodococcus (1.8%) were enriched in CM treatment.

Table 2. Summary of sequencing outputs and diversity indices for bacterial communities in composting experiments of crude oil sludge using different manures.a

Indicesy PM HM CM SM CT
OTU 119 69 179 47 44
Target reads 26,884 25,972 15,377 40,129 17,610
Dominance_D 0.29 (0.282–0.291) 0.51 (0.503–0.518) 0.13 (0.131–0.138) 0.12 (0.119–0.122) 0.11 (0.113–0.119)
Simpson_1-D 0.71 (0.709–0.718) 0.49 (0.482–0.497) 0.87 (0.862–0.0.869) 0.88 (0.878–0.881) 0.88 (0.881–0.887)
Shannon_H 1.91 (1.885–1.926) 1.27 (1.253–1.292) 2.83 (2.809–2.865) 2.65 (2.639–2.664) 2.76 (2.737–2.774)
Evenness_e^H/S 0.06 (0.055–0.058) 0.05 (0.051–0.053) 0.10 (0.098–0.098) 0.30 (0.298–0.305) 0.36 (0.351–0.364)
Chao-1 119 (119.2–128.2) 69 (69.3–80.0) 180 (180.8–196.3) 47 (47–48) 44(44–47)
Good's coverage (%) 99.7 98.5 99.2 98.6 99.0
a
PM – poultry manure; HM – horse manure; CM – cow manure; SM – swine/pig manure; and CT – control (CT ¼ enrichment sample having no manure
supplementation).
y
Chao-1, community richness-higher number represents more richness; Shannon_H, community diversity-higher number represents more diversity; coverage,
sampling depth; OTUs, Operational taxonomic units.

6
O. Ubani et al. Heliyon 8 (2022) e08945

Figure 4. Comparative taxonomic dis-

tribution of bacterial communities in
crude oil sludge composting treatment
with different manures. Diversity at
phylum level (a) and the dominant or-
ders in the three most abundant bacte-
rial phyla is illustrated (b-d). The
classification of the dominant bacterial
orders belonging to phylum Proteobac-
teria into α-, β- and γ-class lineage is
shown in brackets (c). Soil-crude oil
sludge mixture (SSM) þ wood chips
amended with poultry manure (PM);
horse manure (HM); cow manure (CM);
pig manure (SM), and no manure
amendment (CT).

3.5. Venn diagram analysis of the variations in taxonomic groups treated compost was 4.1  103 CFU/g, with PM and CT yielding the
highest (389,089 CFU/g) and lowest counts (1,040 CFU/g), respectively.
According to Venn diagrams consistent overlap patterns of genus A total of 211 bacterial colonies were picked from MSA plates supple-
clusters among different manure-treated co-composting and control mented with crude oil waste sludge as a sole C-source, and were identi-
treatment of crude oil waste sludge were obtained (Figure 5b). The fied based on partial 16S rDNA sequence to putatively belong to 31
shared OTUs between manure-treated and control samples were 34 phylotypes based on OTUs. A heatmap showing the relative abundance of
(12.3%), mainly assigned to genus Pseudomonas, Delftia, Methyl- the bacterial phylotypes recovered from the different manure and control
obacterium, Dietzia, Bacillus, Propionibacterium, Bradyrhizobium, Strepto- samples after 10 months composting of crude oil waste sludge is pre-
myces, Achromobacter, Microbacterium and Sphingomonas. However, sented in Figure 6.
manure-treated had the highest unique OTUs with 233 (84.4%), while Overall, phylotypes belonging to genera Bacillus, Lysinibacillus,
CT had 9 (3.3%). The major unique genera in manure-treated samples Microbacterium, Burkholderia, Dietzia, Rhodococcus, Pseudomonas and
included Proteiniphilum, unclassified Micrococcales, unclassified Lachno- Paeniclostridium were recovered in high frequency (>4% relative abun-
spiraceae, Sphingobium and Stenotrophomonas, whereas Afpia, Klebsiella dance) across all samples. However, only five isolates belonging to 4
and Burkholderia-Paraburkholderia were dominant unique genera in CT. genera: Bacillus; Microbacterium; Rhodococcus; and Paenibacillus were
The Venn diagram of the shared and unique microbiome in manure- recovered in CM. In contrast, members of genera Burkholderia, Bacillus,
treated samples is presented in Figure 5c. Manure-treated compost Gordonia, Enterococcus, Geobacillus, Sanguibacter and Bhargavaea were
treatments shared 11 OTUs (4.1%), with HM, CM, SM and PM having 27, isolated from control (CT) samples. Overall, higher species diversity and
96, 8 and 43 unique OTUs detected, respectively. number of isolates (n ¼ 119) were recovered in PM samples, with phy-
lotypes belonging to genera Dietzia, Burkholderia, Arthrobacter, Lysiniba-
3.6. Isolation of culturable bacteria from co-composting samples cillus, Sporosarcina, Staphylococcus, Sanguibacter, Bhargavaea Rhodococcus
and Streptomyces were unique to PM samples only (Figure 6). In contrast,
To augment metagenomic-based studies, axenic culture studies were HM, SM and CT exhibited moderate species diversity among the isolates,
performed. The average number of culturable bacteria for manure- with CM reporting the least.

7
O. Ubani et al. Heliyon 8 (2022) e08945

Figure 5. Relative taxonomic distribution of genera and common microbiome diversity in the composting treatment of crude oil sludge. a) Heatmap of the normalized
abundance at the genus level for bacteria in the six microcosm metagenomic sequences. Colour code based on higher (yellow) or low (black) relative abundance in
metagenomes (see scale on the top left). Venn diagrams representing unique and shared bacterial OTUs between manure-amended and control treatments (b) and
within the four manure-treated samples (c).

3.7. PAH biodegradation and cell viability of bacterial isolates within a the crude oil sludge could be phylogenetically grouped into 4 clades
microcosm setup (Figure 7). Clade I consisted members of order Micrococcales (genus
Microbacterium, Brevibacterium, Geobacillus, Micrococcus, Arthrobacter,
In order to perform PAH-degrading tests, 93 putative hydro- Sanguibacter and Cellulimicrobacterium) belonging to the phylum Actino-
carbonoclastic bacterial isolates were chosen based on colour and colony bacteria. These isolates were recovered from all treatments, including the
morphology differences. A microcosm-based strategy was implemented control (CT) samples, with exception of swine/pig manure (SM). In clade
to measure the ability of the axenic cultures to grow, utilise and degrade II, six isolates belonging to orders Streptomycetales (genus Streptomyces)
target PAHs, using crude oil waste sludge as the sole carbon or energy and Corynebacteriales (genus Dietzia, Rhodococcus, and Gordonia) within
source. The activity was screened colorimetrically using redox indicator the phylum Actinobacteria. In contrast, Clade III were mostly members of
2,6-dichlorophenol indophenol (2,6-DCPIP). The ability of each axenic th phylum Firmicutes. The bacterial genus Bacillus and Lysinibacillus were
culture to degrade the crude oil waste sludge was considered to be pro- the most recovered group in this study. Others included Clostridium,
portional to the decolourization of DCPIP incorporated into the growth Enterococcus, Staphylococcus, Sporosarcina and Bhargavaea. In clade IV,
media. included isolates members of class Betaproteobacteria (genus Bur-
Axenic cultures of thirty-three isolates were qualitative positive for kholderia) and Alphaproteobacteria (genus Sphingomonas and Ochrobacta-
crude oil waste sludge degradation, with 6, 7, 6, 1 and 13 recovered from rum). Interestingly, one novel PAH-degrading bacterial isolate that
CM, CT, HM, SM and PM microcosms, respectively (Figure 7). These exhibited the highest similarity of ~76.7% to members of genus Bacillus
isolates were also recovered after 30 days treatment using crude oil waste was also recovered from cow manure (CM) sample. These bacterial iso-
sludge as sole carbon source, yielding 0.1-3.12  104 CFU/g culturable lates were the most efficient degraders of the crude oil sludge. The
bacteria. Furthermore, 29 out of 33 isolates were positive for catechol evolutionary relationship of these isolates and their GenBank relatives
2,3-dioxygenase gene (C23O). Cultures found to be efficient degraders of are displayed in Table 3.

8
O. Ubani et al. Heliyon 8 (2022) e08945

Figure 6. The relative abundance of 31 bacterial phylotypes (based on OTUs) isolated from cow (CM), horse (HM), poultry (PM) and pig (SM) manure amended
samples after 10 months composting treatment of crude oil sludge. The putative identity based on 16S rDNA sequence of representative isolate and total number of
isolates recovered for each phylotype is provided in the brackets.

3.8. Microbial bioremediation of crude oil sludge PAHs using the enriched to 0.084 mg/kg, respectively, after 30 days of treatment (Figure 8a). The
bacterial consortium removal rates were 39.6–54.8, 77.8–98.3, 43.2–58.6, and 26.4–44.7%
for ΣPAHs, two-to three, three-to five and six-ring PAHs were observed in
In this study, an enriched consortia of the 33 efficient PAH-degraders BC1 treatment samples after 30 days treatment. In BC2 and BC3 treat-
were added to crude oil sludge (BC1)-, pyrene (BC2)- and anthracene- ments, higher reduction rates of 72.5–81.6 and 87.8–98.6% were
contaminated (BC3) media to test their bioaugmentation capacity. The observed for pyrene and anthracene, respectively.
PAHs were degraded gradually in media; total PAH (ΣPAHs), pyrene and To further gain insight on the viability and antagonistic effects of
anthracene contents dropped from 359.7 to 106.7, 14.0 to 3.36 and 42.0 bacterial isolates during PAHs degradation, a cultivation-independent

9
O. Ubani et al. Heliyon 8 (2022) e08945

Figure 7. The putative PAH-degrading bacteria isolated from compositing treatment of crude oil sludge using different manures. The bacterial isolates were identified
by 16S rDNA sequencing (average length 1450 bp) and used for constructing the cladogram. The percentage of replicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) are shown next to the branches. The four major phylogenetic clades (I, II, III and IV) of PAH-degrading bacteria is
illustrated using different colors. The putative identity at genera level, 2,6-DCPIP tests, plate counts and in the presence of catechol 2,3-dioxygenase is provided. Rating
based on the loss of 2-DCPIP indicator blue color due transfer of electrons from the degradation of PAHs by bacteria: -, no change; þ, minimal; þþ, moderate; and
þþþ, complete color change to colorless. z Plate counts after culturing in MSM-crude oil sludge for 30 days. The assays were performed in biological triplicates.

detection method using 16S rDNA amplicon sequencing was carried out clustering based on weighted UniFrac distance showed a clear separation
for three microcosms (BCI, BC2 and BC3) at day 30. Heatmap of the of pyrene treated samples (BC2) from other two treatments. Overall, BC1
normalized abundance at the genus level for bacteria in the three mi- had higher species richness (Chao-1 ¼ 89.1) than other two treatments
crocosms on day 30 is illustrated in Figure 8b. The result revealed that (Chao-1 ¼ 17 and 28 for BC1 and BC3, respectively). BC2 also had a
OTUs assigned to genus Sphingomonas, Pseudomonas, Microbacterium, higher species diversity and relative abundance of top 40 OTUs recov-
Rhodococcocus, Burkholderia-Paraburkholderia, unclassified Bradyrhizo- ered at day 30. The most abundant genus observed in BC2 included
biaceae, Bacillus, Delftia, Cellulomonas, Enterobacter and Aflpia were rela- Microbacterium, Sphingomonas, Rhodococcus, Burkholderia-Para-
tively viable and dominant across all samples. However, subtle variation burkholderia and unclassified Bradyrhizobiaceae. Interestingly, OTUs
in the enrichment of the bacterial groups was observed. Hierarchical assigned to genera such as Sporosarcina, Pseudoarthrobacter,

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O. Ubani et al. Heliyon 8 (2022) e08945

Table 3. Molecular identification of the putative PAH-degrading bacterial isolates. All sequences were compared with reference 16S rRNA gene sequences available in
the GenBank/EMBL/DDBJ databases using BLAST. The accession number to the NCBI, the closest type strain and the corresponding sequence is listed.
Isolate Accession NCBI Closest Type Strain Reference Sequence Similarity (%) Source of type strain
CO15 MK854828.1 Paenibacillus lautus strain NBRC 13380(T) NR_112724.1 99.8 Soil
C0102 MK854981.1 Bacillus pumilus isolate El-24-8 AJ494726.1 76.8 Marine sediments
CT51 MK854979.1 Microbacterium hominis strain DSM 12509 NR_042480.1 97.3 Lung aspirate
CT55 MK854980.1 Microbacterium hominis strain DSM 12509 NR_042480.1 97.3 Lung aspirate
MC10 MK854858.1 Micrococcus luteus strain NCCP 16831 CP043842.1 99.7 human
H121 MK854848.1 Rhodococcus soli strain DSD51W NR_134799.2 98.8 Park soils
CT121 MK854971.1 Sanguibacter soli strain DCY22 NR_044276.1 99.5 Ginseng field
CT61 MK854986.1 Gordonia amicalis strain IEGM 1273 NR_028735.1 99.5 Oil contaminated soil
H151 MK854850.1 Sphingopyxis bauzanensis strain BZ30 NR_117213.1 99.3 Oil contaminated soil
H3 MK854922.1 Paeniclostridium sordelli strain JCM 3814(T) NR_113140.1 99.5 Marine sediment
H4b MK854850.1 Rhodococcus degradans strain CCM 4446 NR_043535.1 100 Contaminated soil
H93 MK854855.1 Burkholderia lata strain 383 NR_102890.1 99.9 Forest soils
Hc10 MK854858.1 Micrococcus aloeverae strain DSM 27472(T) NR_075062.2 99.8 Aloe vera tissues
H4a MK854951.1 Cellulosimicrobium funkei strain W6122 NR_042937.1 99.3 Blood
PO101 MK854904.1 Bacillus kochii strain WCC 4582(T) NR_117050.1 98.5 Foods, pharmaceutical
manufacturing site
PO341 MK854924.1 Pseudoarthrobacter oxydans strain DSM 20119 NR_026236.1 99.8 Air
PO41 MK854926.1 Dietzia maris strain DSM 43672 NR_037025.1 99.0 Open soil
CO20 MK854831.1 Rhodococcus hoagiii strain ATCC 6939 NR_116691.1 99.9 Horse
PO129 MK854949.1 Bhargavaea beijingensis strain ge10 NR_117988.1 99.1 Ginseng root
CT10 MK854970.1 Enterococcus mundtii strain DSM 4838 CP018061.1 99.4 Soil
PO45 MK854932.1 Staphylococcus succinus strain 14BME20 CP018199.1 100 Fermented soybean food
PO124 MK854908.1 Lysinibacillus fusiformis strain DSM2898 NR_042072.1 100 Open soil
PO1i MK854914.1 Arthrobacter tecti strain LMG 22282 NR_042251.1 99.2 Deteriorated mural paintings
PO35 MK854925.1 Sporosarcina luteola strain NBRC 105378(T) NR_114283.1 99.7 Sea water
PO42d MK854965.1 Dietzia maris strain DSM 436782 NR_118596.1 100 Open soil
PO49a MK854936.1 Lysinibacillus pakistanensis strain NCCP-54 NR_113166.1 99.5 Soybean rhizosphere
PO35 MK854925.1 Sporosarcina luteola strain NBRC 105378 NR_114283.1 99.7 Soy sauce
PO44 MK854931.1 Staphylococcus epidermis strain 10091 NR_0369904 99.3 Skin
CO41 MK854834.1 Bacillis subtilis strain IAM 12118 NR_112116.2 99.9 Open soil
PO47 MK855493.1 Sanguibacter marinus strain 1-19 NR_042311.1 96.6 Coastal sediments
PO62 MK854943.1 Streptomyces pseudogriseolus strain NRRL B-3288 NR_043835.1 99.2 Soil
PO7 MK854944.1 Lysinibacillus pakistanensis strain NCCP-54 NR_113166.1 99.5 Soyabean rhizosphere
CO3i MK854833.1 Bacillus zhangzhouensis strain MCCC 1A08372 NR_148786.1 99.7 Aquaculture water
Pi131b MK854890.1 Ochrobactrum pecoris strain 08RB2639 NR_117053.1 100 Sheep
H131 MK854849.1 Pseudomonas chloritidismutans strain AW-1 NR_115115.1 98.4 Anaerobic chlorate-reducing
bioreactor
CT22 MK854978.1 Burkholderia metallica strain R-16017 NR_042636.1 99.7 Plants
Pi132a MK854889.1 Burkholderia lata strain 383 NR_104978.1 99.7 Forest soil

Cellulomicrobium and Ochrobactarum associated with bacterial con- bacterial community using high-throughput targeted 16S rDNA amplicon
sortium were only observed in BC2. In contrast, only BC3 exhibited sequencing to provide insight on the manure-induced community dy-
enriched abundance of genus Enterobacter. namics in the sludge microbiome during PAH degradation, (iii) carried
out the isolation, identification and partial characterization of 93 puta-
4. Discussion tive hydrocarbonoclastic bacteria and (iv) narrowed on a simplified
33-strains PAH-degrading bacterial consortium which might be useful in
Bioremediation has been established to be a reliable cost-effective designing bioaugmentation/biostimulation strategy for the treatment of
technology for oil spill and crude oil waste remediation [40, 41]. How- crude oil refinery wastes.
ever, its success is generally dependent on the natural microbial com-
munity populations, whose remediation capacity is greatly influenced by 4.1. Microcosm-based crude oil sludge PAH degradation under manure
nutrient availability and other in situ and ex situ physicochemical and treatments
environmental conditions. To improve the natural ability of microor-
ganisms to degrade contaminants in oil associated environments, several In engineered bioremediation for oil-contaminated soils and refinery
successful engineered bioremediation approaches utilizing bio- wastes, addition of N and P containing fertilisers to alleviate nutrient
augmentation (addition of known degraders of the contaminant) and or limitation is key in enhancing microbial activity and concomitant PAHs
biostimulation (addition of nutrients in the form of fertilizers) have been biodegradation [42, 44]. In this study, four animal manures character-
reported [40, 41, 42, 43]. In this study, we (i) investigated PAHs ized by variable levels of TN, TP and TOC (Table 1) were used in
degradation potential of autochthonous microbial community via bio- microcosm-based biostimulation and/or bioaugmentation of crude oil
stimulation and/or bioaugmentation by co-composting crude oil sludge sludge PAHs degradation. Our data demonstrated greater effect of the
with different manures amendments, (ii) performed survey on the total manure amendments in the stimulation of respiration activities and PAHs

11
O. Ubani et al. Heliyon 8 (2022) e08945

Figure 8. Bacterial consortium utility for bioremediation of crude oil sludge waste PAHs. a) PAH content by molecular weight on day 30. Error bars represent the
standard deviation of the mean of triplicate microcosms. b) Heatmap the normalized abundance at the genus level for bacteria in the three microcosms metagenomic
sequences (BCI, BC2 and BC3 included culture media supplemented with crude oil sludge, pyrene and anthracene, respectively, as sole carbon source) on day 30 based
on the weighted UniFrac distance.

degradation rates. This is consistent with findings that addition of Assessment of the nature of the hydrocarbons within the co-
organic waste materials such as sewage sludge and soybean meal [45] composting microcosms was also done to gain information on possible
and farm manures [41] enriched oil contaminated soils and refinery role of manure amendment in the biostimulation of indigenous micro-
wastes with nutrients, such as P and N, whose limitation is known to slow organisms capacity to efficiently degrade different PAH compounds in
down biodegradation processes [46]. Among the manure treatments, the crude oil sludge (Figure 2). GC/MS analysis revealed that the original
poultry manure (PM) treatment had a significantly higher CO2 evolution crude oil sludge was found to primarily consisting of LMW PAHs (2–4
(Figure 1a) and higher temperatures (Figure 1b) during composting, rings; 81.5%) and HMW PAHs (5–6 rings; 18.5%). Following manure
recording ~90% loss in the total PAH (ΣPAHs) after 300 days. The higher amendments, depletion rate between 76.1-99.9% was achieved for LMW-
temperature observed in the PM amendment could be attributed to its PAHs such as naphthalene, acenaphthene, anthracene, fluorene and
high N and P content and the existence of high diversity and density of phenanthrene (Figure 2a) after 300 days. HMW-PAHs such as perylene,
microorganisms therein, which may have stimulated microbial growth dibenzo [a,h] anthacene, benzo [ghi] perylene and benzo [e] acephe-
and PAH degradation activities. These results are consistent with reports nathrylene exhibited lower reduction (39.6–81.7%) (Figure 2b). In
of several researchers that have employed the application of poultry contrast, non-manure microcosm recorded similar depletion rate for
droppings for improved bioremediation of oil-polluted environments LMW PAHs (70.2–97.3%), but relatively very low reduction rate
[41, 47, 48]. Similarly, higher degradation rates leading to total PAH (<51.9%) for 5–6 rings PAHs. The recalcitrance of PAHs attributed to
losses >90% and relatively higher CO2 evolution was observed for other number of aromaticity determines the distinctive behaviors during
manure treatments. In contrast, non-manure microcosm (CT) showed a degradation, with 2–4 rings PAHs undergoing faster initial degradation,
baseline attenuation of 52% for total PAH. and followed by five-to six-rings PAHs soon afterward [49]. In this study

12
O. Ubani et al. Heliyon 8 (2022) e08945

manure amendment promoted considerable biodegradation of HMW plasticity, metabolic versatility and production of extracellular and
PAHs compared to CT. cellular biosurfactants by members of Firmicutes and Actinobacteria, have
been reported to enhance the uptake and biodegradation of hydrophobic
4.2. Shift in microbial community with manure amendments pollutants [60]. This may explain their predominance in CM microcosm.

Based on Baas-Becking hypothesis that “everything is everywhere, but 4.3. Identification of PAH-degrading bacteria and potential applications in
the environment selects” [50], we envisaged that deeper coverage of the bioaugmentation
co-composting microcosms would reveal pertinent manure-induced bio-
stimulation and bioaugmentation of in situ and ex situ bacterial community In this study, we also attempted to isolate and identify PAH-degrading
for the improved crude oil sludge remediation. Metagenomic analysis bacteria from the co-composting microcosms. A total of 33 putative PAH
showed that Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes degraders based on 2,6-DCPIP tests, cell viability and presence of cate-
were the predominant phyla present in all the microcosms at day 300. The chol 2,3-dioxygenase gene (C23O) (Figure 7) were identified. Consistent
shared genera between manure-treated and control microcosm included with metagenomic results, analysis of the partial 16S rDNA sequences
Pseudomonas, Delftia, Methylobacterium, Dietzia, Bacillus, Propionibacterium, showed that these putative PAH degraders belonged to the genera Ba-
Bradyrhizobium, Streptomyces, Achromobacter, Microbacterium and Sphin- cillus, Lysinibacillus, Microbacterium, Burkholderia, Dietzia, Rhodococcus,
gomonas, some of which has been previously reported in oil-contaminated Pseudomonas and Paeniclostridium, taxa that has been previously been
soils [41, 43, 45], with several members known for their ability to degrade isolated from crude oil sludge [61]. The detected GenBank relatives were
aliphatic and aromatic hydrocarbons [51, 52, 53]. Whereas Pseudomonas isolates from sources such as oil contaminated soils, marine sediments,
and Sphingomonas have been previously associated with degradation of rhizosphere, open field and forest soils (Table 3). The bulk of these
LMW PAHs [54, 55], fermentative, CO2-assimilating and methanogenic bacterial isolates were recovered from PM microcosms, but were het-
microorganisms (Bacillus, Methylobacterium and Achromobacter) are erogeneous across the 4 phylogenetic clades (Figure 7). In contrast, only
known to be key players in HMW PAH degradation [55]. In addition, one isolate assigned to genus Micrococcus was recovered SM sample.
Bacillus, Dietzia and Achromobacter are potent biosurfactant producers that Redox indicator 2,6-DCPIP (an electron acceptor) undergoes a colour
promotes efficient emulsification and eventual biodegradation of HMW change from blue (oxidised form) to colourless (reduced form). This color
PAHs [19, 56]. Bacteria from the genera such as Delftia, Bradyrhizobium, change can be used to determine the capability of microorganisms to
Microbacterium and Streptomyces can also decompose various aromatic utilise and to estimate the PAH biodegradation capacities of axenic bac-
compounds during denitrification [53, 55, 57]. Persistence of the terial cultures [41, 61]. In this study, all the bacterial isolates tolerated
above-mentioned groups in all microcosm treatments provided clues of and biodegraded crude oil sludge, positively reacting with the 2,6-DCPIP.
their autochthonous nature to either crude oil sludge, soil or wood chips However, 9 isolates exhibited very intense and rapid reaction with 2,
used for the experiments, and the important role they play in the baseline 6-DCPIP, completely changing the color from blue to colourless within
attenuation of crude oil sludge PAHs in absence of manure amendments. 3 days. This included: 3 isolates recovered from CM microcosm assigned
Interestingly, the manure-amended treatments were associated with to genus Rhodococcus, Paenibacillus and Bacillus; 3 species from HM
comparatively higher bacterial species richness and diversity estimates belonging genus Rhodococcus, Sphingomonas and Pseudomonas; and Lysi-
than CT (Table 2). Besides the shared bacterial groups, manure-treated nibacillus, Bhargavaea and Burkholderia. The elevated activities of these
microcosms were also characterized by presence of additional potential isolates in early stages of PAH degradation, indicate that they are fast PAH
hydrocarbon-degrading bacterial taxa such as Proteiniphilum, unclassified degraders, mainly associated with degradation of LMW PAHs. In support
Micrococcales, unclassified Lachnospiraceae, Sphingobium and Steno- of these findings, Obi et al. [61] also reported the isolation of Pseudomonas
trophomonas [58]. The observed higher relative abundance of the afore- and Bacillus species from crude oil sludge that had characteristic fast PAH
mentioned native and introduced bacterial groups due to degradation (decolourising the 2,6-DCPIP in the shortest possible time).
manure-treatment, therefore, points towards the contribution of manure To complement 2,6-DCPIP test, cell viability test of the isolates after 30
amendments in the improvement of co-composting microcosm microbial days incubation in MSM media supplemented with crude oil sludge as sole
diversity and the associated PAHs degradation. carbon and energy source was undertaken. All isolates yielded between
As shown in Figure 5, subtle variations in the bacterial community 0.1-3.12  104 CFU/g culturable bacteria, indicating their ability to grow
diversity were also observed within the manure-treatments. The rela- and utilize crude oil sludge PAHs. Further, PCR amplification with
tionship among the samples using Weighted UniFrac analysis revealed degenerate primers was also performed to screen for the presence of the
clustering into three distinct groups; the bacterial community structures cathecol-2,3-dioxygenase (C23O) genes in the isolates. The results showed
in PM and HM differing significantly from SM and CM. These results that all the isolates possessed the catabolic C23O genes, providing clues on
substantiated the fact that the microcosm microbial community struc- the their potential capability to degrade the crude oil sludge PAHs.
tures were impacted differently by the manure amendments. Increase in The functional profile of the selected bacterial isolates related to their
the members of class γ-proteobacteria (Pseudomonas, Rhodanobacter, ability to utilize crude oil PAHs suggests that crude oil sludge-soil-
Stenotrophomonas, Pseudoxanthomonas), β-proteobacteria (Achromobacter, woodchips-manure co-composting microcosms is a reservoir for the re-
Massilia, and Delftia), α-proteobacteria (Sphingobium, Methylobacterium covery of important bacterial species that can be exploited for engineered
and unclassified Rhizobiales), were prominent in PM and HM, while Fir- remediation processes. In this study, an enriched consortia of the iden-
micutes (Bacillus, KCM-B-112, Nocardiopsis, Clostridium sensu stricto 1, tified 33 PAH-degraders was used for laboratory scale bioaugmentation
Mycobacterium, Corynebacterium and Rhodococcus) and Actinobacteria study of crude oil sludge in absence of manure amendments. The treat-
(Propionbacterium, Corynebacterium) were found to predominate CM ment resulted in accelerated degradation of total PAHs, anthracene and
sample. As Proteobacteria are known for their catabolic versatility, genetic pyrene within 30 days (Figure 8a), with viability of these strains
plasticity and metabolic diversity leading to broad substrate specificities confirmed by 16S rDNA amplicon high-throughput sequencing. These
for several classes of hydrocarbons in environments such as natural oil results demonstrate the potential application of the bacterial consortia as
deposits, asphalt, crude oil, oil sand, oil contaminated water, soil and a basic microbial agent for the bioaugmentation of animal manure-
sludge [59], their enrichment in PM and HM microcosms system may not treated co-composting bioremediation of crude sludge waste.
be unconnected to the manure amendments. Further, the copiotrophic
nature of the α, β, and γ classes of Proteobacteria, imply that the 5. Conclusion
amendment with manures rich in utilizable C, N and other nutrients
could be responsible for the increase in the relative abundance of the In conclusion, the current study attempted static co-composting
phylum Proteobacteria in PM and HM systems. In contrast, the genetic technique with different manures to understand the bacterial diversity

13
O. Ubani et al. Heliyon 8 (2022) e08945

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